Crystalline forms of therapeutic compounds and uses thereof.

(54) Title: CRYSTALLINE FORMS OF THERAPEUTIC COMPOUNDS AND THEIR USES. (54) Title: CRYSTALLINE FORMS OF THERAPEUTIC COMPOUNDS AND USES THEREOF.

(57) Summary

Certain crystalline forms of Compound 3, as well as pharmaceutical compositions, employing the crystalline forms are described. Particles (eg nanoparticles) comprising such crystalline forms or pharmaceutical compositions are also described. In certain examples, the particles are penetrating mucus particles (MPP). The present invention further relates to methods of treating or preventing disease using crystalline forms or pharmaceutical compositions.

(57) Abstract

Described herein is certain crystalline forms of Compound 3, as well as pharmaceutical compositions employing the crystalline forms. Also provided are particles (eg, nanoparticles) comprising such crystalline forms or pharmaceutical compositions. In certain examples, the particles are mucus penetrating particles (MPPs). The present invention further relates to methods of treating or preventing diseases using crystalline forms or pharmaceutical compositions.

CRYSTALLINE FORMS OF THERAPEUTIC COMPOUNDS AND THEIR

APPLICATIONS

FIELD OF THE INVENTION

This invention relates to crystalline forms of a therapeutic compound useful for treating diseases, including proliferative diseases and angiogenesis related diseases, such as cancer and macular degeneration.

BACKGROUND OF THE INVENTION

Growth factors play an important role in angiogenesis, lymphangiogenesis, and vasculogenesis. Growth factors regulate angiogenesis in a variety of processes, including embryonic development, wound healing, as well as various aspects of female reproductive function. Undesirable or pathological angiogenesis is associated with diseases including diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi's sarcoma, and hemangioma (Fan et al., 1995, Trends Pharmacol. Sel. 16: 57 66; Folkman, 1995, Nature Medicine 1:27, 31). Angiogenic eye conditions represent the main cause of irreversible vision loss in developed countries. For example, in the United States, retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration are the leading causes of blindness in infants, working-age adults, and the elderly, respectively. Efforts have been made to inhibit angiogenesis in the treatment of these conditions (R. Roskoski Jr., Critical Reviews in Oncology / Hematology, 62 (2007), 179-213).

Therefore, there is a need for new therapeutic compounds for the treatment of diseases associated with aberrant signaling of growth factors and diseases related to angiogenesis, such as cancer, macular degeneration and diabetic retinopathy.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to crystalline forms of compound of 7- (3- (4- (4-fluoro-2-methyl-1 H-indole-5-loxy) -6-methoxyquinazole-7- ¡Lox!) Prop! L) -2-oxa-7-azaspiro [3.5] nonane, mentioned here as Compound 3 as shown below:

<img file="MX2016005668A_D0001.tif" />

(compound 3)

In one embodiment, the present invention is compound 3 illustrated above, 7- (3- (4- (4-fluoro-2-methyl-1H-indole-5-yloxy) -6methoxyquinazole-7-yloxy) propyl) -2-oxa-7-azaspiro [3.5] nonane, in crystalline form A. In certain embodiments, the crystalline form is a crystalline form having a powder X-ray diffraction pattern (XRPD) with peaks approximately 6.11, 9.63, 16.41, 18.60, 20.36 and 23.0110.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and

3.8610.3 A in d-separation. In other embodiments, the crystalline form also has XRPD peaks around 11.46, 12.26, 18.16, 19.51, 21.12, and 25.7110.3 degrees two theta or 7.71, 7.22, 4.88, 4.55, 4, 20 and 3.4610.3 A in d-separation. In other embodiments, the crystalline form also has XRPD peaks around 11.10, 15.66, 17.54, 22.31, 24.79, and 28.9010.3 degrees two theta or 7.96, 5.65, 5.05, 3.98, 3.59, and 3.0910.3 A at d-separation. In other modalities, the crystalline form has an XRPD pattern with peaks of around 6.11, 9.63, 11.10, 11.46, 12.26, 15.66, 16.410, 17.54, 18.16, 17.1 1, 19.51, 20.36, 21.12, 22.31, 23.01, 24.79, 28.90 and 25.7110.3 degrees two theta or 14.45, 9.17, 7.96, 7.71, 7.22, 5.65, 5.40, 5.05, 4.88, 4.77 , 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.0910.3 A in separation.

In other embodiments, the present invention provides 7- (3 (4- (4-fluoro-2-methyl-1 H-indole-5-yloxy) -6-methoxy, quinoline-7-yloxy) prop! l) -2-oxa-7-azaspiro [3.5] nonane, in crystalline B form. In certain embodiments, the crystalline form is crystalline form B that has a powder X-ray diffraction pattern (XRPD) with peaks at approximately 7.70, 13.53, 17.27, 18.44, 19.73, 23.10, and 26.07 ± 0 , 3 degrees two theta or 1 1.47, 6.54, 5.13, 4.81, 4.50, 3.85 and 3.41 ± 0.3 A in d-separation. In other embodiments, the crystalline form B also has XRPD peaks around 9.87, 12.88, 14.40, 15.45, 21.14, and

26.8410.3 degrees two theta or 8.96, 6.87, 6.14, 5.73, 4.20 and 3.32 ± 0.3 A in d-separation. In other embodiments, crystalline Form B also has XRPD peaks at approximately 10.69, 16.42, 18.90, 22.56, and 29.12 ± 0.3 degrees two theta or 8.27, 5.39, 4.69 , 3.94 and 3.06 ± 0.3 A in d-separation. In yet other embodiments, crystalline Form B has an XRPD pattern with peaks around 7.70, 9.87, 10.69, 12.88, 13.53, 14.40, 15.45, 16.42, 17.27, 18.44, 18.90, 21, 14, 22.56, 19.73, 23.10, 26.07, 26.84 and 29.12 ± 0.3 degrees two theta or 1 1.47, 8.96, 8.27, 6.87, 6.54, 6.14, 5.73, 5.39, 5.13, 4.81, 4.69, 4.50, 4.20, 3.94, 3.85, 3.41, 3.32 and 3.0610.3 A in d-separation.

In one aspect, the present invention relates to a compound having the formula

H in crystalline form A.

In another aspect, the present invention relates to a crystalline form of a compound having the formula

<img file="MX2016005668A_D0002.tif" />

wherein said crystalline form is crystalline Form A having a powder X-ray diffraction pattern (XRPD) with peaks at approximately 6.11, 9.63, 16.41, 18.60, 20.36 and 23.01 ± 0.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and 3.8610.3 A in separation.

In another embodiment, the present invention relates to a compound having the formula

<img file="MX2016005668A_D0003.tif" />

in crystalline B form.

In another embodiment, the present invention relates to a crystalline form of a compound having the formula

<img file="MX2016005668A_D0004.tif" />

<img file="MX2016005668A_D0005.tif" />

wherein said crystalline form is crystalline form B having a powder X-ray diffraction pattern (XRPD) with peaks at approximately 7.70, 13.53, 17.27, 18.44, 19.73, 23.10, and 26.07 ± 0, 3 degrees two theta or 1 1.47, 6.54, 5.13, 4.81, 4.50, 3.85 and 3.4110.3 A in d-separation.

In another aspect, the present invention relates to a process for preparing a crystalline form of compound 3. In certain embodiments, the present invention relates to a method for preparing a crystalline form of compound 3. In other embodiments, the method of preparation Crystalline Form A comprises wet grinding of a slurry comprising an amorphous form of Compound 3 and a nonionic surfactant to obtain the nanoparticles of the compound. In yet other embodiments, the resulting crystalline Form A nanoparticles have an XRPD pattern with peaks at approximately 6.11, 9.63, 16.41, 18.60, 20.36, and 23.0110.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and

3.8610.3 A in d-separation. In other embodiments, crystalline Form A also has XRPD peaks around 11.46, 12.26, 18.16, 19.51, 21.12, and 25.7110.3 degrees two theta or 7.71, 7.22, 4.88, 4.55, 4 , 20 and 3.4610.3 A in d-separation. In other embodiments, the crystalline form A also has XRPD peaks around 11.10, 15.66, 17.54, 22.31, 24.79, and 28.9010.3 degrees two theta or 7.96, 5.65, 5.05, 3.98, 3.59, and 3.0910.3 A at d-separation. In yet other embodiments, crystalline Form A possesses an XRPD pattern with peaks around 6.11, 9.63, 1 1.10, 1 1.46, 12.26, 15.66, 16.410,

17.54, 18.16, 17.1 1, 19.51, 20.36, 21.12, 22.31, 23.01, 24.79, 28.90 and 25.71 ± 0.3 degrees two theta or 14.45, 9.17, 7.96, 7.71, 7, 22, 5.65, 5.40, 5.05, 4.88, 4.77, 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.09 ± 0.3 A in d-separation.

In other embodiments, the present invention relates to a method for the preparation of crystalline form B of compound 3. In certain embodiments, the method of preparing crystalline form B comprises a) dissolving the amorphous form of compound 3 in water and acetone; b) the crystallization of compound 3 from a mixture of solvent composed of water and acetone; and c) isolation of crystalline form B of compound 3 from the solvent mixture. In certain embodiments, the starting compound 3 is amorphous. In particular embodiments, the crystalline Form B preparation method uses a solvent mixture consisting of 4: 1 acetone: water. In other embodiments, the method of preparing crystalline Form B further comprises the step of heating the solvent mixture to dissolve the compound and / or cooling the solvent mixture to allow crystal formation. In some embodiments, the resulting crystalline Form B has an XRPD pattern with peaks at approximately 7.70, 13.53, 17.27, 18.44, 19.73, 23.10, and 26.07 ± 0.3 degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.50, 3.85 and 3.41 ± 0.3 A in d-separation. In other embodiments, the crystalline form B also has XRPD peaks around 9.87, 12.88, 14.40, 15.45, 21.14, and 26.84 ± 0.3 degrees two theta or 8.96, 6.87, 6.14, 5.73, 4.20, and 3.32 ± 0.3 A at d- separation. In other embodiments, crystalline Form B also has XRPD peaks at approximately 10.69, 16.42, 18.90, 22.56, and 29.12 ± 0.3 degrees two theta or 8.27, 5.39, 4.69 , 3.94 and 3.06 ± 0.3 A in d-separation. In yet other embodiments, crystalline Form B has an XRPD pattern with peaks around 7.70, 9.87, 10.69, 12.88, 13.53, 14.40, 15.45, 16.42, 17.27, 18.44, 18.90, 21, 14, 22.56, 19.73, 23.10, 26.07, 26.84 and 29.12 ± 0.3 degrees two theta or 11.47, 8.96, 8.27, 6.87, 6.54, 6.14, 5.73, 5.39, 5.13, 4.81, 4.69, 4.50, 4.20, 3.94 , 3.85, 3.41, 3.32 and 3.06 ± 0.3 A in separation.

In another aspect, the present invention relates to pharmaceutical compositions and kits for treating diseases, including proliferative diseases, eye diseases, dermatological diseases, inflammatory diseases, autoimmune diseases, auto-inflammatory diseases and metabolic diseases comprising a crystalline form of compound 3 . In another aspect, the present invention provides methods for using a crystalline form of compound 3 to study inhibition of growth factor signaling and / or to treat and / or prevent proliferative diseases, eye diseases, dermatological diseases, inflammatory diseases, diseases autoimmune, auto-inflammatory diseases and metabolic diseases. In certain particular aspects, a crystalline form of compound 3 is used in the treatment of angiogenesis related diseases.

In another aspect, the present invention provides pharmaceutical compositions comprising crystalline forms of compound 3, wherein the pharmaceutical compositions optionally comprise an acceptable pharmaceutical carrier. In certain embodiments, the disclosed pharmaceutical compositions include a therapeutically effective amount of a crystalline form of compound 3. In certain embodiments, the pharmaceutical composition may be useful in treating proliferative diseases (eg, cancers, benign tumors, inflammatory diseases, autoimmune diseases) and / or eye diseases (eg, macular degeneration, glaucoma, diabetic retinopathy, retinoblastoma, edema, uveitis, dry eye, blepharitis, and post-surgical inflammation) in a subject in need thereof. The pharmaceutical composition can also be useful for the inhibition of abnormal angiogenesis and / or aberrant signaling of a growth factor in a subject or cell.

In some embodiments, the crystalline forms of Compound 3 can be intended for delivery to the subject's tissues with mucus (eg, eye, respiratory tract, gastrointestinal tract, genito-urinary tract), which is an adhesive and viscoelastic substance that traps most foreign objects (for example, microorganisms, particles, dust). Compound or particles that are immobilized in the mucus are quickly removed by mucus removal mechanisms; therefore, they are not capable of effectively imparting the desired therapeutic effect. In these tissues, for the compound to be effective, it must penetrate i

Quickly mucus and / or avoid mucus removal mechanisms. Therefore, modifying the mucoadhesive compounds or compound-containing particles with a coating to reduce mucoadhesion, and decreasing the particle size of the compound can allow for efficient delivery and therapeutic effect.

In one aspect of the invention, the crystalline forms of compound 3 of the invention are formulated as mucus penetrating particles or mucus penetrating crystals (collectively, MPPs) suitable for administration (eg, topical, inhalation, injection ) to the tissues of the subjects that have mucus (for example, eye, respiratory tract, gastrointestinal tract, genito-urinary tract). In certain embodiments, the particles comprising a crystalline form of Compound 3 (eg, crystalline Form B) are mucus penetrating. MPPs can include a layer around a core. The nuclei may primarily contain a crystalline form of Compound 3, or the nucleus may be a polymeric nucleus with the crystalline form of Compound 3 encapsulated in the polymer. In certain embodiments, the MPPs are nanoparticles (eg, particles having an average diameter of at least about 10nm and less than about 1pm). MPPs can be useful in the delivery of pharmaceutical agent to a subject. In certain embodiments, MPPs are capable of delivering the crystalline form of Compound 3 to or through the mucosa of a subject.

Another aspect of the invention relates to pharmaceutical compositions comprising particles composed of crystalline forms of compound 3. In a particular embodiment, the particles comprise crystalline Form B of compound 3. In another embodiment, the particles constitute a crystalline Form A of the compound. 3. In certain embodiments, pharmaceutical compositions are useful for delivering crystalline forms of Compound 3 to a subject.

In another aspect of the invention, the present invention provides pharmaceutical compositions comprising a plurality of particles comprising (i) a core composed of a crystalline form of compound 3, and (ii) a layer of surface alteration agent surrounding the nucleus, where the surface alteration agent is present on the outer surface of the nucleus at a density of at least 0.01 surface alteration agent per nm² and optionally at least one pharmaceutically acceptable excipient. In some embodiments, the surface-altering agent is a back-block copolymer of the structure (hydrophilic block) - (hydrophobic block) - (hydrophilic block). In some respects, it is a block copolymer of three PLURONIC or poloxamer. In other aspects, the surface alteration agent is a polyvinyl alcohol or a polysorbate. In a preferred aspect, the core comprises crystalline Form B of compound 3. In another, the core comprises crystalline Form A of Compound 3.

In certain embodiments, the compound, particle, or pharmaceutical composition is formulated to be penetrating for mucus.

Another aspect of the present invention relates to methods of treatment and / or prevention of a disease associated with abnormal angiogenesis in a subject in need thereof.

Another aspect of the present invention relates to methods of treating and / or preventing a disease associated with aberrant signaling of a growth factor signaling pathway in a subject in need thereof.

In another aspect, the present invention provides methods of inhibiting angiogenesis in a subject in need thereof.

In another aspect, the present invention provides methods of inhibiting aberrant signaling of a growth factor signaling pathway in a subject or cell. In certain modalities, growth factor is associated with angiogenesis. In certain modalities, the growth factor is VEGF.

The methods of the present invention include administration to a subject of an effective amount of a crystalline form of compound 3 or pharmaceutical compositions thereof of the invention. The diseases include proliferative diseases, eye diseases, dermatological diseases, inflammatory diseases, autoimmune diseases, autoinflammatory diseases and metabolic diseases. In certain embodiments, the effective amount is a prophylactically effective amount.

In another aspect, the present invention provides kits of a crystalline form of compound 3. Kits of the invention can include a single dose or multiple doses of a crystalline form of compound 3, or pharmaceutical compositions thereof. The kits can be useful for the treatment of proliferative diseases, eye diseases, dermatological diseases, inflammatory diseases, autoimmune diseases, autoinflammatory diseases and metabolic diseases. In certain embodiments, the kits described herein may be useful for treating and / or preventing a disease associated with abnormal angiogenesis and / or with aberrant signaling of a growth factor in a subject in need thereof. The kits may also be useful for the inhibition of abnormal angiogenesis and / or aberrant signaling of a growth factor signaling pathway in a subject in need thereof. In certain embodiments, the kit also includes instructions for administering crystalline forms of compound 3 of the invention. The kits may also include packaging information describing the use or prescribing information for the subject or a healthcare professional. Such information may be required by a regulatory agency such as the United States Food and Drug Administration (FDA). The kit may also optionally include a crystalline form delivery device for Compound 3 or the composition thereof, for example, a dropper for ocular administration or a syringe for parenteral administration.

Details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will become apparent from the detailed description, figures, examples, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a representative X-ray powder diffraction pattern (XRPD) for crystalline Form A of compound 3.

Figure 2 provides a representative standard XRPD of crystalline Form B of compound 3.

Figure 3 provides a representative differential scanning calorimetry (DSC) thermogram of crystalline Form B of compound 3.

Figure 4 provides a representative thermogravimetric analysis (TGA) thermogram of crystalline Form B of compound 3.

Figure 5 provides an XRPD standard for crystalline Form B of compound 3 after formation (bottom fingerprint) and after being suspended for 7 weeks (top fingerprint).

Figure 6 provides an XRPD pattern of crystalline Form B of compound 3 (bottom fingerprint) and an XRPD pattern of a grinding mixture of amorphous compound 3 and crystalline Form B of compound 3 (top fingerprint).

Figure 7 provides XRPD patterns, from bottom to top, for: Crystalline Form A of Compound 3, Crystalline Form B of Compound 3, a mixture of Crystalline Forms A and B that were separately milled and then combined (t = 0), a mixture of Crystalline Forms A and B that were separately milled and then combined and stored at room temperature for&gt; 2 months, and a mixture of crystalline Forms A and B which were separately ground and then combined and stirred at room temperature for 5 weeks.

Figure 8 is a PK profile for compound 3 in Gottingen mini-pig choroid tissue after topical administration.

Figure 9 is a PK profile for compound 3 in Gottingen mini-pig retina tissue after topical administration.

Figure 10 is a PK profile for compound 3 in Gottingen mini-pig plasma after topical administration.

Definitions

Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^to ed., inner cover and specific functional groups are generally defined as described therein. Furthermore, the general principles of organic chemistry, as well as specific functional portions and reactivity, are described in Thomas Sorrell, Organic Chemistry, Universiti Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th edition, John Wiley &amp; Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^or edition, Cambridge Universiti Press, Cambridge, 1987.

As used herein, when referring to powder X-ray diffraction peak positions (XRPD), approximately means ± 0.3, preferably ± 0.2, more preferably ± 0.1, more preferably ± 0.05, and even more, preferably ± 0.02.

Other definitions

The following definitions are more general terms used in the present application.

The term polymorphic refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof) in a given crystal packaging arrangement. All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns (eg XRPD patterns), infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and / or solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause a crystalline form to dominate. A particular method for characterizing different crystalline forms of a compound is X-ray powder diffraction analysis (XRPD), which is a technique that is well known in the art. Various polymorphs of a compound can be prepared by crystallization under different conditions. As used herein, the term crystal form or crystalline form refers to a particular polymorph of a compound that has one or more identifying characteristics, for example, a certain X-ray diffraction or XRPD pattern.

A subject for which administration is contemplated includes, but is not limited to, human beings (i.e., a man or woman of any age, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (eg, young adults, middle-aged, adult, or older adult)) and / or other non-human animals, eg, mammals (eg, primates (eg, Java macaques, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs and birds (eg, commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In certain modalities, the animal is a mammal. The animal can be male or female at any stage of development. The animal can be transgenic animals or genetically animals

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<td colspan="2">human. In certain modalities, the</td><td>animals</td><td>a</td><td>fish.</td><td>A</td>

<td>patient</td><td>refers to a subject</td><td colspan="2">human with</td><td>need</td><td>of</td>

<td>treatment</td><td>of an illness.</td><td></td><td></td><td></td><td></td>

<td>The</td><td>terms manage,</td><td>than</td><td colspan="2">administers,</td><td> 0</td>

Administration, as used herein, refers to implantation, absorption, ingestion, injection, inhalation, or otherwise introduction of a crystalline form of Compound 3, or a pharmaceutical composition thereof, into or on a subject. .

As used herein, the terms "treatment", "treating" and "treating" refer to reversing, mitigating, delaying the onset of, or inhibiting the progression of a disease described herein. In some modalities, treatment may be administered after one or more developed or observed signs or symptoms of the disease. In other modalities, treatment can be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (eg, in light of the history of symptoms and / or in light of genetic or other susceptibility factors) or exposure to a pathogen). Treatment can also be continued after symptoms have resolved, for example to delay or prevent recurrence.

As used herein, the terms condition, disease, and disorder are used interchangeably.

An effective amount of a crystalline form of compound 3 described herein refers to an amount sufficient to elicit a desired biological response, i.e., treatment of the condition. As appreciated by ordinaries of skill in this art, the effective amount of a crystalline form of Compound 3 described herein may vary depending on factors such as the desired biological end point, the pharmacokinetics of the crystalline form of Compound 3, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses prophylactic and therapeutic treatment. For example, in the treatment of cancer, an effective amount of a crystalline form of compound 3 described herein can reduce tumor burden or stop the growth or spread of a tumor. In the treatment of macular degeneration, an effective amount of a crystalline form of compound 3 described herein can improve eyesight, reduce the risk of vision loss, loss of central vision, or prevent them from worsening.

A "therapeutically effective amount" of a crystalline form of compound 3 described herein is an amount sufficient to provide a therapeutic benefit in treating a condition or to delay or reduce one or more symptoms associated with the disease. A therapeutically effective amount of a crystalline form of compound 3 described herein means an amount of a crystalline form of compound 3, alone or in combination with other therapies, that provides a therapeutic benefit in treating the condition. The term "therapeutically effective amount" may encompass an amount that improves therapy, reduces or avoids the signs, symptoms, or causes of the condition, and / or improves the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount of the crystalline form of compound 3 or the composition thereof is the amount necessary to inhibit angiogenesis in a subject.

A prophylactically effective amount of a crystalline form of compound 3 described herein is an amount sufficient to prevent a condition, or one or more of the symptoms associated with the disease, or to prevent its recurrence. A prophylactically effective amount of a crystalline form of compound 3 described herein means an amount of a crystalline form of compound 3, alone or in combination with other agents, that provides a prophylactic benefit in preventing disease. The term "prophylactically effective amount" may encompass an amount that improves prophylaxis or improves the prophylactic efficacy of another prophylactic agent.

As used herein, the term "growth factor" refers to a naturally occurring substance (eg, a protein or spheroid hormone) capable of stimulating cell growth, proliferation, and / or cell differentiation. Growth factors can act as signaling molecules between cells and / or promote cell maturation and differentiation.

Proliferative disease refers to a disease that occurs due to abnormal growth or spread by multiplication of cells (Walker, Cambridge Dictionary of Biology, Cambridge Universiti Press: Cambridge, UK, 1990). A proliferative disease can be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the migration of pathological cells from their normal location (eg, metastasis of neoplastic cells); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (eg collagenases, gelatinases, and elastases); or 4) pathological angiogenesis as in proliferative tumor retinopathy and metastasis. Illustrative proliferative diseases include cancers (i.e., malignant neoplasms), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

As used herein, the term physiological angiogenesis refers to the process through which new blood vessels are formed from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels of a developing embryo are formed through vasculogenesis, after angiogenesis is responsible for most of the blood vessels during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a critical step in the transition of tumors from a benign to a malignant state, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis can be chemically stimulated by angiogenic proteins, such as growth factors (eg, VEGF).

The terms neoplasm and tumor are used interchangeably herein and refer to an abnormal mass of tissue, in which the growth of the mass exceeds and is not coordinated with the growth of normal tissue. A neoplasm or tumor can be benign or malignant, depending on the following characteristics: degree of cell differentiation (including morphology and functionality), growth rate, local invasion, and metastasis. A benign neoplasm is generally well differentiated, typically has a slower growth than a malignant neoplasm, and remains localized at the site of origin. Furthermore, a benign neoplasm does not have the ability to infiltrate, invade, or metastasize to distant sites. Illustrative benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, skin tags, senile angiomas, seborrheic keratosis, lentigines, and sebaceous hyperplasias. In some cases, certain benign tumors can later give rise to malignant neoplasms, which may be the result of additional genetic changes in a subpopulation of tumor neoplastic cells, and these tumors are called pre-malignant neoplasms. An illustrative pre-malignant neoplasm is a teratoma. In contrast, a malignant neoplasm is generally poorly differentiated (anaplasia) and typically has rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the ability to metastasize to distant sites. The term metastatic, metastatic, or metastasizing refers to the spread or migration of cancer cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a secondary tumor or secondary cell mass. of the original or primary tumor tissue type and not of the organ or tissue in which the secondary (metastatic) tumor is located. For example, prostate cancer that has migrated to bone is said to be metastatic prostate cancer and includes prostate cancer cancer cells that grow in bone tissue.

As used herein, the term cancer refers to a malignancy (Stedman's Medical Dictionary, 25^to ed .; Hensil ed .; Williams &amp; Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to: acoustic neuroma; adenocarcinoma; adrenal cancer; anal cancer; angiosarcoma (eg, limfangiosarcoma, limfangioendotel osarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (for example, cholangiocarcinoma); bladder cancer, breast cancer (eg, adenocarcinoma of the breast, papillary carcinoma of the breast, breast cancer, medullary carcinoma of the breast); brain cancer (eg, meningioma, gIioblastomas, glioma (eg, astrocytoma, oligodendrogliorna), medulloblastoma); bronchial cancer; carcinoid tumor; cervical cancer (eg, cervical adenocarcinomas); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (eg, colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (eg Kaposi's Sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (eg, cervical cancer, uterine sarcoma); esophageal cancer (eg esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (eg, intraocular melanoma, retinoblastoma); hi pe reos in of the family; gallbladder cancer; gastric cancer (for example, adenocarcinoma of the stomach); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (eg, squamous cell carcinoma of the head and neck, oral cancer (eg, oral squamous cell carcinoma), throat cancer (eg, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, cancer of the the oropharynx); hematopoietic cancers (eg, leukemia such as acute lymphocytic leukemia (ALL) (eg, B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (eg, B-cell AML, T-cell AML) , chronic myelocytic leukemia (CML) (eg, B-cell CML, T-cell CML) and chronic lymphocytic leukemia (CLL) (eg, B-cell CLL, T-cell CLL); lymphoma, such as Hodgkin lymphoma (HL) (eg, B-cell HL, T-cell HL), and non-Hodgkin lymphoma (NHL) (eg, B-cell NHL such as diffuse large cell lymphoma (DLCL) (eg, diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (LLC / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (eg, lymphomas of mucosa-associated lymphoid tissue (MALT), Marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma) primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), lymphoma of immunoblastic large cells, precursor lymphoblastic B lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (eg, cutaneous T-cell lymphoma (CTCL) (eg, mycosis fungiode, Sezary syndrome), lymphoma of noblastic T cells, extranodal natural killer T cell lymphoma, enteropathy-like T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM), heavy chain disease (eg, alpha chain disease, gamma chain disease, mu chain disease);

hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (eg nephroblastoma aka Wilms tumor, renal cell carcinoma); liver cancer (eg, hepatocellular cancer (HCC), malignant hepatoma); lung cancer (eg, bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (eg, systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (eg polycythemia vera (PV), essential thrombocytosis (ET), angiogenic myeloid metaplasia (AMM) aka myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL) ), hypereosinophilic syndrome (HES), neuroblastoma; neurofibroma (eg, neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine carcinoma (eg, gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (eg, bone cancer); ovarian cancer (eg, cystadenocarcinoma, embryonal ovarian carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (eg, pancreatic adenocarcinoma, introductory papillary muscle neoplasm (IMP), islet cell tumors); cancer of the penis (for example, Pagel disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT);

plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (for example, prostate adenocarcinoma); rectal cancer; Rhabdomyosarcoma; salivary gland cancer; skin cancer (eg, squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC); cancer of the small intestine (eg, cancer of the appendix); soft tissue sarcoma (eg, malignant fibrous histiocytoma (HFM), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, myrosarcoma fibrosarcoma); sebaceous gland carcinoma; small intestine cancer; carcinoma of the sweat gland; synovioma; testicular cancer (eg seminoma, testicular embryonal carcinoma); thyroid cancer (eg, papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid cancer); cancer of the urethra; vaginal cancer; and cancer of the vulva (for example, Paget's disease of the vulva).

As used herein, the term "inflammatory disease or inflammation" refers to a disease caused by, resulting from, or resulting in inflammation. The term inflammatory disease can also refer to a poorly regulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and / or T lymphocytes that leads to damage to the abnormal tissue and / or cell death. An inflammatory disease can be an acute or chronic inflammatory condition and can be the result of infections or non-infectious causes. Inflammatory diseases include, without limitation, atherosclerosis, arteriesclerosis, autoimmune disorders, multiple sclerosis, lupus erythematosus, polymyalgia rheumatica (PMR), gout, arthritis, osteoarthritis, tendinitis, 5 bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis , Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (eg Type I), myasthenia gravis, Hashimoto's thyroiditis 10, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory dermatosis, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, 15 berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, interstitial giant cell pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of angiitis (arteritis of temporal and polyarteritis nodosa), delayed-type dermatosis (delayed-type hypersensitivity reactions) for example, poison ivy dermatitis), pneumonia, inflammation of the respiratory tract, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hay fever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemic lesions (ischemia), reperfusion injury, allograft rejection, host rejection- versus graft, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryodenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, pancreatitis, parotitis, pericarditis, pharyngitis , proctitis, pneumonitis, prostatitis, rhinitis, sinusitis, salpingitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomilitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fascilitis and necrotizing enterocolitis. Inflammatory eye diseases include, but are not limited to, eye allergy, uveitis (eg, anterior uveitis, intermediate uveitis, and posterior uveitis), conjunctivitis, panuveitis, cyclitis, scleritis, episcleritis, retrobulbar optic neuritis, optic neuritis, keratitis ( for example, immune keratitis and infectious keratitis), blepharitis, meibomian gland disease or dysfunction, corneal ulcer, conjunctival ulcer and symptoms caused by them, inflammatory eye diseases caused by eye disorders, inflammatory eye diseases caused by physical injury, post-surgical inflammation, and dry eye (eg, dry eye syndrome).

As used herein, an autoimmune disease refers to a disease derived from an inadequate immune response of a subject's body against substances and tissues normally present in the body. In other words, the immune system makes a mistake somewhere in the body as a pathogen and attacks its own cells. This may be restricted to certain organs (for example, in autoimmune thyroiditis) or involves a certain tissue in different places (for example, Goodpasture's disease that can affect the basement membrane in the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppressants, for example, drugs that decrease the immune response. Illustrative autoimmune diseases include, but are not limited to, glomerulonephritis, Goodspature syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, lupus erythematosus systemis, psoriasis, ulcerative colitis, , nt ifosfo I ide antibody syndrome, scleroderma perfigus vulgaris, ANCA-associated vasculitis (for example, Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lime's arthritis, GuillainBarre syndrome, Hashimoto's thyroiditis, and cardiomyopathy a.

The term autoinflammatory disease refers to a category of diseases that are similar but different from autoimmune diseases. Autoinflammatory and autoimmune diseases share common characteristics in that both groups of disorders result from the immune system attacking the subject's own tissues and resulting in increased inflammation. In autoinflammatory diseases, a subject's innate immune system causes inflammation for unknown reasons. The innate immune system reacts even though it has never found antibodies or antigens in the subject. Autoinflammatory disorders are characterized by intense episodes of inflammation that result in symptoms such as fever, rash, or swelling. These diseases also carry the risk of amyloidosis, a life-threatening accumulation of a protein in the blood in vital organs. Autoinflammatory diseases include, but are not limited to, Familial Mediterranean Fever (FMF), Neonatal Onset Multisystemic Inflammatory Disease (NOMID), Periodic Periodic Syndrome Receptor Tumor Necrosis Factor (TNF) (TRAPS), Interleukin Receptor Antagonist Deficiency -1 (DIRA), and BehQet disease.

The term biological sample refers to any sample of tissue samples (such as tissue sections and tissue biopsies); cell samples (eg, smears (such as PAP or blood smears) or microdissection cell samples); whole organism samples (such as bacteria or yeast samples); or cell portions, fragments or organelles (such as that obtained by lysis of cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, tissue biopsy (for example, obtained by surgical biopsy or needle biopsy), aspirates of nipple, milk, vaginal fluids, saliva, swabs (such as oral swabs), or any material containing biomolecules derived from a first biological sample. Biological samples also include those biological samples, which are transgenic, such as transgenic oocytes, sperm, blastocyst, embryo, fetus, donor cells, or cell nucleus.

The term eye disease or eye disorder refers to any eye disease and / or disorder. For example, eye diseases can be disorders of the lacrimal system, the eyelids and the orbit, disorders of the conjunctiva, disorders of the sclera, cornea and iris, ciliary body, disorders of the choroid, disorders of the retina, glaucoma , diseases of the optic nerve and visual trajectories, diseases or disorders of ocular neovascularization, inflammatory eye diseases, or disorders of the eye muscles. In addition, eye disease can also refer to discomfort after injury, surgery, or laser treatment. Eye diseases and disorders or eye diseases include, but are not limited to, retinopathy, diabetic retinopathy, retinal vein occlusion, macular degeneration, age-related macular degeneration, dry eye syndrome, blepharitis, inflammatory gland disease Meibomian, uveitis, allergic conjunctivitis, glaucoma, macular edema, diabetic macular edema, cystoid macular edema, and rosacea (of the eye). Dry eye syndrome (DES), also known as keratoconjunctivitis sicca (KCS), keratitis sicca, sicca syndrome, or xerophthalmia, is an eye disease caused by decreased tear production or increased tear film evaporation commonly found in humans and some animals.

The term age-related macular degeneration, or AMD, is an eye disease that usually affects older adults and results in loss of vision in the center of the visual field (the macula) due to damage to the retina. It occurs in "dry" and "wet" forms. It is a major cause of blindness and visual impairment in older adults (&gt; 50 years old).

Macular degeneration can make it difficult or impossible to read or recognize faces, although peripheral vision is still enough to allow other activities of daily life. The macula is the central area of ​​the retina, which provides the most detailed information on central vision. In the dry (non-exudative) form, cellular debris called drusen accumulates between the retina and choroid, the retina may detach. In the wet (exudative) form, which is more severe, blood vessels grow from the choroid behind the retina, and the retina may also detach. It can be treated with laser coagulation, and with medication that stops and sometimes reverses the growth of blood vessels. Macular degeneration includes some macular dystrophies that affect younger patients, as well as age-related macular degeneration (AMD or ARMD), which is more commonly known. AMD begins with characteristic yellow deposits (drusen) in the macula, between the retinal pigment epithelium and the underlying choroid. Most patients with these early changes (known as age-related maculopathy) have good vision. Drusen patients may develop advanced AMD. The risk is much higher when drusen are large and numerous, and associated with alterations in the layer of pigmented cells under the macula. Recent research suggests that large, smooth drusen are associated with elevated cholesterol deposits and may respond to cholesterol-lowering agents.

The term macular edema refers to eye diseases, cystoid macular edema (CME) or diabetic macular edema (DME). CME is an eye disease that affects the central retina or the macula of the eye. When this condition is present, multiple cyst-like (cystic) areas of fluid appear in the macula and cause retinal inflammation or edema. CME can accompany a variety of diseases such as retinal vein occlusion, uveitis, and / or diabetes. CME commonly occurs after cataract surgery. DME occurs when blood vessels in the retina of diabetes patients begin to leak into the macula. These leaks cause the macula to thicken and swell, progressively distorting sharp vision. Although swelling cannot lead to blindness, the effect can cause severe loss of central vision.

The term glaucoma refers to an eye disease in which the optic nerve is damaged in a characteristic pattern. This can permanently damage vision in the affected eye and lead to blindness if not treated. It is normally associated with increased pressure within the eye (aqueous humor). The term ocular hypertension is used for patients with constantly elevated infraocular pressure (OIP) without any damage to the optic nerve. In contrast, the term normal tension or low-tension glaucoma is used for those with damage to the optic nerve and associated visual field loss but normal or low infraocular pressure. Nerve damage is the loss of retinal ganglion cells in a characteristic pattern. There are many different subtypes of glaucoma, but they can all be considered a type of optic neuropathy. Elevated infraocular pressure (eg, above 21 mmHg or 2.8 kPa) is the single most important modifiable risk factor for glaucoma. However, some can have high eye pressure for years and never develop damage, while others can develop nerve damage at relatively low pressure. Untreated glaucoma can lead to permanent damage to the optic nerve and consequent loss of the visual field, which can progress to blindness over time.

The term uveitis refers to an inflammatory disease of the uvea, the vascular layer of the eye caught between the retina and the white part of the eye (sclera). The uvea extends to the front of the eye and is made up of the iris, ciliary body, and choroid layer. Uveitis includes anterior uveitis, intermediate uveitis, and posterior uveitis. The most common type of uveitis is an inflammation of the iris called iritis (anterior uveitis). Uveitis can also occur in the posterior segment of the eye (for example, in the choroid). Inflammation of the uvea can be recurrent and can cause serious problems, such as blindness if not treated (represents 10% of blindness worldwide). Early diagnosis and treatment are important to avoid complications of uveitis.

The term dry eye or dry eyes refers to an eye disease in which there are not enough tears to lubricate and nourish the eye. Tears are necessary to maintain the health of the front surface of the eye and to provide clear vision. Patients with dry eyes do not produce enough tears or have poor quality tears. Dry eye is a common and often chronic problem, especially in older adults. With each blink of the eyelid, tears are spread over the front surface of the eye, known as the cornea. Tears provide lubrication, reduce the risk of eye infection, wash the foreign body in the eye, and keep the surface of the eyes smooth and clear. Excess tears in the eyes flow into the small drainage ducts, in the inner corners of the eyelids, which drain into the back of the nose. Tears are produced by various glands (for example, the lacrimal gland) in and around the eyelids. Tear production tends to decrease with age, with various medical conditions, or as a side effect of certain medications. Environmental conditions such as wind and dry weather can also affect tear volume by increasing tear evaporation. When the normal amount of tear production decreases or the tears evaporated too quickly from the eyes, dry eye symptoms may develop. The most common form of dry eyes is due to insufficient amount of tear water layer. This condition, called keratoconjunctivitis sicca (KCS), is also known as dry eye syndrome.

The term diabetic retinopathy refers to retinopathy (i.e., a disease of the retina) caused by complications of diabetes, which can lead to blindness. Diabetic retinopathy may not cause symptoms, mild vision problems, or even blindness. Diabetic retinopathy is the result of complications of microvascular retinal changes. Intramural pericyte death induced by hyperglycemia and thickening of the basement membrane lead to incompetence of the vascular walls. These damages change the formation of the blood-retinal barrier and also make the blood vessels more permeable. Pericyte death is caused when hyperglycemia persistently activates protein kinase C-δ (PKCδ-, encoded by Prkcd) and mitogen-activated protein kinase p38 (MAPK) to increase expression of a previously unknown target of PKC-δ signaling , phosphatase-1 containing the Src homology-2 domain (SHP-1), a protein tyrosine phosphatase. This signaling cascade leads to dephosphorylation of the PDGF receptor and a reduction in subsequent signaling of this receptor, resulting in pericyte apoptosis. Small blood vessels, such as those in the eye, are especially vulnerable to poor blood sugar control. An accumulation of glucose and / or fructose damages the tiny blood vessels in the retina. During the initial stage, called nonproliferative diabetic retinopathy (NPDR), most patients do not notice any changes in their vision. The first changes that are reversible and do not threaten central vision are sometimes called simple retinopathy or background retinopathy. As the disease progresses, severe nonproliferative diabetic retinopathy enters an advanced stage of "proliferating diabetic retinopathy (PDR) when the blood vessels proliferate. Lack of oxygen in the retina causes new fragile blood vessels to develop, along the retina and in the clear, gelatinous vitreous humor that fills the inside of the eye, which can cause bleeding, blurred vision, retinal damage, or fractional retinal detachment.

The term VEGF is used herein interchangeably with vascular endothelial growth factor. VEGF includes, but is not limited to, VEGF related proteins such as placental growth factor (PIGF), VEGF-A, VEGF-B, VEGF-C, VEGFD, VEGF-E, and VEGF-F. The term VEGF also covers a number of proteins from two families that result from the alternating division of mRNA from a single VEGF gene, exon 8. The two families are known based on their terminal exon splice site (exon 8) - the proximal splice site (denoted VEGF_XXX) or distal splice site (VEGF_xxxb). Furthermore, alternative splicing of exon 6 and 7 alters its heparin binding affinity, and the number of amino acids (in humans: VEGF₁₂₁, VEGFi_21b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF189, VEGF₂₀₆; Rodent orthologs of these proteins contain less amino acid.) These domains have important functional consequences for VEGF splice variants, as the terminal splice site (exon 8) determines whether the proteins are pro-angiogenic (proximal splice site, expressed during angiogenesis) or anti-angiogenic (site of distal splice, expressed in normal tissues). Furthermore, the inclusion or exclusion of exons 6 and 7 mediate interactions with heparan sulfate proteoglycans (HSPG) and neuropilin co-receptors on the cell surface, improving their ability to bind and activate VEGF receptors (VEGFR). The term VEGF also includes VEGF receptors. There are three main VEGFR subtypes, numbered 1, 2 and 3. In addition, they can be membrane linked (mbVEGFR) or soluble (sVEGFR), depending on the alternative division.

The term particle refers to a small object, 'fragment or piece of a substance that can be a single element, inorganic material, organic material, or a mixture of these. Examples of particles include polymeric particles, single emulsion particles, double emulsion particles, coacervates, liposomes, microparticles, nanoparticles, macroscopic particles, pellets, 10 crystals (eg crystalline forms of compounds or active pharmaceutical agent), aggregates, composite materials. , powdered, bleached, or otherwise unbalanced matrices and entangled protein particles or polysaccharides, each particle has an average characteristic dimension less than 15 1 mm and at least 1 nm, where the characteristic dimension, or critical dimension, of the particle is the smallest transverse dimension of the particle. A particle can be made up of a single substance or several substances. In certain embodiments, the particle is not a viral particle. In other embodiments, particle 20 is not a liposome. In certain embodiments, the particle is not a micelle. In certain embodiments, the particle is substantially solid. In certain embodiments, the particle is a nanoparticle. In certain embodiments, the particle is a microparticle.

The term nanoparticle refers to a particle having a characteristic dimension of less than about 1 micron and at least 1 nanometer, where the characteristic dimension of the particle is the smallest transverse dimension of the particle. A crystalline nanoparticle is called a nanocrystal.

The term microparticle refers to a particle that has a characteristic dimension of less than about 1 millimeter, and at least 1 micrometer, where the characteristic dimension of the particle is the smallest transverse dimension of the particle.

The term nanostructure refers to a structure having at least one characteristic region or dimension with a dimension of less than about 1000 nm, for example, less than about 300 nm, less than about 200 nm, with less than about 100 nm, or less than about 50nm. Typically, the characteristic region or dimension will be along the minor axis of the structure. Examples of such structures include nanowires, nanowires, nanotubes, branched nanocrystals, nanotetrapods, tripods, bipods, nanpoints, nanocrystals, quantum dots, nanoparticles, branched tetrapods (eg, inorganic dendrimers), and the like. The nanostructures can be substantially homogeneous in material properties, or in certain embodiments they can be heterogeneous (eg, heterostructures). The nanostructures can be, for example, substantially crystalline, substantially monocrystalline, polycrystalline, amorphous, or a combination thereof. In one aspect, each of the three dimensions of the nanostructure has a dimension of less than about 1000 nm, for example, or even less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. The nanostructures can include one or more surface ligands (eg, surfactants).

The terms crystalline or substantially crystalline, when used with respect to nanostructures, refer to the fact that nanostructures typically exhibit long-range ordering through one or more of the dimensions of the structure. It will be understood by a person skilled in the art that the term long-range arrangement will depend on the absolute size of the nanostructures, since the arrangement of a single crystal cannot extend beyond the limits of the crystal. In this case, long-range ordering will mean a substantial order across at least most of the nanostructure dimension. In some cases, a nanostructure may carry an oxide or other coating, or it may be composed of a core and at least one shell. In such cases, it is appreciated that the oxide, shell (s), or any other necessary coating does not exhibit such an arrangement (eg, it may be amorphous, polycrystalline, or otherwise). In such cases, the phrase crystalline, substantially crystalline, substantially monocrystalline, or monocrystalline refers to the central core of the nanostructure (excluding the cladding or covering layers). The terms crystalline or substantially crystalline as used herein are also intended to encompass structures composed of various defects, stacking failures, atomic substitutions, and the like, provided that the structure exhibits substantial long-range ordering (for example, the order for at least about 80% of the length of at least one axis of the nanostructure or its nucleus). Furthermore, it is appreciated that the adjoining surface between the core and the exterior of a nanostructure or between an adjacent core and shell or between an adjacent shell and second shell may contain non-crystalline regions and may even be amorphous. This does not prevent the nanostructure from being crystalline or substantially crystalline as defined herein. The term monocrystalline when used with respect to a nanostructure indicates that the nanostructure is substantially crystalline and comprises substantially single crystal. When used with respect to a heterostructure nanostructure comprising a core and one or more shells, monocrystalline indicates that the core is substantially crystalline and is substantially composed of a single crystal. When not used with respect to a nanostructure, the term monocrystalline for materials that is substantially composed of a single crystallite of virtually the same size and orientation.

Nanocrystal is a nanostructure that is substantially monocrystalline. Such a nanocrystal has at least one characteristic region or dimension with a dimension of less than about 1000 nm, for example, less than about

300 nm, less than about 200 nm, less than about 100 nm or less than about 50 nm. Typically, the characteristic region or dimension will be along the minor axis of the structure. Examples of such structures include nanowires, nanowires, nanotubes, nanowires, branched, nanotripods, nanotetrapods, nanobipods, nanocrystals, nanopoints, quantum dots, nanoparticles, nanolistons, and the like. The nanostructures can be substantially homogeneous in material properties, or in certain embodiments they can be heterogeneous (eg, heterostructures). Optionally, a nanocrystal can include one or more surface ligands (eg, surfactants). The nanocrystal is optional and substantially single crystal in structure (a single crystal nanostructure or a monocrystalline nanostructure). While the nanostructures for use in the present invention can be manufactured from basically any suitable material or material, preferably the nanostructure is prepared from an inorganic material, for example, an inorganic semiconductor or conductive material. A conductive or semiconductor nanostructure often shows 1dimensional quantum confinement, for example, an electron can often travel along a single dimension of the structure. Nanocrystals can be substantially homogeneous in material properties, or in certain embodiments they can be heterogeneous (eg, heterostructures). The term nanocrystall is intended to encompass substantially single crystal nanostructures comprising various defects, stacking failures, atomic substitutions, etc., as well as substantially monocrystalline nanostructures without such defects, failures, or substitutions. In the case of nanocrystal heterostructures comprising a core and one or more shells, the core of the nanocrystal is typically substantially monocrystalline, but the shell (s) is not required. Nanocrystals can be made from basically any suitable material or materials.

The term polycrystalline does not refer to materials that are made up of many crystallites of different sizes and orientations. When used with respect to nanostructures, the term polycrystalline refers to a crystalline nanostructure that is not monocrystalline.

A biocompatible material refers to a material that does not normally induce a negative response when inserted or injected into a subject. The negative response includes significant inflammation and / or acute rejection of the material by the subject's immune system, for example, through a T cell mediated response. It is recognized that biocompatibility is a relative term and that some degree of immune response is expected even for materials that are highly compatible with the subject's living tissues. However, as used herein, biocompatibility refers to the acute rejection of a material by at least a part of the immune system, that is, a material that lacks biocompatibility (i.e. not biocompatible) in a subject elicits an immune response in the subject that is serious enough, such that the rejection of the material by the immune system cannot be adequately controlled and is often of such a degree that the material must be removed from the subject in order for the subject to be as it was before non-biocompatible material is introduced to the subject. One test to determine the biocompatibility of a material is to expose the material to cells (eg, fibroblasts or epithelial cells) in vitro; the material is considered biocompatible if it does not result in significant cell death at moderate concentrations, for example at concentrations of around 50 micrograms / 10⁶ cells. In certain modalities, there is no significant cell death if less than about 20% of the cells are dead, even if they are phagocytosed or otherwise taken up by the cells. In some embodiments, a material is biocompatible if contact with cells in vitro results in less than 20% of cell death and if administration of the material in vivo does not induce unwanted inflammation or other adverse effects. In certain embodiments, a biocompatible material is biodegradable. A non-limiting example of biocompatible materials is biocompatible polymers (including biocompatible copolymers).

A biodegradable material refers to a material that is capable of being chemically and / or biologically degraded (eg, by hydrolysis or enzymatic activity), within a physiological environment, such as within the body or when introduced into cells. For example, the material may be one that spontaneously hydrolyzes upon exposure to water (eg, within a subject) and / or may degrade upon exposure to heat (eg, at a temperature of approximately 37 ° C). Degradation of a material can occur at different speeds, depending on the material used. For example, the half-life of the material (the time when 50% of the material breaks down into smaller components) may be on the order of days, weeks, months, or years. The material can be biologically degraded, for example, by enzymatic activity or cellular machinery, for example, through exposure to a lysozyme. In some embodiments, the material can be divided into smaller components that cells can reuse or dispose of without any toxic effect on cells (for example, less than about 20% of cells are killed when the components are added to cells in vitro) . Non-limiting examples of biodegradable materials are biodegradable polymers (including biodegradable copolymers). Examples of biodegradable polymers include, but are not limited to, three-block copolymers of poly (ethylene glycol) -poIi (propylene oxide) -poly (ethylene glycol), polyvinyl alcohol (PVA), poly (lactide) ( o poly (lactide) acid, poly (glycolic acid) (or poly (glycolic acid), poly (orthoesters), poly (caprolactones), polylysine imine, poly (ethylene), poly (acrylic acid), poly (urethanes ), poly (anhydrides), poly (esters), po I i (trim et i le n carbonate), poly (ethyleneimine), poly (acrylic acid), poly (urethane), poly (beta amino esters), and copolymers thereof (eg, poIi (Ictide-co-glycylide) (PLGA)).

As used herein, the terms pharmaceutical composition and formulation are used interchangeably.

As used herein, the terms pharmaceutical agent and drug are used interchangeably.

DESCRIPTION OF SOME MODALITIES OF THE INVENTION

The present invention provides crystalline forms of the compound of 7- (3- (4- (4-fluoro-2-methyl-1 H-indole-5-yloxy) -6methoxyquinazolin-7-yloxy!) Propyl) -2-oxa- 7-azaspiro [3.5] nonane, referred to herein as Compound 3 as shown below:

Compound (3)

In particular embodiments, the crystalline form is a crystalline form A, having a powder X-ray diffraction pattern (XRPD) with peaks at approximately 6.1 1, 9.63, 16.41, 18.60, 20.36, and 23.01 ± 0.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and

3.8610.3 A in d-separation. In other embodiments, the crystalline form A further has XRPD peaks around 1.46, 12.26, 18.16, 19.51, 21.12, and 25.71 ± 0.3 degrees two theta or 7.71, 7.22, 4.88, 4, 55, 4.20 and 3.4610.3 A in d-separation. In other embodiments, the crystalline form A also has XRPD peaks around 11.10, 15.66, 17.54, 22.31, 24.79, and 28.9010.3 degrees two theta or 7.96, 5.65, 5.05, 3.98, 3.59, and 3.0910.3 A at d-separation. In still other modalities, crystalline form A possesses an XRPD pattern with peaks around 6.11, 9.63, 1 1.10, 11.46, 12.26, 15.66, 16.410, 17.54, 18.16, 17.1 1, 19.51, 20.36, 21.12, 22.31, 23.01, 24.79, 28.90 and

25.7110.3 degrees two theta or 14.45, 9.17, 7.96, 7.71, 7.22, 5.65, 5.40, 5.05, 4.88, 4.77, 4.55 , 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.0910.3 A in d-separation.

In other particular embodiments, the crystalline form is crystalline form B, which has an XRPD pattern with peaks at approximately 7.70, 13.53, 17.27, 18.44, 19.73, 23.10, and 26.0710.3 degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.50, 3.85 and 3.4110.3 A in d-separation. In other embodiments, the crystalline form B also has XRPD peaks around 9.87, 12.88, 14.40, 15.45, 21.14, and

26.8410.3 degrees two theta or 8.96, 6.87, 6.14, 5.73, 4.20 and 3.3210.3 A in d-separation. In other embodiments, the crystalline Form B also has XRPD peaks at approximately 10.69, 16.42, 18.90, 22.56, and 29.1210.3 degrees two theta or 8.27, 5.39, 4.69, 3 , 94 and 3.0610.3 A in d-separation. In yet other embodiments, crystalline Form B has an XRPD pattern with peaks around 7.70, 9.87, 10.69, 12.88, 13.53, 14.40, 15.45, 16.42, 17.27, 18.44, 18.90, 21, 14,

22.56, 19.73, 23.10, 26.07, 26.84 and 29.12 ± 0.3 degrees two theta or 1 1.47, 8.96, 8.27, 6.87, 6.54, 6.14, 5.73, 5.39, 5.13, 4.81, 4.69, 4.50, 4.20, 3.94, 3.85, 3.41, 3.32 and 3.06 ± 0.3 A in d-separation.

In one aspect, the present invention relates to a compound having the formula in crystalline form A.

In another aspect, the present invention relates to a crystalline form of a compound having the formula wherein said crystalline form is a crystalline form A having a powder X-ray diffraction pattern (XRPD) with peaks at approximately 6.11, 9.63, 16.41, 18.60, 20.36 and 23.0110.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and 3.8610.3 A in separation.

In another embodiment, the present invention relates to a compound having the formula

<img file="MX2016005668A_D0006.tif" />

in crystalline B form.

In another embodiment, the present invention relates to a crystalline form of a compound having the formula

<img file="MX2016005668A_D0007.tif" />

wherein said crystalline form is crystalline form B having a powder X-ray diffraction pattern (XRPD) with peaks at approximately 7.70, 13.53, 17.27, 18.44, 19.73, 23.10, and 26.0710.3 degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.50, 3.85 and 3.4110.3 A in d-separation.

The present invention also relates to a process for preparing a crystalline form of compound 3. In certain embodiments, the present invention relates to a method for preparing a crystalline form A of compound 3. In other embodiments, the method of preparing form A crystalline comprises the wet grinding of a slurry composed of an amorphous form of compound 3 and a nonionic surfactant to obtain the nanoparticles of the compound. In yet other embodiments, the result of the crystalline-shaped nanoparticles have an XRPD pattern with peaks at approximately 6.11, 9.63, 16.41, 18.60, 20.36, and 23.01 ± 0.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and

3.8610.3 A in d-separation. In yet other embodiments, the result of the crystalline Form A nanoparticles further has an XRPD pattern with peaks at approximately 11.46, 12.26, 18.16, 19.51, 21.12, and 25.7110.3 degrees two theta or 7.71, 7.22, 4.88, 4.55, 4.20 and

3.4610.3 A at d-clearance or around 11.10, 15.66, 17.54,

22.31, 24.79 and 28.910.3 degrees two theta or 7.96, 5.65, 5.05, 3.98, 3.59 and 3.0910.3 A in d-separation, or both. In other embodiments, the resulting crystalline Form A nanoparticles have an XRPD pattern with peaks around 6.1 1, 9.63, 11.10, 11.46, 12.26, 15.66, 16.410, 17.54, 18.16, 17.1 1, 19.51, 20.36, 21.12, 22.31,

23.01, 24.79, 25.71 and 28,910.3 degrees two theta or 14.45, 9.17, 7.96, 7.71, 7.22, 5.65, 5.40, 5.05, 4.88 , 4.77, 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.0910.3 A in d-separation.

In other embodiments, the present invention relates to a method for the preparation of crystalline form B of compound 3. In certain embodiments, the method of preparing crystalline form B consists of the crystallization of the amorphous form of compound 3 from a mixture of solvent comprising water and acetone. In particular embodiments, the crystalline Form B preparation method uses a compound of a 4: 1 solvent mixture of acetone: water mixture. In other embodiments, the method of preparing crystalline Form B further comprises heating the solvent mixture to dissolve the compound and / or cooling the solvent mixture to allow crystal formation. In some embodiments, the resulting crystalline Form B has an XRPD pattern with peaks at about 7.7, 13.53, 17.27, 18.44, 19.73,

23.1 and 26,710.3 degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.5, 3.85 and 3.41 ± 0.3 A in d-separation. In other embodiments, the resulting crystalline form B also has an XRPD pattern with peaks around 9.87, 12.88, 14.4, 15.45, 21.14, and 26.8410.3 degrees two theta or 8.96, 6.87, 6.14, 5.73, 4.2, and 3.3210.3 A in d-separation, or at approximately 10.69, 16.42, 18.9, 29.12 and 22.5610.3 degrees two theta or 8.27, 5.39, 4.69, 3.94 and 3.0610.3 A in d-separation, or both. In other embodiments, the resulting crystalline form B has an XRPD pattern with peaks around 7.7, 9.87, 10.69, 12.88, 13.53, 14.4, 15.45, 16.42, 17.27, 18.44, 18.9, 19.73, 21.14, 22.56,

23.1, 26.07, 26.84 and 29.1210.3 degrees two theta or 11.47, 8.96, 8.27,

6.87, 6.54, 6.14, 5.73, 5.39, 5.13, 4.81, 4.69, 4.5, 4.2, 3.94, 3.85, 3.41, 3.32 and 3.0610.3 A in d-separation.

Methods of using the crystalline forms of compound 3 to treat diseases are also provided, including proliferative diseases, eye diseases, dermatological diseases, inflammatory diseases, autoimmune diseases, autoinflammatory diseases and metabolic diseases. The present invention further provides methods of using crystalline form A crystalline Form A or crystalline form B of compound 3 as a therapeutic, for example, in the treatment and / or prevention of diseases associated with abnormal angiogenesis and / or aberrant signaling of a activity growth factor (eg vascular endothelial growth factor (VEGF) or angiogenesis. In certain embodiments, the disease being treated and / or prevented by a crystalline form A or crystalline form B of compound 3, pharmaceutical compositions, equipment, uses, and methods include proliferative diseases (eg, cancers, benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, autoimmune diseases) and diseases, ocular (eg, macular degeneration, glaucoma, diabetic retinopathy, retinoblastoma, edema, macular edema, corneal neovascularization, uveitis, dry eye, blepharitis, and post-surgical inflammation).

In certain embodiments, the crystalline forms of the invention are monocrystalline. In certain embodiments, the compounds of the invention are polycrystalline.

The crystalline forms of the invention may also have a relatively low aqueous solubility (i.e., a solubility in water, optionally with one or more pH regulators). For example, the crystalline forms of Compound 3 can have an aqueous solubility of less than or equal to 3 mg / mL, less than about 1 mg / mL, less than about 0.3 mg / mL, less than about 0 0.1 mg / mL, less than about 0.03 mg / mL, less than about 0.01 mg / mL, less than about 1 pg / mL, less than about 0.1 pg / mL, less about 0.01 pg / mL, just under 1 ng / mL, at less than about 0.1 ng / mL or less than 0.01 ng / mL at 25 ° C. In some embodiments, the crystalline forms of Compound 3 have an aqueous solubility of at least 1 pg / mL, at least about 10 pg / mL, at least about 0.1 ng / mL, to less than 1 ng / mL , at least about 10 ng / mL, at least about 0.1 pg / mL, at least about 1 pg / mL, at least about 3 pg / mL, at least about 0.01 mg / mL , at least about 0.03 mg / mL, at least about 0.1 mg / mL, at least about 0.3 mg / mL, at least about 1.0 mg / mL, or at least about 3 mg / mL at 25 ° C. Combinations of the scales mentioned above are possible (eg, an aqueous solubility of at least about 10 pg / mL and less than 1 mg / mL). Other scales are also possible. The crystalline forms of Compound 3 can have these or other aqueous solubility scales at any point throughout the entire pH range (eg, at about pH 7 or from pH 1 to pH 14).

The crystalline forms of Compound 3 may be suitable for processing into pharmaceutical mucus-penetrating compositions (eg, particles or crystals). In certain embodiments, the crystalline forms of Compound 3 are suitable for grinding (eg, nano-grinding). In certain embodiments, the crystalline forms of compound 3 are suitable for precipitation (eg, microprecipitation, nanoprecipitation, crystallization, or controlled crystallization). In certain embodiments, the crystalline forms of Compound 3 are suitable for emulsification. In certain embodiments, the crystalline forms of Compound 3 are suitable for freeze drying.

Compound 3 can be prepared using any suitable method. In certain embodiments, Compound 3 can be prepared using a method as shown in Scheme 1:

Scheme 1: Method A to synthesize compound 3.

<img file="MX2016005668A_D0008.tif" />

<img file="MX2016005668A_D0009.tif" />

2

CI

Br

K₂CO₃, DMF

<img file="MX2016005668A_D0010.tif" />

(COOH)₂

K₂C0₃, KBr, DMF

<img file="MX2016005668A_D0011.tif" />

In certain embodiments, Compound 3 can also be prepared by Method B, as shown in Scheme 2:

Scheme 2: Method B of synthesizing compound 3.

<img file="MX2016005668A_D0012.tif" />

Pharmaceutical compositions, equipment, and methods of use and administration

The present invention provides pharmaceutical compositions comprising a crystalline form A of compound 3, and optionally a pharmaceutically acceptable carrier, or crystalline form B of compound 3 and, optionally, a pharmaceutically acceptable carrier. In certain embodiments, a compound described herein is provided in an effective amount of the pharmaceutical composition. In certain embodiments, the amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an effective amount to treat and / or prevent disease. In certain embodiments, the effective amount is an effective amount for treating a disease. In certain embodiments, the effective amount is an effective amount to treat and / or prevent a disease associated with aberrant growth factor signaling. In certain embodiments, the effective amount is an effective amount for the treatment of a disease associated with aberrant growth factor signaling. In certain embodiments, the effective amount is an effective amount to treat and / or prevent a disease associated with aberrant signaling of vascular endothelial growth factor (VEGF). In certain modalities, the effective amount is an effective amount to treat and / or prevent a disease associated with angiogenesis abnormalities, such as cancer, benign neoplasm, atherosclerosis, hypertension, inflammatory disease, rheumatoid arthritis, macular degeneration, choroidal neovascularization, retinal neovascularization and diabetic retinopathy. In certain embodiments, the effective amount is an effective amount to treat cancer (eg, eye cancer). In certain modalities, the effective amount is an effective amount to treat macular degeneration.

An effective amount of crystalline form of compound 3 of the invention can range from about 0.001 mg / kg to 000 mg / kg in one or more doses administrations for one or several days (depending on the mode of administration). In certain modalities, the effective amount per dose ranges from about 0.001 mg / kg to 000 mg / kg, from about 0.01 mg / kg to 750 mg / kg, from about 0.1 mg / kg to 500 mg / kg, from about 1.0 mg / kg to 250 mg / kg, and from about 10.0 mg / kg to 150 mg / kg.

An effective amount of the crystalline form of Compound 3 of the invention can inhibit abnormal angiogenesis and / or aberrant growth factor signaling of at least 10%, at least 20%, at least about 30%, by less than about 40%, at least 50%, at least 60%, at least about 70%, at least about 80%, or at least about 90%. An effective amount of a compound 3 of the invention can inhibit abnormal angiogenesis and / or aberrant growth factor signaling by less than about 90%, less than 80%, less than about 70%, less than 60%, less approximately 50%, less than approximately 40%, less than 30%, less than 20%, or less than 10%. The combinations of the ranges described (eg, at least 20% and less than 50%) are also within the scope of the invention. In certain embodiments, an effective amount of a compound of the invention 3 inhibits abnormal angiogenesis and / or aberrant signaling of a growth factor by a percentage or percentage range described herein, compared to normal angiogenesis and / or signaling.

The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, these methods include the preparatory steps of bringing a crystalline form of compound 3 described herein (i.e., the active ingredient) in association with a vehicle or excipient, and / or one or more accessory ingredients, and then, if it is necessary and / or convenient, modeling and / or packaging of the desired product in a single dose unit or multiple doses.

The pharmaceutical compositions can be prepared, packaged and / or sold in bulk, as a single dose unit and / or as a plurality of single dose unit. As used herein, a unit dose is a discrete amount of the pharmaceutical composition that comprises a given amount of active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject and / or a portion of those convenient doses such as, for example, half or a third of said dose.

The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any other additional ingredient in a pharmaceutical composition of the invention will vary, depending on the identity, size and / or condition of the subject treated and continue to depend on the route by which the composition is to be administered. The composition can comprise between 0.001% and 100% (w / w) of active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of the pharmaceutical compositions provided include inert diluents, dispersing and / or granulating agents, surface active and / or emulsifying agents, disintegrating agents, binding agents, preservatives, pH regulating agents, lubricating agents and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavors and proportioning agents may also be present in the composition.

Illustrative diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium acid phosphate, sodium phosphate, sucrose, lactose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride. , dry starch, cornstarch, powdered sugar, and mixtures thereof.

Illustrative granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation exchange resins, calcium carbonate, sodium carbonate, silicates, crosslinked polyvinyl pyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, interlaced sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (1500 starch), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and the same.

Illustrative surface active agents and / or emulsifiers include natural emulsifiers (eg, acacia, agar, sodium alginate, alginic acid, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool, fat. , cholesterol, wax and lecithin), colloidal clays (for example, bentonite (aluminum silicate) and Veegum (magnesium and aluminum silicate), derivatives of long chain amino acids, high molecular weight alcohols (for example, stearyl alcohol, cetyl alcohol, oleic alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (for example, polymethyl carboxy, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer) , carrageenan, cellulosic derivatives (eg sodium carboxymethyl cellulose, cellulose powder, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, meth Icelu slab), fatty acid esters of sorbitan (for example, polyoxyethylene sorbitan monolaurate (TWEEN® 20), polyoxyethylene sorbitan (TWEEN® 60), polyoxyethylene sorbitan monooleate (TWEEN® 80), sorbitan monopalmitate (SPAN® 40) sorbitan also monostearate (SPAN® 60), sorbitan triestearate (SPAN® 65), glycerol monooleate, sorbitan monooleate (SPAN® 80), polyoxyethylene esters (for example, polyoxyethylene monostearate (MYRJ®45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (for example, CREMOPHOR®), polyoxyethylene ethers, (for example, polyoxyethylene) lauryl ether (BRIJ® 30), poly ^ vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F-68, Poloxamer P-188, cet i I pyr id inio bromide, cetrimonium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

Illustrative binding or binding agents include starch (eg, cornstarch and starch paste), gelatin, sugars (eg, sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), gums natural and synthetic (eg acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol husk mucilage, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl pi I ce I u I osa, hydroxy ro pi I meti I ce I u Iose, microcrystalline cellulose, cellulose acetate, poly (vinyl pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, salts of inorganic calcium, silicic acid, polymethacrylates, waxes, water, alcohol and / or mixtures thereof.

Illustrative preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain modalities, the preservative is an antioxidant. In other modalities, the conservator is a chelating agent.

Illustrative antioxidants are alpha tocopherol, ascorbic acid, acybil palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

Chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (eg, sodium edetate, disodium edetate, trisodium edetate, disodium calcium edetate, dipotassium edetate edetate, and the like), citric acid, and salts and hydrates thereof (for example, citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and its salts and hydrates thereof, and tartaric acid and its salts and hydrates. Illustrative antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorozylenol, cresol, ethyl alcohol, 15 glycerol, hexetidine, phenol, hexididine, phenol , phenylmercuric nitrate, propylene glycol, and thimerosal.

Illustrative antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Illustrative alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Illustrative acidic preservatives are vitamin A, vitamin 25 C, vitamin E, beta-carotene, citric acid, acetic acid, acid

<img file="MX2016005668A_D0013.tif" />

dehydroacetic, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethyleneodiamine, sodium lauryl sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLIDANT® Plus, PHENONIP®, methylparaben, GERMALL® 115® II, NEOLONE GERMABEN®, KATON® and EUXIL®.

Illustrative pH regulators include citrate pH buffer solutions, acetate pH buffer solutions, phosphate pH buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, gluceptate calcium, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, mixtures of potassium phosphate, sodium acetate, sodium bicarbonate , sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, mixtures of sodium phosphate, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

Illustrative lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, po I iet i le ng I ico I, sodium benzoate, sodium acetate, chloride sodium, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

Illustrative natural oils include almonds, apricot kernel, avocado, babassu, bergamot, black seeds, borage, Cade, chamomile, cañola, caraway, carnauba, castor, cocoa butter, coconut, cod liver, coffee, corn , cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, pumpkin, grapeseed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui, lavandin, lavender, lemon, Litsea cubeba, macademia nut, mallow, mango seeds, Prairie Grass Seeds, Mink, Nutmeg, Olive, Orange, Palm, Palm Center, Peach Center, Peanut, Poppy Seeds, Pumpkin Seeds, Rapeseed, Rice Bran, Rosemary, Safflower, Sandalwood , sasquana, tasty, sea buckthorn, sesame, shea butter, silicone, soy, sunflower, tea tree, thistle, tsubaki, vetiver, walnut and wheat germ oils. Illustrative synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capic triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleic alcohol, silicone oil, and mixtures of the themselves.

Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients, liquid dosage forms may encompass inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate. , benzyl alcohol, benzyl benzoate, propylene g I i co 1, 1,3-Butylene glycol, dimethylformamide, oils (eg cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavors, and perfume-providing agents. In certain modalities for parenteral administration, the conjugates of the invention are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

A pharmaceutical composition of the invention can be formulated for administration by injection in any acceptable form, including intravenous, subcutaneous, intramuscular, intraperitoneal, parenteral, epidural or infraocular administration. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a parenterally acceptable non-toxic diluent or solvent, for example as a 1,3-butanediol solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any fixed soft oil can be used including synthetic mono- or di-glycerides. Furthermore, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be, for example, sterilized by filtration through a bacterial retention filter, or incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or any other sterile injectable medium prior to use. The formulation can also be prepared under aseptic conditions or sterilized with heat or radiation.

An injectable formulation or pharmaceutical composition of the invention may also be formulated for ophthalmic administration by injection in any acceptable form, including, but not limited to, intravitreal, intrastromal, intracamerular, sub-retinal, conjunctive, subconjunctival, sub-tenon (eg, anterior or posterior ), circumcorneal, scleral, episcleral, posterior, peri-juxtascleral, peri-bulbar, retro-bulbar, supracorodial and lacrimal duct. A pharmaceutical composition of the invention may also be formulated for ophthalmic administration by implant or the use of reservoirs (eg, biodegradable delivery system, non-biodegradable delivery system and other extended implants or slow release device or formulation).

Compositions for vaginal or rectal administration are typically suppositories that can be prepared by formulating a crystalline form of Compound 3 of this invention with suitable non-irritant excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax that are solid at room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient or vehicle, such as sodium citrate or dicalcium phosphate and / or (a) fillers or extension agents such as starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as, for example, carboxymethyl cellulose, alginates, gelatin po I ivini I pir ro I id in on a, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, pills the dosage form may include a pH regulating agent.

Similar solid compositions can be used as fillers in soft and hard filled gelatin capsules with excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like. Solid dosage forms of tablets, dragees, capsules, lozenges and granules can be prepared with coatings and coatings such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opaque agents and may be of a composition that release the active ingredient (s) only, or preferably, to a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions that can be used include polymeric substances and waxes. Similar type solid compositions can be used as fillers in soft and hard filled gelatin capsules with excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.

The active ingredient may be in a microencapsulated form with one or more excipients as indicated above. Solid dosage forms of tablets, dragees, capsules, lozenges and granules can be prepared with coatings and coatings such as enteric coatings, release control coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active ingredient can be mixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is customary practice, additional substances other than inert diluents, for example, tabletting lubricants and other tabletting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may include pH regulating agents. They may optionally comprise opaque agents and may be of a composition that release the active ingredient (s) only, or preferably, to a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents that can be used include polymeric substances and waxes.

Dosage forms for topical and / or transdermal administration of a compound of the present invention may include ointments, pastes, creams, lotions, gels, powders, solutions, suspensions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and / or any necessary preservative and / or pH regulator as may be required. Furthermore, the present invention contemplates the use of transdermal patches, which often have the advantage of offering controlled delivery of an active substance to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispersing the active ingredient in the suitable medium. Alternatively or additionally, the rate can be controlled by a rate control membrane and / or by dispersing the active ingredient in a polymeric matrix and / or gel.

Devices suitable for use in the intradermal delivery of disclosed pharmaceutical compositions include short needle devices such as those described in US Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; 5,417,662. The intradermal compositions can be administered by devices limiting the effective penetration length 20 of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Alternatively or additionally, conventional syringes can be used in the classic Mantoux method of intradermal administration. Jet injection devices are suitable that deliver liquid vaccines to the dermis through a liquid jet injector and / or through a needle that penetrates the stratum corneum and produces a jet that reaches the dermis. Jet injection devices are described, for example, in US Pat. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704.91 1; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder / particle delivery devices are used that use compressed gas to accelerate the powdery compound through the outer layers of the skin into the dermis.

Formulations suitable for topical administration (including ocular or dermal) include, but are not limited to, liquid and / or semi-liquid preparations such as ointments, lotions, oil-in-water and / or water-in-oil emulsions. such as creams, ointments and / or pastes, and / or solutions and / or suspensions. Topically administrable formulations may, for example, comprise about 0.001% to about 50% (w / w) of active ingredient, although the concentration of the ingredient

<td>active can</td><td>to be so</td><td>high</td><td>As the</td><td>limit</td><td>of</td><td>solubility</td><td>of the</td>

<td colspan="2">active ingredient in</td><td>the</td><td>solvent.</td><td>The</td><td colspan="2">formulations</td><td>for</td>

<td>administration</td><td>topical</td><td colspan="2">may include</td><td>of</td><td>one</td><td>or more than</td><td>the</td>

additional ingredients described here. In one aspect, the present invention relates to pharmaceutical formulations or compositions suitable for topical administration comprising a form

A crystalline of compound 3 or crystalline form B of compound 3.

A pharmaceutical composition of the invention can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration. Such a formulation can include dry particles comprising the active ingredient and having a diameter in the range of from about 0.5 to about 7 microns, or from about 1 to 6 microns. Such compositions are conveniently in powder form for administration by a device consisting of a dry powder reservoir so that a flow of propellant can be directed to disperse the powder and / or by using a self-propelled solvent / powder dispenser such as a composite device by the active ingredient dissolved or suspended in a low boiling point propellant in an airtight container. This type of powder comprises particles where at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the number of the particles have a diameter of less than 20 microns. Alternatively, at least 95% of the weight of the particles have a diameter greater than 1 nanometer and at least 90% of the number of the particles have a diameter of less than 15 microns. The dry powder compositions can include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants with a boiling point below 18.8 ° C at atmospheric pressure. Generally, the propellant can make up from 50 to 99.9% (w / w) of the composition, and the active ingredient can make up from 0.001 to 20% (w / w) of the composition. The propellant may also comprise additional ingredients such as a liquid nonionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same order as the particles that make up the active ingredient).

The pharmaceutical compositions of the invention formulated for pulmonary delivery can provide the active ingredient in the form of drops of a solution and / or suspension. Such formulations can be prepared, packaged and / or sold as optionally sterile aqueous and / or dilute alcoholic solutions and / or suspensions, comprising the active ingredient, and can be conveniently administered using any nebulization and / or atomization device. Such formulations may consist of one or more additional ingredients including, but not limited to, a flavoring agent, such as sodium saccharin, a volatile oil, a pH regulating agent, a surface active agent, and / or preservative such as methylhydroxybenzoate. Drops delivered by this route of administration can have an average diameter in the range of about 0.01 to about 200 microns. Alternatively, the formulations for pulmonary administration may comprise a powder and / or an aerosol and / or spray solution and / or suspension comprising the active ingredient. Such powder, aerosol, and / or spray formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.01 to about 200 microns, and may include one or more of the additional ingredients. described here.

The formulations herein described as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder composed of the active ingredient and having an average particle size of about 0.2 to 500 microns. This formulation is administered by rapid inhalation through the nasal passage from a powder container kept close to the nostrils. Formulations for nasal administration may, for example, comprise from as little as 0.001% (w / w) to as much as 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged and / or sold in a formulation for oral administration. Such formulations may be, for example, in the form of tablets and / or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w / w) of active ingredient, the remainder comprising a composition that can be dissolved orally and / or degraded and, optionally, one or more of the additional ingredients described herein.

The formulations described herein can also be supplied through buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges made using conventional methods, and may contain, for example, 0.001 to 50% (w / w) of active ingredient, the remainder comprise a composition which can be absorb orally and / or degradable and, optionally, one or more of the additional ingredients described here.

A pharmaceutical composition of the invention can be prepared, packaged and / or sold in a formulation for ophthalmic administration. Such formulations may be, for example, in the form of eye drops, for example, 0.001 / 10.0% (w / w) of a solution and / or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Those drops may also comprise pH regulating agents, salts and / or one or more of the additional ingredients described herein. Other ophthalmically administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form and / or in a liposomal preparation.

A pharmaceutical composition of the invention may also be formulated for administration via the ophthalmic mucosa membrane, such as, for example, eye drops, ointments, or gels. These formulations can be prepared by conventional means and, if desired, the present compositions can be mixed with any conventional additive, such as a pH regulating or pH adjusting agent, tonicity adjusting agents, viscosity modifiers, stabilizers of suspension, preservatives, and other pharmaceutical excipients. Furthermore, in certain embodiments, compositions described herein can be lyophilized or subjected to another suitable drying technique such as spray drying. Ear drops are also contemplated to be within the scope of this invention.

Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by experts that those compositions are generally suitable for administration to animals of all kinds. Modification of pharmaceutical compositions suitable for administration to humans to process compositions suitable for administration to various animals is well understood, and the experienced veterinary pharmacologist can usually design and / or perform such modification with common experimentation.

The compositions provided herein are typically formulated in unit dose form to facilitate administration and uniform dosage. It will be understood, however, that the total daily use of the compositions of the present invention will be decided by the treating physician within the scope of the sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the specific active ingredient used; the specific composition used; the subject's age, body weight, general health, sex, and diet; the time of administration, route of administration, and the rate of excretion of the specific active ingredient employed; the duration of treatment; medicines used in combination or coincident with the specific active ingredient used; and similar factors known in the medical art.

The compositions provided herein can be administered by any route, including the enteral (eg oral), parenteral, infraocular, intramuscular, intravenous, intraarterial, intramedullary, intrathecal, subcutaneous, transdermal, interdermal intraventricular, rectal, intravaginal, intraperitic route. neal, topical (including dermal or ocular, such as powders, ointments, creams and / or drops), buccal, nasal, sublingual mucosa; by intratracheal instillation, bronchial instillation and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, injections, including intravenous administration (eg, systemic intravenous injection) and infraocular administration, regional administration through the blood or lymph, delivery and / or direct administration to an affected site including topical administration (by example, dermal and / or ocular). In general, the most suitable route of administration will depend on a variety of factors, including the nature of the agent (eg, its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (eg, whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition of the invention is suitable for administration to the eyes of a subject. In another embodiment, the pharmaceutical composition or compound in a crystalline form of compound 3 is suitable for topical administration to a subject's eye.

The exact amount of a crystalline form of Compound 3 of the invention necessary to achieve an effective amount will vary from subject to subject, depending, for example, on the species, age and general condition of the subject, on the severity of the effects secondary or disorder, mode of administration, and the like.

The desired dosage can be delivered three times a day, twice a day, once a day, every day, every three days, every week, every two weeks, every three weeks or every four weeks. In certain embodiments, the desired dose can be delivered using various administrations (eg, two, three, 20, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations).

In certain embodiments, an effective amount of a crystalline form of compound 3 of the invention for administration one or more times a day for a 70 kg human adult can comprise from about 0.0001 mg to about

<td colspan="3">3000 mg, approximately</td><td>0.0001 mg a</td><td> 2000</td><td>mg,</td><td>of</td>

<td>about 0.0001</td><td>mg</td><td colspan="3">up to about 1000</td><td>mg,</td><td>of</td>

<td>about 0.001</td><td>mg</td><td>to</td><td>approximately</td><td> 1000</td><td>mg,</td><td>of</td>

<td>about 0.01</td><td>mg</td><td>to</td><td>approximately</td><td> 1000</td><td>mg,</td><td>of</td>

about 0.1 mg to about 1,000 mg, 1 mg to about 1,000 mg, about 1 mg to 100 mg, about 10 mg to about 1,000 mg, about 10 mg to 100 mg, or about 100 mg to about 1,000 mg, of the compound of 3 per unit dose form.

In certain embodiments, the crystalline forms of Compound 3 described herein may be at dose levels sufficient to deliver about 0.001 mg / kg to 000 mg / kg, from about 0.01 mg / kg to 500 mg / kg, and preferably from about 0.1 mg / kg to 400 mg / kg, and preferably from about 0.5 mg / kg to 300 mg / kg, from about 0.01 mg / kg to 100 mg / kg, from about 0.1 mg / kg to 10 mg / kg, and more preferably from about 1 mg / kg to 25 mg / kg of subject body weight per day, one or more times a day, to obtain the desired prophylactic and / or therapeutic effect.

It will be appreciated that the dose ranges, as described herein, provide guidance for the administration of pharmaceutical compositions to an adult. The amount administered to, for example, a child or adolescent may be determined by a physician or a person skilled in the technical art and may be less than or equal to that administered to an adult.

It will also be appreciated that a crystalline form of compound 3 or the composition thereof, as described herein, can be administered in combination with one or more pharmaceutical agents (eg, therapeutically or prophylactically active agents). The crystalline forms of compound 3 or compositions can be administered in combination with other drugs that enhance their activity (eg, activity in the prevention and / or treatment of a disease associated with aberrant signaling of a growth factor (eg, VEGF) or with abnormal angiogenesis in a subject, in the inhibition of aberrant growth factor signaling (eg, VEGF) in a subject or cell or in the inhibition of abnormal angiogenesis in a subject), bioavailability, reduce and / or modify its metabolism, inhibit its excretion, and / or modify its distribution within a subject's body. It will also be appreciated that the therapy employed can achieve a desired effect for the same disorder, and / or can achieve different effects.

The crystalline form of compound 3 or the composition of the invention can be administered in conjunction with, before or after one or more additional pharmaceutical agents, which may be useful as, for example, combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactic active agents. Pharmaceutical agents include small organic molecules such as from pharmacological compounds (for example, compounds approved for human or veterinary use by the US Food and Drug Administration. in accordance with the Code of Federal Regulations (CFR), peptides, proteins, carbohydrates, monosaccharides, polysaccharide oligosaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA , RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing a disease described herein. Each additional pharmaceutical agent can be administered at a dose and / or at a specific time for that pharmaceutical agent. Additional pharmaceutical agents can also be administered to each other and / or with the described compound or composition in a single dose or administered separately in different doses. The particular combination for employing a regimen will take into account the compatibility of the compound of the invention with the additional pharmacological agent (s) and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, it is expected that the additional pharmacological agent (s) used in combination can be used at levels that do not exceed the levels at which they are used individually. In some modalities, the levels used in combination will be lower than those used individually.

Additional pharmaceutical agents include, but are not limited to, anti-proliferative agents (eg, anti-cancer agents), anti-angiogenesis agents, anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral, anti-diabetic agents, anti-agents -allergics, and pain relief agents. In certain embodiments, the additional pharmaceutical agent is a growth factor inhibitor. In certain embodiments, the additional pharmaceutical agent is a VEGF inhibitor. In certain embodiments, the additional pharmaceutical agent is an angiogenesis inhibitor. In certain embodiments, the additional pharmaceutical agent is an endogenous angiogenesis inhibitor (eg, vascular endothelial growth factor receptor 1 (VEGFR-1, eg, pazopanib (VOTRIENT®), cediranib (RECENTIN®), tivozanib (AV-951), INLITA (axitinib®), semaxanib), HER2 (lapatinib (TIVERB TICURB®®), linifanib (ABT-869), MGCD265 and KRN-633), VEGFR-2 (eg regorafenib ( BAY 734506), telatinib (BAY 57-9352), vatalanib (PTK787, PTK / ZK), MGCD265, OSI-930 and KRN-633), NPR-1, angiopoietin 2, TSP-1, TSP-2, angiostatin, endostatin, vasostatin, calreticulin, platelet factor-4 and TIMP-1, CDAI, Met-1 , Met-2, IFN-a, IFN-β, IFN-y, CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle-2 domain), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, a proliferin-related protein, sorafenib (Nexavar®), and restin). In certain embodiments, the additional pharmaceutical agent is an exogenous angiogenesis inhibitor (eg, bevacizumab, itraconazole, carboxyamidotriazole, PNT-470, CM101, IFN-α, IL-12, platelet factor-4, suramin, SU5416, thrombospondin , a VEGFR antagonist, an angiostatic spheroid, an angiostatic spheroid + heparin, a cartilage derivative, the angiogenesis-inhibiting factor, an extracellular matrix metalloprotease, angiostatin inhibitor, endostatin, 2-methoxystradiol, tecogalan, tetrathiol i bdato, thalidomide, thrombospondin, prolactin, an ανβ3 inhibitor, linomide, and tasquinimod). In certain embodiments, the additional pharmaceutical agent is a corticosteroid, a receptor tyrosine kinase inhibitor (RTK), a cyclooxygenase inhibitor (COX), a prostaglandin analog, a non-steroidal anti-inflammatory drug (NSAID), a beta blocker, or an inhibitor. carbonic anhydrase. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing AMD, such as verteporfin (for example, CHLORIN®, VISUDYNE®), thalidomide (for example, AMBIODRY®, SYNOVIR®, TALOMID®), sodium talaporfin (for example, APTOCINE®, LASERPHYRIN®, LITX®), ranibizumab (for example, LUCENTIS®), petaptanib octasodium (for example, MACUGEN®, MACUVERSE®), isopropyl unoprostone (for example, OCUSEVA®, RESCULA®), inferred beta (eg FERON®), fluocinolone acetonide (for example, ENVISION TD®, RETISERT®), everolimus (for example, AFINITOR®, CERTICAN®, VOTUBIA®, ZORTRESS®), eculizumab (for example, Solaris®, SOLIRIS®), dexamethasone (for example, OSURDEX ®, OZURDEX®, POSURDEX®, SURODEX®), canakinumab (for example, ILARIS ®), bromfenac (BROMDAY®), ophthalmic (for example, BRONAC®, BRONUCK®, XIBROM®, YELLOX®), brimonidine (for example, ALPHAGAN®, BROMOXIDINE®, ENIDIN®), anecortavo acetate (for example, RETAANE®, EDEX®, RIGIDUR PROSTAVASIN®®®, VIRIDAL VASOPROST®), aflibercept ophthalmic solution (for example, EYELEA®, EILEA®, VEGF-TRAP-EYE®), ocriplasmin (for example, ILUVIEN®, MEDIDUR®, MEDIDUR FA®), sirolimus ( eg PERCEIVA®), NT-501, KH-902, phosbretabulin tromethamine (eg ZYBRESTAT®), AL-8309, aganirsen (eg NORVESS®), volociximab (eg OPTOTEC®), triamcinolone (eg example, Icono Bioscience), TRC-105, Burixafor (for example, TG-0054), TB-403 (for example, R-7334), squalamine (for example, EVIZON®), SB-623, S-646240, RTP-8011-14 (for example, PF4523655), RG-7417 (for example, FCFD-4514S), AL-78898A (for example, POT -4), PG-1 1047 (for example, CGC-1 1047), pazopanib hydrochloride, sonepcizumab (for example, ASONEP®, SPHINGOMAB®), padeliporfin (for example, STAKEL®), OT-551 onteclzumab, NOX- A12, hCNS-SC, Neu-2000 NAFB001, MA09-hRPE, LFG-316, ¡Co-007 (for example, ISIS-13650), hl-con1, GSK-933776A, GS-6624 (for example, AB-0024 ), ESBA-1008, epitalon, E-10030 (eg ARC-127), dalantercept, MP-0112, CNTO-2476, CERE-120, AAV-NTN, CCX-168, brimonidine-DDS, bevasiranib sodium (eg Cy5), bertilimumab, AVA -101, ALG-1001, AL-39324, AGN-150998, ACU4429, A6 (for example, PARALIT®), TT-30, sFLT-01 gene therapy RETINOSTAT®, PRS-050 (for example, ANGIOCAL®), PF -4382923,

Palomid-529, MC-1101, GW-824575, Dz13 (for example, TRC-093), D93 CDX-1135 (for example, TP10), ATL-1103, ARC-1905, XV-615, wet-AMD antibodies (eg pSivida), VEGF / rGel, VAR, VAL566-620-10200-MULTI, TKI, TK-001, STP-601, dry AMD stem cell therapy (eg), OpRegen EyeCyte, SMT-D004 , SAR-397769, RTU-007, RST-001, RGNX-004, RFE-007-CAI, retinal degeneration program (for example, ORPHAGEN), cells of the retina (for example, ISCO), ReN003, PRM-167 , ProDex, photoelectric switches (eg, Photoswitch Biosciences switches), Parkinson's disease therapy, OMS-721, OC-10X, NV. AT.08, NT-503, NAFB002, NADPH-oxidase inhibitors (for example, Alimera Sciences), MC-2002, anti-angiogenic protein licium, IXSVEGF, integrin inhibitors, GW-771806, GBS-007, Eos-013, EC-400, dry AMD therapy (for example, Neurona Systems), CGEN-25017, CERE-140, AP-202, AMD therapy (for example,

Valens Terapeutics), AMD therapy (eg Amarna Therapeutics), AMD RNAi therapy (eg RXi), ALK-001, AMD therapy (eg Aciont), AC-301, 4-IPP, complexes zincmonocysteine ​​(eg Adeona), vatalanib, TG, prinomastat-100344, PMX-53, Neovastat, mecamylamine, JSM-6427, JPE-1375,

CereCRIB, BA-285, ATX-S10, AG-13958, verteporfin / alphavB3 conjugate, VEGF / rGel, VEGF-saporin, VEGF-R2 (eg, Allostera antagonists), VEGF inhibitors (eg, Santen antagonists), VEGF (for example, Arca), VANGIOLUX®, Triphenylmethanes (for example, Alimera), TG TG-100-572-100-801 ,, TA88

106, T2-TrpRS, SU-0879, stem cell therapy (eg, Pfizer and UCL), SOD mimetics (eg,), SHEF lnotek-1, rostaporfin (eg, PHOTREX®, PURLITIN®, SnET2) , RNA interference (eg Idera and Merck), rhCFHp (eg Opterion), retino-NPY, retinitis pigmentosa therapy (eg Mimetogen), AMD gene therapy (eg Novartis), retinal gene therapy (eg Genzyme), AMD gene therapy (eg Copernicus), ther retinal dystrophy (eg Fovea and Genzyme), Ramot project no. K-734B, PRS-055, Porcine Retinal Pigment Epithelium Cells (eg, GenVec), PMI002, PLG-101 (eg, BiCentis®), PJ-34, PI3K conjugates (eg, Semafore), PhotoPoint, Pharmaprojects No. 6526, Pegaptanib Sodium (eg SurModics®), PEDF ZFP TF, PEDF Gene Therapy (eg GenVec), PDS-1.0, PAN-90806, Opt-21, OPK-HVB OPK-010-HVB-004, Ophtalmologicals (eg, NetwoRx cell), ophthalmic compounds (eg, and AstraZenca Alcon), OcuXan, NTC-200, NT-502, NOVA, NEUROSOLVE-21012®, neuroprotectors (eg, BDSI), MEDI-548, MCT -355, MCEYE®, LENTIVUE®, LIN-002 LX-213, lutetium texaphyrin (for example,

ANTRIN®), LG-339 inhibitors (eg Lexicon), KDR kinase inhibitors (eg Merck), ISV-616, INDUS-815C, ICAM1 aptamer (eg Eyetech), hedgehog antagonists (eg example, Opthalmo), GTx-822, GS-102, Granzyme B / VEGF®, gene therapy (e.g., EyeGate), GCS-100 analog programming, FOV-RD-27, fibroblast growth factor (e.g.,

Ramot), fenretinide, F-200 (eg EOS-200-F), PANZEM SR®, ETX, ETX-6201 -6991, EG-3306, Dz-13, disulfiram (eg ORA-102), diclofenac (eg Ophtalmopharma), ACU-02, CLT-010, CLT-009, CLT-008, CLT-007, CLT-006, CLT-005, CLT-004, CLT-003 (eg CHIROVIS®) , CLT-001, CETRIN® (eg BA-210), celecoxib, CD91 antagonist (eg Ophtalmophar), CB-42, BNC-4 bestrophin, batimastat, BA-1049, AVT2, AVT-1, ATU012 , Ape1 (for example, the ApeX-2 program), antiVEGF (eg Gryphon), AMD ZFPs (eg ToolGen), AMD therapy (eg Opterion), AMD therapy (eg IterX), dry AMD therapy (eg Opko), AMD therapy ( eg CSL), AMD therapies (eg Pharmacopeia and Allergan), AMD therapeutic proteins (eg IterX), AMD RNAi therapy (eg BioMolecular Therapeutics), AM-1101, ALNVEG01, AK-1003, AGN-211745, ACU-XSP-001 (for example,

EXCELLAIR®), ACU-HTR-028, HHY-ACU-011, ACT-MD (for example, NewNeural), ABCA4 modulators (for example, Active Pass), A36 (for example, Angstrom), 267268 (for example, SB-267268), bevacizumab (for example VASTIN®), aflibercept (for example EILEA®), 131-I-TM-601, vandetanib (for example CAPRELSA®, ZACTIMA®, ZICTIFA®), sunitinib malate ( for example, SUTENE® SUTENT®), sorafenib (for example, NEXAVAR®), pazopanib (for example, ARMALA®, PATORMA®, VOTRIENT®), axitinib (for example, INLITA®), tivozanib, XL-647, RAF-265, pegdinetanib (for example ANGIOCEPT®), pazopanib, MGCD-265, icrucumab, foretinib, ENMD90

2076, BMS-690514, regorafenib, ramucirumab, plitidepsin (for example, APLIDIN®), orantinib, nintedanib (for example, VARGATEF®), motesanib, midostauriña, linifanib, telatinib, lenvatinib, elpamotide dovitinib, cedi ), JI-101, cabozantinib, brivanib, apatinib, ANGIOZYME®, X-82, SSR, rebastinib-106462, PF-337210, IMC-3C5, CYC116, AL-3818, VEGFR-2 inhibitor (eg AB Science ), VEGF / rGel (eg Clayton Biotechnologies), TLK-60596, TLK-60404, R84 antibody (eg Peregrine), MG-516, FLT4 kinase inhibitors (eg Sareum) kinase inhibitors flt-4, Sareum, DCC-2618, CH-330331, XL-999, XL-820, vatalanib, SU-14813, semaxanib, KRN633, CEP-7055, CEP-5214, ZK-CDK, ZK-261991, YM-359445, YM231 146, VEGFR-2 kinase inhibitors (eg Takeda), inhibitors of VEGFR-2 kinase (eg Hanmi), VEGFR-2 antagonist (eg Affymax), VEGF / rGel (eg Targa), VEGF-TK inhibitors (eg AstraZeneca), tyrosine kinase inhibitors (eg Abbott), tyrosine kinase inhibitors (eg Abbott), Tie2 kinase inhibitors (eg GSK), their- 0879, SP-5.2, sorafenib lanyard (for example, NEXAVAR® lanyard), SAR-131675, Ro-4383596, R-1530,

Pharmaprojects No. 6059, OSI-930, OSI-817, OSI-632, MED-A300, L000021649, KM-2550, kinase inhibitors (for example,

MethylGene), kinase inhibitors (eg Amgen), Ki-8751, KDR kinase inhibitors (eg Celltech), KDR kinase inhibitors (eg Merck), KDR kinase inhibitors (eg , Amgen), KDR inhibitors (eg Abbott), KDR KDR inhibitor (eg LGLS), JNJ-17029259, IMC-1C11, anticancer Flt 3/4 (eg Sentinel), EG-3306, DP-2514 , DCC-2157, CDP-791, CB-173, c-kit inhibitors (eg Deciphera), BIW-8556, anticancer (eg Braceo and Dyax), MAbs anti-Flt-1 (eg ImClone), AGN-211745, AEE788, or AB-434. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in treating and / or preventing dry eye, such as cyclosporine (RESTASIS®). In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing cystatic macular edema (CME), such as a

<td>NSAID (for</td><td>example, bromfenac (BROMDAY®)). In certain</td>

<td>modalities,</td><td>the additional pharmaceutical agent is an agent</td>

Pharmaceutical useful to treat and / or prevent diabetic macular edema

<td>(DME), such</td><td>such as ranibizumab (LUCENTIS®). In certain</td>

<td>modalities,</td><td>the additional pharmaceutical agent is an agent</td>

<td>pharmacist</td><td>useful to treat and / or prevent uveitis, such as</td>

<td>TOBRADEX®</td><td>(dexamethasone 0.1% / 0.3% tobramycin), ZILET®</td>

(0.5% loteprednol etabonate / 0.3% tobramycin), triamcinolone acetonide (TRIVARIS® and TRIESENCE®), fluocinolone (RETISERTt®) and dexamethasone (OZURDEX®). In certain embodiments, the additional pharmaceutical agent is a useful pharmaceutical agent for treating and / or preventing glaucoma, such as latanoprost (Xalatan®), bimatoprost (LUMIGAN®), travoprost (Travatan Z®), timolol (TIMOPTIC®), tartrate of brimonidine ALPHAGAN®), dorzolamide (TRUSOPT®) and pilocarpine (ISOPTO®). In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing an inflammatory eye disease (eg, post-surgical inflammation), such as steroids (eg, (loteprednol etabonate LOTEMAX®), difluprednate (DUREZOL ®), prednisolone acetate (PRED MILD® and OMNIPRED®) and NSAID (for example, bromfenac (BROMDAY®), nepafenac (NEVANAC®), ketorolac tromethamine (ACULAR LS®, ACUVAIL®, TORADOL® and SPRIX®), diclofenac (VOLTRAN®, ACLONAC® and CATAFLAM®).

The invention also encompasses equipment (eg, pharmaceutical packages). The kits provided may include a pharmaceutical composition or crystalline form of compound 3 of the invention and a container (eg, a vial, ampoule, bottle, syringe and / or dispenser package or other suitable container). In some embodiments, kits may optionally further include a second container comprised of a pharmaceutical carrier for dissolution or suspension of a pharmaceutical composition or crystalline form of compound 3. In certain embodiments, a pharmaceutical composition or crystalline form of compound 3 provided in the The first container and the second container combine to form a unit dose form.

Thus, in one aspect, kits are provided including a first container comprising a crystalline form of compound 3 described herein, or a pharmaceutical composition thereof. In certain embodiments, the kits described herein are useful for preventing and / or treating a disease described here. In certain embodiments, the kits described herein are useful for preventing and / or treating a disease associated with aberrant signaling of a growth factor (eg, VEGF) in a subject in need thereof. In certain embodiments, the kits described herein are useful for preventing and / or treating a disease associated with abnormal angiogenesis in a subject in need thereof. In certain embodiments, the kits described herein are useful in preventing and / or treating proliferative diseases (eg, cancer, benign neoplasms, inflammatory diseases, autoimmune diseases) and / or eye diseases (eg, macular degeneration, glaucoma, diabetic retinopathy, retinoblastoma, edema, uveitis, dry eye, or postsurgical inflammation). In certain embodiments, the kits described herein are useful for inhibiting aberrant signaling of a growth factor (eg, VEGF) in a subject or cell in need thereof. In certain embodiments, the kits described herein are useful for inhibiting abnormal angiogenesis in a subject in need thereof. In certain embodiments, the kits further include instructions for administering the crystalline form of Compound 3, or the pharmaceutical composition thereof. Teams may also include information required by a regulatory agency such as the United States Food and Drug Administration (FDA). In certain modalities, the information included in the kits is the prescription information. In certain modalities, providing instructions and equipment to treat and / or prevent a disease described here. In certain embodiments, kits and instructions are provided to prevent and / or treat a disease associated with aberrant growth factor signaling (eg, VEGF) in a subject in need thereof. In certain embodiments, kits and instructions are provided to prevent and / or treat a disease associated with abnormal angiogenesis in a subject in need thereof. In certain embodiments, kits and instructions are provided to inhibit aberrant signaling of a growth factor (eg, VEGF) in a subject or cell in need thereof. In certain embodiments, kits and instructions are provided for the inhibition of abnormal angiogenesis in a subject in need thereof. The kit of the invention may include one or more additional pharmaceutical agents described herein as a separate composition.

Also the present invention provides particles comprising a crystalline form of compound 3 described herein that can penetrate mucus, pharmaceutical compositions thereof, kits, and methods of using and preparing the particles and their pharmaceutical compositions. Pharmaceutical compositions, equipment, and methods may involve a modification of the surface coating of the particles, such as pharmaceutical agent particles having low aqueous solubility. Such pharmaceutical compositions, kits, and methods can be used to achieve efficient transport of the particles that make up the inventive crystalline forms of Compound 3 across mucus barriers in a subject.

In certain embodiments, the crystalline forms, particles, pharmaceutical compositions, kits, and methods of the invention are useful for applications to the eye, such as treating and / or preventing eye disease (eg, macular degeneration, diabetic retinopathy, edema. macular, retinal vein occlusion, dry eye syndrome, uveitis, glaucoma, allergic conjunctivitis and rosacea).

The particles (eg, nanoparticles and microparticles) of the invention comprise a crystalline form of compound 3. In a particular aspect, the particles that make up crystalline form B of compound 3. The particles of the invention also include an alteration agent. surface that modifies the surface of the particles to reduce the adherence of the particles to the mucus and / or to facilitate the penetration of the particles through the mucus.

The present invention also provides pharmaceutical compositions comprising the particles of the invention. In certain embodiments, the pharmaceutical compositions of the invention can be administered topically to a subject's eye. Topical pharmaceutical compositions are advantageous in pharmaceutical compositions that are administered by injection or orally.

Particles

The present invention also provides pharmaceutical compositions comprising a plurality of particles or crystals of the invention, which can be mucus penetrating particles or crystals (MPP). MPPs comprising crystalline form A or crystalline form B of compound 3 useful in the present invention can be made as described, for example, in US Patent Publication No. 2013/0316001, 2013/0316006, 2013/0323179, 2013/0316009, 2012/0121718, 2010/0215580 and 2008/0166414, all of which are incorporated herein by reference in their entirety. Such pharmaceutical compositions may be suitable for administration through various routes described herein. In one embodiment, pharmaceutical compositions comprising a plurality of particles comprising a crystalline form A or crystalline form B of compound 3, wherein the particles are mucus penetrating particles, are formulated to be delivered to the eyes of a subject or to treating and / or preventing an eye disease of a subject. In a preferred embodiment, the mucus penetrating particles comprise crystalline form B of compound 3.

In some embodiments, the particles of the invention have a core shell type configuration. The nucleus may comprise a crystalline form of compound 3, a polymeric vehicle, a lipid, and / or a protein. The core can also comprise a gel or liquid.

In some embodiments, the core is a solid. The solid may be, for example, a crystalline form of compound 3 (eg, crystalline form B). In certain embodiments, the core is a gel or liquid (for example, an oil-in-water or water-oil emulsion).

A crystalline form of Compound 3 (eg, crystalline form B) may be present in the nucleus in any suitable amount, for example, at least about 0.01% by weight, at least about 0.1% by weight , at least about 1% by weight, at least about 5% by weight, at least about 10% by weight, at least about 20% by weight, at least 30% by weight, at least about 40% in weight, at least about 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least about 95% by weight, or at least about 99% in core weight. In one embodiment, the core consists of 100% by weight of a crystalline form of compound 3. In some cases, the crystalline form of Compound 3 (eg, crystalline form B) may be present in the center at less than or equal to about 100% by weight, less than or equal to about 95% by weight, less than or equal to 90% in

<td>weight,</td><td>lower</td><td> 0</td><td>same</td><td>to the</td><td> 85</td><td> %</td><td>in</td><td>weight,</td><td>lower</td><td> 0</td><td>same</td><td>to the</td><td> 80</td><td> %</td><td>in</td>

<td>weight,</td><td>lower</td><td> 0</td><td>same</td><td>to the</td><td> 70</td><td> %</td><td>in</td><td>weight,</td><td>lower</td><td> 0</td><td>same</td><td>to the</td><td> 60</td><td> %</td><td>in</td>

<td>weight,</td><td>lower</td><td> 0</td><td>same</td><td>to the</td><td> 50</td><td> %</td><td>in</td><td>weight,</td><td>lower</td><td> 0</td><td>same</td><td>to the</td><td> 40</td><td> %</td><td>in</td>

weight, less than or equal to approximately 30% by weight, less than or equal to 20% by weight, less than or equal to 10% by weight, less than or equal to approximately 5% by weight, less than or equal to 2% by weight, or less than or equal to 1% by weight of the core. Combinations of the aforementioned ranges are also possible (eg, present in an amount of at least 80% by weight and less than or equal to about 100% by weight of the core). Other ranges are also possible. In one embodiment, a crystalline form of compound 3 (eg, crystalline form B) comprises at least 90% by weight of the core of a particle of the invention. In another embodiment, a crystalline form of compound 3 (eg, crystalline form B) comprises at least 95% by weight of the core of a particle of the invention.

When a polymer is present in the core, the polymer can be present in the core in any suitable amount, for example, less than 100% by weight, less than 80% by weight, less than 60% by weight, less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, less than 10% by weight, less than 5% by weight or less than about 1% by weight. In some cases, the polymer may be present in an amount of at least 1% by weight, at least about 5% by weight, at least about 10% by weight, at least about 20% by weight, at least 30 % by weight, at least about 40% by weight, at least about 50% by weight, at least 75% by weight, at least 90% by weight, or at least about 99% by weight in the core . Combinations of the aforementioned ranges are also possible (eg, present in an amount of at least 1% by weight and less than about 20% by weight). Other ranges are also possible. In some embodiments, the core is substantially free of a polymer component.

The cores can be of any convenient shape and / or size. For example, the core may be substantially spherical and non-spherical, oval, rod-shaped, pyramidal, cube-type, disk-shaped, cable-like, or irregularly shaped. The nuclei may have a cross-sectional dimension greater or less than, for example, less than 10 pm, less than 3 pm, less than 1 pm, less than 500 nm, less than 400 nm, less than 300 nm, less from about 200nm, with less than about 100nm, to less than about 30nm or less than about 10nm. In some cases, the core may have a transverse dimension greater or less than, for example, at least about 10nm, at least about 30nm, at least about 100nm, at least about 200nm, at least about 300nm, at least about 400nm, at least about 500nm, at least about 1pm, or at least about 3pm. Combinations of the aforementioned ranges are also possible (eg, a cross-sectional dimension greater or less than at least about 30nm and less than 500nm). Other ranges are also possible. In some embodiments, the sizes of the nuclei formed by a process described in this document have a Gaussian type distribution. Unless otherwise noted, size measurements of

100 Particle or core sizes refer to the smallest transverse dimension.

Techniques for determining particle sizes (eg, larger or smaller cross-sectional dimensions) are known in the art. Examples of appropriate techniques include Dynamic Light Diffusion (DLS), Transmission Electron Microscopy, Scanning Electron Microscopy, Electroresistance Counting, and Laser Diffraction. Although many methods for determining particle sizes are known, the sizes described (eg, average particle sizes and thickness) refer to those measured by DLS.

In some embodiments, a substantial portion of the core is formed from a crystalline form of Compound 3 as described herein, which can lead to certain beneficial and / or therapeutic effects. The nuclei can be, for example, a nanocrystal (i.e., a nanocrystalline particle) of a crystalline form of compound 3. In certain embodiments, the core includes a polymeric vehicle, optionally, with a crystalline form of compound 3 encapsulated or otherwise related to the core. In certain embodiments, the core includes a gel, protein, lipid, liquid, and / or other appropriate material to be delivered to a subject. The core includes a surface where one or more surface alteration agents can be attached.

In some embodiments, the core is surrounded by a liner, which includes an inner surface and a surface

101 Exterior. The coating may be formed, at least in part, of one or more surface alteration agents, such as a polymer (eg, a block copolymer and / or a polymer with pendant hydroxyl groups), which can associate with the surface of the nucleus. The surface alteration agent may be associated with core particles, for example, being covalently bound to core particles, non-covalently bound to the core particle, adsorbed to the core, or bound to the core through ionic interactions , hydrophobic and / or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or combinations thereof.

The coating and / or surface alteration agent of the particles of the invention can include any suitable material, such as a hydrophobic material, a hydrophilic material, and / or an amphiphilic material. In some embodiments, the liner includes a polymer. In certain embodiments, the polymer is a synthetic polymer (i.e., a polymer not produced in nature). In other embodiments, the polymer is a natural polymer (eg, a protein, polysaccharide, or rubber). In certain embodiments, the polymer is a surface active polymer. In certain embodiments, the polymer is a nonionic polymer. In certain embodiments, the polymer is a linear, nonionic synthetic polymer. In certain embodiments, the polymer is a nonionic block copolymer. The polymer can be a copolymer. In certain embodiments, a repeating unit of the copolymer is

102 relatively hydrophobic and another repeat unit of the copolymer is relatively hydrophilic. The copolymer can be, for example, a two-block, three-block, alternating or random copolymer. The polymer can be loaded or unloaded.

5 Non-limiting examples of suitable polymers of the coating may include polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polietieniminas, polyisocyanates, polyacrylates, polymethacrylates, po I ia cri I on itri I os and polyarylates. Non-limiting examples of specific polymers include poly (caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly (lactide acid) (PLA), poly (Llactide acid) (PLLA), poly (glycolic acid) (PGA) ), poly (lactide-co-glycolic acid) (PLGA), poly (L-lactide-co-glycolic acid) (PLLGA), poly (D, L-lactide) (PDLA), poly (L-lactide) (PLLA ), poly (D, L-lactide-co-caprolactone) and poly (D, L-lactide-co-caprolactone-cogl icolide), poly (D, L-lactide-co-PEO-co-D, L-lactide) , poly (D, L-lactidoco-PPO-co-D, L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-Lysine (PLL), hydroxypropyl methacrylate (HPMA), poly (ethylene glycol), 20 poly-L-glutamic acid , poly (hydroxy acids), polyanhydrides, polyorthoesters, poly (ester amides), polyamides, poly (ester esters), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol) (PEG), polyalkylene terephthalates such as poly (ethylene terephthalate), 25 polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as

103 poly (vinyl acetate), polyvinyl halides such as poly (vinyl) chloride (PVC), po I i vi ni I pirro I idona, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic acid polymers, such as poly (methyl (meth) acrylate) (PMMA), poly (ethyl (meth) acrylate), poly (butyl (met) acrylate), poly (isobutyl (meth) acrylate), poly (hexyl (meth) acrylate), poly (isodecyl (meth) acrylate), poIi (lauryl (meth) acrylate), poly (pheniI (meth) acrylate), poly (methyl acrylate), poIi (IsopropiI acrylate), poIi (isobutiI acrylate), poly (octadecyl acrylate) (collectively referred to herein as polyacrylic acids), and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyol poloxamers, poly (ortho) esters, poly (butyric acid), poly (valeric acid), poly (l-acido-co-caprolactone) and trimethylene carbonate.

The molecular weight of the coating polymer can vary. In some embodiments, the molecular weight of the coating polymer is at least 0.5 kDa, at least about 1 kDa, at least about 1.8 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 8 kDa, at least about 10 kDa, at least about 12 kDa, at least about 15 kDa , at least about 20 kDa, at least about 30 kDa, at least about 40 kDa, or at least about 50 kDa. In some

104 modalities, the molecular weight of the coating polymer is less than about 50 kDa, less than about 40 kDa, less than about 30 kDa, less than about 20 kDa, less than about 12 kDa, less than about 10 kDa , less than 8 kDa, less than 6 kDa, less than 5 kDa, or less than 4 kDa. Combinations of the mentioned ranges are possible (eg, a molecular weight of at least about 2 kDa and less than about 15 kDa). Other ranges are also possible. The molecular weight of the coating polymer can be determined using any known technique such as light diffusion and gel permeation chromatography. Other methods are known in the art. Although the particles of the invention, and the coating thereof, can include both polymers, in some embodiments, the particles of the invention comprise a hydrophobic material, which is not a polymer or pharmaceutical agent. Non-limiting examples of non-polymeric hydrophobic materials include, for example, metals, waxes, and organic materials (eg, silanes and perfluorinated or fluorinated organic materials).

In some embodiments, surface alteration agents, or their portions, are chosen to facilitate the transport of the particles through a mucosal barrier (eg, the mucosa or mucous membrane). In certain embodiments described below, one or more surface altering agents are oriented in a particular configuration on the coating. In

105 Some embodiments, when a surface alteration agent is a three block copolymer, such as having a three block copolymer having a configuration of (hydrophilic block) (hydrophobic block) - (hydrophilic block), a hydrophobic block may be oriented toward the core surface, and the hydrophilic blocks may be oriented away from the core surface (eg, towards the outside of the particle). Hydrophilic blocks may have characteristics that facilitate the transport of the particles through a mucosal barrier. The particular chemical composition and / or components of coating and surface alteration agent (s) can be chosen to impart certain functions to the particles, such as for example the improvement of transport through mucosal barriers.

In some embodiments, at least one particle of the invention includes a core and a coating surrounding the core. A particle that includes a core and a coating on the core is called a coated particle. In some embodiments, at least one particle of the invention includes a core but not a coating on the core. A particle that includes a core but not a coating on the core is called an uncoated particle.

It should be understood that a coating surrounding a core need not completely surround the core, although such modalities may be possible. For example, the coating can surround at least about 10%, at least about 30%, when

106 minus 50%, at least about 70%, at least about 90%, or at least about 99% of the core surface. In some cases, the cladding substantially surrounds a core. In other cases, the lining completely surrounds the core. In other embodiments, a cladding surrounds less than about 100%, less than about 90%, less than 70%, or less than about 50% of the core surface. Combinations of the aforementioned ranges are also possible (eg, surrounding at least 70% and less than 100% of the core surface).

The cladding material can be evenly distributed over the entire core surface in some cases, and in other cases unevenly. For example, the liner may include portions (eg, holes) that do not include any materials. If desired, the coating can be designed to allow penetration and / or transport of certain molecules and components into or out of the layer, but can prevent penetration and / or transport of other molecules and components into or out of the layer. cap. The ability of some molecules to penetrate and / or be transported in and / or through a coating may depend, for example, on the packing density of the surface alteration agents forming the coating and the chemical and physical properties of the components that form the lining. As described here, the liner may include a layer of material (i.e., a monolayer) or multilayer of

107 materials. A single type or several types of surface alteration agents may be present.

The coated particles of the invention can be of any suitable thickness. For example, the coating may have an average thickness of at least 1nm, at least about 3Nm, at least about 10nm, at least about 30nm, at least about 100nm, at least about 300nm, at minus about 1 pm, or at least around 3 pm. In some cases, the average layer thickness is less than 3 pm, less than 1 pm, less than about 300 nm, less than about 100 nm, less than about 30 nm, less than about 10 nm or less of approximately 3 Nm. Combinations of the aforementioned ranges are also possible (eg an average thickness of at least 1nm and less than 100nm). Other ranges are also possible. For particles that have multiple coatings, each layer can have one of the thicknesses described here.

The pharmaceutical compositions of the invention can allow the coated particles of the invention with hydrophilic surface alteration portions without the need for covalent association of surface alteration portions to the core surface. In some embodiments, the core having a hydrophobic surface is coated with a polymer described herein, thereby causing a plurality of surface alteration portions to be placed on the surface of the nucleus without alteration of

108 substantially the characteristics of the core itself. For example, the surface alteration agent may be present on (eg, adsorbed on) the outer surface of the core. In other embodiments, a surface-altering agent is covalently bound to the nucleus.

In certain embodiments where the surface alteration agent is adsorbed on the core surface, the surface alteration agent may be in equilibrium with other molecules of the surface alteration agent in solution, optionally with 10 other components (eg, in a pharmaceutical composition).

In some cases, the adsorbed surface-altering agent may be present on the core surface at a density described here. The density can be an average density since the surface alteration agent is in equilibrium with 15 other components of the solution.

In certain embodiments, the present invention is directed to coated particles comprising a composite core of crystalline form A or a crystalline form B of compound 3 described herein and a coating surrounding the core. In some 20 embodiments, the liner is made of a hydrophilic material.

The coating may consist of one or more surface alteration agents described herein, such as a polymer and / or a surfactant (eg PVA, poloxamer, polysorbate (eg TWEEN 80®). Other coatings or coating agents Surface alteration useful in the present invention are described, by

109 example, in US Patent Publication No. 2013/0316001,

2013/0316006, 2013/0323179, 2013/0316009, 2012/0121718,.

2010/0215580 and 2008/0166414, which have been incorporated by reference in their entirety here.

In some embodiments, the compositions and methods involve the use of poloxamers that aid in the transport of particles in the mucus. Poloxamers are typically three-block copolymers comprising a central hydrophobic block (eg, a poly (propylene oxide) block) flanked by two hydrophilic blocks (eg, poly (ethylene oxide) blocks). Poloxamers have the trade name PLURONIC®. Examples of PLURONIC® polymers that may be useful in the described embodiments include, but are not limited to, F127 (poloxamer 407), F38, F108 (poloxamer 338), F68, F77, F87, F88, F98, L101, L121, L31 , L35, L43, L44, L61, L62, L64, L81, L92, N3, P103, P104, P105, P123, P65, P84 and P85. In certain embodiments, the molecular weight of the hydrophobic block of the three-block copolymer of the (hydrophilic block) - (hydrophobic block) - (hydrophilic block) configuration of is at least about 2 kDa, and the two hydrophilic blocks constitute at least the 15% by weight of the three block copolymer. In certain embodiments, the compositions and methods involve the use of polysorbates that aid in the transport of particles in the mucus. Polysorbates are normally derived from fatty acid esterified pegylated sorbitan (a sorbitol derivative). Common brand names for polysorbates include

110

TWEEN®, ALKEST®, CANARCEL®. Examples of the polysorbates include polyoxyethylene sorbitan monooleate (eg TWEEN 80®), polyoxyethylene sorbitan monostearate (eg TWEEN 60®), polyoxyethylene sorbitan monopalmitate (eg TWEEN 40®) and polyoxyethylene sorbitan monolaurate (for example TWEEN 20®).

It should be understood that components and configurations other than those described herein may be suitable for certain pharmaceutical particles and compositions, and that not all of the components described are necessarily present in some embodiments.

In some embodiments, the particles of the invention, when introduced into a subject, can interact with one or more components in the subject such as mucus, cells, tissues, organs, particles, liquids (eg, blood), microorganisms, and portions or combinations thereof. In some embodiments, the coated particles of the invention can be designed to include surface-altering agents or other components with properties that allow favorable interactions (eg, transport, binding, and adsorption) with one or more materials from the subject. For example, the coating may include surface-altering agents or other components that have a certain hydrophilicity, hydrophobicity, surface charge, functional group, specificity for binding, and / or density to facilitate or reduce particular interactions in the subject. . An example is a

111 choice of hydrophilicity, surface charge, hydrophobicity, functional group, specificity for binding, and / or the density of one or more surface alteration agents to reduce physical and / or chemical interactions between the particles and the mucus of the subject, as well as to improve the mobility of particles through mucus. Other examples are described in more detail below.

In some embodiments, once a particle is successfully transported in and / or through a mucosal barrier (eg, mucus or mucous membrane) in a subject, new interactions between the particles and the subject can take place. In some embodiments, where the nucleus is comprised of a pharmaceutical agent or compound of the invention, the conversion, cleavage, release, and / or transport of the pharmaceutical agents from the particles can lead to certain and / or beneficial therapeutic effects in the subject . Therefore, the particles of the invention can be used for the treatment and / or prevention of certain diseases.

Examples for the use of the particles of the invention are presented below, in the context of being suitable for administration to a mucosal barrier (eg, mucus or mucous membrane) in a subject. It should be noted that while many of the modalities herein are described in this context and in the context of providing a benefit for diseases involving the transport of materials through a mucosal barrier, the invention is not limited as such, and particles , compositions

112 pharmaceuticals, and the kits of the invention can be used to treat and / or prevent other diseases.

In some embodiments, the pharmaceutical compositions of the invention comprise MPPs that include a crystalline form of compound 3 and, optionally, at least one additional pharmaceutical agent, each of which is associated with polymer carriers through encapsulation or other processes. In other embodiments, the pharmaceutical compositions of the invention comprise MPP without polymeric carriers or with minimal use of polymeric carriers. Polymer-based MPPs may have one or more limitations inherent in some embodiments. In particular, taking into account drug delivery applications, these limitations may include one or more of the following. A) Low efficiency of drug encapsulation and low drug load: encapsulation of drugs in polymeric particles is often ineffective, generally less than 10% of the total amount of drug used is encapsulated in particles during manufacturing; in addition, drug loads above 50% are rarely achieved. B) Convenience of use: Pharmaceutical compositions based on drug-laden polymeric particles generally need to be stored as dry powder to avoid premature drug release and therefore require point-of-use re-constitution or a sophisticated dosing device. C) Biocompatibility: accumulation of slowly degrading polymer carriers

113 after repeated doses and their long-term toxicity are of major concern to polymeric drug transporters. D) Physical and chemical stability: Polymer degradation can compromise the stability of encapsulated drugs. In many encapsulation processes, the drug undergoes a transition from a solution phase to a solid phase, which is not well controlled physically in terms of the emerging solid phase (i.e. amorphous vs. crystalline vs. crystalline polymorphs); this is a concern for various aspects of the performance of the pharmaceutical composition, including physical and chemical stability and release kinetics. E) Manufacturing Complexity: Manufacturing, especially the scalability of drug-laden polymeric MPP is a fairly complex process that may involve several steps and a considerable amount of toxic organic solvents. Therefore, by avoiding or reducing the need to encapsulate pharmaceutical agents in polymeric carriers, certain limitations of polymeric MPP with respect to drug loading, ease of use, biocompatibility, stability, and / or manufacturing complexity can be addressed.

It should be noted, however, that in other modalities, pharmaceutical agents can associate with polymer carriers through encapsulation or other processes. Thus, the description provided here is not limited in this regard. For example, despite the aforementioned drawbacks of certain particles

114 penetrating mucus including a polymeric vehicle, in certain embodiments such particles may be preferred. For example, it may be preferable to use polymer carriers for controlled release and / or to encapsulate certain pharmaceutical agents that are difficult to formulate into particulates. As such, in some embodiments described herein, particles are described that include a carrier polymer. In some embodiments, the pharmaceutical compositions of the invention involve the use of alcohols po I i (vi η í I i eos) (PVA), a non-ionic synthetic polymer soluble in water, to facilitate the transport of particles in the mucus, such as described in US Patent Publication No. 2013/0316009, which is incorporated herein by reference in its entirety. Pharmaceutical compositions may involve making MPP or MPC, for example, an emulsification process in the presence of certain PVAs. In certain embodiments, the pharmaceutical compositions and methods involve making MPP or MPC from pre-fabricated particles by non-covalent coating with specific PVAs. In some embodiments, the pharmaceutical compositions and methods involve making MPP in the presence of certain PVAs without polymeric carriers or with minimal use of polymeric carriers. It should be noted, however, that in other modalities, polymeric carriers can be used.

Particles with reduced mucoadhesion

The particles of the invention comprising forms

115 Crystalline Compound 3 (eg crystalline Form B) may have reduced mucoadhesion. A material that requires increased diffusivity through mucus may be hydrophobic, may include many hydrogen bond donors or acceptors, and / or may be highly charged. In some cases, the material may include a crystalline or amorphous solid material. The core-serving material may be coated with a suitable polymer described herein, forming a particle with a plurality of surface-altering portions on the surface, resulting in reduced mucoadhesion. Particles of the invention that have reduced mucoadhesion can be characterized by having increased transport through mucus, being mobile in mucus or mucus-penetrating (i.e., particles that penetrate mucus), meaning that the particles are transported through of mucus faster than a negative control particle. The negative control particle can be a particle that is known to be mucoadhesive, for example, an unmodified particle or core that is not coated with a coating described herein, such as 200nm of carboxylated polystyrene particles.

The particles of the invention can be adapted for delivery (eg, ocular delivery) to mucus or a mucous surface of a subject. Particles with altered surface portions may be delivered to the subject's mucosal surface, may cross the mucosa barrier in the subject and / or retain

116 prolonged and / or increased uniform distribution of particles on mucosal surfaces, for example due to reduced mucoadhesion.

Furthermore, in some embodiments, the particles of the invention that have reduced mucoadhesion facilitate better particle distribution on the surface of a subject's tissues and / or have a prolonged presence on the tissue surface, compared to particles that they are more mucoadhesive. For example, a luminal space such as the gastrointestinal tract is surrounded by a mucus-coated surface. Mucoadhesive particles delivered to that space are normally removed from the luminal space and from the mucus-coated surface by the subject's natural removal mechanisms. The particles of the invention with less mucoadhesion will be able to remain in the luminal space for relatively long periods compared to mucoadhesive particles. This prolonged presence can prevent or reduce the separation of the particles and / or can allow a better distribution of the particles on the tissue surface. The prolonged presence can also affect the transport of particles through the luminal space, for example, the particles can be distributed in the mucous layer and can reach the underlying epithelium.

In certain embodiments, the core of the particles of the invention coated with the coating polymer can pass through mucus or a mucous barrier in a subject, exhibiting

117 prolonged retention, and / or increases the uniform distribution of particles from mucosal surfaces, for example, such substances are removed more slowly (for example, at least about 2 times, 5 times, about 10 times, or even at least a few 20 times slower) from a subject's body compared to a negative control of the particles of the invention.

The mobility of the particles of the invention in mucus can be characterized by, for example, the relative velocity and / or the diffusivity of the particles. In certain embodiments, the particles of the invention have a certain relative velocity, &lt;V_Igives^&gt;rel, which is defined as follows:

V_I¡_to&gt; sample V_mec| ia&gt; negative control ^&lt;V_media^&gt;rei = ___________________________________________ (Equation 1) ^&lt;Vmed¡a^&gt;positive control ~ ^&lt;Vmedia^&gt;negative control ^&lt;V_Iday^&gt; is the average speed of the average assembly path;

Vmedía is the speed of an individual particle averaged throughout its trajectory.

the sample is the particle of interest;

the negative control is 200 Nm carboxylated polystyrene particles; and the positive control is one of the densely pegylated 200 nm polystyrene particles with 2-5 kDa PEG.

Relative speed can be measured using a multiple particle tracking technique. For example, a

118 Fluorescence equipped with a CCD camera can be used to capture 15 s movies with a temporal resolution of 66.7 ms (15 frames / s) with 100 ^x the enlargement of various zones within each sample for each type of particle: sample, negative control, and positive control. The sample, negative control, and positive control can be fluorescent particles to observe the trace. Alternatively, the non-fluorescent particles can be coated with a fluorescent molecule, a fluorescently labeled surface agent, or a fluorescently labeled polymer. Advanced image processing software (eg Image Pro or MetaMorph) can be used to measure the individual paths of multiple particles on a time scale of at least 3,335 s (50 frames).

In some embodiments, a particle described herein has a relative velocity of greater than or equal to 0.3, greater than or equal to 0.4, greater than or equal to approximately 0.5, greater than or equal to 0.6, greater than or equal to approximately 0.7 , greater than or equal to approximately 0.8, greater than or equal to approximately 0.9, greater than or equal to 1.0, greater than or equal to approximately 1.1, greater than or equal to approximately 1.2, greater than or equal to about 1.3, greater than or equal to 1.4, greater than or equal to approximately 1.5, greater than or equal to 1.6, greater than or equal to 1.7, greater than or equal to approximately 1.8, greater than or equal to approximately 1.9 or greater than or equal to 2.0 in mucus . In some embodiments, a particle described herein has a relative velocity of less than or

119 equal to about 10.0, less than or equal to 8.0, less than or equal to 6.0, less than or equal to 4.0, less than or equal to 3.0, less than or equal to 2.0, less than or equal at 1.9, less than or equal to 1.8, less than or equal to 1.7, less than or equal to 1.6, less than or equal to 1.5, less than or equal to 1.4, less than or equal to 1 , 3, less than or equal to 1.2, less than or equal to 1.1, less than or equal to approximately 1.0, less than or equal to 0.9, less than or equal to 0.8, or less than or equal to 1.7 in the mucus. Combinations of the mentioned ranges are possible (for example, a relative speed of greater than or equal to 0.5 and less than or equal to 6.0). Other ranges are also possible. The mucus can be, for example, human cervicovaginai mucus.

In certain embodiments, a particle described herein can diffuse through mucus or a mucus barrier at a higher rate or diffusivity than a control particle or a corresponding particle (eg, a corresponding particle that is unchanged and / or is not coated with a described coating). In some cases, a particle described here can pass through mucus or a mucus barrier at a diffusivity rate, which is at least about 10 times, 20 times, 30 times, 50 times, 100 times, 200 times, 500 times , 1000 times, 2000 times, 5000 times, 10000 times or more, greater than a control particle or a corresponding particle. In some cases, a particle described here can pass through mucus or a mucus barrier at the rate of diffusivity that is less than or equal to about 10,000 times higher, less than or equal to about 5,000 times

120 greater than or less than 2000 times greater than or less than

<td>approximately</td><td> 1000</td><td>times</td><td>higher,</td><td>lower</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 500</td><td>times</td><td>higher,</td><td>less</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 200</td><td>times</td><td>higher,</td><td>lower</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 100</td><td>times</td><td>higher,</td><td>lower</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 50</td><td>times</td><td>higher,</td><td>lower</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 30</td><td>times</td><td>higher,</td><td>lower</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 20</td><td>times</td><td>older, or</td><td>less</td><td> 0</td><td>same</td><td>to</td>

approximately 10 times larger than the control particle or a corresponding particle. Combinations of the aforementioned ranges are also possible (for example, at least about 10 times and less than or equal to 1000 times greater than a control particle or a corresponding particle). Other ranges are also possible.

For the purposes of the comparisons described herein, the corresponding particles may be approximately the same size, shape, and / or density as the particles of the invention, but the lack of coating causes the particles of the invention to move in the mucus. In some embodiments, the measurement of the geometric least squares displacement and the velocity of diffusivity of the particles (for example, the corresponding particles and the particles of the invention) is based on a time scale of about 1 second, about 3 seconds, or about 10 seconds. Methods for determining the geometric least squares displacement and the velocity of

121 Diffusive displacement are known in the art. The particles of the invention can pass through mucus or a mucus barrier with a geometric least squares shift that is at least 10 times, about 30 times, about 100 times, about 300 times, about 1000 times, about 3000 times, about 10,000 times greater than corresponding particles or negative control particles. In some embodiments, the particles of the invention pass through the mucosa or a mucosal barrier with a geometric least squares shift that is less than about 10,000 times higher, less than about 3,000 times higher, less than about 1,000 times higher, lower to about 300 times higher, less than about 100 times higher, less than 30 times higher, or less than about 10 times larger than negative control particles or corresponding particles. Combinations of the aforementioned ranges are also possible (for example, at least about 10 times and less than about 1000 times greater than negative control particles or corresponding particles). Other ranges are also possible.

In some embodiments, the particles of the invention diffuse through a mucosal barrier at a rate approaching the rate or diffusivity at which the particles can diffuse through water. In some embodiments, the particles of the invention pass through a mucosal barrier to a

122 velocity or diffusivity less than about 1/100, less than 1/300, less than about 1/1000, less than about 1/3000, less than 1 / 10,000 of the diffusivity at which particles diffuse through of water under similar conditions. In some embodiments, the particles of the invention pass through a mucosal barrier at a rate or diffusivity that is greater than or equal to approximately 1 / 10,000, greater than or equal to approximately 1/3000, greater than or equal to approximately 1 / 1000, greater than or equal to approximately 1/300, or greater than or equal to 1/100 of the diffusivity at which the particles diffuse through water under similar conditions. Combinations of the aforementioned ranges are also possible (eg, greater than or equal to about 1/3000 and less than 1/300 of the diffusivity that the particles diffuse through water under similar conditions). Other ranges are also possible. The diffusivity measurement can be based on a time scale of about 1 second, or about 0.5 seconds, or about 2 seconds, or about 5 seconds, or about 10 seconds.

In some embodiments, the particles of the invention diffuse through human cervicovaginal mucus at a diffusivity that is less than about 1/500 of the diffusivity in which the particles diffuse through water. In some embodiments, the diffusivity measurement is based on a time scale of about 1 second, or about 0.5 seconds, or

123 about 2 seconds, or about 5 seconds, or about 10 seconds.

In certain embodiments, the present invention provides particles that travel through mucus, such as human cervicovaginal mucus, at absolute diffusivities. For example, the particles described here can travel at diffusivities of at least 1 x 10 '⁴ 2 x 10 'pm / s⁴ pm / s, 5 x 10 '⁴ pm / s, 1 x 10 '³ 2 x 10 'pm / s³ pm / s, 5 x 10 '³ pm / s, 1 x 10 '² 2 x 10 'pm / s² pm / s, 4 x 10 '² pm / s, 5 x 10 '² pm / s, 6 x 10 '² pm / s, 8 x 10 '² pm / s, 1 x 10 '¹ 2x10 pm / s ¹ pm / s, 5 x 10 '¹ pm / s, 1 pm / s or 2 pm / s. In some cases, the particles can travel at diffusivities less than or equal to

<td>2 pm / s,</td><td colspan="2">lower</td><td> 0</td><td colspan="3">equal to 1</td><td>pm / s,</td><td>lower</td><td> 0</td><td>same</td><td>to 5</td><td>X</td><td> 10’¹</td><td>pm / s,</td>

<td>lower</td><td> 0</td><td>same</td><td>to</td><td> 2</td><td>X</td><td> 10’¹</td><td>pm / s,</td><td>lower</td><td> 0</td><td>same</td><td>to 1</td><td>X</td><td> 10’¹</td><td>pm / s,</td>

<td>lower</td><td> 0</td><td>same</td><td>to</td><td> 8</td><td>X</td><td> 10’²</td><td>pm / s,</td><td>lower</td><td> 0</td><td>same</td><td>to 6</td><td>X</td><td> 10’²</td><td>pm / s,</td>

<td>lower</td><td> 0</td><td>same</td><td>to</td><td> 5</td><td>X</td><td> 10-2</td><td>pm / s,</td><td>lower</td><td> 0</td><td>same</td><td>to 4</td><td>X</td><td> 10-2</td><td>pm / s,</td>

less than or equal to 2 x

10'² pm / s, less than or equal to approximately x 10 '² pm / s, less than or equal to 5 x 10 '³ pm / s, less than or equal to 2 x 10 '³ pm / s, less than or equal to 1 x 10 '³ pm / s, less than or equal to 5 x 10 '⁴pm / s, less than or equal to 2 x 10 '⁴ pm / s or less than or equal to 1 x 10 '⁴ pm / s. Combinations of the aforementioned ranges are also possible (for example, greater than or equal to 2 x 10 '⁴ pm / se less than or equal to 1 x 10 '¹ pm / s). Other ranges are also possible. In some cases, the measurement is based on a time scale of about 1 second, or about 0.5 seconds, or about 2 seconds, or about 5 seconds, or about 10 seconds.

It should be noted that while mobility (for example, speed

124 relative and diffusivity) of the particles of the invention can be measured in human cervicovaginal mucus, mobility can be measured in other types of mucus.

In certain embodiments, a particle described herein is comprised of surface alteration portions at a given density. The surface alteration portions may be portions of a surface alteration agent that, for example, are exposed to the solvent containing the particle. As an example, the hydrolyzed PVA units / blocks may be surface alteration portions of the PVA surface alteration agent. In another example, the PEG segments may be portions of the PEG-PPO-PEG surface altering agent. In some cases, the surface alteration portions and / or surface alteration agents are present at a density of at least 0.001 units or molecules per nm², at least about 0.002, at least about 0.005, at least about 0.01, at least about 0.02, at least about 0.05, at least about 0.1, at least about 0.2, at least about 0.5, at least 1, at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100 units or molecules per nm², or more units or molecules per nm². In some cases, the surface alteration portions and / or surface alteration agents are present at a density less than or equal to 100 units or molecules per nm², less than or equal to about 50, less than or equal to

125 approximately 20, less than or equal to 10 and less than or equal to 5 and less than or equal to 2 and less than or equal to 1 and less than or equal to 0.5, less than or equal to 0.2, less than or equal to 0.1 , less than or equal to 0.05, less than or equal to 0.02, or less than or equal to about 0.01 units or molecules per nm². Combinations of the mentioned ranges are possible (for example, a density of at least 0.01 and less than or equal to 1 units or molecules per nm²). Other ranges are also possible. In some embodiments, the density values ​​described above may be an average density since the surface alteration agent is in equilibrium with other components of the solution.

Those skilled in the art may be aware of methods for estimating the average density of surface alteration portions (see, eg, Budijono et al., Colloids and Surfaces A: Physicochem. Eng. Aspects 2010, 360, 105-110; Joshi et al., Anal. Chim. Acta 1979, 704,153-160). For example, as described herein, the average density of surface alteration portions can be determined by HPLC quantification and DLS analysis. A suspension of particles for which the determination of surface density is of interest is first dimensioned using DLS: a small volume is diluted to an appropriate concentration (eg, about 100 pg / mL), and the average diameter z is taken as a representative measure of particle size. The remainder of the suspension is then divided into two aliquots. Using HPLC, the first aliquot is

126 analyzes for the total concentration of core material and for the total concentration of the surface alteration portion. Again by HPLC, the second aliquot is analyzed for the concentration of free or unaltered surface alteration portion. In order to obtain only the free or unbound surface alteration portion from the second aliquot, the particles, and thus any bonded surface alteration portion, are removed by ultracentrifugation. By subtracting the concentration of the unbound surface alteration portion from the total concentration of the surface alteration portion, the concentration of the attached surface alteration portion can be determined. Since the total concentration of core material is also determined from the first aliquot, the mass ratio between the core material and the surface alteration portion can be determined. Using the molecular weight of the surface alteration portion the mass material surface alteration portion number of the core can be calculated. To activate this number in a measurement of surface density, the surface area per mass of core material must be calculated. The volume of the particle was approximated as a sphere with the diameter obtained from DLS that allows the calculation of the surface area per mass of core material. In this way the number of surface alteration portions per surface area can be determined.

In certain embodiments, the particles of the invention

127 they comprise portions of surface alteration and / or agents that affect the zeta potential of the particle. The zeta potential of the particle can be, for example, at least about -100 mV, at least about -30 mV, at least about -10 mV, at least about -3 mV, at least about 3 mV, at least about 10 mV, at least about 30 mV, or at least about 100 mV. The zeta potential of the particles can also be, for example, less than about 100mV, less than 30mV, less than 10mV, less than 3mV, less than about -3mV, less than about -10mV, at less than about -30 mV, or less than about -100 mV. Combinations of the mentioned ranges are possible (for example, a zeta potential of at least -30 mV and less than about 30 mV). Other ranges are also possible.

The coated particles described herein can have any convenient shape and / or size. In some embodiments, a coated particle is substantially similar in shape to the shape of the nucleus. In some cases, a coated particle described herein may be a nanoparticle, that is, the particle has a characteristic dimension of less than 1 micron, where the characteristic dimension of the particle is the diameter of a perfect sphere with the same volume as the of the particle. In other embodiments, larger sizes are possible (eg, about 1 to 10 microns). A plurality of particles, in some embodiments, can also be characterized by a

128 average size (eg, a larger average cross-sectional dimension, or a smaller average cross-sectional dimension for the plurality of particles). A plurality of particles have an average size of, for example, less than or equal to 10 pm, less than or equal to 5 pm, less than or equal to 1 pm, less than or equal to about 800 nm, less than or equal to about 700 nm, less than or equal to about 500 nm, less than or equal to 400 nm, less than or equal to 300 nm, less than or equal to about 200 nm, less than or equal to about 100 nm, less than or equal to 75 nm, less than or equal to 50 nm, less than or equal to 40 nm, less than or equal to about 35nm, less than or equal to 30nm, less than or equal to 25nm, less than or equal to 20nm, less than or equal to 15nm, or less than or equal to about 5nm. In some cases, a plurality of particles have an average size of, for example, at least about 5nm, at least about 20nm, at least about 50nm, at least about 100nm, at least about 200nm , at least about 300 nm, at least about 400 nm, at least about 500 nm, at least about 1 pm, at least, or at least around 5 pm. Combinations of the aforementioned ranges are also possible (eg an average size of at least about 50nm and less than or equal to 500nm). Other ranges are also possible. In some embodiments, the sizes of the nuclei formed by a process described in this document have a Gaussian type distribution.

129

Pharmaceutical agents

A particle or pharmaceutical composition of the invention may comprise at least one pharmaceutical acceptable crystalline form of compound 3. In one embodiment, a particle or pharmaceutical composition comprises crystalline form A. In another embodiment, a particle or pharmaceutical composition comprises crystalline form B. The crystalline form of compound 3 may be present in the core and / or one or more coatings of the particle (eg, dispersed in the core and / or coating). In some embodiments, the crystalline form of Compound 3 may be disposed on the surface of the particles (eg, on the outside or inside of the surface of one or more coatings or on the surface of the core). The crystalline form of Compound 3 can be contained within the particle and / or arranged in a portion of the particle by commonly known techniques (eg, coating, adsorption, covalent bonding, and encapsulation). In some embodiments, the crystalline form of compound 3 is present during nucleation. In other embodiments, the crystalline form of compound 3 is not present during nucleation. In certain embodiments, the crystalline form of Compound 3 is present in the core shell.

In some embodiments, the crystalline form of Compound 3 contained in a particle or pharmaceutical composition of the invention has a therapeutic value and / or prophylactic effect on a mucosal tissue that will be the target. Non-limiting examples of tissues

130 Mucous include ophthalmic, respiratory (eg, including nasal, pharynx, trachea, bronchi, and membranes), oral (eg, including oral and esophageal membranes, and tonsil surface), gastrointestinal (eg, including stomach, small intestine, intestine thick, colon or rectum), nasal, and genital tissues (eg, including vaginal, cervical, and urethral membranes).

Any suitable number of pharmaceutical agents can be present in a particle or pharmaceutical composition of the invention. For example, in addition to a crystalline form of compound 3, at least 1, at least 2, at least 3, at least 4, at least 5 or more pharmaceutical agents may be present in the particle or pharmaceutical composition of the invention. In certain embodiments, less than 10 pharmaceutical agents are present in the pharmaceutical particle or composition of the invention.

In certain embodiments, the pharmaceutical agent in the pharmaceutical particles or compositions of the invention is a crystalline form of compound 3. In one embodiment, the pharmaceutical agent in the pharmaceutical particles or compositions of the invention is a crystalline form A of compound 3. In In another embodiment, the pharmaceutical agent in the particles or pharmaceutical compositions of the invention forms crystalline B of compound 3. The pharmaceutical agent described herein (eg, a crystalline form of compound 3) can be encapsulated in a polymer, a lipid, a protein, or a combination thereof.

131

Pharmaceutical compositions

In another aspect, the present invention provides pharmaceutical compositions comprising at least one particle of the invention. Pharmaceutical compositions described herein and for use in accordance with the articles and methods described herein may include a pharmaceutical acceptable carrier or excipient. A pharmaceutically acceptable excipient or pharmaceutically acceptable carrier may include a non-toxic, inert solid filler, diluent, encapsulating material, or formulation aid of any suitable type. Some examples of materials that can serve as a pharmaceutically acceptable carrier are sugars such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; jelly; talcum powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN 80; pH regulating agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate pH buffers, as well as other compatible non-toxic lubricants such as sodium lauryl sulfate and stearate

132 Magnesium, as well as coloring agents, releasing agents, coating agents, sweeteners, flavors and perfume-providing agents, preservatives and antioxidants, may also be present in the composition, at the discretion of the formulator. As may be appreciated by one of skill in the art, excipients may be chosen based on the route of administration, as described below, the pharmaceutical agent being delivered, the course of agent delivery time, etc.

The pharmaceutical compositions containing the particles described herein can be administered to a subject by any route known in the art. These include, but are not limited to, oral, nasal, sublingual (eg, intravenous, subcutaneous, intramuscular, intradermal injection), rectal, vaginal, intraarterial, intracisternally, intraperitoneal, intravitreal periocular, topical (eg, ocular or dermal , such as powders, creams, ointments or drops), buccal, inhalational administration. In some embodiments, the compositions described herein can be administered parenterally by injections (such as intravenous, intramuscular, or subcutaneous), drop infusion preparations, or suppositories. As would be appreciated by one of skill in this art, the route of administration and the effective dose to achieve the desired biological effect can be determined by the agent being administered, the target organ, the preparation being administered, the

133 course of administration time, disease being treated, intended use, etc.

In certain embodiments, pharmaceutical compositions are useful for delivery of a crystalline form of Compound 3 herein described through either mucus or a mucous surface in a subject. The pharmaceutical compositions may be delivered to the mucosal surface in the subject and may pass through a mucosal barrier in the subject (eg, mucus), and / or may exhibit prolonged retention and / or increased uniform distribution of the particles of the invention on the mucous surface, for example, due to reduced mucoadhesion.

In certain embodiments, the pharmaceutical compositions are bioavailable for Compound 3 useful for increasing it in the subject. In certain embodiments, pharmaceutical compositions are useful for increasing the concentration of compound 3 in the subject. In certain embodiments, the pharmaceutical compositions are useful for increasing the exposure of compound 3 in the subject. In addition, the pharmaceutical compositions could be useful for treating and / or preventing a disease (eg, eye disease) in a subject.

In addition, the pharmaceutical compositions can be administered parenterally as injections (intravenous, intramuscular, or subcutaneous), drop infusion preparations, or suppositories. For ophthalmic applications, the pharmaceutical compositions can be administered by injection (eg,

134 Infraocular, conjunctival, subconjunctival, intrastromal, or intravitreal intravitreal), or by the local or ophthalmic mucous membrane pathway, pharmaceutical compositions can be administered topically, such as solutions, suspensions (eg, eye drops), gels, or ointments.

In some embodiments, the particles described herein that can be administered in aerosol or inhalant formulations comprise one or more pharmaceutical agents, such as adjuvants, diagnostic agents, imaging agents, or therapeutic agents useful in inhalation therapy. The particle size of the drug should be such as to allow inhalation of substantially all of the drug to the lungs after administration of the aerosol formulation and may be, for example, less than 20 microns, for example, in the range of 1 at 10 microns, for example, about 1 to 5 microns, although other ranges are also possible. The size of the drug particles can be reduced by conventional means, for example, through milling or micronization. Alternatively, the particulate drug can be administered to the lungs through nebulization of a suspension. The final aerosol formulation may contain, for example, between 0.005-90% w / w, between 0.00550%, between 0.005-10%, at approximately 0.005-5% w / w, or between 0.01-1.0% w / w, of medication in relation to the total weight of the formulation. Other ranges are also possible.

It is desirable, but not necessary, that the formulations here

135 described do not contain components that can cause degradation of stratospheric ozone. In particular, in some embodiments, propellants are selected not to contain or consist essentially of chlorofluorocarbons such as CCI.₃F, CCI2F2 and CF3CCI3.

The aerosol may consist of a propellant. The propellant may optionally contain an adjuvant of higher polarity and / or a higher boiling point than the propellant. Polar adjuvants that can be used include aliphatic alcohols (eg, C₂.₆) and polyols, such as ethanol, isopropanol, and propylene glycol, preferably ethanol. In general, only small amounts of polar adjuvants (eg 0.05-3.0% w / w) may be necessary to improve dispersion stability, use of amounts in excess of 5% w / w may tend to dissolve the drug . Formulations in accordance with the modalities described herein may contain less than 1% w / w, eg, about 0.1% w / w, of polar adjuvant. However, the formulations described herein can be substantially free of polar adjuvants, especially ethanol. Suitable volatile aids include saturated hydrocarbons such as propane, nbutane, isobutane, pentane, and isopentane, and alkyl ethers such as dimethyl ether. In general, up to 50% w / w of the propellant can include a volatile adjuvant, for example, up to 30% w / w of a saturated volatile Ci-Ce hydrocarbon. Optionally, the aerosol formulations according to the invention can include one or more of the agents

136 surfactants. Surfactants can be physiologically acceptable on administration by inhalation. This category includes surfactants such as L-af or sf atidi I co I ina (PC), 1,2-di pa I m it oi If osf at id I co I ina (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, natural lecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether, lauryl polyoxyethylene ether, lauryl polyoxyethylene ether Synthetic Lecithin, Diethylene Glycol Dioleate, Tetrahydrofurfuryl Oleate, Ethyl Oleate, Isopropyl Myristate, Glycerol Monooleate, Glyceryl Monostearate, Glyceryl Monoricinoleate, Cetyl Alcohol, Stearyl Alcohol, Polyethylene Glyceryl Ilyl, Polyethylene Glyceryl olive oil, glyceryl monolaurate, corn oil, cottonseed oil, and sunflower oil.

The formulations described herein can be prepared by dispersing the particles in the selected propellant and / or co-propellant in a suitable container, for example, with the aid of sound application. The particles can be suspended in a co-propellant and placed as fillers in a suitable container. The container valve is then sealed in place and the propeller is pressed in by filling through the valve in the conventional manner. The particles can be suspended or dissolved in a liquefied propeller, sealed in a container with a metering valve, and attached to an actuator. Such dose inhalers

137 Such measures are well known in the art. The metering valve can dose from 10 to 500 pL and preferably from 25 to 150 pl. In certain embodiments, dispersion can be achieved by using dry powder inhalers (eg, rotary inhaler) for the particles (which remain as dry powders). In other embodiments, the nanospheres may be suspended in an aqueous liquid and nebulized in very fine droplets that volatilize into the lungs.

Sonic nebulizers can be used because they minimize the agent's exposure to shear stress, which can result in particle degradation. Ordinarily, an aqueous aerosol solution is made by formulating an aqueous solution or suspension of the particles together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary according to compositional requirements, but typically include nonionic surfactants (TWEENS, PLURONIC®, or polyethylene glycol), harmless proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids like glycine , pH regulators, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

The compositions and / or formulations described herein can have any suitable osmolarity. In certain embodiments, a composition and / or formulation described herein can have an osmolarity of at least about 0 mOsm / L, at least about 5

138 mOsm / L, at least about 25 mOsm / L, at least about 50 mOsm / L, at least about 75 mOsm / L, at least about 100 mOsm / L, at least about 150 mOsm / L , at least around 200 mOsm / L, at least around 250 mOsm / L, or at least around 310 mOsm / L. In certain embodiments, a composition and / or formulation described herein may have an osmolarity less than or equal to about 310 mOsm / L, less than or equal to about 250 mOsm / L, less than or equal to about 200 mOsm / L, less than or equal to about 150 mOsm / L, less than or equal to about 100 mOsm / L, less than or equal to about 75 mOsm / L, less than or equal to about 50 mOsm / L, less than or equal to about 25 mOsm / L, or less than or equal to 5 mOsm / L. Combinations of the above mentioned ranges are also possible (eg, an osmolarity of at least about 0 mOsm / L and less than or equal to about 50 mOsm / L). Other ranges are also possible. The osmolarity of the composition and / or formulation can be varied by changing, for example, the concentration of salts present in the solvent of the composition and / or formulation.

The pharmaceutical composition of the invention may include one or more pharmaceutical agents described herein, as a crystalline form of compound 3. In certain embodiments, the pharmaceutical composition includes a plurality of particles of the invention that comprise one or more pharmaceutical agents in the core and /or the

139 coated particles. In some embodiments, the ratio of the surface alteration agent to the pharmaceutical agent (or salt thereof) can be at least 0.001: 1 (weight ratio, molar ratio, or p: v ratio), at least 0.01: 1, 0.01: 1, at least at least 1: 1, at least 2: 1, less than 3: 1, at least 5: 1, at least 10: 1, at least 25: 1 , 50: 1, at least a minimum of 100: 1, or at least 500: 1. In some cases, the ratio of surface altering agent to pharmaceutical agent (or salt thereof) may be less than or equal to 1000: 1 (weight ratio or molar ratio), less than or equal to 500: 1, less than or equal to 100: 1, less than or equal to 75: 1, less than or equal to 50: 1, less than or equal to 25: 1, less than or equal to 10: 1, less than or equal to 5: 1, less than or equal to 3: 1, less than or equal to 2: 1, less than or equal to 1: 1, or less than or equal to 0.1: 1. Combinations of the mentioned ranges are possible (for example, a ratio of at least 5: 1 and less than or equal to 50: 1). Other ranges are also possible. In some embodiments, the pharmaceutical composition of the invention includes the aforementioned ranges for the weight ratio of each of the pharmaceutical agents to the weight of each of the one or more surface alteration agents during a formation process and / or a dilution process described here. In certain embodiments, the pharmaceutical composition includes the aforementioned ranges for the weight ratio of each of the pharmaceutical agents to the weight of each of the one or more surface alteration agents immediately before the pharmaceutical composition is administered to a subject or contact

140 with a biological sample. The pharmaceutical agent may be present in the pharmaceutical composition of the invention in any suitable amount, for example, at least about 0.01% by weight, at least about 0.1% by weight, at least about 1% in weight, at least about 5% by weight, at least about 10% by weight, at least 30% by weight of the pharmaceutical composition. In some cases, the pharmaceutical agent may be present in the pharmaceutical composition at less than about 30% by weight, less than 10% by weight, less than 5% by weight, less than 2% by weight, or less than about 1 % by weight of the pharmaceutical composition. Combinations of the aforementioned ranges are also possible (eg, present in an amount of at least 0.1% by weight and less than 10% by weight of the pharmaceutical composition). Other ranges are also possible. In certain embodiments, the pharmaceutical agent is approximately 0.1-2% by weight of the

<td>composition</td><td>pharmaceutical.</td><td>In certain</td><td>modalities,</td><td>the</td><td>agent</td>

<td>pharmacist</td><td colspan="2">is approximately</td><td>2-20% in</td><td>weight</td><td>of the</td>

<td>composition</td><td>pharmaceutical.</td><td>In certain</td><td>modalities,</td><td>the</td><td>agent</td>

<td>pharmacist</td><td>It is</td><td colspan="2">about 0.2%</td><td>in</td><td>weight,</td>

about 0.4% by weight, about 1% by weight, about 2% by weight, about 5% by weight, or about 10% by weight of the pharmaceutical composition.

In a set of modalities, a composition and / or

141 formulation includes one or more chelating agents. A chelator used herein refers to a chemical compound that has the ability to react with a metal ion to form a complex through one or more bonds. One or more bonds are typically ionic or coordination bonds. The chelating agent can be an organic or inorganic compound. A metal ion capable of catalyzing certain chemical reactions (for example, oxidation reactions) can lose its catalytic activity when the metal ion is bound to a chelating agent to form a complex. Therefore, a chelating agent can show conservative properties when bound to a metal ion. Any suitable chelating agent that has preservative properties can be used, such as phosphonic acids, aminocarboxylic or hydroxycarboxylic acids, acids, polyamines, amino alcohols, polymers and chelating agents. Specific examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), Nh id roxi et i I and i I acid in diaminetriacetic (HEDTA), tetraborates, triethylamine diamine, and salts and derivatives thereof. In certain modalities, the chelating agent is EDTA. In certain embodiments, the chelating agent is an EDTA salt. In certain embodiments, the chelating agent is disodium EDTA.

In certain embodiments, the pharmaceutical composition includes a plurality of particles of the invention that make up the agent.

142 chelator in the formulation containing the particles. In certain embodiments, the chelating agent concentration is greater than or equal to 0% by weight, greater than or equal to approximately 0.0001% by weight, greater than or equal to approximately 0.003% by weight, greater than or equal to approximately 0.01% by weight, greater than or equal to 0.03% by weight, greater than or equal to 0.05% by weight, greater than or equal to

<td>approximately</td><td> 0.1%</td><td>in</td><td colspan="2">weight,</td><td>higher</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 0.3%</td><td>in</td><td colspan="2">weight,</td><td>higher</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 1%</td><td>in</td><td>weight</td><td> 0</td><td>higher</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 3%</td><td>in</td><td>weight.</td><td>In</td><td>certain</td><td colspan="3">modalities,</td><td>the</td>

<td>concentration of</td><td colspan="2">agent</td><td colspan="2">chelator</td><td colspan="2">is less</td><td> 0</td><td>same</td><td>to</td>

about 3% by weight, less than or equal to about

1% by weight, less than or equal to about 0.3% by weight, less than or equal to about 0.1% by weight, less than or equal to 0.05% by weight, less than or equal to 0.03% by weight, less which equals approximately

0.01% by weight, less than or equal to approximately

0.003% by weight, less than or equal to approximately

0.001% by weight or less or equal approximately

0.0003% by weight. Combinations of the mentioned ranges are possible (eg, a concentration greater than or equal to about 0.01% by weight and less than or equal to about 0.3% by weight). Other ranges are also possible. In certain embodiments, the concentration of the chelating agent is approximately 0.001-0.1% by weight. In certain embodiments, the concentration of the chelating agent is approximately 0.005% in

143 weight. In certain embodiments, the concentration of the chelating agent is about 0.01% by weight. In certain embodiments, the concentration of the chelating agent is about 0.05% by weight. In certain embodiments, the concentration of the chelating agent is about 0.1% by weight.

In some embodiments, an antimicrobial agent can be included in a composition and / or formulation including the coated particles described herein. An antimicrobial agent used herein refers to a bioactive agent effective in inhibiting prevention or protection against microorganisms, such as bacteria, microbes, fungi, viruses, spores, yeasts, molds, and others, generally associated with infections. Examples of antimicrobial agents include, clindamycin, cephalosporins, carbapenems, minocyclines, oramfean ico I, rifampin, penicillins, monobactams, quinolones, tetracyclines, macrolides, sulfa antibiotics, trimethoprim, fusidic acid, amphotericin, amphotericin, amphotericin. , cilofungin, bactericidal nitrofuran compounds, metallic silver nanoparticles or a silver alloy containing approximately 2.5% / w copper, silver citrate, silver acetate, silver benzoate, bismuth pyrithione, zinc pyrithione, zinc percarbonates, zinc perborates, bismuth salts, parabens (eg, methyl, ethyl-, propyl, butyl, and octyl esters of benzoic acid), citric acid, benzalkonium chloride ( BAC), rifamycin and sodium percarbonate.

In certain embodiments, the pharmaceutical composition includes

144 a plurality of particles of the invention that make up the antimicrobial agent in the formulation containing the particles. In certain embodiments, the concentration of the antimicrobial agent may be greater than or equal to approximately 0% by weight, greater than or equal to approximately 0.0001% by weight, greater than or equal to approximately 0.003% by weight, greater than or equal to approximately 0.01% by weight, greater than or equal to 0.03% by weight, greater than or equal to approximately 0.1% by weight greater than or equal to approximately 0.3% by weight, greater than or equal to approximately 1% by weight or greater that or approximately

3% by weight. In certain embodiments, the concentration of the antimicrobial agent may be less than or equal to about 3% by weight, less than or equal to about

1% by weight, less than or equal to about 0.3% by weight, less than or equal to about 0.1% by weight, less than or equal to 0.03% by weight, less than or equal to about 0.01% by weight , less than or equal to approximately 0.003% by weight, less than or equal to approximately 0.001% by weight or less than or equal to approximately 0.0003% by weight. Combinations of the mentioned ranges are possible (for example, a concentration greater than or equal to approximately 0.001% by weight and less than or equal to approximately 0.1% by weight). Other ranges are also possible. In certain embodiments, the concentration of the antimicrobial agent is approximately 0.001-0.05% by weight. In certain modalities, the concentration of the antimicrobial agent is

145 about 0.002% by weight. In certain embodiments, the concentration of the antimicrobial agent is approximately 0.005% by weight. In certain embodiments, the concentration of the antimicrobial agent is approximately 0.01% by weight. In certain embodiments, the concentration of the antimicrobial agent is approximately 0.02% by weight. In certain embodiments, the concentration of the antimicrobial agent is around 0.05% by weight.

In some embodiments, a tonicity agent can be included in a composition and / or formulation including the coated particles described herein. A tonicity agent used herein refers to a compound or substance that can be used to adjust the composition of a formulation to the desired osmolarity range. In certain embodiments, the desired osmolarity range is a blood compatible isotonic solution range. In certain modalities, the desired osmolarity range is hypotonic. In certain modalities, the desired osmolarity range is hypertonic. Examples of tonicity agents include glycerin, lactose, dextrose, mannitol, sodium chloride, sodium sulfate, sorbitol, saline-sodium citrate (SSC), and the like. In certain embodiments, a combination of one or more tonicity agents can be used. In certain embodiments, the tonicity agent is glycerin. In certain embodiments, the tonicity agent is sodium chloride.

A tonicity agent (such as those described here) may be

146 present in a suitable concentration in a composition and / or formulation including the coated particles described herein. In certain embodiments, the concentration of the tonicity agent is greater than or equal to 0% by weight, greater than or equal to approximately 0.001% by weight, greater than or equal to 0.03% by weight, greater than or equal to approximately 0.1% in weight, greater than or equal to approximately 0.3% by weight, greater than or equal to approximately 1% by weight, greater than or equal to approximately 3% by weight, greater than or equal to approximately 10% by weight, greater than or equal to at 20% by weight, or greater than or equal to about 30% by weight. In certain embodiments, the concentration of the tonicity agent is less than or equal to about 30% by weight, less than or equal to 10% by weight, less than or equal to 3% by weight, less than or equal to 1% by weight, less than or equal to about 0.3% by weight, less than or equal to about 0.1% by weight, less than or equal to 0.03% by weight, less than or equal to about 0.01% by weight, or less or equal to about 0.003% by weight. Combinations of the mentioned ranges are possible (eg, a concentration greater than or equal to about 0.1% by weight and less than or equal to about 10% by weight). Other ranges are also possible. In certain embodiments, the concentration of the tonicity agent is approximately 0.1-1%. In certain embodiments, the concentration of the tonicity agent is approximately 0.53%. In certain modalities, the concentration of the tonicity agent

147 it is about 0.25%. In certain embodiments, the concentration of the tonicity agent is approximately 0.45% by weight. In certain embodiments, the concentration of the tonicity agent approaches 0.9% by weight. In certain modalities, the concentration of the tonicity agent approaches 1.2% by weight. In certain embodiments, the concentration of the tonicity agent approaches 2.4% by weight. In certain embodiments, the concentration of the tonicity agent is approximately 5% by weight.

In certain embodiments, a composition and / or formulation described herein can have an osmolarity of at least about 0 mOsm / L, at least about 5 mOsm / L, at least about 25 mOsm / L, at least about 50 mOsm / L, at least about 75 mOsm / L, at least around 100 mOsm / L, at least around 150 mOsm / L, at least around 200 mOsm / L, at least around 250 mOsm / L , at least around 310 mOsm / L, or at least around 450 mOsm / L. In certain embodiments, a composition and / or formulation described herein may have an osmolarity of less than or equal to about 450 mOsm / L, less than or equal to about 310 mOsm / L, less than or equal to

<td>approximately</td><td> 250</td><td>mOsm / L,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 200</td><td>mOsm / L,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 150</td><td>mOsm / L,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 100</td><td>mOsm / L,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

about 75 mOsm / L, less than or equal to about mOsm / L, less than or equal to about 25 mOsm / L, or

148 less than or equal to 5 mOsm / L. Combinations of the above mentioned ranges are also possible (eg, an osmolarity of at least about 0 mOsm / L and less than or equal to about 50 mOsm / L). Other ranges are also possible.

It is appreciated in the art that the ionic resistance of a pharmaceutical composition of the invention comprising a plurality of particles of the invention can affect the polydispersity of the plurality of the particles. Ionic resistance can also affect the colloidal stability of the plurality of particles. For example, a relatively high ionic resistance of the pharmaceutical composition can cause the plurality of particles to clot and thus can destabilize the pharmaceutical composition. In some embodiments, the pharmaceutical composition is stabilized by repulsive forces between the particles. For example, the plurality of particles can be electrically or electrostatically charged. Two charged particles repel each other, avoiding collision and aggregation. When the repulsive forces between the particles weaken or become attractive, the plurality of particles can begin to add. For example, when the ionic resistance of the pharmaceutical composition is increased to a certain level, the charges (eg, negative charges) of the plurality of the particles can be neutralized by the oppositely charged ions present in the pharmaceutical composition (eg. example the Na ions⁺ in the solution). As a result, the plurality of particles can

149 collide and link together to form larger aggregates (eg, clusters or flocculations). The aggregates formed from particles can also vary in size and therefore the polydispersity of the pharmaceutical composition can also increase. For example, a pharmaceutical composition of the invention comprising particles of similar size can be converted to a pharmaceutical composition comprising particles having different sizes (for example, due to aggregation) when the ionic strength of the pharmaceutical composition increases above a certain level. In the course of aggregation, the aggregates can grow in size and eventually settle to the bottom of the container, and the pharmaceutical composition is considered colloidally unstable. Once the plurality of particles in a pharmaceutical composition form aggregates, it is generally difficult to break the aggregates down into individual particles.

Some pharmaceutical compositions of the invention show unexpected properties in that, among other things, the presence of one or more ionic tonicity agents (for example, a salt, such as NaCI) in pharmaceutical compositions at certain concentrations actually decreases or maintains the degree aggregation of the particles present in the pharmaceutical compositions and / or not significantly increase the aggregation. In certain embodiments, the polydispersity of the pharmaceutical composition decreases, is relatively constant, or does not change

150 an appreciable amount upon addition of one or more ionic tonicity agents to the pharmaceutical composition. For example, in some embodiments, the polydispersity of a pharmaceutical composition is relatively constant in the presence of added ionic resistance and / or when added, the ionic resistance of the pharmaceutical composition remains relatively constant or higher (for example, during formation and / or or described dilution process). In certain embodiments, when the ion resistance increases by at least 50%, polydispersity increases by less than about 300%, less than about 100%, less than 30%, less than about 10%, less than 3% , or less than approximately 1%. In certain embodiments, when the ion resistance increases by at least 50%, the polydispersity increases by greater than or equal to 1%, greater than or equal to approximately 3%, greater than or equal to approximately 10%, greater than or equal to approximately 30%, or equal to or greater than 100%. Combinations of the mentioned ranges are possible (for example, an increase in polydispersity of less than 30% and greater than or equal to 3%). Other ranges are also possible.

The ionic resistance of a pharmaceutical composition of the invention can be controlled (eg, increased, decreased, or maintained) through a variety of means, such as the addition of one or more ionic tonicity agents (eg, a salt, such as NaCI) to the pharmaceutical composition. In certain

151 Modalities, the ion resistance of a pharmaceutical composition of the invention is greater than or equal to approximately 0.003 M, greater than or equal to approximately 0.001 m, greater than or equal to approximately 0.003 m, greater than or equal to approximately 0.01 M , greater than or equal to approximately 0.03 M, greater than or equal to approximately 0.1 M, greater than or equal to approximately 0.3 M, greater than or equal to approximately 1 M, greater than or equal to 3 m , or greater than or equal to about 10 M. In certain embodiments, the ionic resistance of a pharmaceutical composition of the invention is less than about 10m, less than about 3M, less than about 1m, less than about 0.3M, less than about 0.1 m, less than about 0.03 m, less than about 0.01 M, less than about 0.003 m, less than about 0.001 m, or less than about 0.0003 M. Combinations of the aforementioned ranges are possible (for example, an ion resistance of greater than or equal to 0.01 m and less than 1 m). Other ranges are also possible. In certain embodiments, the ion resistance of a pharmaceutical composition of the invention is about 0.1M, about 0.15m, or about 0.3M.

In certain embodiments, polydispersity of a pharmaceutical composition does not change upon the addition of one or more ionic tonicity agents to the pharmaceutical composition. In certain modalities, polydispersity does not increase significantly after

152 addition of one or more ionic tonicity agents in the pharmaceutical composition. In certain embodiments, polydispersity increases to a level described after the addition of one or more ionic tonicity agents to the pharmaceutical composition.

The polydispersity of a pharmaceutical composition of the invention comprising a plurality of particles of the invention can be measured by the polydispersity index (PDI). In certain embodiments, the PDI of the pharmaceutical composition is less than 1, less than about 0.8, less than 0.6, less than about 0.4, less than about 0.3, less than about 0.2 , less than about 0.15, less than about 0.1, less than about 0.05, less than about 0.01 or less than about 0.005. In certain embodiments, the PDI of the pharmaceutical composition is greater than or equal to approximately 0.005, greater than or equal to approximately 0.01, greater than or equal to 0.05, equal to or greater than 0.1, greater than or equal to approximately 0.15, greater than or equal to approximately 0.2, greater than or equal to approximately 0.3, greater than or equal to 0.4, greater than or equal to 0.6, greater than or equal to 0.8, or greater than or equal to 1 . Combinations of the ranges mentioned above are possible (for example, a POI of greater than or equal to 0.1 and less than 0.5). Other ranges are also possible. In certain embodiments, the PDI of the pharmaceutical composition is about 0.1, 0.15, or about 0.2. In certain embodiments, the pharmaceutical composition is highly dispersible and does not tend to

153 form aggregates. Even when the particles do not form aggregates, the aggregates can easily be divided into individual particles without rigorously shaking the pharmaceutical composition.

For example, in some embodiments, the polydispersity of a composition and / or formulation is relatively constant in the presence of added ionic resistance and / or when added, the ionic resistance of the composition and / or formulation remains relatively constant or increased (for example , during the formation and / or dilution process). In certain embodiments, when the ion resistance increases by at least 50%, the polydispersity increases by less than or equal to about 200%, less than or equal to about 150%, less than or equal to about 100% , less than or equal to approximately 75%, less than or equal to approximately 50%, less than or equal to approximately 30%, less than or equal to approximately 20%, less than or equal to approximately 10%, less than or equal to approximately 3%, or less than or equal to approximately 1%. In certain embodiments, when the ion resistance is increased by at least 50%, the polydispersity increases by greater than or equal to approximately 1%, greater than or equal to approximately 3%, greater than or equal to approximately 10%, greater than or equal to approximately 30%, or greater than or equal to approximately 100%. The combinations of the ranges mentioned above are

154 possible (for example, a polydispersity increase of less than or equal to 50% and greater than or equal to 1%). Other ranges are also possible.

The ionic resistance of a formulation described here can be controlled (eg, increased) through a variety of means, such as the addition of one or more ionic tonicity agents (eg, a salt such as NaCI) to the formulation. . In certain embodiments, the ion resistance of a formulation described herein is greater than or equal to approximately 0.0005 M, greater than or equal to approximately 0.001 m, greater than or equal to approximately 0.003 m, greater than or equal to approximately 0, 01 M, greater than or equal to approximately 0.03 M, greater than or equal to approximately 0.1 M, greater than or equal to approximately 0.3 M, greater than or equal to approximately 1 M, greater than or equal to 3 m, or greater than or equal to about 10 M. In certain embodiments, the ion resistance of a formulation described herein is less than or equal to about 10m, less than or equal to about 3M, less than or equal to about 1m, less than or equal to about 0.3m, less than or equal to at about 0.1m, less than or equal to about 0.03m, less than or equal to about 0.01m, less than or equal to about 0.003m, less than or equal to about 0.001m, or less than or equal to about 0.0005 M. Combinations of the ranges mentioned above are possible (for example, an ionic resistance of greater than or equal to about 0.01 M e

155 less than or equal to about 1 m). Other ranges are also possible. In certain embodiments, the ion resistance of a formulation described herein is about 0.1M. In certain embodiments, the ion resistance of a formulation described herein is approximately 0.15M. In certain embodiments, the ion resistance of an Formulation described here is approximately 0.3 M.

Generally, it is desirable for a formulation to be sterile before or after administration to a subject. A sterile formulation is essentially free of pathogenic microorganisms, such as bacteria, microbes, fungi, viruses, spores, yeasts, molds, and others, generally associated with infections. In some embodiments, the compositions and / or formulations including the coated particles described herein may be subject to an aseptic and / or other sterilization process. An aseptic process normally involves sterilization of the components of the formulation, the final formulation, and / or closure of the packaging of a product through a process such as heat, gamma irradiation, ethylene oxide, or filtration and then combination in a sterile environment. In some cases, an aseptic process is preferred. In other embodiments, terminal sterilization is preferred.

Examples of other sterilization methods include radiation sterilization (eg, gamma radiation, electrons, or X-rays), heat sterilization, sterile filtration, and

156 sterilization with ethylene oxide. The terms radiation and irradiation are used interchangeably here. Unlike other sterilization methods, radiation sterilization has the advantage of high penetrating capacity and instantaneous effects, without the need to control temperature, pressure, vacuum or humidity on some occasions. In certain embodiments, the radiation used to sterilize the coated particles described herein is gamma radiation. Gamma radiation can be applied in an amount sufficient to kill most or substantially all microbes in or on the coated particles. The temperature of the coated particles described here and the radiation rate can be relatively constant throughout the gamma radiation period. Gamma irradiation can be performed at any suitable temperature (eg, room temperature, about 40 ° C, between about 30 ° C to about 50 ° C). Unless otherwise indicated, the gamma radiation measurements described herein refer to those made at approximately 40 ° C.

In embodiments where a sterilization process is used, it may be desired that the process does not: (1) significantly change the particle size of the coated particles described herein; (2) significantly change the integrity of the active ingredient (as a drug) of the coated particles described herein; and (3) generate unacceptable concentrations of impurities during or after the process. In certain

157 In other ways, the impurities generated during or after the process are degradation agents of the active ingredient of the coated particles described herein.

In certain embodiments, a process used to sterilize a composition and / or formulation described herein results in the presence of one or more degradation agents in the formulation of less than or equal to 10% by weight (relative to the weight of the drug not degraded), less than or equal to about 3% by weight, less than or equal to 2% by weight, less than or equal to 1.5% by weight, less than or equal to 1% by weight, less than or equal to 0, 9% by weight, less

<td colspan="5">that or equal to about 0.8% by weight,</td><td colspan="4">less than or equal to</td>

<td>approximately</td><td> 0,7%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 0,6%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 0.5%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td>or</td><td>same</td><td>to</td>

<td>approximately</td><td> 0,4%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 0,3%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td>or</td><td>same</td><td>to</td>

<td>approximately</td><td> 0,2%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>approximately</td><td> 0,15%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td> 0</td><td>Equal</td><td>to</td>

<td>approximately</td><td> 0,1%</td><td>in</td><td>weight,</td><td>less</td><td>than</td><td>or</td><td>Equal</td><td>to</td>

<td>approximately</td><td> 0.03%,</td><td colspan="2">smaller than</td><td>or the same</td><td colspan="4">to approximately</td>

<td colspan="3">0.01% by weight, less than oi</td><td>equal to</td><td colspan="3">about 0</td><td> .003%</td><td>in</td>

<td>weight or less than</td><td>or alike</td><td colspan="3">to approximately</td><td> 0,001%</td><td>in</td><td>weight.</td><td>In</td>

<td colspan="2">some modalities, the</td><td colspan="2">process</td><td>result</td><td>in a</td><td colspan="2">agent</td><td>of</td>

degradation in the formulation in greater than or equal to approximately

0.001% by weight, greater than or equal to approximately 0.003% by weight,

158 greater than or equal to approximately 0.01% by weight, greater than or equal to

<td>to about 0.03%</td><td>in</td><td>weight,</td><td>higher</td><td> 0</td><td>same</td><td>to</td>

<td>about 0.1% in</td><td>weight,</td><td>higher</td><td>than</td><td> 0</td><td>same</td><td>to</td>

<td>about 0.3% in</td><td>weight,</td><td>higher</td><td>than</td><td> 0</td><td>same</td><td>to</td>

about 1% by weight, greater than or equal to about 3% by weight or greater than or equal to about 10% by weight. Combinations of the aforementioned ranges are also possible (eg, less than or equal to about 1% by weight and greater than or equal to about 0.01% by weight). Other ranges are also possible.

When gamma irradiation is used in a sterilization process, the accumulated amount of the gamma radiation used may vary. In certain embodiments, the cumulative amount of gamma radiation is greater than or equal to approximately 0.1 kGy, greater than or equal to approximately 0.3 kGy, greater than or equal to approximately 1 kGy, greater than or equal to approximately 3 kGy, greater than or equal to approximately 10 kGy, greater than or equal to approximately 30 kGy, greater than or equal to approximately 100 kGy, or greater than or equal to approximately 300 kGy. In certain embodiments, the cumulative amount of gamma radiation is less than or equal to about 0.1 kGy, less than or equal to about 0.3 kGy, less than or equal to about 1 kGy, less than or equal to about 3 kGy, less than or equal to about 10 kGy, less than or equal to about 30 kGy, less than or equal to

159 approximately 100 kGy, or less than or equal to approximately 300 kGy. Combinations of the aforementioned ranges are possible (eg, greater than or equal to about 1 kGy and less than or equal to about 30 kGy). Other ranges are

<td colspan="2">also possible. In</td><td>certain modalities,</td><td>multiple</td><td>dose of</td>

<td>radiation</td><td>are used</td><td>to get a</td><td>dose of</td><td>radiation</td>

<td>accumulated</td><td>desired.</td><td></td><td></td><td></td>

<td>The</td><td colspan="2">compositions and / or formulations</td><td>described</td><td>in this</td>

<td>document</td><td>they can have</td><td colspan="3">adequate pH values. The term pH,</td>

<td>unless</td><td>be arranged</td><td>something else refers</td><td>at a pH</td><td>measured at</td>

ambient temperature (for example, around 20 ° C, about 23 ° C or 25 ° C). The compositions and / or formulations have, for example, an acidic pH, a neutral pH or a basic pH and may depend, for example, on where the compositions and / or formulations are to be supplied into the body. In certain embodiments, the compositions and / or formulations have a physiological pH. In certain embodiments, the pH value of the composition and / or formulations is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, per at least about 6.2, at least about 6.4, at least about 6.6, at least about 6.8, at least about 7, at least about 7.2, at least about 7.4, at least about 7.6, at least about 7.8, at

160 minus about 8, at least about 8.2, at least about 8.4 to 8.6, at least a minimum of about 8.8, at least about 9, at least about 10, at least about 11, or at least about 12. In certain embodiments, the pH value of the compositions and / or formulations is less than or equal to about 12, less than or equal to about 11, less than or equal to about 10, less than or equal to about 9, and less than or equal to approximately 8.8, less than or equal to approximately 8.6, less than or equal to approximately 8.4, less than or equal to approximately 8.2, less than or equal to approximately 8 and less than or equal to about 7.8, less than or equal to about 7.6, less than or equal to about 7.4, less than or equal to about 7.2, less than or equal to about 7, less than or equal to about 6.8, less than or equal to about 6.6, less than or equal to about 6.4, less than or equal to about 6.2, less than or equal to approximately 6 and less than or equal to approximately 5 and less than or equal to approximately 4 and less than or equal to approximately 3 and less than or equal to approximately 2, or less than or equal to approximately 1. Combinations of the aforementioned ranges are possible (for example, a pH value of at least about 5 and less than or equal to about 8.2). Other ranges are also possible. In certain modalities, the

161 pH value of the composition and / or formulations described in this document is at least about 5 and less than or equal to about 8.

In some embodiments, the particles, compositions, and / or formulations described herein increase the ocular bioavailability of Compound 3 by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least a 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 5 times, at least about 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 500 times, or at least about 1000 times. In certain embodiments, the particles, compositions, and / or formulations described herein increase the ocular bioavailability of Compound 3 by less than or equal to about 1000 times, less than or equal to about 500 times, less than or equal to about

100 times less than or equal to about 50 times less than or equal to about 20 times less than or equal to about 10 times less than or equal to about 5 times less than or equal to about 200% less than or equal to about 150%, less than or equal to

162 about 100%, less than or equal to about 90%, less than or equal to about 80%, less than or equal to 70%, less than or equal to about 60%, less than or equal to about 50%, less than or equal to approximately 40%, less than or equal to approximately 30%, less than or equal to approximately 20%, or less than or equal to approximately 10%. Combinations of the aforementioned ranges are also possible (for example, an increase of at least 10% and less than or equal to approximately 10 times). Other ranges are also possible. In some cases, the AUC of compound 3 increases in tissue and / or fluid in the front of the eye. In other cases, the AUC of compound 3 increases in tissue and / or fluid in the back of the eye.

In general, an increase in ocular bioavailability can be calculated by taking the difference in AUC measured in an eye tissue of interest (eg, in aqueous humor) between that of a test composition and a control composition, and dividing the difference between the bioavailability of the control composition. A test composition can include particles that comprise a crystalline form of Compound 3, and the particles can be characterized as penetrating mucus (for example, they have a relative mucus velocity greater than about 0.5, or another relative velocity here. described). A control composition can include particles comprising the same crystalline form of compound 3 as that present in the test composition, the

163 particles substantially similar in size as those in the composition test, but not penetrating mucus (for example, having a relative velocity in mucus of less than or equal to about 0.5, or another relative velocity described herein).

The ocular bioavailability of Compound 3 can be measured in a suitable animal model (eg, in a New Zealand white rabbit model, or a Gottingen mini-pig model). The concentration of compound 3 and, where appropriate, its metabolite (s), in the appropriate tissues or eye fluids is measured as a function of time after administration. Other methods of measuring the ocular bioavailability of compound 3 are possible.

In some embodiments, the concentration of compound 3 in an eye tissue and / or fluid can be increased when the crystalline form of compound 3 is delivered (eg, via topical administration to the eye) using the particles, compositions, and / or formulations described here compared to when the crystalline form of compound 3 is supplied using some of the existing objects, Compositions and / or formulations containing the same crystalline form of Compound 3 (or compared to supplying the same crystalline form of Compound 3 (eg, similar size) as the coated particles in question, but not including the coating). In certain embodiments, a dose of the particles, compositions, and / or formulations is administered, followed by measurement of the

164 concentration of the crystalline form of compound 3 in a tissue or fluid of the eye. For comparison purposes, the amount of the crystalline form of compound 3 included in the administered dose of the particles, compositions and / or formulations described herein may be similar or substantially equal to the amount of the crystalline form of compound 3 included in the dose. administered of the existing particles, compositions and / or formulations. In certain embodiments, the concentration of compound 3 in an eye tissue and / or fluid is measured at a specified time after administration (post-dose time) of a dose of the particles, compositions and / or formulations described herein or of the existing particles, compositions and / or formulations. In certain modalities, the time when measuring its concentration is about 1 min, about 10 min, about 30 min, about 1 h, about 2 h, about 3 h, about 4 h, about 5 h, about 6h, about 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours, approximately 36 hours, or approximately 48 hours after the dose.

In some embodiments, the concentration of Compound 3 in a tissue and / or fluid may be increased due, at least in part, to a core coated particles comprising the crystalline form of Compound 3 representing the mucus penetrating particles, compared to particles of the same crystalline form the

165 Compound 3 (eg similar in size) as the coated particles in question, but which does not include the coating. In some embodiments, the particles, compositions, and / or formulations described herein increase the concentration of Compound 3 in a tissue and / or fluid by at least about 10%, at least about 20%, at least about 30%, by minus about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 1000 times, at least about 10⁴ times at least about 10⁵ times, or at least about 10⁶ times. In some cases, the particles, compositions and / or formulations described herein increase the concentration of compound 3 in a tissue and / or fluid by at least or equal to about 10⁶ times less than or equal to about 10⁵ times less than or equal to about 10⁴ times 1000 times less than or equal to about 100 times less than or equal to about 10 times less than or equal to about 500% less than or equal to about

166

400%, less than or equal to approximately 300%, less than or equal to approximately 200%, less than or equal to approximately 100%, less than or equal to approximately 90%, less than or equal to approximately 80%, less than or equal to approximately 70%, less than or equal to approximately 60%, less than or equal to approximately 50%, less than or equal to approximately 40%, less than or equal to approximately 30%, less than or equal to approximately 20%, or less than or equal to about 10%. Combinations of the aforementioned ranges are also possible (eg, an increase greater than or equal to about 10% and less than or equal to about 90%). Other ranges are also possible. In some cases, the concentration of compound 3 increases in a tissue and / or fluid in the front of the eye. In other cases, the concentration of compound 3 increases in tissue and / or fluid in the back of the eye.

The ocular concentration of compound 3, and, when appropriate, its metabolite (s) in suitable tissues or eye fluids can be measured as a function of time in vivo using a suitable animal model. One method of determining the ocular concentration of compound 3 involves dissecting the eye to isolate the tissues of interest (eg, in an animal model comparable to the subject). The concentration of compound 3 in the tissues of interest is determined by HPLC or LC / MS analysis.

In certain embodiments, the period of time between administration of the particles described herein

167 document and obtaining a sample for concentration or AUC measurement is less than about 1 hour, less than or equal to about 2 hours, less than or equal to about 3 hours, less than or equal to about 4 hours, less or equal to approximately 6 hours, less than or equal to approximately 12 hours, less than or equal to approximately 36 hours, or less than or equal to approximately 48 hours. In certain embodiments, the time period is at least about 1 hour, at least about hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 36 hours, or at least about 48 hours. Combinations of the aforementioned ranges are also possible (eg, a time period between consecutive doses of greater than or equal to approximately approximately hours and less than or equal to approximately approximately 12 hours). Other ranges are also possible.

Other methods of measuring the concentration of compound 3 in an eye of a subject or animal model are also possible. In some embodiments, the concentration of compound 3 can be measured in the subject's eye directly or indirectly (eg, taking a fluid sample, such as the vitreous humor of a subject's eye).

In general, an increase in the concentration of compound 3 in

168 An eye site can be calculated by taking the measured concentration difference between those of a test composition and a control composition, and dividing the difference between the concentration of the control composition. A test composition may include particles comprising a crystalline form of Compound 3, and the particles may be characterized as penetrating mucus (eg, with a relative speed of more than 0.5 composition control or another relative speed described herein). A control composition can include particles that comprise the same crystalline form of compound 3 as that present in the test composition, particles that are substantially similar in size as those in the composition test, but that are not penetrating mucus (eg. , with a relative speed of less than about 0.5, or another relative speed described here).

As described herein, in some embodiments, the particles, compositions and / or formulations described herein, or a component thereof, is present in an amount sufficient to increase the bioavailability and / or the concentration of compound 3 in a eye tissue, compared to the crystalline form of compound 3 administered to eye tissue in the absence of the particles, compositions, and formulations described herein, or a component thereof.

The eye tissue may be an anterior eye tissue (eg, a palpebral conjunctiva, a bulbar conjunctiva, or a cornea).

169

In certain embodiments, the core particle of a formulation comprising a crystalline form of compound 3 is present in an amount sufficient to increase the bioavailability and / or concentration of compound 3 in an eye tissue. In certain embodiments, the core-coated particles a formulation comprising a crystalline form of compound 3 is present in an amount sufficient to increase the bioavailability and / or the concentration of compound 3 in an eye tissue. In certain embodiments, the core particle coating of a formulation comprising a crystalline form of Compound 3 is present in an amount sufficient to increase the concentration of Compound 3 in an eye tissue after at least 10 minutes, at least 20 minutes , at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 9 hours, at least 12 hours, at least 18 hours, or at least 24 hours after administration of the formulation to the eye tissue. In certain embodiments, the core particle coating of a formulation comprising a crystalline form of Compound 3 is present in an amount sufficient to increase the concentration of Compound 3 in an eye tissue after less than or equal to 24 hours, less or equal to 18 hours, less than or equal to 12 hours, less than or equal to 9 hours, less than or equal to 6 hours, less than or equal to 4 hours, less than or equal to 3 hours, less than or equal to 2 hours, less than or equal to at 1 hour, less than or equal to 30 minutes, less than or equal

170 20 minutes or less or equal to 10 minutes after administration of the formulation to the eye tissue. Combinations of the aforementioned ranges are also possible (for example, the concentration of compound 3 increases after at least 10 minutes and less than or equal to 2 hours). Other ranges are also possible. In certain embodiments, the core particle coating of a formulation comprising a crystalline form of Compound 3 is present in an amount sufficient to increase the concentration of Compound 3 in an eye tissue after about 30 minutes after administration of the formulation to the eye tissue.

In some embodiments, the particles, compositions, and / or formulations described herein can be administered topically to a subject's eye in various dosage forms. For example, the particles, compositions, and / or formulations described herein can be administered in a single unit dose or repeatedly administered in a plurality of individual unit doses. A unit dose is a discrete amount of the particles, compositions, and / or formulations described herein that comprise a predetermined amount of a pharmaceutical agent. In some embodiments, fewer dose numbers (eg, 1/2, 1/3, or 1/4 of the dose number) are required using the particles described herein with a penetrating mucus coating compared to particles that do not have that type of coating.

The exact amount of the particles, compositions and / or

171 Formulations described in this document required to achieve a prophylactically or therapeutically effective amount will vary from subject to subject, depending, for example, on the species, age and general condition of the subject, on the severity of side effects or disorder, the mode of administration, and the like. The particles, compositions and / or formulations described herein can be supplied with repeated administrations where there is a period of time between consecutive doses. Repeated administration may be advantageous in that it can allow the eye to be exposed to a prophylactic or therapeutically effective amount of compound 3 for a period of time long enough for the eye condition to be treated, prevented, or administered. In certain embodiments, the time period between consecutive doses is less than or equal to about 1 hour, less than or equal to about 2 hours, less than or equal to about 3 hours, less than or equal to about 4 hours, less than or equal to about 6 hours, less than or equal to approximately 12 hours, less than or equal to approximately 36 hours, or less than or equal to approximately 48 hours. In certain embodiments, the time period between consecutive doses is at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 36 hours, or at least about 48 hours.

172

Combinations of the aforementioned ranges are also possible (eg, a period of time between consecutive doses of greater than or equal to approximately 3 hours and less than or equal to approximately 12 hours). Other ranges are also possible.

Delivery of the particles, compositions and / or formulations described herein to an eye tissue can cause ophthalmically effective levels of compound 3 in the eye tissue for an extended period of time after administration (eg, topical administration or direct injection administration) . An ophthalmically effective level of Compound 3 refers to an amount sufficient to obtain the desired biological response of an eye tissue, that is, the treatment of an eye disease. As will be appreciated by those skilled in the art, the ophthalmically effective level of Compound 3 may vary depending on factors such as the desired biological end point, the pharmacokinetics of Compound 3, the eye disease to be treated, the mode of administration , and the age and health of the subject. In certain embodiments, the ophthalmically effective level of Compound 3 is an amount of Compound 3, alone or in combination with other therapies, that provide a therapeutic benefit in treating the eye condition. The ophthalmically effective level of Compound 3 can encompass a level that improves total therapy, reduces or avoids the symptoms or causes of the eye condition, or improves the therapeutic efficacy of another therapeutic agent.

173

In some embodiments, an ophthalmically effective level of Compound 3 can be measured, at least in part, by the maximum concentration (C_max) of compound 3 in the eye tissue after administration.

In some embodiments, the ophthalmically effective level of Compound 3 is measured, at least in part, by minimally effective concentrations of Compounds 3, eg IC₅₀ or IC₉₀, as known in the art.

In certain modalities where ophthalmically effective levels (or C_max IC₅₀, or IC_9C) of compound 3 are present in the eye tissue for an extended period of time after administration, the prolonged period of time after administration may vary from hours to days. In certain modalities, the prolonged period of time after administration is at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 9 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, with a minimum of 4 days, at least 5 days, at least 6 days, or at least 1 week. In certain modalities, the prolonged period of time after administration is less than or equal to 1 week, less than or equal to 6 days, less than or equal to 5 days, less than or equal to 4 days, less than or equal to 3 days, less than or equal to equal to 2 days, less than or equal to 1 day, less than or equal to 12 hours, less than or equal to 9 hours, less than or equal to 6 hours, less than or equal to 4 hours, less than or equal to 2 hours, less than or equal within 1 hour. The combinations of the ranges before

174 These are also possible (for example, a long period of time of at least 4 hours and less than or equal to approximately 1 week). Other ranges are also possible.

In certain embodiments, the particles, compositions, and / or formulations described herein can be at doses sufficient to deliver an effective amount of Compound 3 to a subject's eye to obtain a desired therapeutic or prophylactic effect. In certain embodiments, an effective amount of compound 3 that is delivered to a suitable eye tissue is at least about 10 '³ ng / g, at least about 10 '² ng / g, at least about 10 '¹ ng / g, at least about 1 ng / g, at least about 10¹ ng / g, at least around 10² ng / g, at least around 10³ ng / g, at least about 10⁴ ng / g, at least about 10⁵ ng / g, or at least 15 10⁶ ng / g tissue weight. In certain embodiments, an effective amount of Compound 3 delivered to the eye is less than or equal to 10⁶ ng / g, less than or equal to 10⁵ ng / g, less than or equal to 10⁴ ng / g, less than or equal to 10³ ng / g, less than or equal to 10² ng / g, less than or equal to about 10¹ ng / g less than or equal to 1 ng / g less than or equal to about 10 '¹ ng / g, less than or equal to about 10 '² ng / g, or less than or equal to about 10 '³ ng / g tissue weight. Combinations of the above ranges are also possible (eg, an effective amount of compound 3 of at least 10 '² ng / ge less than or equal to 25 10³ ng / g tissue weight). Other ranges are also possible. In

175 Certain modalities, the particles, compositions, and / or formulations described herein can be at doses sufficient to deliver an effective amount of Compound 3 to the back of a subject's eye to obtain a desired therapeutic or prophylactic effect.

It will be appreciated that dose ranges as described herein provide guidance for the administration of particles, compositions, and / or formulations to an adult. The amount to be administered to, for example, a child or adolescent may be determined by a physician or a person skilled in the technical art and may be less than or equal to that administered to an adult.

The particles, compositions and / or formulations described herein can be topically administered (eg, ocular or dermal) by any method, eg, such as drops, powders, ointments, or creams. Other approaches or forms of topical administration are also possible.

In certain embodiments, the compositions and / or formulations described herein are packaged as a ready-to-use shelf stable suspension. Drop formulations are traditionally liquid formulations (solutions or suspensions), which can be packaged in dropper bottles (which dispense a standard volume of drop liquid) or in single-use droppers (normally used for preservative-free drops; they are used once and discarded). They can be stored in suspension and can retain the characteristics that allow the particles to avoid sticking to mucus.

176

Particle preparation methods and pharmaceutical compositions thereof

In one aspect, the present invention provides methods of preparing the particles of the invention. Similar particle preparation methods have been described in US Patent Publications Nos. 2013/0316001, 2013/0316006, 2013/0316009 and 20130323179, each of which are incorporated herein by reference in their entirety.

The nucleus of the particle can be formed by any suitable method. Suitable methods may include, for example, top-down techniques, i.e. techniques based on the reduction of relatively large particle size into smaller particles (eg, grinding or homogenization) or bottom-up techniques, is that is, techniques based on the growth of small particle particles or individual molecules (for example, precipitation or freezing by liquid spray).

In some embodiments, the core of the particle may be coated with a coating. For example, the cores can be provided or formed in a first step, and then the core can be coated in a second step. In some embodiments, the core particle is formed and coated substantially simultaneously (eg, in a single step).

In some embodiments, the particle is formed by a method that involves the use of a formulation process, a process of

177 grinding, and / or a dilution process. In certain embodiments, a particle formation method includes a grinding process, optionally with a formulation process and / or a dilution process. A formulation process can be used to form a suspension composed of a core material, one or more surface-altering agents, and other components, such as solvents, tonicity agents, chelating agents, salts, and / or pH regulators (eg example, a pH regulator for sodium citrate and citric acid), each of which is described here. The formulation process can be performed using a formulation container. The core material and other components can be added to the formulation container at the same time or at different times. A mixture of the core material and / or one or more other components can be removed and / or stirred, or otherwise stirred in the container to facilitate suspension of the components to form the suspension. The temperature and / or pressure of the core material, other components and / or mixture can also be individually increased or decreased to facilitate the suspension process. In some embodiments, the core material and other components were processed as described herein in the formulation vessel under an inert atmosphere (eg, nitrogen or argon) and / or protected from light. The suspension obtained from the formulation container may be subsequently subjected to a grinding process, which may be followed by a dilution process.

178

In some embodiments of a core composed of a solid material (eg, crystalline compound of the invention), a grinding process can be used to reduce the size of the solid material to form particles from a range of micrometer to nanometer size. The grinding process can be carried out using a mill or other suitable apparatus. Dry and wet milling processes such as jet milling, cryo-milling, ball milling, milling media, sound application, and homogenization are known and can be used in the methods of the invention. For example, in a wet milling process, a suspension of solid material that is used to form the core (core material) is stirred with or without excipients to reduce the size of the core that was formed. Dry grinding is a process in which the core material is mixed with grinding media with or without excipients to reduce the size of the core that was formed. In a cryogenic grinding process, a suspension of the core material is mixed with grinding media with or without excipients under cold temperatures. In certain embodiments, when surface alteration agents are used, a suspension composed of coated particles is obtained from the grinding process. In certain embodiments, when no surface altering agents are used, a suspension composed of uncoated particles is obtained from the grinding process.

The particle suspension (coated or uncoated) of the invention obtained from a grinding process can be

179 processed with a dilution process. A dilution process can be used to achieve a target dosage concentration by diluting a suspension of the particles that formed during a grinding process, with or without surface-altering agents and / or other components. In certain embodiments, when a coated particle suspension comprising a first surface alteration agent is processed with a dilution process involving a second surface alteration agent, a suspension of coated particles comprising the second surface alteration agent It is obtained from the dilution process. In certain embodiments, when a coated particle suspension comprising a surface alteration agent is processed with a dilution process without the participation or the same surface alteration agent, a suspension of coated particles that make up the surface alteration agent It is obtained from the dilution process. In certain embodiments, when a suspension of uncoated particles is processed with a dilution process with a surface alteration agent, a suspension of coated particles comprising the surface alteration agent is obtained from the dilution process. The dilution process can be performed using a fresh product or any other suitable device. In certain embodiments, the suspension of the particles is diluted, that is, mixed or otherwise processed with a diluent, in the product container. The diluent may contain solvents,

180 surface altering agents, tonicity agents, chelating agents, salts, antimicrobial agents, or a combination thereof, as described herein. The suspension and diluent can be added to the product container at the same time or at different times. In certain embodiments when the suspension is obtained by a grinding process with grinding media, the grinding media may be separated from the suspension before the suspension is added to the product container. The suspension, diluent, or mixture of suspension and diluent can be removed and / or whipped, or otherwise stirred, to form the particles and / or pharmaceutical compositions of the invention. The temperature and / or pressure of the suspension, diluent, or mixture can also be individually increased or decreased to form the coated particles. In some embodiments, the suspension and diluent are processed in the product container under an inert atmosphere (eg, nitrogen or argon) and / or protected from light.

In some embodiments, the core and / or coated particles can be produced by grinding a solid material (eg, a pharmaceutical agent) in the presence of one or more surface alteration agents. Small particles of a solid material may require the presence of one or more surface alteration agents, which can function as a stabilizer in some embodiments, in order to stabilize a suspension of particles without agglomeration or aggregation in a liquid solution. In

181 In some of these embodiments, the stabilizer can act as a surface alteration agent, forming the coated particles of the invention.

As described herein, a method of forming the core and / or coated particles may involve choosing a surface-altering agent that is suitable for both grinding and layering on the core, where the coating causes the particle to penetrate the mucus.

In a wet milling process, milling can be performed in a dispersion (eg, an aqueous dispersion) containing at least one surface disturbance agent, a milling medium, a solid to be ground (eg, a pharmaceutical agent solid), and a solvent. The solvent described here includes a single solvent or a mixture of different solvents. Any suitable amount of a surface alteration agent can be included in the solvent. In some embodiments, the surface altering agent may be present in the solvent in an amount of at least 0.001% (% / po% weight to volume (w: v), at least about 0.01%, by at least about 0.1%, at least about 1%, at least about 3%, at least 10%, at least about 30%, or at least 60% solvent. In some cases, the surface alteration agent may be present in the solvent in an amount of approximately 100% (for example, in an instance where the surface alteration agent is the solvent). In others

182 modalities, the surface altering agent may be present in the solvent in an amount of less than 100%, less than about 60%, and less than 30%, less than 10%, less than 3% or less approximately 1% of the solvent. Combinations of the aforementioned ranges are also possible (eg, an amount of less than 3% and a minimum of approximately 1% of the solvent). Other ranges are also possible. In certain embodiments, the surface alteration agent is present in the solvent in an amount of about 0.01-2%, about 0.2-20%, about 0.1%, 0.4%, about 1%, about 2%, about 5%, or about 10% of the solvent.

The particular scale chosen can influence factors that may affect the ability of the particles to penetrate the mucus such as the stability of the coating of the surface alteration agent on the surface of the particle, the average thickness of the coating of the surface alteration agent. on the particles, the orientation of the surface alteration agent on the particles, the density of the surface alteration agent on the particles, the ratio of the surface alteration agent to the pharmaceutical agent, the concentration of the pharmaceutical agent, the size, the dispersibility, and polydispersity of the formed particles, and the morphology of the formed particles.

The pharmaceutical agent may be present in the solvent in

183 any suitable quantity. In some embodiments, the pharmaceutical agent is present in an amount of at least 0.001% (% / po% weight to volume (w: v), at least about 0.01%, at least about 0.1% , at least about 1%, at least about 3%, at least about 10%, at least about 30%, or at least about 60% of the solvent. In some cases, the pharmaceutical agent may be present in the solvent in an amount of less than about 100%, less than 60%, less than 30%, less than about 10%, less than 3%, or less than 1 % of solvent. Combinations of the aforementioned ranges are also possible (eg, an amount of less than 30% and a minimum of approximately 1% of the solvent).

The ratio of the surface alteration agent to the pharmaceutical agent in a solvent can also vary. In some embodiments, the ratio of the surface altering agent to the pharmaceutical agent is at least about 0.001: 1 (weight ratio, molar ratio, op: v), at least about 0.01: 1, at least about 0.01 : 1, at least about 1: 1, at least about 2: 1, at least about 3: 1, at least about 5: 1, at least about 10: 1, at least about 30: 1, at least around 100: 1, or at least around 1000: 1. In some embodiments, the ratio of the surface altering agent to the pharmaceutical agent is less than 1000: 1 (weight ratio, molar ratio, op: v), less than about

184 100: 1, less than about 30: 1, less than about 10: 1, less than about 5: 1, less than about 3: 1, less than about 2: 1, less than about 1 : 1, or less than 0.1: 1. Combinations of the mentioned ranges are possible (eg, a ratio of at least 5: 1 and less than about 30: 1). Other ranges are also possible.

The surface alteration agents described herein that can act as stabilizers can be, for example, polymers or surfactants. Examples of polymers are suitable for use in the coated particles of the invention, such as polyvinyl alcohol and PLURONICS®. Examples of the surfactants include La-phosphatidylcholine (PC), 1,2d i pa I mi to i If osf at id i I co I ina (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, monolaurate of sorbitan, polyoxylene sorbitan fatty acid esters (TWEENS), polysorbates (eg, polyoxyethylene sorbitan monooleate) (eg, TWEEN 80®), polyoxyethylene sorbitan monostearate (eg, TWEEN 60®), polyoxyethylene sorbitan monopalmitate ( for example, TWEEN 40®), polyoxyethylene sorbitan monolaurate (for example, TWEEN 20®), natural lecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether, lauryl polyoxyethylene ether, polyoxylene alkyl ethers, polyethylene and oxypropylene block copolymers, polyethylene esters, polyoxyethylene esters derivatives, Vitamin-PEG and its derivatives, synthetic lecithin, diethylene glycol dioleate, oleate of

185 tetrahydrofurf uri lo, ethyl oleate, isopropyl myristate, glycerol monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol, cetyl pyridinium chloride, benzalkonium chloride, olive oil, monolaurate of glycerol corn, cottonseed oil, and sunflower seed oil. Derivatives of the mentioned compounds are also possible. The combinations of the mentioned compounds and others described herein can also be used as surface alteration agents of the particles of the invention. As described herein, in some embodiments, a surface-altering agent can act as a stabilizing agent, a surface-active agent, and / or an emulsifier. In some embodiments, the surface-altering agent aids in the transport of particles in the mucus.

A stabilizer used for grinding can form the coating of a particle of the invention, in which the coating causes the particles to penetrate the mucus. The stabilizer can also be exchanged with one or more surface alteration agents after the particle has formed. For example, a first stabilizer / surface alteration agent can be used during a grinding process, and can form a first coating of the particle of the invention, and all or part of the first stabilizer / surface alteration agent can be exchanged with a second stabilizer / surface alteration agent to form a

186 second coating of the particle. In some embodiments, the second stabilizer / surface alteration agent can cause the particle to penetrate the mucus more than the first stabilizer / surface alteration agent. In some embodiments, a particle made up of multiple coatings including various surface alteration agents is formed by a method of the invention.

Any suitable grinding media can be used for grinding. In some embodiments, a ceramic and / or polymeric materials and / or a metal can be used. Examples of suitable materials include zirconium oxide, silicon carbide, silicon oxide, silicon nitride, zirconium silicate, yttrium oxide, glass, aluminum, alpha-alumina, aluminum oxide, polystyrene, poly (methyl methacrylate) , titanium and steel. A grinding medium can be of any suitable size. For example, the grinding media can have an average diameter of at least 0.1mm, at least about 0.2mm, at least about 0.5mm, at least about 0.8mm, so minus about 1 mm, at least about 2 mm, or at least about 5 mm. In some cases, the grinding media may have an average diameter of less than about 5mm, less than about 2mm, less than 1mm, less than about 0.8, less than 0.5mm, or less than 0.2mm . Combinations of the aforementioned ranges are also possible (for example, an average diameter of at least 0.5 millimeters and less than 1 mm). Other ranges are also

187 possible.

A solvent can be used for grinding. The choice of the proper solvent for grinding may depend on factors such as the solid material (eg, a solid pharmaceutical agent) being ground, the particular type of stabilizer / surface disturbance agent (eg, one that can cause the particle penetrates the mucus), and the grinding materials. The solvent suitable for grinding may be one of the solvents that does not substantially dissolve the solid material or grinding material, but dissolve the stabilizer / surface alteration agent to a suitable degree. Examples of solvents suitable for milling include water, aqueous solutions, alcohols, pH-regulated solutions (eg, ethanol, methanol, and butanol) and mixtures thereof, each of which may optionally include other components, such as one or more pharmaceutical excipients, polymers, pharmaceuticals, salts, preservatives, viscosity modifiers, tonicity modifiers, flavor masking agents, antioxidants, and pH modifiers. In some embodiments, the proper solvent for grinding is an organic solvent.

A pharmaceutical agent described herein (eg, a crystalline form of compound 3) may have adequate solubility in a solvent suitable for milling, such as solubility in one or more ranges described for aqueous solubility or solubility in a coating solution. A pharmaceutical agent that has

188 a relatively low solubility in a solvent (for example, water or a coating solution) may be preferable because a milling process described here normally requires a material (for example, a pharmaceutical agent) that is in solid form, in order to that the material is ground. In some cases, if the material to be ground has a relatively high solubility in a solvent (for example, water or a coating solution) used in the grinding process, grinding cannot be done because significant or complete dissolution will occur of the material to be ground in the solvent. In certain embodiments, a relatively high solubility of a solid material (eg, a solid pharmaceutical agent) in a solvent is at least about 1 mg / mL, at least about 3 mg / mL, or at least about 10 mg / mL at 25 ° C. In certain embodiments, a relatively low solubility of a substance (eg, a pharmaceutical agent) in a solvent is less than 1 mg / mL, less than about 0.3 mg / mL, less than about 0.1 mg / mL, less than about 0.03 mg / mL, less than about 0.01 mg / mL, less than 0.003 mg / mL, or less than 0.001 mg / mL at 25 ° C. Solid materials can have these or other ranges of solubilities at any point throughout the entire pH range (for example, from pH 1 to pH 14).

In other embodiments, the core and / or coated particles can be formed by an emulsification process or technique (emulsification) known in the art. See, for example, Publication of

189 US Patent No. 20130316006. Generally, emulsification techniques may involve dissolving or dispersing a material to be used as the core of a solvent; This solution or dispersion is then emulsified in a second non-miscible solvent, thus constituting a plurality of particles that comprise the material. Suitable emulsification techniques may include the formation of oil-in-water emulsions, water-in-oil emulsions, water-oil-water emulsions, oil-in-oil oil emulsions, solid-in-oil-in-water emulsions, and solid emulsions -in-water-in-oil, etc., with or without subsequent solvent extraction, for example, by evaporation or extraction. Emulsification techniques are versatile and can be useful for the preparation of core particles comprising pharmaceutical agents that have a relatively low aqueous solubility as well as pharmaceutical agents that have a relatively high aqueous solubility.

In some embodiments, the core particles described herein can be produced by emulsification in the presence of one or more surface alteration agents. In some of these embodiments, the stabilizer can act as a surface-altering agent, forming a coating on the particle (i.e., the emulsification and coating steps can be performed substantially simultaneously).

In some embodiments, a method of forming core particles by emulsification involves choosing a stabilizer that is suitable for both emulsifying and forming a

190 coating on the particle and the representation of penetrating mucus particles. For example, as described in detail below, it has been shown that 200-500 nm of nanoparticles of model PLA polymers produced by emulsification in the presence of certain PVA polymers resulted in particles that can penetrate physiological mucosa samples at the same rate So well established of polymeric pegylated MPP. Interestingly, it was observed that only a subset of PVA polymers were tested to fit the criteria to be suitable for both emulsification and for the formation of a coating on the particle that causes the particles to penetrate the mucus, as described in more detail below.

In other embodiments, the particles were first formed using an emulsification technique, followed by coating the particles with a surface-altering agent.

Any suitable solvent and solvent combinations can be used for emulsification. Some examples of solvents that can serve as the oil phase are organic solvents such as chloroform, dichloromethane, ethyl acetate, ethyl ether, petroleum ether (hexane, heptane) and oils such as peanut oil, cottonseed oil, safflower, sesame oil; olive oil; corn oil, soybean oil and silicone oil. Some examples of solvents that can serve as a water phase are water and pH regulators. Other solvents are also possible.

191

The core and / or coated particles can also be formed by a precipitation process or technique (precipitation). Precipitation techniques (eg, microprecipitation, nanoprecipitation, crystallization, and controlled crystallization) may involve forming a first solution composed of the material that will form the core (eg, a pharmaceutical agent) and a first solvent, where the material has a relatively high solubility in the first solvent. The first solution can be added to a second solution that comprises a second solvent that is an anti-solvent, where the material has a relatively low solubility, thus constituting a plurality of particles that make up the material. In certain modalities, the second solvent is miscible with the first solvent. In some embodiments, one or more surface altering agents and / or surfactants may be present in the first and / or second solutions. A coating can be formed during the process of precipitating the core (eg, the particle coating can be formed substantially simultaneously when precipitation is performed) to form the coated particles of the invention.

In other embodiments, the core of the particles of the invention is first formed using a precipitation technique, followed by coating the core with a surface-altering agent to form the coated particles of the invention.

192

In some embodiments, a precipitation technique can be used to form the polymeric core of the particles of the invention with or without a pharmaceutical agent. Generally, a precipitation technique involves dissolving a polymer that is to form the core in a first solvent, in the presence or absence of a pharmaceutical agent, to form a solution. The solution is then added to a second solvent which is an anti-solvent and is miscible with the first solvent, in the presence or absence of one or more excipients, to form the nucleus of the particles. In some embodiments, precipitation is very useful for the preparation of a polymeric core composed of one or more pharmaceutical agents that have a relatively low aqueous solubility.

The precipitations described here involve the use of a solvent. Examples of suitable first solvents for precipitation include organic solvents (eg, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, / \ / - methyl-2pyrrolidone, 2-pyrrolidone, and tetrahydrofuran) and inorganic solvents.

The precipitation described here also involves the use of a second solvent. In certain embodiments, the second suitable solvent for precipitation is an anti-solvent. Examples of suitable second solvents for precipitation include the solvents described herein that can be used for grinding. In some embodiments, the second suitable solvent for precipitation is water, an aqueous solution (for example, a

193 pH regulated solution), an alcohol (eg, methanol, ethanol, propanol, or butanol), or a mixture of these, optionally may include one or more other components, such as pharmaceutical excipients, polymers, and pharmaceutical agents.

The surface alteration agents for emulsification and precipitation described herein can be polymers or surfactants, including the surface alteration agents described here, which can be used for grinding.

Examples of polymers suitable for forming all or part of the core of the particles of the invention by emulsification or precipitation may include polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethi logs, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, polyarylates, polypeptides, polynucleotides and polysaccharides. Non-limiting examples of specific polymers include poly (caprolactone) (PCL), ethylene vinyl acetate (EVA) polymer, poly (lactide acid) (PLA), poly (Llactide acid) (PLLA), poly (glycolic acid) ( PGA), poly (lactide-co-glycolic acid) (PLGA), poly (L-lactide-co-glycolic acid) (PLLGA), poly (D, L-lactide) (PDLA), poly (L-lactide) ( PLLA), poly (D, Llactide-co-caprolactone), poly (D, L-lactide-co-caprolactone-coglycolide), poly (D, L-lactide-co-PEO-co-D, L-lactide), poly (D, L-lactidac-PPO-co-D, L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-Llisin (PLL), hydroxypropyl methacrylate (HPMA), poly (ethylene glycol),

194 poly-L-glutamic acid, poly (hydroxy acids), polyanhydrides, polyorthoesters, poly (ester amides), polyamides, poly (ester esters), polycarbonates, polyalkylols such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol ) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly (ethylene terephthalate), alcohols po I ivi η í I i eos (PVA), polyvinyl ethers, polyvinyl esters such as poly (vinyl acetate) , polyvinyl halides such as poly (vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, esters of cellulose, nitrocellulose, hydroxypropylcellulose, carboxy acrylic acid polymers, such as poly (methyl (meth) acrylate) (PMMA), poly (ethyl (meth) acrylate), poly (butyl (meth) acrylate), poly (isobutyl (meth) acrylate), poly ( hexyl (meth) acrylate), poly (isodecyl (meth) acrylate), poIi (IauriI (meth) acrylate), poly (phenyl (meth) acrylate), poly (methyl acrylate), poIi (IsopropiI acrylate), poly (isobutiI acrylate), poly (octadecyl acrylate) (collectively referred to herein as polyacrylic acids), and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate), polyoxymethylene, poloxamers, poly (ortho) esters, poly (butyric acid), poly (valeric acid), pol i (lact ida-co-caprolactone) and trimethylene carbonate, polyvinylpyrrolidone, bovine serum albumin, human serum albumin, collagen, DNA, RNA, carboxymethyl cellulose, chitosan, dextran.

195

Suitable polymers to form all or part of a core and / or surface-altering agent may also include a poly (ethylene glycol) -vitamin E conjugate (hereinafter PEG-VitE conjugate). The particles, compositions and / or formulations including a PEG-VitE conjugate, and the methods of making and using the particles, compositions and / or formulations, are presented in more detail in PCT International Publication Application WO2012 / 061703, the which is incorporated herein by reference in its entirety for all purposes. In some cases, the molecular weight of the PEG portion of the PEG-VitE conjugate is greater than 2 kDa. The molecular weight of the PEG portion of the PEG-VitE conjugate can be selected to aid in the formation and / or transport of the particles through a mucosal barrier as described herein. In some embodiments, the use of a PEG-VitE conjugate with a PEG moiety with a molecular weight greater than about 2 kDa may allow greater particle penetration through a mucosal barrier compared to the use of the PEG conjugate. -VitE with a portion of PEG with a molecular weight less than about 2 kDa. In addition, in some embodiments, a higher molecular weight PEG portion can facilitate drug encapsulation. The combined ability to act as a surfactant and reduce mucoadhesion offers significant advantages compared to other surfactants commonly used for drug encapsulation. In some cases, the molecular weight of

196 the PEG portion of the PEG-VitE conjugate is between about 2 kDa and about 8 kDa, or between about 3 kDa and about 7 kDa, or between 4 kDa and 6 kDa, or between about 4.5 kDa and about 6, 5 kDa, or about 5 kDa.

In some embodiments, a precipitation technique can be used to form particles composed predominantly of a pharmaceutical agent (eg, a crystalline form of compound 3). In certain embodiments, the particles of the invention formed by the precipitation technique primarily comprise a crystalline form of Compound 3 which is a nanocrystal. Generally, this precipitation technique involves dissolving the compound 3 that will form the nucleus in a first solvent, which is then added to a second solvent that is an anti-solvent, where the crystalline form of compound 3 has a relatively soluble Low, in the presence or absence of one or more pharmaceutical excipients, to form the core or uncoated particles. In some embodiments, this technique may be useful for preparing, for example, pharmaceutical agent particles that are slightly soluble (1-10 mg / mL), very slightly soluble (0.1-1 mg / mL), or virtually insoluble (&lt; 0.1 mg / mL) in aqueous solutions (for example, agents that have a relatively low aqueous solubility).

A pharmaceutical agent described herein (eg, a crystalline form of compound 3) may have adequate solubility in the first and second solvents suitable for precipitation, such as

197 a solubility in one or more ranges described for aqueous solubility or solubility in a coating solution. A pharmaceutical agent with relatively high solubility in the first solvent (eg, an organic solvent) may be preferred. In certain embodiments, the pharmaceutical agent is substantially or completely dissolved in the first solvent. A pharmaceutical agent that has a relatively low solubility in the second solvent (eg, water or a coating solution) may also be preferred. In certain embodiments, the solubility of the pharmaceutical agent in a mixture of the first and second solvents is less than the solubility of the pharmaceutical agent in the first solvent agent. Relatively high solubility and relatively low solubility are as described here.

Another exemplary method of forming the core and / or coated particles is a freeze-drying process or technique known in the art. See, for example, US Patent Publication No. 2013/0316006. In this technique, Compound 3 can be dissolved in an aqueous solution, optionally with a surface-altering agent. The solution can be immediately frozen by vaporization and freeze-dried. The dry powder can be reconstituted in a suitable solvent (eg, an aqueous solution such as water) at a desired concentration.

If the surface alteration agent is present in the solvent prior to freeze drying, it can be present in any suitable concentration, such as a concentration of al

198 minus 0.001% (w / v), at least about 0.005% (w / v), at least about 0.01% (w / v), at least about 0.05% (w / v), at least about 0.1 % (w / v), at least about 0.5% (w / v), at least about 1% (w / v), or at least about 5% (w / v) in the aqueous solution. In some cases, the surface alteration agent is present in the solvent at a concentration less than or equal to about 5% (w / v), less than or equal to about 1% (w / v), less than or equal at approximately 0.5% (w / v), less than or equal to approximately 0.1% (w / v), less than or equal to approximately 0.05% (w / v), less than or equal to approximately 0.01 % (w / v), or less than or equal to approximately 0.005% (w / v). Combinations of the aforementioned ranges are also possible (eg, a concentration of at least about 0.01% (w / v) and less than or equal to about 1% (w / v). Other ranges are also possible.

The concentration of the surface alteration agent present in the solvent can be above or below the critical micellar concentration (CMC) of the surface alteration agent, depending on the surface alteration agent used. In other embodiments, stable particles can be formed by adding excess counterions to a solution containing a pharmaceutical agent. The precipitate can then be washed by various methods, such as centrifugation. The resulting grout can be sound applied. One or more agents

199 Surface alteration can be added to stabilize the resulting particles.

Other methods of nucleic particle formation are also possible. For example, additional techniques for forming the core and / or coated particles include coacervation phase separation, melt dispersion, adjoining surface deposition, in situ polymerization, self-assembly of macromolecules (eg, formation of polyelectrolyte complexes or polyelectrolyte-surfactant complexes) and spray drying and spray freezing, electro-spray, air suspension coating, tray and spray coating, freeze drying, air drying, vacuum drying, fluidized bed drying, precipitation (eg, nanoprecipitation, microprecipitation), critical fluid extraction, and lithographic approaches (eg, soft lithography, step and flash printing, interference lithography and photolithography). Combinations of the methods described here are also possible. In some embodiments, a core of a pharmaceutical agent is first formed by precipitation, and then the core size is reduced by a grinding process, optionally a coating is formed on the core by the grinding process.

Upon formation of the nucleus of the particles, including a pharmaceutical agent, the nucleus may optionally be exposed to a solution composed of a (second) alteration agent.

200 surface that can associate and / or cover the nucleus. In embodiments where the pharmaceutical agent already includes a coating of a first surface alteration agent, all or part of the first surface alteration agent can be exchanged with a second surface alteration agent. In some embodiments, the second surface alteration agent causes the mucus penetrating particle to have more penetration than the first surface alteration agent. In some embodiments, a particle having a coating is formed including various surface altering agents (eg, in a single layer or multiple layers). In some embodiments, a particle with multiple coatings can be formed (eg, each coat optionally comprised of different surface altering agents). In some embodiments, the coating is in the form of a monolayer of a surface disturbing agent. Other configurations are also possible.

In any of the methods described herein, a coating consisting of a surface alteration agent can be formed in a nucleus of the particles of the invention by incubating the nucleus in a solution that includes a surface alteration agent for a period of at minus about 1 minute, at least about 3 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 60 minutes or more. In some cases, incubation can take place

201 over a period of less than about 10 hours, less than about 3 hours, or less than about 60 minutes. Combinations of the ranges indicated above are also possible (eg, an incubation period lasting less than about 60 minutes and at least about 1 minute).

Treatment methods and uses

A scale of disease can arise when a subject's body loses control of angiogenesis, that is, new blood vessels grow abnormally (i.e., excessively or insufficiently) or grow as a result of a tumor. Excessive angiogenesis is often observed in subjects with diseases such as proliferative diseases (eg, cancer, benign neoplasms, inflammatory diseases, autoimmune diseases), and eye diseases, especially with cancer, diabetic retinopathy, macular degeneration, rheumatoid arthritis, and psoriasis. In these diseases, new blood vessels feed abnormal tissues and / or destroy normal tissues. Excess angiogenesis can occur when abnormal amounts of angiogenic growth factors are present, overwhelming the effects of natural angiogenesis inhibitors. Therefore, inhibition of the growth of new blood vessels may be useful in treating diseases associated with excessive angiogenesis. Insufficient angiogenesis is observed

202 typically in individuals with diseases such as coronary artery disease, stroke, or chronic wounds. In these diseases, the growth of blood vessels is inadequate and circulation is not restored properly, which can lead to tissue death.

VEGFs have been found to play an important role in angiogenesis, for example, by increasing the number of capillaries in a given network. In vitro studies have shown that bovine capillary endothelial cells proliferated and showed signs of tube structures after stimulation with VEGF. VEGF up-regulation is an important component of the physiological response to exercise and its role in angiogenesis is suspected to be a possible treatment for vascular lesions. In vitro studies have shown that VEGFs are a potent stimulator of angiogenesis, because, among other things, in the presence of this growth factor, plaque endothelial cells will proliferate and migrate, eventually forming capillary-like tube structures. VEGFs can cause a huge signaling cascade in endothelial cells. Binding to the VEGF receptor-2 initiates a tyrosine kinase signaling cascade that stimulates the production of factors that indiscriminately stimulate vessel permeability, proliferation / survival, migration, and ultimately differentiation in mature blood vessels. Mechanically, VEGF is over-regulated with

203 muscle contractions as a result of increased blood flow to the affected areas. The increased flux also causes a large increase in the production of VEGF receptor 1 and 2 mRNA. Increased receptor production indicates that muscle contractions could cause upregulation of the signaling cascade relative to angiogenesis.

In one aspect, the present invention provides methods of treating and / or preventing a disease associated with angiogenesis abnormalities, comprising administering an effective amount of compound 3 to a subject in need thereof. In certain embodiments, the disease associated with abnormal angiogenesis is treated and / or avoided by the methods of the invention. In certain embodiments, the disease being treated and / or avoided by the methods of the invention is associated with excessive and / or pathological angiogenesis.

In another aspect, the present invention provides methods of treating and / or preventing a disease associated with aberrant signaling of a growth factor in a subject in need thereof. In certain embodiments, the disease associated with aberrant growth factor signaling is treated and / or avoided by the methods of the invention. In certain modalities, the disease is associated with excess growth factor signaling. In certain embodiments, the disease being treated and / or avoided by the methods of the invention is associated with aberrant VEGF signaling. In certain modalities, the

204 disease is associated with excess signaling or aberration of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-F, and / or placental growth factor (PGF). In certain embodiments, the disease associated with aberrant VEGF signaling is treated and / or avoided by the methods of the invention.

As used herein, the term growth factor-associated disease means any disease in which growth factors are known to play an important role. Consequently, in some modalities, the current description refers to the treatment of diseases in which growth factors are known to play an important role. These diseases include proliferative diseases, eye diseases, dermatological diseases, inflammation and metabolic diseases.

In certain embodiments, the present disclosure provides methods of treating a disease comprising contacting a biological sample with an effective amount of compound 3. In certain embodiments, the biological sample includes a cell or tissue. In some embodiments, the methods include inhibiting growth factor signaling in a cell, tissue, or subject. In some embodiments, the biological sample is an ocular tissue. In certain embodiments, the method is an in vitro method. In certain modalities, the method is an in vivo method. It will be understood by a person skilled in the art that the levels of inhibition do not necessarily have to be 100%. Inhibition levels

205 may be at least 10% inhibition, about 10% to about 25% of inhibition, about 25% to about 50% of inhibition, about 50% to about 75% of inhibition, at least 50% inhibition, at least 75% inhibition, about 80% inhibition, about 90% inhibition, or greater than 90% inhibition.

In certain embodiments, the disease being treated and / or avoided by the methods of the invention is a proliferative disease. All the types of proliferative diseases described here can be treated and / or avoided by the methods of the invention. In certain embodiments, proliferative disease is treated and / or avoided by the methods of the invention. In certain embodiments, the disease that is being treated and / or avoided by the methods of the invention is cancer. All the types of cancer described in this document can be treated and / or prevented by the methods of the invention. In certain modalities, the cancer is an eye cancer. In certain modalities, retinoblastoma eye cancer, medulloepithelioma, melanoma, ciliary body melanoma, or primary intraocular lymphoma. In certain embodiments, cancer is treated and / or avoided by the methods of the invention. In certain embodiments, the disease being treated and / or avoided by the methods of the invention is a benign neoplasm. All the types of benign neoplasm described here can be treated and / or avoided by the methods of the invention. In certain modalities, the benign neoplasm is a benign ocular neoplasm. In certain

206 modalities, the benign neoplasm is orbital dermoid cysts. In certain embodiments, the benign neoplasm is treated and / or prevented by the methods of the invention.

In certain embodiments, the disease being treated and / or avoided by the methods of the invention is an inflammatory disease. All types of inflammatory diseases described herein can be treated and / or avoided by the methods of the invention. In certain modalities, inflammatory disease is an inflammatory eye disease. In certain modalities, inflammatory eye disease is post-surgical inflammation. In certain embodiments, the inflammatory disease is treated and / or avoided by the methods of the invention. In certain embodiments, the disease being treated and / or avoided by the methods of the invention is an autoimmune disease. All types of autoimmune diseases described herein can be treated and / or avoided by the methods of the invention. In certain modalities, rheumatoid arthritis is an autoimmune disease. In certain embodiments, autoimmune disease is treated and / or avoided by the methods of the invention. In certain embodiments, the disease being treated and / or avoided by the methods of the invention is diabetes. In certain modalities, the disease is type 1 diabetes. In certain modalities, the disease is type 2 diabetes. In certain modalities, the disease is gestational diabetes. In certain embodiments, diabetes is treated and / or avoided by the methods of the invention.

207

The disease being treated and / or avoided by the methods of the invention may be an eye disease. In some embodiments, the eye disease that is treated and / or avoided by the methods of the invention is an anterior eye disease that occurs in the "front or front portion" of a subject's eyes. The anterior portion of the eye includes the cornea, iris, conjunctiva, tear film, corneal epithelium, anterior chamber, lens, ciliary body, ciliary zone, posterior chamber, retina, macula, sclera, an optic nerve, choroid, and vitreous chamber. In certain embodiments, the anterior eye disease that is treated and / or avoided by the methods of the invention is allergy, post-surgical inflammation, uveitis, an infection (eg, a viral, bacterial, or fungal infection), aphakia, pseudophakia, astigmatism, blepharospasm, cataracts, a disease of the conjunctiva, conjunctivitis, a corneal disease, corneal edema, blepharitis, meibomian gland disease, corneal transplant surgery, corneal ulcer, dry eye (for example, dry eye syndrome), a disease of the eyelid, a disease of the lacrimal apparatus, obstruction of the lacrimal duct, laser-induced exudation, myopia, presbyopia, pterygium, pupil disorders, corneal neovascularization, a disorder of refraction, strabismus, or glaucoma. In some embodiments, the eye disease that is treated and / or avoided by the methods of the invention is a posterior eye disease that occurs in the back or rear of the eye. The posterior portion of the eye includes the choroid,

208 sclera, vitreous humor, vitreous chamber, retina, macula, optic nerve, and blood vessels and nerves that supply or vascularize a posterior ocular region or site. In certain embodiments, the posterior eye disease that is treated and / or avoided by the methods of the invention is intraocular melanoma, acute macular neuroretinopathy, exudative eye disease, Behcet's disease, exudative retinopathy, macular edema, retinopathy of prematurity, a disorder epirethmal membrane, choroidal neovascularization, diabetic uveitis uveitis, histoplasmosis, an infection (for example, a viral, bacterial, or fungal infection), macular degeneration (eg, acute macular degeneration and age-related macular degeneration (AMD, such as non-exudative age-related macular degeneration and exudative age-related macular degeneration), edema (eg, macular edema, such as edema macular cystoid (CME) and diabetic macular edema (DME), multifocal choroiditis, ocular trauma affecting a posterior ocular site or location, ocular cancer, a disorder of the retina (for example, occlusion of the central retinal veins), proliferative retinopathy (for example, diabetic retinopathy and non-proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal occlusive arterial disease, retinal detachment, uveitic retinal disease, sympathetic ophthalmia, Vogt Koyanagi syndrome - Harada (VKH), uveal diffusion, a posterior eye condition caused or influenced by laser eye treatment, a

209 posterior ocular condition caused or influenced by photodynamic therapy, photocoagulation, radiation retinopathy, an epiretinal membrane disorder, branching retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal dysfunction without retinopathy, retinitis pigmentosa, retinoblastoma, or glaucoma . In certain modalities, the eye disease that is avoided and / or treated by the methods of the invention is macular degeneration. In certain modalities, eye disease is age-related macular degeneration (AMD). In 10 certain modalities, eye disease is glaucoma. In certain modalities, eye disease is diabetic retinopathy. In certain modalities, the eye disease is retinoblastoma. In certain modalities, the eye disease is edema. In certain modalities, the ocular disease is cystoid macular edema 15 (CME). In certain modalities, the eye disease is diabetic macular edema (DME). In certain modalities, eye disease is an inflammatory eye disease. In certain modalities, eye disease is post-surgical inflammation.

In certain modalities, the ocular disease is uveitis (for example, anterior uveitis, intermediate uveitis, and posterior uveitis). In certain modalities, the eye disease is blepharitis. In certain

<td>modalities,</td><td>the</td><td>disease</td><td>ocular</td><td>it is panuveitis.</td><td>In</td><td>certain</td>

<td>modalities,</td><td>the</td><td>disease</td><td>ocular</td><td>it is scleritis.</td><td>In</td><td>certain</td>

<td>modalities,</td><td>the</td><td>disease</td><td>ocular</td><td>it is dry eye.</td><td>In</td><td>certain</td>

modalities, eye disease is Sjógren's syndrome. In

210 certain modalities, eye disease is eye surgery. In certain embodiments, eye disease is treated and / or avoided by the methods of the invention.

In certain embodiments, the compounds, particles, compositions, and / or formulations described herein are packaged as a ready-to-use shelf stable suspension. Eye drop formulations are traditionally liquid formulations (solutions or suspensions), which can be packaged in dropper bottles (which dispense a standard drop volume of liquid) or in single-use droppers (normally used for preservative-free drops, once used and discarded). These formulations are ready for use and can be self-administered. In some cases the bottle must be shaken before use to ensure homogeneity of the formulation, but no further preparation may be necessary. This may be the simplest and most convenient method of eye supply. The compositions and / or formulations described herein can be packaged in the same way as traditional eye drop formulations.

Another aspect of the present invention relates to methods of inhibiting aberrant signaling of a growth factor (eg, VEGF) in a subject or cell. In certain embodiments, aberrant growth factor signaling is inhibited by the methods of the invention.

In another aspect, the present invention provides methods

211 for the inhibition of abnormal or pathological angiogenesis in a subject in need thereof. In certain embodiments, abnormal or pathological angiogenesis is inhibited by the methods of the invention.

In certain modalities, the subject described here is a human being. In certain modalities, the subject is an animal. The animal can be of either sex and can be at any stage of development. In certain modalities, the subject is a fish. In certain modalities, the subject is a mammal. In certain modalities, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain modalities, the subject is a livestock animal such as cows, pigs, horses, sheep, or goats. In certain modalities, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent (eg, mouse, rat), dog, pig, or non-human primate. In certain modalities, the animal is a genetically manipulated animal. In certain modalities, the animal is a transgenic animal.

In some embodiments, the crystal forms described herein are useful for the treatment of cancer, including, but not limited to, acoustic neuroma, adenocarcinoma, cancer of the adrenal gland, anal cancer, angiosarcoma (eg, lymphangiosarcoma, limfangioendotheliosarcoma, hemangiosarcoma) , appendix cancer, benign monoclonal gammopathy, biliary cancer

212 (eg, cholangiocarcinoma), bladder cancer, breast cancer (eg, adenocarcinoma of the breast, papillary carcinoma of the breast, breast cancer, medullary carcinoma of the breast), brain cancer (eg, meningioma, glioma; eg, astrocytoma, oligodendroglioma, medulloblastoma;), bronchial cancer, carcinoid tumor, cervical cancer (eg, cervical adenocarcinomas), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (eg, colon cancer, rectal cancer, colorectal adenocarcinoma ), epithelial carcinoma, ependymoma, endotheliosarcoma (for example, Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (for example, uterine cancer, uterine sarcoma), esophageal cancer (for example, adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (for example, intraocular melanoma, retinoblastoma), familial hypereosinophilia, gastric cancer (for example, adenocarcinoma of the stomach), gastrointestinal stromal tumor (GIST), head and neck cancer (eg, squamous cell carcinoma of the head and neck, oral cancer (eg, oral squamous cell carcinoma (OSCC), throat cancer (for example, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer), hematopoietic cancers (for example, leukemia such as acute lymphocytic leukemia (ALL) (for example, ALL of cell B, ALL of T-cell), acute myeloid leukemia (AML) (for example, B-cell AML, T-cell AML), myelocytic leukemia Leukemia (CML) (for example,

213

B-cell CML, T-cell CML) and chronic lymphocytic leukemia (CLL) (eg, B-cell CLL, T-cell CLL); lymphoma, such as Hodgkin lymphoma (HL) (for example, B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (for example, B-cell NHL such as diffuse large cell lymphoma (DLCL) ( for example, diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MOL), marginal zone B-cell lymphoma (for example, mucosa-associated lymphoid tissue (MALT), B-cell lymphoma of the nodal marginal zone, B-cell lymphoma of the splenic marginal zone), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL) , immunoblastic large cell lymphoma, precursor Blinfoblastic lymphoma and lymphoma of the main central nervous system (CNS); and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (eg, cutaneous T-cell lymphoma (LCCT) (eg, mycosis fungiodes, Sezary syndrome), cell lymphoma Angioimmunoblastic T, extranodal natural annihilating T-cell lymphoma, enteropathy-like T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM), heavy chain disease (eg alpha chain disease,

214 gamma chain disease, mu chain disease), hemangioblastoma, inflammatory broblastic tumors, immunocytic amyloidosis, kidney cancer (eg nephroblastoma aka Wilms tumor, renal cell carcinoma), liver cancer (eg cancer hepatocellular (CHC), malignant hepatoma), lung cancer (for example, bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (eg, systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (eg, polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM), also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES), neuroblastoma, neurofibroma (for example, neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine carcinoma (eg, gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (eg, cystadenocarcinoma, embryonal ovarian carcinoma, adenocarcinoma ), papillary adenocarcinoma, pancreatic cancer (for example, pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumors), penile cancer (for example, Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT),

215 prostate cancer (eg, prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (eg, squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma ( BCC), cancer of the small intestine (for example, cancer of the appendix), soft tissue sarcoma (for example, malignant fibrous histiocytoma (HFM), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovitis, testicular cancer (eg, testicular seminoma, embryonal carcinoma), thyroid cancer (eg, papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, and vulvar cancer (for example, Paget's disease of the vulva).

In certain embodiments, the cell described herein is in vivo. In certain modalities, the cell is in vitro. In certain modalities, the cell is ex vitro.

In certain embodiments, the methods of the invention include administering to a subject in need thereof an effective amount of compound 3, particles, or pharmaceutical composition of the invention. In certain embodiments, the methods of the invention include contacting a cell with an effective amount of a compound, particles, or pharmaceutical composition of the invention.

216

In certain embodiments, the methods of the invention are the in vivo methods. In certain embodiments, the methods of the invention are in vitro methods. In certain embodiments, the methods of the invention are ex vitro methods.

In another aspect, the present invention provides the crystalline forms of Compound 3, particles, and pharmaceutical compositions of the invention for use in treating and / or preventing a disease described herein in a subject in need thereof.

In another aspect, the present invention provides the crystalline forms of Compound 3, particles, and pharmaceutical compositions of the invention for use in inhibiting abnormal angiogenesis in a subject in need thereof.

In another aspect, the present invention provides the crystalline forms of compound 3, particles, and pharmaceutical compositions of the invention for use in inhibiting aberrant signaling of a growth factor in a subject or cell in need thereof.

Examples

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as a limitation.

217 of this invention in any form.

Example 1: Synthesis of compound 3, Method A

Compound 1: 4- (4-fluoro-2-methyl-1 H-indole-5-yloxy) -7- (benci loxy) -6methoxyquinazoline

<img file="MX2016005668A_D0014.tif" />

Scheme 1A

4-Fluoro-2-methyl-1H-indole-5-ol (0.53 g, 3.2 mmol) was dissolved in N, N-dimethylfomamide (25 mL). The suspension was purged with nitrogen and potassium carbonate (0.92 g, 6.7 mmol) was added. 7- (Benzyloxy) -4-chloro-6-methoxyquinazoline (1.0 g, 3.3 mmol) was added and the suspension was purged with nitrogen again. The suspension was heated to 85 ° C overnight in an oil bath. Solvent evaporated. The residue was treated with water (100 mL) and sound was applied. The solid was filtered, washed with water and hexanes, and dried in a high vacuum overnight leaving compound 1 as a gray solid (1.4 g, 100%). m / z: 430 (M + H, 100%) (positive ionization mode).

218

Compound 2:

4- (4-f I or oro-2-meth 1-1 H-indole-5-i loxy) -6 methoxyquinazoli η-7-ol

<img file="MX2016005668A_D0015.tif" />

Scheme 1 B

4- (4-Fluoro-2-methyl-1H-indole-5-yloxy) -7- (benzyloxy) -6methoxyquinazoline (compound 1, 0.46 g, 1.1 mmol) was dissolved in N, Ndimethylformamide (10 mL ). Catalyst palladium hydroxide (250mg, 10% carbon) was added, followed by ammonium formate (0.67g, 10.6mmol). The reaction solution is stirred for 2 hours at room temperature. The catalyst is filtered through a CELITE pad, then the solution was evaporated, then dried in a high vacuum overnight to generate compound 2 as a brown solid (0.36 g, 100% m / z: 340 (M + H, 100%) (positive ionization mode).

219

Compound 3: 7- (3- (4- (4-fluoro-2-methyl-1H-indole-5-yloxy) -6methoxyquinazolin-7-yloxy) propyl) -2-oxa-7-azaspiro [3.5] no na not

<img file="MX2016005668A_D0016.tif" />

3

Scheme 1C

4- (4-Fluoro-2-methyl-1 H-indole-5-yloxy) -6methoxyquinazolin-7-ol (compound 2, 0.36 g, 1.1 mmol) was dissolved in N, N-dimethylformamide (10 mL) . Potassium carbonate (0.90 g, 6.5 mmol) was added, followed by 1-bromo-3-chloropropane (0.34 g, 2.2 mmol). The suspension was heated at 45 ° C for 2 hours. The solvent was evaporated and the residue was suspended in dichloromethane (20 mL). The suspension was applied on a pad of silica gel. Impurities were eluted with dichloromethane and the compound was eluted with ethyl acetate (Rf = 0.7 in ethyl acetate). The solvent was evaporated and the residue was dried under high vacuum leaving a yellow foam (0.35 g, 80%), m / z: 416 (M + H, 100%) (Positive ionization mode), which was dissolved in N, N-dimethylformamide (5 mL). Potassium bromide (0.12 g, 1.0 mmol) was added followed by potassium carbonate (0.90 g, 7.8 mmol) and 2-oxa-7azaspiro [3.5] nonane oxalate (0.35 g, 1.9 mmol) ). The suspension was heated at 85 ° C for 4 hours. The solvent was evaporated and the residue was

220 Suspended in an aqueous solution of sodium bicarbonate (50 mL) and sound was applied. The precipitate was filtered. Drying under high vacuum gave a brown solid (0.34 g). Reverse phase HPLC purification provided compound 3 as a white solid (20mg). m / z: 507 (M + H, 100%) (Positive ionization mode). 1H NMR: (chloroform-d): 8.60 (s, 1H), 8.10 (s, 1H), 7.65 (s, 1H); 7.35 (s, 1H), 7.10 (D, J = 9.0 Hz 1H), 7.00 (dd, J = 8.0; J = 9.0 Hz, 1H), 6.35 (s, 1H), 4.45 (s, 4H), 4.35 (t, J = 7.0 Hz, 2H), 4.15 (s, 3H), 2.55 (t, J = 7.0 Hz, 2H), 2.46 (s, 3H), 2.40- 2.35 (m, 4H), 2.15-2.10 (m, 2H), 1.90-1.85 (m, 4H).

Example 2: Synthesis of compound 3, Method B

<td rowspan="2"></td><td colspan="2"> 0</td>

<td></td><td></td>

<td>Cl ^</td><td></td><td>^^ nh₂</td>

NH₄OAc Methyl Orthoformate Methanol

<img file="MX2016005668A_D0017.tif" />

Scheme 2A

To a solution of 2-amino-4- (3-chloropropoxy) -5-methoxybenzoic acid methyl ester (48 g, 175 mmol) in methanol (150 mL) was added methyl orthoformate (46.4 g, 438 mmol), acetate ammonium (33.7 g, 438 mmol). The reaction mixture was stirred at reflux for 5 hours. Water (200 mL) was added to the reaction mixture to precipitate the product, which was collected by filtration, washed with water (200 mL) and methanol (50 mL), and then dried under reduced pressure to give 44 g (93.4%) of

221 compound 4 as a white solid.

<img file="MX2016005668A_D0018.tif" />

POCI₃toluene

<img file="MX2016005668A_D0019.tif" />

Scheme 2B

A mixture of compound 4 (75 g, 279 mmol) and POCI₃ (100 mL) in toluene (500 mL) was refluxed until the solution became apparent. The solution was concentrated under reduced pressure, and the residue was emptied into ice-cold water. After filtration, the solid was washed with water (500 mL χ 2) and dried to give 65 g (81.2%) of compound 5 as a yellow solid.

<img file="MX2016005668A_D0020.tif" />

s 6

Scheme 2C

The mixture of compound 5 (11 g, 38.33 mmol) and 4-fluoro hydroxy-5-2-methylol (9.49 g 57.50 mmol), CS₂CO₃ (25 g, 76.66 mmol) in tetrahydrofuran (200 mL) was washed at 50 ° C overnight. The reaction mixture was extracted with ethyl acetate (200 mL x 2) and the organic layers were combined and washed with water (200 mL) and brine (200 mL) successively. The organic layer was dried with sodium sulfate, concentrated to dryness. The residue is

222 Purified by flash chromatography (petroleum ether: ethyl acetate = 10: 1 to 2: 1) to give 13 g (81.5%) of compound 6 as a brown solid.

<img file="MX2016005668A_D0021.tif" />

To the solution of Compound 6 (11 g, 26.44 mmol) in N, Ndimethylformamide (100 mL) was added 2-oxa-7-azaspiro [3.5] nonane (8.73 g, 68.75 mmol), iodide tetrabutylammonium (9.76 g, 26.44 mmol) and diisopropylethylamine (10.23 g, 79.33 mmol). The solution was heated to a temperature of 60 ° C overnight, then diluted with ethyl acetate (200 mL). The mixture was washed with brine (100 mL χ 5), dried over sodium sulfate, and then the solvent was evaporated under reduced pressure to give the crude product as a black solid. The crude product was purified by flash chromatography (dichloromethane: methanol = 50: 1 to 10: 1) to give 6.5 g (48.5%) of compound 3 as a light yellow solid. XRPD analysis showed that the isolated compound was amorphous.

223

Example 3: Compound 3 formulated as mucus penetration particles (MPP)

Compound 3 was formulated as the Mucus Penetration Particles (MPP). Specifically, Compound 3 of Example 2 was ground in the presence of PLURONIC F127 (F127) to determine if F127 1) aids in particle size reduction to several hundred nanometers and 2) physically (non-covalently) covers the Nanoparticle surface generated with a mucoinert coating that minimizes particle interactions with mucus constituents and prevents mucus adherence.

A grinding procedure was employed in which an aqueous dispersion containing coarse drug particles and PLURONIC F127 (F127) was ground with grinding media until the particle size was reduced to approximately 270 nm (z-averaged) as measured by light scattering dynamic. These particles were found to have a polydispersity index (a measure of the amplitude of the particle size distribution) of 0.142. In this example the suspensions were pH regulated using DPBS (Phosphate Regulated Saline) which produces a suspension that is both isotonic and has a physiologically relevant pH.

In order to determine whether the generated particles have reduced interactions with mucins and are therefore capable of moving within mucus without being trapped, the particles were incubated with human cervicovaginal mucus (CVM) and observed through a

224 dark field microscope. 1 pL or less of the nanoparticle suspension was added to 20 μΙ_ CVM. Observations were made in a minimum of three different areas selected at random from the CVM sample. Control particles with known behavior were used to qualify the CVM sample as appropriate for the assay. Mobility in mucus was observed and therefore nanoparticles are considered effective MPP.

Example 4: crystalline forms of Compound 3

The crystalline forms of compound 3 were prepared and analyzed by XRPD, Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA).

For XRPD, standards were obtained using a Rigaku MiniFlex 600 bench X-ray diffractometer equipped with a Cu X-ray tube (Cu / Κα = Á 1.54059), a six-position sample changer, and a D / teX Ultra detector.

Sample preparation, procedure A

As described below for the preparation of a crystalline Form A through milling, the particles were isolated from the bulk formulation by centrifugation at 55,000 rpm for 15 minutes and deposited in a thin, uniform layer on an XRPD sample holder of flat zero bottom (Rigaku 906165 Flush, IS510). The sample was allowed to dry under gentle air flow, generally up to 3 minutes, until visually dry.

225

Sample preparation, procedure B

As described below for the preparation of the crystalline net form B by crystallization, milligram amounts of solid sample were tightly packed into the 5mm x 0.2mm depression of a zero-bottom sample holder (Rigaku 906166 5mm χ 0 , 2 mm Well, IS510).

XRPD standards of 3-40 ° two theta were acquired in step size of 0.02 ° and capture rate of 5 ° / min by configuring the following instruments: 40 kV-15mA X-ray generator, 2.5 ° Soller slot, 10mm HIS, 0.625 ° Divergence slot, 8mm Dispersion slot with Κβ filter, and one open Receive slot. Diffraction patterns were viewed and analyzed using PDXL analysis software provided by the instrument manufacturer. Using the sample preparation procedures outlined above, a reference standard silicon powder in (NIST 640d Standard Reference Material) generated a 28.44 ° and 28.38 ° two theta peak using Procedure A and Procedure B, respectively.

For DSC, approximately 2 mg of sample was loaded into a standard aluminum sample tray. The sample tray was loaded into the apparatus (Q1000 differential analysis calorimeter, TA Instruments), which was equipped with an autosampler. A thermogram was obtained by individually heating the sample at a rate of 10 ° C / min from room temperature to approximately 250-300 ° C using an aluminum tray

226 empty standard for reference. Dry nitrogen was used as a gas sample purge from and set at a flow of 50 mL / min. Thermal transitions were seen and analyzed using the analysis software provided with the instrument.

By TGA, approximately 6 mg of the sample was transferred onto an aluminum sample tray. The tray was placed on the loading platform and then automatically loaded into the apparatus (Q500 Thermogravimetric Analyzer, TA Instruments) using the control software. Thermograms were obtained by individually heating the sample to 10 ° C / min from room temperature to 300 ° C under dry nitrogen, with a sample purge flow rate of 25 mL / min and an equilibrium purge flow rate 10 mL / min. Thermal transitions (eg, weight changes) were seen, and analyzed using the analysis software provided with the instrument.

Preparation of penetrating mucus particles comprising crystalline form A

According to Example 3, penetrating mucus particles comprising crystalline Form A were prepared by wet milling the amorphous compound 3 produced in Example 2. A mixture containing 5% of amorphous compound 3 and 5% of PBS F127 (0 , 0067 M PO₄³), pH 7.1 was added to an equal 1-mm bulk volume of ceria stabilized zirconium oxide stabilized beads in a glass bottle (eg, 2 mL of slurry per 2 mL of

227 pearls). A magnetic stir bar was used to stir the beads, stirring at approximately 500 rpm. The sample was ground for 2 days. Nanoparticles were generated that were approximately 200nm (z-averaged) in diameter, measured by dynamic light scattering (DLS). During the milling process, Compound 3 was converted from amorphous to crystalline Form A as confirmed by XRPD.

XRPD analysis of crystalline Form A of the resulting compound 3 was carried out. The crystalline form A XRPD pattern is illustrated in Figure 1 and the reflections within its XRPD pattern are listed in Table 1.

Table 1: List of XRPD peaks for crystalline Form A of compound 3

<td>No.</td><td>Position ± 0.3 [° 2Θ]</td><td>d-separation ± 0.3 [A]</td><td>Relative intensity [%]</td>

<td> 1</td><td> 6.11</td><td> 14.45</td><td> 60.14</td>

<td> 2</td><td> 9.63</td><td> 9.17</td><td> 52.72</td>

<td> 3</td><td> 11.10</td><td> 7.96</td><td> 11.17</td>

<td> 4</td><td> 11.46</td><td> 7.71</td><td> 22.60</td>

<td> 5</td><td> 12.26</td><td> 7.22</td><td> 10.66</td>

<td> 6</td><td> 15.66</td><td> 5.65</td><td> 3.25</td>

<td> 7</td><td> 16.41</td><td> 5.40</td><td> 30.14</td>

<td> 8</td><td> 17.54</td><td> 5.05</td><td> 7.29</td>

<td> 9</td><td> 18.16</td><td> 4.88</td><td> 44.67</td>

228

<td> 10</td><td> 18.60</td><td> 4.77</td><td> 100</td>

<td> 11</td><td> 19.51</td><td> 4.55</td><td> 32.84</td>

<td> 12</td><td> 20.36</td><td> 4.36</td><td> 52.26</td>

<td> 13</td><td> 21.12</td><td> 4.20</td><td> 12.65</td>

<td> 14</td><td> 22.31</td><td> 3.98</td><td> 7.49</td>

<td> 15</td><td> 23.01</td><td> 3.86</td><td> 24.04</td>

<td> 16</td><td> 24.79</td><td> 3.59</td><td> 7.64</td>

<td> 17</td><td> 25.71</td><td> 3.46</td><td> 30.32</td>

<td> 18</td><td> 28.90</td><td> 3.09</td><td> 8.29</td>

<td> 19</td><td> 30.81</td><td> 2.90</td><td> 1.99</td>

<td> 20</td><td> 31.64</td><td> 2.83</td><td> 3.28</td>

Preparation of net crystalline form B

Crystalline Form B of Compound 3 was prepared by thermal crystallization of the amorphous form of Compound 3 from a binary mixture of acetone and water. Specifically, Compound 3 (80 mg) from Example 2 was added to an 8 mL scintillation bottle containing a 7x2 mm stir bar, followed by the addition of a hot 4: 1 mixture of acetone: water (4 mL total). The flask was heated on a hot plate, shaking to completely dissolve compound 3. After spontaneous cooling to room temperature, Form B slowly crystallized from the solution. After allowing crystallization to continue overnight, the solvent was discarded and the solid crystals that remained in the flask were collected and allowed to dry overnight under vacuum. As discussed below, XRPD analysis generated unique reflections, indicating the formation of a new crystalline form.

229

An XRPD analysis of the resulting crystalline form B of compound 3 was carried out. The crystalline Form B XRPD pattern is illustrated in Figure 2 and the reflections comprised in its XRPD pattern are listed in Table 2.

Table 2: List of XRPD peaks of crystalline form B of the

Compound 3

<td>No.</td><td>Position ± 0.3 [° 2θ]</td><td>d-separation ± 0.3 [A]</td><td>Relative intensity [%]</td>

<td> 1</td><td> 7.7</td><td> 11.47</td><td> 7</td>

<td> 2</td><td> 9.87</td><td> 8.96</td><td> 5</td>

<td> 3</td><td> 10.69</td><td> 8.27</td><td> 4</td>

<td> 4</td><td> 12.88</td><td> 6.87</td><td> 1</td>

<td> 5</td><td> 13.53</td><td> 6.54</td><td> 30</td>

<td> 6</td><td> 14.4</td><td> 6.14</td><td> 12</td>

<td> 7</td><td> 14.97</td><td> 5.91</td><td> 5</td>

<td> 8</td><td> 15.45</td><td> 5.73</td><td> 12</td>

<td> 9</td><td> 16.42</td><td> 5.39</td><td> 3</td>

<td> 10</td><td> 17.27</td><td> 5.13</td><td> 42</td>

<td> 11</td><td> 18.44</td><td> 4.81</td><td> 100</td>

<td> 12</td><td> 18.9</td><td> 4.69</td><td> 3</td>

<td> 13</td><td> 19.73</td><td> 4.5</td><td> 20</td>

<td> 14</td><td> 21.14</td><td> 4.2</td><td> 8</td>

<td> 15</td><td> 21.86</td><td> 4.06</td><td> 2</td>

<td> 16</td><td> 22.56</td><td> 3.94</td><td> 14</td>

<td> 17</td><td> 23.1</td><td> 3.85</td><td> 67</td>

<td> 18</td><td> 26.07</td><td> 3.41</td><td> 72</td>

<td> 19</td><td> 26.84</td><td> 3.32</td><td> 8</td>

<td> 20</td><td> 29.12</td><td> 3.06</td><td> 9</td>

DSC and TGA were also performed on crystalline form B of compound 3. Figure 3 shows that the DSC thermogram measured at

230 From 25 ° C to 250 ° C, increased to 10 ° C / min, it exhibits a wide dehydration endothermic event at 117 ° C followed by crystallization then fusion of the presumed anhydrous form at 188 ° C. Figure 4 shows that the TGA thermogram measured from 25 ° C to 300 ° C, increased to 10 ° C / min, was found to exhibit a 6% mass loss from 25 ° C to 120 ° C. Presumably, the loss in mass corresponds to two water molecules (theoretical weight loss of dihydrate = 6.6%), so crystalline form B becomes a dihydrate.

Preparation of penetrating mucus particles comprising crystalline form B

In accordance with Example 3, penetrating mucus particles comprising crystalline Form B were prepared by wet-grinding the net crystalline Form B. A slurry containing 5% crystalline Form B of the 3 and 5% compound of F127 in DPBS (Dulbecco's Phosphate pH Regulated Saline) was added to an equal 1-mm bulk volume of zirconium oxide beads stabilized with ceria in a glass bottle (for example, 0.5 mL of slurry for 0.5 mL of pearls). A magnetic stir bar was used to stir the beads, stirring at approximately 500 rpm. The sample was ground for 2 days. Nanoparticles approximately 200nm in diameter (z-mean) were generated, as measured by DLS. After grinding, XRPD analysis (not shown) confirmed that the crystal shape was unchanged, indicating that

231 crystalline form B remained stable during milling.

Stability of MPP formulations containing crystalline forms A and / or B

To characterize the stability of the resulting crystal forms of compound 3, changes in XRPD profiles were determined after long-term storage. A suspension of MPP composed of crystalline Form A was stored at room temperature for 7 weeks, while a second suspension of MPP also composed of crystalline Form A was stored at room temperature for 7 weeks, followed by an additional 1.5 weeks of agitation. After these periods, XRPD analyzes revealed that the compounds still possessed the crystalline Form A XRPD profile, indicating that the material is perishable in solution for at least 7 weeks. To further test the potential for long-term storage, two other MPP samples comprising Form A, which were formulated at pH 5.8 and pH 7.4, incorporating different pH regulators in the grinding slurry, were they were stored at room temperature for 8 months and then analyzed by XRPD. Again, XRPD analysis (not shown) revealed that the crystals still possessed the XRPD profile of crystalline Form A, indicating that the material is perishable as suspensions for at least 8 months.

To confirm long-term stability, a suspension of crystalline Form B MPP was stored for 7 weeks at

232 room temperature and then characterized by XRPD. The results of this analysis are shown in Figure 5, which provides the XRPD pattern of the original crystalline Form B material in the bottom fingerprint and the XRPD pattern of the material after seven weeks of storage in the top footprint. The material was maintained as crystalline form B, demonstrating that crystalline form B is physically stable during storage.

Seeding of crystalline Form B during milling of Amorphous Compound 3

In the absence of crystalline material, the amorphous material from Example 2 was converted to a crystalline form A during milling, as described above. As such, the inventors wanted to determine whether the presence of crystalline form B during milling could seed the formation of crystalline form B from amorphous compound 3 during milling. A mixture of crystalline Form B and amorphous material was ground as follows: milling media, specifically 1-mm ceria stabilized zirconium oxide beads, was added to a scintillation glass bottle. Separately, a grinding slurry containing 2.5% crystalline Form B of Compound 3, 2.5% of Amorphous Compound 3, and 5% F127 was generated in DPBS (pH-regulated saline with Dulbecco phosphate) . The grinding slurry was added to the glass bottle in the same bulk volume to the beads (eg 0.5 mL of slurry for 0.5 mL of beads). A stir bar was used

233 to stir the beads, stirring at approximately 500 rpm. The sample was ground for 3 days. Nanoparticles that were approximately 150 nm (z-averaged) in diameter, measured by DLS, were generated. After grinding, XRPD analysis was performed, the results of which are shown in Figure 6, which provides the XRPD pattern of crystalline form B of compound 3 in the bottom tread and the XRPD pattern of the grinding mixture of the crystalline form. and amorphous B of compound 3 in the upper footprint. This analysis confirmed that when some crystalline form B is present during the milling process, the amorphous material is converted to crystalline form B as opposed to a crystalline form A during milling.

Competition between crystalline forms A and B to determine the most stable or preferred MPP

In order to assign a form as more stable, or more preferred, in the current formulation of conditions, a competition experiment was carried out. A suspension of the crystalline Form A nanoparticles was generated as described through wet milling of the amorphous material. A suspension of the Form B nanoparticles was generated through the wet grinding of net Form B crystals as described. The two suspensions were mixed in a 1: 1 ratio and incubated at room temperature. After 11 days, an XRPD analysis was performed, which estimated that the ratio of forms A and B was maintained

2. 3. 4 unchanged in the mix (not shown). A fraction of the mixture was then stirred with a magnetic stir bar to provide a higher input of energy to accelerate the results of the competition experiment. After 5 weeks of stirring, Compound 3 in the stirred formulation became crystalline Form B while the crystals in the formulation without stirring remained a mixture, as shown in Figure 7. This result indicates that crystalline form B is more stable under current formulation conditions.

Example 5: Drug exposure in the back of the eye of topical instillation of a MPP comprising a crystalline form A of compound 3

A pharmacokinetic (PK) study of crystalline Form A of Compound 3 formulated as MPP was performed in accordance with Example 4 to demonstrate that topical instillation of MPP formulations of these compounds results in exposure to the drug in the back of the eye. The study design is shown in Table 3.

235

Table 3. Composite PK Evaluation Study Design

3, MPP form A

<td>Group</td><td>Test article</td><td>Number of animals (n / time point)</td><td>Dose volume</td><td>Frequency / Duration</td><td>End time points (hours)</td>

<td> 1</td><td>3, MPP Form A, 2.0%</td><td> 3</td><td>35 pL</td><td>IDB / 5 days</td><td> 0.5</td>

<td> 2</td><td>3, MPP Form A, 2.0%</td><td> 3</td><td>35 pL</td><td>IDB / 5 days</td><td> 1</td>

<td> 3</td><td>3, MPP Form A, 2.0%</td><td> 3</td><td>35 pL</td><td>IDB / 5 days</td><td> 2</td>

<td> 4</td><td>3, MPP Form A, 2.0%</td><td> 3</td><td>35 pL</td><td>IDB / 5 days</td><td> 4</td>

IDB = twice a day

Gottingen female mini-pigs were used in these studies. Animals received a single topical ocular dose in the right eye twice daily, at approximately 12 hours (± 1 hour), for 4 consecutive days; on the fifth day the animals received a single topical eye dose in the morning only for a total of 9 doses for the duration of the study.

All animals were sacrificed with sodium pentobarbital and blood was collected by cardiac puncture in tubes containing K₂EDTA and centrifuged to obtain plasma. Both eyes were then enucleated, frozen by vaporization, and stored at -70 ° C for at least 2 hours. Within approximately 2 days, the frozen matrices were collected as left and right eye of choroid and retina.

The resulting drug exposures in the plasma and in the back of the eye are shown in Figures 8 to 10. These

236 Results demonstrate that topical instillation of crystalline Form A of Compound 3 as MPP resulted in exposure to the drug in the retina and choroid in vivo.

Equivalents and scope

In articles of the claims such as one, one and the, "the" may mean one or more than one, unless otherwise indicated, or is otherwise apparent from the context. Claims or descriptions that include or among one or more members of a group are considered satisfied if one, more than one, or all members of the group are present in, employed in or relevant to a certain product or process, unless indicate otherwise, or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present, employed in or relevant to a certain product or process. The invention includes modalities where more than one or all the members of the group are present in, employed in or relevant to a certain product or process.

Furthermore, the invention encompasses all variations, combinations and permutations where one or more limitations, elements, clauses and descriptive terms of one or more of the listed claims is introduced in another claim. For example, any claim that is dependent on another claim can be modified to include one or more

237 limitations found in any other claim that is dependent on the same base claim. Where items are presented as lists, for example, in Markush group format, each subgroup of items is also described, and any item (s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as being composed of particular elements and / or features, some embodiments of the invention or aspects of the invention consist of, or consist primarily of, such elements and / or characteristics. For the sake of simplicity, those modalities have not been specifically stated in haec verba here. It is also noted that the terms it includes and contains are intended to be open and allow the inclusion of other elements or steps. Where extreme ranges are given, endpoints are included. Furthermore, unless otherwise indicated or not evident by context and the understanding of one skilled in the art, values ​​expressed as ranges may assume any specific value or sub-range within the limits set in different modalities. of the invention, to one tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

This application relates to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the references

238 incorporated and this specification, the specification will exercise control. Furthermore, any particular embodiment of the present invention that falls within the prior art can be explicitly excluded from one or more of the claims. Since such incorporations are considered to be known to a person skilled in the art, they can be excluded, although the exclusion is not expressly established herein. Any particular embodiment of the invention may be excluded from any claim, for whatever reason, whether or not they are related to the existence of prior art.

Those skilled in the art will recognize or be able to determine without using further routine experimentation many equivalents to the specific modalities described herein. The scope of the present modalities described herein is not intended to be limited to the foregoing Description, but rather is set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the present invention, as defined in the following claims.

239