Therapeutic nanoparticles comprising a therapeutic agent and methods of making and using same

THERAPEUTIC NANOPARTICLES INCLUDING A HYDROPHOBIC ACID AND A THERAPEUTIC AGENT, USEFUL FOR DIFFERENT TYPES OF CANCER AND THE PREPARATION PROCESS OF THE

SAME.

RELATED REQUESTS

This application claims the benefit of US Provisional Application 61 / 953,628, filed March 14, 2014, incorporated into the present application by reference in its entirety.

BACKGROUND

Systems that deliver certain drugs to a patient (for example, targeting a particular cell type or tissue, or targeting a specific diseased tissue, but not normal tissue) or controlling drug release have long been recognized. as beneficial.

For example, therapeutic agents that include an active drug and that, for example, target a particular tissue or cell type, or that target a specific diseased tissue, but not normal tissue, can reduce the amount of the drug. in tissues of the organism to which they are not directed. This is particularly important when treating a condition such as cancer, where it is desirable that a cytotoxic dose of the drug be delivered to the cancer cells, without killing the surrounding non-cancerous tissue. Effective drug targeting can reduce the undesirable and sometimes life-threatening side effects common in cancer therapy. Furthermore, such therapeutic agents can allow drugs to reach certain tissues that they could not otherwise reach.

Therapeutic agents that offer controlled release and / or targeted therapy must also be able to deliver an effective amount of drug, which is a known limitation in other nanoparticle delivery systems. For example, it can be a challenge to prepare nanoparticle systems that have an appropriate amount of drug associated with each nanoparticle, while keeping the size of the nanoparticles small enough to possess convenient delivery properties.

Therapeutic agents that contain at least one basic nitrogen atom (ie, protonatable nitrogen-containing therapeutic agents) represent an important group of therapeutic agents. However, nanoparticle formulations of this class of drugs are often hampered by undesirable properties, for example, unfavorable sudden release profiles and poor drug loading.

Accordingly, there is a need to formulate nanoparticle therapeutics and methods for making such nanoparticles that are capable of delivering therapeutic levels of protonatable nitrogen-containing therapeutic agents for the treatment of diseases such as cancer, while at the same time , reduce side effects on the patient.

SYNTHESIS

The present invention relates to a therapeutic nanoparticle of the therapeutic drug, 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} fenii) —3— [4— (4,6-dimorpholin— 4-I-1,3,5-triazin-2-yl) phenyl] urea or its pharmaceutically acceptable salts. More specifically, the present invention relates to a therapeutic nanoparticle comprising 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonl} phenyl I) —3— [4— ( 4,6-di morpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea or its pharmaceutically acceptable salts and further comprising a substantially hydrophobic acid. Additionally, the present invention relates to a therapeutic nanoparticle comprising 1— (4 - {[4— (dimethylamino) piperidine-1 — l] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4 —I — 1,3,5— triazin-2-yl) phenyl] urea or its pharmaceutically acceptable salts, a substantially hydrophobic acid, and a polymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer , a diblock copolymer of poly (lactic acid-co-glycolic acid) -poly (ethylene) glycol and combinations thereof, where the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol. The present invention further relates to a pharmaceutical composition comprising said nanoparticles, which include a plurality of said nanoparticles, and a pharmaceutically acceptable excipient. Furthermore, the present invention relates to a therapeutic nanoparticle comprising from about 0.05 to about 30 percent by weight of a substantially hydrophobic acid, about 0.2 to about 25 weight percent 1- (4 - {[4- (dimethylamine) piperidine-1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 —1—1,3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof and about 50 to about 99.75 percent by weight of a copolymer selected from poly (acid diblock copolymer). lactic) —poly (ethylene) glycol, a diblock copolymer of poly (lactic acid-co-glycolic acid) —poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol, as well as a pharmaceutical composition comprising the therapeutic nanoparticle and a pharmaceutically acceptable excipient. The present invention further relates to a therapeutic nanoparticle comprising about 0.05 to about 30 percent by weight of a substantially hydrophobic acid, about 0.2 to about 20 percent by weight of 1- (4 - {[4- (dimethylamine) piperdin-1-yl] carbonyl} phenyl) -3- [4- (4,6-dimorpholin-4-il-1,3,5-triazin-2-yl) phenyl] urea or one of its pharmaceutically acceptable salts, and about 50 to about 99.75 weight percent of a copolymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer, a poly (lactic acid-co-glycolic acid) -poly diblock copolymer. (ethylene) glycol and combinations thereof, where the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol, as well as a pharmaceutical composition comprising the therapeutic nanoparticle and a pharmaceutically acceptable excipient.

Described in this application are polymeric nanoparticles that include the therapeutic agent, 1— (4 - {[4— (dimethylamine) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4, 6-dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof. This compound is basic, and is a protonatable nitrogen-containing therapeutic agent, as defined below. Methods for the preparation and use of said therapeutic nanoparticles are also described in the present application.

In one aspect, a therapeutic nanoparticle is provided. In this regard, the therapeutic nanoparticle comprises about 0.05 to about 30 weight percent of a substantially hydrophobic acid, about 0.2 to about 25 weight percent of 1- (4 - {[4- (dimethylamino) piperidin1 —Yl] carbonl} phenyl) -3— [4— (4,6-dimorpholin-4-i-1,3,5-triazin-2-yl) phenyl] urea or one of its pharmaceutically acceptable salts, where the pK value_to of the protonated form of the therapeutic agent is at least about 1.0 pKa units greater than the pK_to of the hydrophobic acid, and about 50 to about 99.75 percent by weight of a copolymer selected from poly (lactic acid) diblock copolymer - poly (ethylene) glycol, a diblock copolymer of poly (lactic acid-co-acid). glycolic) - poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol. In one embodiment, the therapeutic nanoparticle comprises about 0.05 to about 30 weight percent of a substantially hydrophobic acid, about 0.2 to about 20 weight percent of 1— (4 - {[4— (dimet ¡Lamino) piper¡din-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-¡I-1,3,5-triazin-2-yl) phenyl] urea or a of its pharmaceutically acceptable salts, where the pK value_to of the protonated form of the therapeutic agent is at least about 1.0 pK units_to greater than pK_to of hydrophobic acid, and about 50 to about 99.75 weight percent of a copolymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer, poly (lactic acid-co-glycolic acid diblock copolymer ) —Poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol.

In certain embodiments, the therapeutic nanoparticle comprises 1- (4 - {[4— (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — II— 1,3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 7 (therapeutic agent: PLA-PEG). In certain embodiments, the therapeutic nanoparticle comprises 1- (4 - {[4— (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4 — ¡1— 1,3,5— triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 5 (therapeutic agent: PLA-PEG). In certain embodiments, the therapeutic nanoparticle comprises 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl —1,3,5— triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 4 (therapeutic agent: PLA-PEG). In certain embodiments, the therapeutic nanoparticle comprises 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl-1 , 3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1:14 (therapeutic agent: PLA-PEG). In certain embodiments, the therapeutic nanoparticle comprises 1- (4 {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-II-1 , 3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 3 (therapeutic agent: PLA-PEG).

In another aspect, the therapeutic nanoparticle comprises a substantially hydrophobic acid, where the molar ratio of the substantially hydrophobic acid to the named therapeutic agent ranges from about 0.25: 1 to about 2: 1, about 0.2 to about 25 weight percent. of the mentioned therapeutic agent, where the pK_to of the protonated therapeutic agent is at least about 1.0 pKa units greater than the pK_to of hydrophobic acid, and about 50 to about 99.75 weight percent of a polymer selected from poly (lactic acid) poly (ethylene) glycol diblock copolymer, poly (lactic acid-coglycolic acid) diblock copolymer - poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol. In one embodiment, the therapeutic nanoparticle comprises a substantially hydrophobic acid, where the molar ratio of the substantially hydrophobic acid to the aforementioned therapeutic agent ranges from about 0.25: 1 to about 2: 1; about 0.2 to about 20 weight percent of the aforementioned therapeutic agent, where the pK value_to of the protonated therapeutic agent is at least about 1.0 pK units_to greater than pK_to of the hydrophobic acid, and about 50 to about 99.75 weight percent of a polymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer, poly (lactic acid-co-glycolic acid diblock copolymer ) poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol.

In certain embodiments, the therapeutic nanoparticle comprises a substantially hydrophobic acid and the aforementioned therapeutic agent, where the pK_to of the protonated therapeutic agent is at least about 1.0 pKa units greater than the pK_to of hydrophobic acid, and a polymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer or a poly (lactic acid-coglycolic acid) -poly (ethylene) glycol diblock copolymer and combinations thereof.

In some embodiments, the therapeutic nanoparticle comprises the said therapeutic agent, a substantially hydrophobic acid, where the molar ratio of the substantially hydrophobic acid to the therapeutic agent ranges from about 0.25: 1 to about 2: 1 and where the pK_to The protonated therapeutic agent is at least about 1.0 pKa units greater than the pKa of the hydrophobic acid, and a polymer selected from poly (lactic acid) diblock copolymer - poly (ethylene) glycol or a poly (acid) diblock copolymer lactic-co-glycolic acid) poly (ethylene) glycol and their combinations.

In some embodiments, the molar ratio of the substantially hydrophobic acid to the named therapeutic agent is from about 0.5: 1 to about 1.5: 1. In certain embodiments, the molar ratio of the substantially hydrophobic acid to the named therapeutic agent is from about 0.75: 1 to about 1.25: 1. In certain embodiments, the molar ratio of substantially hydrophobic acid to named therapeutic agent is from about 0.25: 1 to about 1: 1.

In certain embodiments, the pK_to of the protonated form of the aforementioned therapeutic agent is at least about 2.0 pKa units greater than the pK_to of hydrophobic acid. In other embodiments, the pK_to of the protonated form of the aforementioned therapeutic agent is at least about 4.0 pKa units greater than the pK_toof hydrophobic acid.

In another aspect, the therapeutic nanoparticle comprises a hydrophobic ion pair comprising a hydrophobic acid and the aforementioned therapeutic agent; where the difference between the pK_to of the protonated form of the aforementioned therapeutic agent and the hydrophobic acid is at least about 1.0 pKa units, and about 50 to about 99.75 percent by weight of a poly (lactic acid) -polyethylene diblock copolymer. )glycol, where the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight of about 15 kDa to about 20 kDa of poly (lactic acid) and a number average molecular weight of about 4 kDa to about 6 kDa of poly (ethylene) glycol. In certain embodiments of this aspect of the invention, the difference between the pK_to of the protonated form of the aforementioned therapeutic agent and the hydrophobic acid is at least about 2.0 pKa units. In other embodiments, the difference between the pK_to of the protonated form of the aforementioned therapeutic agent and the hydrophobic acid is at least about 4.0 pKa units.

In certain embodiments, the therapeutic nanoparticle comprises from about 0.05 to about 20 percent by weight of the hydrophobic acid.

In some embodiments, the substantially hydrophobic acid has a P log of from about 2 to about 8, where P is the octanol / water partition coefficient of the hydrophobic acid. In some embodiments, the substantially hydrophobic acid has a P log of about 4 to about 8. In some embodiments, the substantially hydrophobic acid has a P log of about 2 to about 7.

In some embodiments, the substantially hydrophobic acid has a pK_to in water from about -1.0 to about 5.0. In other embodiments, the substantially hydrophobic acid has a pK_to in water from about 2.0 to about 5.0.

In certain embodiments, the substantially hydrophobic acid and the aforementioned therapeutic agent form a hydrophobic ion pair on the therapeutic nanoparticle.

In some embodiments, the hydrophobic acid is a fatty acid. For example, in certain embodiments, the fatty acid is a saturated fatty acid, including, without limitation, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecylic acid, myristic acid. , pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosilic acid, behenic acid, tricosilic acid, lignoceric acid, pentacosyl acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melisic acid, henatriacontilic acid, lacceroic acid, psilic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, or their combinations. In other embodiments, the fatty acid is an omega-3 fatty acid, including, without limitation, hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic, tetracosapentaenoic acid, tetracosahexaenoic acid, or combinations thereof. In still other embodiments, the fatty acid is an omega-6 fatty acid, including, without limitation, linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid. , docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, or combinations thereof. In certain other embodiments, the fatty acid is an omega-9 fatty acid, including, without limitation, oleic acid, eicosenoic acid, Mead acid, erucic acid, nervonic acid, or combinations thereof. In other embodiments, the fatty acid is a polyunsaturated fatty acid, including, without limitation, rumenic acid, α-calendic acid, β-calendic acid, jacaric acid, aleostearic acid, β-eleostearic acid, catalypic acid, punicic acid. , rumelenic acid, α-parinaric acid, β-parinaric acid, bossa pentaenoic acid, pinolenic acid, podocarpic acid, or combinations thereof.

In certain embodiments, the hydrophobic acid is a bile acid. For example, in some embodiments, bile acid includes, without limitation, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, hicolic acid, beta-muricolic acid, cholic acid, lithocholic acid, an amino acid conjugated bile acid, or combinations thereof. . In some embodiments, the bile acid is cholic acid. In other embodiments, the amino acid-conjugated bile acid is a glycine-conjugated bile acid, or a taurine-conjugated bile acid.

In certain embodiments, the hydrophobic acid includes without limitation dioctyl sulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, pamoic acid, undecanoic acid, or combinations thereof.

In other embodiments, the hydrophobic acid has a molecular weight of between about 200 Da and about 800 Da.

In certain embodiments, the hydrophobic acid is pamoic acid. In other embodiments, the hydrophobic acid is oleic acid. In some embodiments, the weight ratio of 1- (4 - {[4- (dimethylamino) piper¡d¡n1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4— yl-1,3,5-triazin-2-yl) phenyl] urea an oleic acid is about 6: 1. In some embodiments, the weight ratio of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6— dimorpholin— 4-yl-1,3,5-triazin-2-yl) phenyl] urea to pamoic acid is about 1.8: 1.

In some embodiments, the therapeutic nanoparticle comprises from about 1 to about 20 weight percent of the aforementioned therapeutic agent. In other embodiments, the therapeutic nanoparticle comprises from about 1 to about 15 percent by weight of the aforementioned therapeutic agent. In other embodiments, the therapeutic nanoparticle comprises from about 2 to about 20 weight percent of the aforementioned therapeutic agent. In other embodiments, the therapeutic nanoparticle comprises about 2 to about 15 percent by weight of the aforementioned therapeutic agent. In still other embodiments, the therapeutic nanoparticle comprises about 4 to about 20 weight percent of the aforementioned therapeutic agent. In still other embodiments, the therapeutic nanoparticle comprises from about 4 to about 15 weight percent of the aforementioned therapeutic agent. In certain other embodiments, the therapeutic nanoparticle comprises from about 5 to about 20 weight percent of the aforementioned therapeutic agent. In certain other embodiments, the therapeutic nanoparticle comprises about 5 to about 10 percent by weight of the aforementioned therapeutic agent.

In some embodiments, the therapeutic nanoparticle substantially retains the therapeutic agent for at least 1 minute when placed in a 37 ° C phosphate buffer solution. In certain embodiments, the therapeutic nanoparticle immediately releases substantially less than about 30% of the therapeutic agent when placed in a phosphate buffer solution at 37 ° C. In certain other embodiments, the therapeutic nanoparticle releases about 10 to about 45% of the therapeutic agent over about 1 hour when placed in a phosphate buffer solution at 37 ° C. In some embodiments, the therapeutic nanoparticle releases about 0.01 to about 15% of the therapeutic agent over about 4 hours when placed in a phosphate buffer solution at 37 ° C. In some embodiments, the therapeutic nanoparticle releases about 0.01 to about 15% of the therapeutic agent over about 10 hours when placed in a phosphate buffer solution at 37 ° C. In some embodiments, the therapeutic nanoparticle releases about 0.01 to about 25% of the therapeutic agent over about 20 hours when placed in a phosphate buffer solution at 37 ° C. In some embodiments, the therapeutic nanoparticle releases about 1 to about 40% of the therapeutic agent over about 40 hours when placed in a phosphate buffer solution at 37 ° C. In still other embodiments, the therapeutic nanoparticle has a release profile that is substantially the same as a release profile for a control nanoparticle that is substantially the same as the therapeutic nanoparticle, except that it does not contain a fatty acid or bile acid.

In certain embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a poly (lactic acid) number average molecular weight fraction of from about 0.6 to about 0.95. In certain other embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.6 to about 0.8. In still other embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a poly (lactic acid) number average molecular weight fraction of from about 0.75 to about 0.85. In other embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a poly (lactic acid) number average molecular weight fraction of from about 0.7 to about 0.9.

In certain embodiments, the therapeutic nanoparticle comprises about 10 to about 25 weight percent poly (ethylene) glycol. In certain other embodiments, the therapeutic nanoparticle comprises about 10 to about 20 weight percent poly (ethylene) glycol. In still other embodiments, the therapeutic nanoparticle comprises about 15 to about 25 weight percent poly (ethylene) glycol. In other embodiments, the therapeutic nanoparticle comprises about 20 to about 30 weight percent poly (ethylene) glycol.

In certain embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight of about 15 kDa to about 20 kDa poly (lactic acid) and a number average molecular weight of about 4 kDa to about 6 kDa of poly (ethylene) glycol.

In some embodiments, the therapeutic nanoparticle further comprises about 0.2 to about 30 weight percent poly (lactic acid) -poly (ethylene) glycol copolymer functionalized with a targeting ligand. In other embodiments, the therapeutic nanoparticle further comprises about 0.2 to about 30 weight percent poly (lactic acid) -co-poly (glycolic acid) -poly (ethylene) glycol copolymer functionalized with a targeting ligand. . In certain embodiments, the rectifying ligand is covalently linked to poly (ethylene) glycol.

In certain embodiments, the hydrophobic acid is a polyelectrolyte. For example, in some embodiments, the polyelectrolyte includes, without limitation, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, or combinations thereof.

In certain embodiments, a contemplated therapeutic nanoparticle further comprises a mixture of two or more substantially hydrophobic acids. For example, in some embodiments, a contemplated therapeutic nanoparticle comprises a mixture of two substantially hydrophobic acids, a mixture of three substantially hydrophobic acids, a mixture of four substantially hydrophobic acids, or a mixture of five substantially hydrophobic acids. In some embodiments, the substantially hydrophobic acid mixture comprises oleic acid and cholic acid. In other embodiments, the mixture of two substantially hydrophobic acids are oleic acid and cholic acid.

In another aspect, the therapeutic nanoparticle is prepared by emulsifying a first organic phase comprising a first polymer, the aforementioned therapeutic agent, and a substantially hydrophobic acid, so as to form an emulsion phase; quenching the emulsion phase, so as to form a quenched phase, and finally, filtering the quenched phase to recover the therapeutic nanoparticles.

In some embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle is a fatty acid. For example, in certain embodiments, the fatty acid used in the preparation of the therapeutic nanoparticle is a saturated fatty acid including, without limitation, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, acid lauric, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosilic acid, behenic acid, tricosilic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melisic acid, henatriacontyl acid, lacceroic acid, psilic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, or their combinations. In other embodiments, the fatty acid used in the preparation of the therapeutic nanoparticle is an omega-3 fatty acid including, without limitation, hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, or combinations thereof. In still other embodiments, the fatty acid used in the preparation of the therapeutic nanoparticle is an omega-6 fatty acid that includes, without limitation, linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, or combinations thereof. In certain other embodiments, the fatty acid used in the preparation of the therapeutic nanoparticle is an omega-9 fatty acid including, without limitation, oleic acid, eicosenoic acid, Mead acid, erucic acid, nervonic acid, or combinations thereof. In other embodiments, the fatty acid used in the preparation of the therapeutic nanoparticle is a polyunsaturated fatty acid that includes, without limitation, rumenic acid, α-calendic acid, β-calendic acid, jacaric acid, α-eleostearic acid, β- eleostearic, cathalpic acid, punicic acid, rumelenic acid, α-parinaric acid, βparinaric acid, bosseo pentaenoic acid, pinolenic acid, podocarpic acid, or combinations thereof.

In certain embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle is a bile acid that includes, without limitation, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, hicolic acid, beta-muricolic acid, cholic acid, lithocholic acid, an amino acid conjugated bile acid, or combinations thereof. In some embodiments, the bile acid is cholic acid. In other embodiments, the amino acid-conjugated bile acid is a glycine-conjugated bile acid or a taurine-conjugated bile acid.

In certain embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle includes, without limitation, dioctyl sulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid, naphthalene-1,5-disulfonic acid, Naphthalene-2-sulfonic acid, pamoic acid, undecanoic acid, or combinations thereof.

In other embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle has a molecular weight of between about 200 Da and about 800 Da.

In certain embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle is pamoic acid. In other embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle is oleic acid. In some embodiments, the weight ratio of 1 - (4 - {[4— (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6— dimorpholin — 4 — ¡I —1,3,5-triazin-2-yl) phenyl] urea an oleic acid used in the preparation of the therapeutic nanoparticle is about 6: 1. In some embodiments, the weight ratio of 1- (4 - {[4- (dimethylamino) piper¡d¡n17

1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea a pamoic acid used in the preparation of the nanoparticle therapeutic is around 1.8: 1. In some embodiments, the weight ratio of 1 (4 - {[4- (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3- [4- (4,6-dimorpholin-4-yl1,3 , 5-triazin-2-yl) phenyl] urea an oleic acid used in the preparation of the therapeutic nanoparticle is 6: 1. In some embodiments, the weight ratio of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6— dimorpholin-4-yl— 1,3,5-triazin-2-yl) phenyl] urea a pamoic acid used in the preparation of the therapeutic nanoparticle is 1.8: 1.

In certain embodiments, the first polymer used in the preparation of the therapeutic nanoparticle is poly (lactic acid) -poly (ethylene) glycol copolymer. In other embodiments, the first polymer is poly (lactic acid) -co-poly (glycolic acid) -poly (ethylene) glycol copolymer. In certain embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.6 to about 0.95. In certain other embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a poly (lactic acid) number average molecular weight fraction of from about 0.6 to about 0.8. In still other embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.75 to about 0.85. In other embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer has a poly (lactic acid) number average molecular weight fraction of from about 0.7 to about 0.9.

In certain embodiments, the therapeutic nanoparticle is prepared using about 10 to about 25 weight percent poly (ethylene) glycol. In certain other embodiments, about 10 to about 20 weight percent poly (ethylene) glycol is used. In still other embodiments, about 15 to about 25 weight percent poly (ethylene) glycol is used. In other embodiments, about 20 to about 30 weight percent poly (ethylene) glycol is used.

In certain embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer used in the preparation of the therapeutic nanoparticle has a number average molecular weight of from about 15 kDa to about 20 kDa of lactic polyphacid) and a number average molecular weight of about 4 kDa to about 6 kDa of poly (ethylene) glycol.

In some embodiments, the therapeutic nanoparticle is prepared by additional functionalization of about 0.2 to about 30 weight percent of poly (lactic acid) -poly (ethylene) glycol copolymer with a targeting ligand. In other embodiments, the therapeutic nanoparticle is prepared by further functionalizing from about 0.2 to about 30 weight percent of poly (lactic acid) -copoly (glycolic acid) -polyphethylene) glycol copolymer with a targeting ligand. In certain embodiments, the targeting ligand is covalently linked to poly (ethylene) glycol.

In certain embodiments, the hydrophobic acid used in the preparation of the therapeutic nanoparticle is a polyelectrolyte. For example, in some embodiments, the polyelectrolyte includes, without limitation, a poly (styrene sulfonic acid), polyacrylic acid, polymethacrylic acid, or combinations thereof.

In certain embodiments, the therapeutic nanoparticle is prepared using a mixture of two or more substantially hydrophobic acids. For example, in some embodiments, a mixture of two substantially hydrophobic acids, a mixture of three substantially hydrophobic acids, a mixture of four substantially hydrophobic acids, or a mixture of five substantially hydrophobic acids can be used to prepare a therapeutic nanoparticle. In some embodiments, the substantially hydrophobic acid mixture comprises oleic acid and cholic acid. In other embodiments, the mixture of two substantially hydrophobic acids are oleic acid and cholic acid.

In certain embodiments, the therapeutic nanoparticle comprises the PLA-PEG polymer, and the molar ratio of PLA-PEG is about 5: 1.

In some embodiments, a therapeutic nanoparticle is prepared through the process of combining a first organic phase with a first aqueous solution to form a second phase; emulsifying the second phase to form an emulsion phase, wherein the emulsion phase comprises a first polymer, therapeutic agent, and a substantially hydrophobic acid; quenching the emulsion phase so as to form a quenched phase; and filtration of the quenched phase in order to recover the therapeutic nanoparticles, where the therapeutic agent is 1- (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4 , 6-dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea, the first organic phase comprises the therapeutic agent and pamoic acid in a weight ratio of therapeutic agent to pamoic acid of about 11: 1, and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 1: 3, in an organic solvent comprising benzyl alcohol and ethyl acetate in a weight ratio of benzyl alcohol to ethyl acetate of about 1.25, and the first aqueous solution comprises a polyoxyethylene (100) stearyl ether dissolved in benzyl alcohol in a weight ratio of 0.005: 1; and combining the first organic phase and the first aqueous phase in a weight ratio of about 1: 5 to form a second phase; and the emulsion of the second phase formed therein and the quenching of the emulsion phase with citric acid, 0.1 M, in aqueous solution at pH 4.5; and the concentration of the resulting product.

In other embodiments, the therapeutic nanoparticle comprises the aforementioned therapeutic agent or a pharmaceutically acceptable salt thereof and a polymer selected from a poly (lactic) acid diblock copolymer - poii (ethylene) glycol or a poly (acid) diblock copolymer. co-glycolic lactic acid) -poly (ethylene) glycol and their combinations.

In certain embodiments, the therapeutic nanoparticle has an additionally present targeting ligand, and the ligand is PLAPEG-GL, where GL has the following structure:

'NH

<img file="CU24488B1_D0001.tif" />

In some embodiments, the therapeutic nanoparticle further comprises a solubilizer. In certain embodiments, the solubilizer is polysorbate 80. In other embodiments, the solubilizer is polyoxyethylene (100) stearyl ether.

In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4— ¡I-1,3,5-triazin-2-yl) phenyl] urea, pamoic acid in a weight ratio of therapeutic agent to pamoic acid of about 1.8: 1, PLAPEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 1: 3, and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL of about 44: 1. In other embodiments, the therapeutic nanoparticle additionally comprises a solubilizer. In certain such embodiments, the solubilizer is chickenxyethylene (100) stearyl ether.

In certain embodiments, the therapeutic nanoparticle comprises therapeutic agent 1— (4 - {[4— (dimethylamine) piperidin-1-yl] carbonyl} pheny) —3— [4 - (4,6-dimorpholin-4-I-1,3,5-triazin-2-yl) phenyl] urea, oleic acid in a weight ratio of therapeutic agent to oleic acid of about 6: 1, PLA -PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 1: 7, and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL of about 46: 1. In some embodiments, the therapeutic nanoparticle additionally comprises cholic acid. In other embodiments, the therapeutic nanoparticle further comprises a solubilizer. In certain such embodiments, the solubilizer is polysorbate 80.

In still another aspect, there is provided a pharmaceutical composition comprising a therapeutic nanoparticle described in this application and a pharmaceutically acceptable excipient. The pharmaceutical composition can comprise a plurality of contemplated therapeutic nanoparticles.

In certain embodiments, the pharmaceutical composition further comprises a saccharide. For example, in some embodiments, the saccharide is a disaccharide selected from the group consisting of sucrose or trehalose, or a mixture thereof.

In certain embodiments, the pharmaceutical composition further comprises a cyclodextrin. For example, in some embodiments, cyclodextrin includes, without limitation, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, heptakis- (2,3,6-tri-O-benzyl) -p-cyclodextrin, heptakis - (2,3,6— tri-O-benzoyl) -p-cyclodextrin, or their mixtures.

In another aspect, a method of treating cancer in a subject in need is provided. The method comprises administering, to the subject, a therapeutically effective amount of a pharmaceutical composition as described in the present application. In some embodiments, the cancer is chronic myelogenous leukemia. In certain embodiments, cancer includes, without limitation, chronic myelomonocytic leukemia, hypereosinophilic syndrome, renal cell carcinoma, hepatocellular carcinoma, Philadelphia chromosome positive acute lymphoblastic leukemia, non-small cell lung cancer, pancreatic cancer, cancer. breast, a solid tumor, mantle cell lymphoma, gastrointestinal stromal tumor, or head and neck cancer. In some embodiments, the cancer is breast cancer.

In yet another aspect, a method of treating a gastrointestinal stromal tumor in a subject in need is provided by administering to the subject a therapeutically effective amount of a pharmaceutical composition as described in this application.

In yet another aspect, a process is provided for the preparation of a therapeutic nanoparticle, the process comprises combining a first organic phase with a first aqueous solution to form a second phase; emulsifying the second phase to form an emulsion phase, wherein the emulsion phase comprises a first polymer, the aforementioned therapeutic agent, and a substantially hydrophobic acid; followed by quenching of the emulsion phase, so as to form a quenched phase, and finally, filtration of the quenched phase in order to recover the therapeutic nanoparticles.

In some embodiments, the process further comprises combining the aforementioned therapeutic agent and the substantially hydrophobic acid in the second phase prior to emulsifying the second phase. In certain embodiments, the aforementioned therapeutic agent and the substantially hydrophobic acid form a hydrophobic ion pair prior to second phase emulsion. In certain other embodiments, the aforementioned therapeutic agent and the substantially hydrophobic acid form a hydrophobic ion pair during second phase emulsification. In certain embodiments, the process further comprises combining the aforementioned therapeutic agent and the substantially hydrophobic acid in the second phase in substantially concurrent form with the emulsion in the second phase. For example, in some embodiments, the first organic phase comprises the mentioned therapeutic agent, and the first aqueous solution comprises the substantially hydrophobic acid.

In some embodiments, the aforementioned therapeutic agent, when protonated, has a first pK_to, the substantially hydrophobic acid has a second pK_to, and the emulsion phase is quenched with an aqueous solution having a pH equal to one unit of pK_tobetween the first pK_to and the second pK_to. For example, in certain embodiments, the quenched phase has a pH equal to one unit of pK._to between the first pK_to and the second pK_to. In other embodiments, the aforementioned therapeutic agent, when protonated, has a first pK_to, the substantially hydrophobic acid has a second pK_to, and the first aqueous solution has a pH equal to one unit of pK_to between the first pK_to and the second pK_to. In certain other embodiments, the pH equals one unit of pK._to which is approximately equidistant between the first pK_to and the second pK_to.

In some embodiments, the aforementioned therapeutic agent, when protonated, has a first pK_to, the substantially hydrophobic acid has a second pK_to, and the emulsion phase is quenched with an aqueous solution having a pH equal to one unit of pK_tobetween the first pK_to and the second pK_to. For example, in certain embodiments, the quenched phase has a pH equal to one unit of pK._to between the first pK_to and the second pK_to. In other embodiments, the aforementioned therapeutic agent, when protonated, has a first pK_to, the substantially hydrophobic acid has a second pK_to, and the first aqueous solution has a pH equal to one unit of pK_to between the first pK_to and the second pK_to. In certain other embodiments, the pH equals one unit of pK._to which is equidistant between the first pK_to and the second pK_to.

In another aspect, a therapeutic nanoparticle is provided as described in the present application for use as a medicament in a subject.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in producing an antiproliferative effect in a subject.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in a subject as an anti-invasive agent in the containment and / or treatment of solid tumor disease.

In yet another aspect, there is provided the use of a therapeutic nanoparticle as described in the present application in the prevention or treatment of cancer in a subject.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in the prevention or treatment of cancer in a subject.

In yet another aspect, the use of a therapeutic nanoparticle as described in the present application is provided in the manufacture of a medicament for the prevention or treatment of cancer in a subject.

In yet another aspect, the use of a therapeutic nanoparticle as described in the present application for the production of an antiproliferative effect in a subject is provided.

In still another aspect, the use of a therapeutic nanoparticle as described in the present application is provided in the manufacture of a medicament for use in producing an antiproliferative effect in a subject.

In still another aspect, the use of a therapeutic nanoparticle as described in the present application is provided in the manufacture of a medicament for use in a subject as an anti-invasive agent in the containment and / or treatment of a solid tumor disease. .

In yet another aspect, there is provided a method for producing an antiproliferative effect in a subject in need of such treatment, comprising administering to said subject an effective amount of a therapeutic nanoparticle as described in the present application.

In still another aspect, there is provided a method for producing an anti-invasive effect by containing and / or treating a solid tumor disease in a subject in need of such treatment, comprising administering, to said subject, an amount effectiveness of a therapeutic nanoparticle as described in the present application.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in the prevention or treatment of a solid tumor disease in a subject.

In yet another aspect, there is provided the use of a therapeutic nanoparticle as described in the present application in the manufacture of a medicament for use in the prevention or treatment of a solid tumor disease in a subject.

In yet another aspect, there is provided a method for the prevention or treatment of a solid tumor disease in a subject in need of such treatment, comprising administering, to said subject, an effective amount of a therapeutic nanoparticle as described in the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow chart for an emulsion process for forming a disclosed nanoparticle.

Figures 2A and 2B show flow charts for a disclosed emulsion process.

Figure 3 depicts the in vitro nanoparticle release profiles of three formulations as described in this application identified below as Formulations A, B, and C, respectively, where each comprises 1- (4 - {[4 (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-l -1,3,5-triazin-2-yl) phenyl] urea. The error bars indicate the standard deviation. Water bath temperature = 37 ° C.

Figure 4 represents the pharmacokinetics of the nanoparticles of three formulations as described in this application and identified as the

Formulations A, B, and C, respectively, each comprising 1— (4— {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4— il-1,3,5-triazin-2-yl) phenyl] urea, in Wistar Han rats; (a) shows the pharmacokinetics of the nanoparticle in relation to free therapeutic agent, while (b) shows the same information with the omitted free therapeutic agent.

Figure 5 represents the in vitro release profiles of Formulation A of drug 1— (4 - {[4— (dimethylamino) piperidin-1— yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin —4-yl-1,3,5-triazin-2-yl) phenyl] urea. The error bars indicate the standard deviation. Water bath temperature = 37 ° C.

Figure 6 represents the in vitro release profiles of Formulation C of drug 1— (4 - {[4— (dimethylamine) piperidin-1— yl] carbonyl} phenyl) —3— [4— (4,6 —Dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea using a citric acid quench, pH 4.5.

Figure 7 represents an MDAMB361 xenograft programming study in female SCID / bg rats dosed with Formulation B nanoparticles or 1— (4 - {[4— (dimethylamino) piperidin — 1 — yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4 — ¡1—1,3,5-triazin-2-yl) phenyl] urea (API (active pharmaceutical ingredient) naked), once each 8 days, compared to once every 4 days.

Figures 8A, 8B and 8C depict an MDAMB361 xenograft programming study in female SCID / bg mice dosed with Formulation A, B, or C nanoparticles or naked API, once every 8 days, compared to once every 4 days. , and in vivo modulation studies of pS6 with nanoparticles of Formulation A, B or C or 1— (4 - {[4— (dimethylamino) piperidin — 1 —¡l] carbon¡l} phen¡l) —3 - [4— (4,6-dimorpholin-4-yl-1,3,5-triazin-2-yljphenyljurea (naked API). TA = therapeutic agent.

Figure 9 represents a tumor growth inhibition study of the WM266-4 model with nu / nu female mice treated with nanoparticles of Formulation B or C or 1— (4 - {[4— (dimethylamino) piperidin — 1 —il ] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-II-1,3,5-triazin-2-yl) phenyl] urea (naked API). TA = therapeutic agent.

Figure 10 represents an analysis of glucose and insulin levels in mice or rats after treatment with nanoparticles of Formulation B or C or 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl ) —3— [4— (4,6— dimorpholin — 4 — 1—1,3,5-triazin-2-yl) phenyl] urea (naked API).

DETAILED DESCRIPTION

Definitions.

The definitions set out in this application are intended to clarify terms used throughout the application.

The term of the / in the present application means the entire application.

Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by one of ordinary skill in the art to which these inventions belong. While methods and materials similar or equivalent to those as described in this application may be used, in the practice or evaluation of inventions, suitable methods and materials are described below. The materials, methods, and examples are illustrative only, and are not intended to be limiting. All publications, patents and other documents mentioned in this application are incorporated by reference in their entirety.

Each embodiment of the inventions as described in this application may be taken alone or in combination with any one or more other embodiments of the inventions.

Throughout this application, the word "a" or "an" shall be understood to imply the inclusion of one or more of members modified by the article "a" or "an".

Throughout this application, the word "comprise" or variations such as "comprises" or "comprising / n" shall be understood to imply the inclusion of an established member or group of members, but not the exclusion of any other member or group of members.

Throughout the application, when the compositions are described as having, including, or comprising specific components, it is contemplated that the compositions further may consist essentially of, or consist of, the recited components. Similarly, when the methods or processes are described so as to have, to include, or to comprise specific process steps, the processes may further consist essentially of, or consist of, the cited process steps. Additionally, it should be understood that the order of steps, or the order to perform certain actions, is immaterial as long as the compositions and methods as described in this application remain operable. Furthermore, two or more stages or actions can be conducted simultaneously.

The term "or" as used in the present application is to be understood to mean "and / or", unless the context clearly indicates otherwise.

The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an attached oxygen. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

Furthermore, the term alkyl (or lower alkyl), as used in the present application, is intended to include both unsubstituted alkyls and substituted alkyls, where the latter refer to alkyl moieties having substituents that replace a hydrogen on one or more carbons from the spinning of the hydrocarbon. Such substituents, if not otherwise specified, may include, for example, a halogen, a hydroxyl, a carbonite (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an amino, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio , a sulfate, a sultanate, a sulfamoyl, a sulfonamide, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic moiety. Those skilled in the art will understand that the substituted moieties in the hydrocarbon chain may themselves be substituted, if appropriate. For example, substituents on a substituted alkyl can include substituted and unsubstituted forms of amino, azido, amino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl, and sultanate), silyl groups. , as well as ethers, alkylthiols, carbonites (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like.

The term "C_x"", When used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups containing from x to 8 carbons in the chain. For example, the term “C_x"" Alkyl "refers to saturated, substituted or unsubstituted hydrocarbon groups, including straight chain alkyl groups and branched alkyl groups containing from x to 2 carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2, 2-trifluoroethyl, etc. C_or alkyl indicates a hydrogen where the group is in a terminal position, a bond, if internal.

The term "amide", as used in the present application, refers to a group

TO

R³⁰ where each R³⁰ independently represents a hydrogen or hydrocarbyl group, or two Rs³⁰ they are taken together with the N atom to which they are attached to complete a heterocycle having 4 to 12 atoms in the ring structure.

The term "aryl" as used in the present application includes substituted or unsubstituted single-ring aromatic groups, where each ring atom is carbon. Preferably the ring is a 5 to 7 membered ring, more preferably a 6 membered ring. The term "aryl" further includes polycyclic ring systems that have two or more cyclic rings in which two or more carbons or heteroatoms are common to two adjacent rings, where at least one of the rings is aromatic, for example, the others Cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls.

The terms "arylalkyl" or "aralkyl", as used in the present application, refer to an alkyl group substituted with an aryl group.

The term "azido" is recognized in the art, and refers to the group —N = N⁺= N.

The term "carboxy", as used in the present application, refers to a group represented by the formula -CO₂H.

The terms "halo" and "halogen" as used in the present application mean halogen and include chlorine, fluorine, bromine and iodine.

The term "hydrocarbyl", as used in the present application, refers to a group that is linked through a carbon atom that does not have a = 0 or = S substituent, and usually has at least one carbon bond. -hydrogen and a primarily carbon backbone, although it may optionally include heteroatoms. Therefore, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for the purposes of this application, while substituents such as acetyl (which has a = 0 substituent on the bonding carbon) and ethoxy (which is bound through oxygen, not carbon) are not. Hydrocarbyl groups include, without limitation, aryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term "hydroxy", as used in the present application, refers to a -OH group.

The term "substituted" refers to moieties that have substituents that replace a hydrogen on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit condition that said substitution is in accordance with the allowed valence of the substituted atom and the substituent, and that the substitution achieves a stable compound, eg, not spontaneously undergoing transformation, such as by rearranging, cycling, deletion, etc. As used in the present application, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic, substituents of organic compounds. The permissible substituents can be one or more, the same or different for the appropriate organic compounds. For the purposes of this application, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible organic compound substituents, as described in this application, that satisfy the valences of the heteroatoms.

Substituents can include any substituent as described in this application, for example, without limitation, a halogen, a hydroxyl, a carbonite (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphorite, a phosphate, a phosphonate, a phosphinate, an amino, an amide, an amidine, an amino, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sultanate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic moiety. Those skilled in the art will understand that the substituents may themselves be substituted, if appropriate. Unless specifically stated as "unsubstituted", references to chemical moieties in this application are understood to include substituted variants. For example, reference to an "aryl" moiety or group implicitly includes both substituted and unsubstituted variants.

The terms "optional" or "optionally" mean that the subsequently described circumstance may or may not occur, so that the application includes cases where the circumstance occurs, and cases where it does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and consequently the application includes structures where a non-hydrogen substituent is present and structures where a non-hydrogen substituent is not present. .

The terms "healthy" and "normal" are used interchangeably in this application to refer to a particular subject or tissue or cell that is devoid (at least to the limit of detection) of a disease.

Unless otherwise indicated, the term "basic therapeutic agent" or "therapeutic agent" refers to the therapeutic agent 1- (4 - {[4 (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3- [ 4- (4,6-dimorpholin-4-II-1,3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof. It has the structure that is exposed below:

NN

It is described in US Patent No. 8,039,469, the contents of which are incorporated by reference. The calculated log of partition coefficient for octanoksaline (clogP) = 1.24 (calculated partition coefficient). The logD, the distribution constant, at pH 6.5 = 0.212, while logD at pH 7.4 = 1.08. As stated above, it is a base. It is a therapeutic agent that contains protonatable nitrogen. As used in the present application, a "protonatable nitrogen-containing therapeutic agent" includes any pharmaceutically active agent that contains at least one nitrogen-containing functional group that is capable of being protonated. In other words, the therapeutic agent has a nitrogen atom on it, which has only one pair of electrons that could potentially accept a proton. The pKa refers to the acid dissociation constant on a logarithmic scale of the corresponding protonated form of the therapeutic agent. In other words, if a proton (H +) were to be present at nitrogen atoms where there is an indicated arrow, the therapeutic agent will have the pKa indicated below:

<img file="CU24488B1_D0002.tif" />

ACD- 9.06

The pKa information is shown above for the more basic (lower) nitrogen and a less basic, but still protonatable (upper) nitrogen.

ACD is a number calculated using standard techniques known in the art, such as those described in Liao CZ reference, Nicklaus MC. "Comparison of nine programs predicting pK (a) values ​​of pharmaceutical substances". J. Chem. Inform. Model. 49 (12): 2801-2812, 2009. The pKa of the therapeutic agent should be understood to refer to its protonated form.

The therapeutic agent of the present invention possesses one or more chiral centers, and the present invention includes each separate enantiomer of said compounds, as well as mixtures of enantiomers. When there are multiple chiral centers, the invention includes each combination, as well as their mixtures. All chiral, diastereomeric and racemic forms of a structure are proposed, unless the specific stereochemistry or isomeric form is specifically indicated. How to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials, is well known in the art.

The term "substantially", when used in reference to a compound such as "hydrophobic acid", refers to the compound present in at least 1% by weight, or refers to a hydrophobic acid with a logP greater than 2. A Hydrophobic acid with a logP greater than 2 has a greater tendency to partition in the organic phase.

The term "hydrophobic acid" refers to a lipophilic acid that has a log of

-7 or higher, that is, -6, -5, -4, -3, -2, -1.0, 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

Nanoparticles as described in this application may further contain pharmaceutically acceptable salts of the therapeutic agent. Representative "pharmaceutically acceptable salts" include, without limitation, water-soluble and water-insoluble salts, such as the salts of acetate, aluminum, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzathine (N , Ndibenzylethylenediamine), benzenesulfonate, benzoate, bicarbonate, bismuth, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate (camphor sulfonate), carbonate, chloride, choline, citrate, clavulariate, diethanolamine, dihydrochloride diphosphate, edetate, edisylate (camphor sulfonate), esilate (ethanesulfonate), ethylenediamine, fumarate, gluceptate (glucoheptonate), gluconate, glucuronate, glutamate, hexafluorophosphate, hexylresorcinate, hydrabaamine (N, Ν'-bis (from ethhydrofoxydiamine), ethylenehydro-bromine) hydrochloride, hydroxynaphthoate, 1-hydroxy-2-naphthoate, 3-hydroxy-2-naphthoate, iodide, isothionate (2-hydroxyethanesulfonate), lactate, lactobionate, laurate, lauryl sulfate, lithium, magnesium, malate, maleate, mandelate, meglumine (1-deoxy-1- (methylamino) D-glucitol), mesylate, methyl bromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, palmitate, pamoate (4.4 ' -methylenebis-3-hydroxy-2-naphthoate, or embonate), pantothenate, phosphate, picrate, polygalacturonate, potassium, propionate, p-toluenesulfonate, salicylate, sodium, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate , theoclate (8-chloro-3,7-dihydro-1,3-dimethyl-1H-purine-2,6dione), triethiodine, tromethamine (2-amino-2- (hydroxymethyl) -1,3-propanediol) valerate and zinc.

When used in this application below, unless otherwise indicated,% is% by weight.

Polymeric nanoparticles that include the basic therapeutic agent and methods for the manufacture and use of said therapeutic nanoparticles are described in this application.

In some embodiments, the inclusion (ie, smoothing) of a substantially hydrophobic acid (eg, a fatty acid and / or a bile acid) in a disclosed nanoparticle, and / or its inclusion in a nanoparticle preparation process can achieve nanoparticles that include improved drug loading. Additionally, in certain embodiments, nanoparticles that include and / or are prepared in the presence of the hydrophobic acid may exhibit improved controlled release properties. For example, the disclosed nanoparticles may release the therapeutic agent more slowly, compared to nanoparticles prepared in the absence of the hydrophobic acid.

Without wishing to be bound by theory, it is believed that the disclosed nanoparticle formulations that include a hydrophobic acid (eg, fatty acid and / or bile acid) have significantly improved formulation properties (eg, drug loading and / or profile. release) through the formation of a hydrophobic ion pair (HIP), between the therapeutic agent on the single electron pair on one or more of the nitrogen atoms on the therapeutic agent indicated above, for example on the amine moiety and an acid. As used in the present application, a HIP is a pair of oppositely charged ions held together by the Coulomb attraction. Furthermore, without wishing to be bound by theory, in some embodiments, HIP can be used to increase the hydrophobicity of the therapeutic agent containing ionized groups (eg, amines). When the therapeutic agent has higher hydrophobicity, this is beneficial for nanoparticle formulations, as the formation of HIP is achieved, which can provide greater solubility of the therapeutic agent in organic solvents. The formation of HIP, contemplated in this application, can produce nanoparticles that have, for example, higher drug loading. The slower release of the therapeutic agent from the nanoparticles may also occur, for example, in some embodiments, due to a decrease in the solubility of the therapeutic agent in aqueous solution. Additionally, complexing of the therapeutic agent with large hydrophobic counterions can slow the diffusion of the therapeutic agent within the polymeric matrix. Conveniently, HIP formation occurs without the need for covalent conjugation of the hydrophobic group to the therapeutic agent.

Without wishing to be bound by theory, it is believed that the concentration of HIP affects the drug loading and release rate of the contemplated nanoparticles. For example, the concentration of HIP can be increased by increasing the magnitude of the difference between the pK_toof the protonated form of the therapeutic agent and the pK_to hydrophobic acid, as described in more detail below. Furthermore, without wishing to be bound by theory, it is believed that the conditions for ion pair formation affect the drug charge and release rate of the contemplated nanoparticles.

The nanoparticles disclosed in this application include one, two, three or more biocompatible and / or biodegradable polymers. For example, a contemplated nanoparticle may include from about 35 to about 99.75 percent by weight in some embodiments; about 50 to about 99.75 percent by weight, in some other embodiments; about 50 to about 99.5 weight percent, in some embodiments; about 50 to about 99 percent by weight in still other embodiments; about 50 to about 98 percent by weight in additional embodiments; about 50 to about 97 percent by weight in still other embodiments; about 50 to about 96 percent by weight in additional embodiments; about 50 to about 95 weight percent in other embodiments, about 50 to about 94 weight percent in still other embodiments; about 50 to about 93 percent by weight in other embodiments; about 50 to about 92 percent by weight in still other embodiments; about 50 to about 91 percent by weight, in some embodiments, about 50 to about 90 percent by weight; in some embodiments, about 50 to about 85 percent by weight; in some embodiments, about 60 to about 85 percent by weight; in some embodiments, about 65 to about 85 percent by weight; and in some embodiments, about 50 to about 80 weight percent, of one or more block copolymers including a biodegradable polymer and poly (ethylene glycol) (PEG), and about 0 to about 50 percent by weight of a biodegradable homopolymer.

In some embodiments, a contemplated nanoparticle may include 35 to 99.75 percent by weight in some embodiments; 50 to 99.75 percent by weight, in some other embodiments; 50 to 99.5 percent by weight, in some embodiments; 50 to 99 percent by weight in still other embodiments; 50 to 98 percent by weight in additional embodiments; 50 to 97 percent by weight in still other embodiments; fifty at 96 percent by weight in additional embodiments; 50 to 95 percent by weight in other embodiments, 50 to 94 percent by weight in still other embodiments; 50 to 93 percent by weight in other embodiments; 50 to 92 percent by weight in still other embodiments; 50 to 91 percent by weight, in some embodiments, 50 to 90 percent by weight; in some embodiments, 50 to 85 percent by weight; in some embodiments, 60 to 85 percent by weight; in some embodiments, 65 to 85 percent by weight; and in some embodiments, 50 to 80 percent by weight of one or more block copolymers including a biodegradable polymer and poly (ethylene glycol) (PEG), and 0 to 50 percent by weight of a biodegradable homopolymer.

In some embodiments, the disclosed nanoparticles may include about 0.2 to about 35 weight percent, about 0.2 to about 25 weight percent, about 0.2 to about 20 weight percent, about 0.2. to about 10 weight percent, about 0.2 to about 5 weight percent, about 0.5 to about 5 weight percent, about 0.75 to about 5 weight percent, about 1 to about 5 percent by weight, about 2 to about 5 percent by weight, about 3 to about 5 percent by weight, about 1 to about 20 percent by weight, about 2 to about 20 weight percent, about 3 to about 20 weight percent, about 5 to about 20 weight percent, about 1 to about 15 weight percent, about 2 to about 15 percent by weight, about 3 to about 15 percent by weight, about 4 to about 15 percent by weight, about 5 to about 15 percent by weight, about 1 to about 10 percent by weight, about 2 to about 10 weight percent, about 3 to about 10 weight percent, about 4 to about 10 weight percent, about 5 to about 10 weight percent, about 10 to about 30 percent by weight, or about 15 to about 25 percent by weight of the therapeutic agent. In some embodiments, the disclosed nanoparticles include around 0.2, around 0.3, around 0.4, around 0.5, around 0.6, around 0.7, around 0.8, around 0.9, around 1, around 2, around 3, around 4, around 5, around 6, around 7, around 8, around 9, around 10, around 11, around 12, around 13, around 14, around 15, around 16, around 17, around 18, around 19, around 20, around 21, around 22, around 23, around 24, around 25, around 26, around 27, around 28, around 29, or around 30 percent by weight of the therapeutic agent.

In certain embodiments, the disclosed nanoparticles may include 0.2 to 35 percent by weight, 0.2 to 25 percent by weight, 0.2 to 20 percent by weight, 0.2 to 10 percent by weight, 0.2 to 5 percent by weight. , 0.5 to 5 percent by weight, 0.75 to 5 percent by weight, 1 to 5 percent by weight, 2 to 5 percent by weight, 3 to 5 percent by weight, 1 to 20 percent by weight, 2 to 20 percent by weight, 3 to 20 percent by weight, 5 to 20 percent by weight, 1 to 15 percent by weight, 2 to 15 percent by weight, 3 to 15 percent by weight, 4 to 15 percent by weight, 5 to 15 percent by weight, 1 to 10 percent by weight, 2 to 10 percent by weight, 3 to 10 percent by weight, 4 to 10 percent by weight, 5 to 10 percent by weight, 10 to 30 percent by weight, or 15 to 25 percent by weight of the therapeutic agent. In some embodiments, the disclosed nanoparticles include 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weight percent therapeutic agent.

In certain embodiments, the disclosed nanoparticles comprise a hydrophobic acid (eg, a fatty acid and / or bile acid) and / or are prepared by a process that includes a hydrophobic acid. Such nanoparticles can have a higher drug loading than nanoparticles prepared by a process without a hydrophobic acid. For example, the drug loading (eg, by weight) of the disclosed nanoparticles prepared by a process comprising the hydrophobic acid can be between about 2 times and about 10 times higher, or even higher, than the disclosed nanoparticles. Prepared by a process without hydrophobic acid. In some embodiments, the drug loading (by weight) of the disclosed nanoparticles prepared by a first process comprising the hydrophobic acid can be at least about 2 times higher, at least about 3 times higher, at least about 4 times higher, at least about 5 times higher, or at least about 10 times higher than the disclosed nanoparticles prepared by a second process, where the second process is identical to the first process, except that the second process does not include hydrophobic acid.

In certain embodiments, the drug loading (eg, by weight) of the disclosed nanoparticles prepared by a process comprising the hydrophobic acid may be 2 to 10 times higher, or even higher, than the disclosed nanoparticles prepared by a process without hydrophobic acid. In some embodiments, the drug loading (by weight) of the disclosed nanoparticles prepared by a first process comprising the hydrophobic acid may be at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, or at least 10 times higher than the disclosed nanoparticles prepared by a second process, where the second process is identical to the first process, Except that the second process does not include hydrophobic acid.

Any suitable hydrophobic acid is contemplated. In some embodiments, the hydrophobic acid can be a carboxylic acid (eg, a monocarboxylic acid, a dicarboxylic acid, tricarboxylic acid, or the like), a sulfinic acid, a sulfenic acid, or a sultanic acid. In some cases, a contemplated hydrophobic acid may include a mixture of two or more acids. For example, in certain embodiments, the hydrophobic acid may comprise a mixture of two substantially hydrophobic acids, in some embodiments, a mixture of three substantially hydrophobic acids, in some embodiments, a mixture of four substantially hydrophobic acids, or in some embodiments, five substantially hydrophobic acids. In some embodiments, the substantially hydrophobic acid mixture comprises oleic acid and cholic acid. In other embodiments, the mixture of two hydrophobic acids is oleic acid and cholic acid.

In some cases, a salt of a hydrophobic acid can be used in a formulation.

For example, a disclosed carboxylic acid can be an aliphatic carboxylic acid (eg, a carboxylic acid having a cyclic or acyclic, branched or unbranched hydrocarbon chain). The disclosed carboxylic acids, in some embodiments, may be substituted with one or more functional groups including, without limitation, halogen (ie, F, Cl, Br, and I), sulfonyl, nitro, and oxo. In certain embodiments, a disclosed carboxylic acid may be unsubstituted.

Examples of carboxylic acids can include a substituted or unsubstituted fatty acid (e.g., C₆-C5q). In some cases, the fatty acid can be a C10-C20 fatty acid. In other cases, the fatty acid can be a C15-C20 fatty acid. The fatty acid in some cases can be saturated. In other embodiments, the fatty acid can be unsaturated. For example, the fatty acid can be a monounsaturated fatty acid or a polyunsaturated fatty acid. In some embodiments, a double bond from an unsaturated fatty acid group may be in the cis conformation. In some embodiments, a double bond of an unsaturated fatty acid can be in the trans configuration. Unsaturated fatty acids include, without limitation, omega-3, omega-6, or omega-9 fatty acids.

Non-limiting examples of saturated fatty acids include caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, lignoceric acid, pentacosanoic acid, cerotic acid, heptacosanoic acid, montanic acid, nonacosanoic acid, melisic acid, henatriacontanoic acid, lacceroic acid, psilic acid, gedic acid, ceroplastic acid, hexatriacontanoic acid, or their combinations.

Non-limiting examples of unsaturated fatty acids include hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, linolenic acid, tetracosapentaenoic acid, linoleic acid. , eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, oleic acid (pK_to = ~ 4-5; logP = 6.78), eicosenoic acid, Mead acid, erucic acid, nervonic acid, rumenic acid, α-calendic acid, β-calendic acid, jacaric acid, α-eleostearic acid, β-eleostearic acid, catalypic acid, punicic acid, rumelenic acid, α-parinaric acid, βparinaric acid, bosseo pentaenoic acid, pinolenic acid, podocarpic acid, palmitoleic acid, vaccenic acid, gadoleic acid, erucic acid, or combinations thereof.

Other non-limiting examples of hydrophobic acids include aromatic acids such as 1-hydroxy-2-naphthoic acid (i.e. xinaphoic acid) (pK_to = ~ 2-3; log P = 2.97), naphthalene-1,5-disulfonic acid (pK_to = -2; logP = 1.3), naphthalene-2-sulfonic acid (pK_to = -1.8; logP = 2.1), pamoic acid (pK_to= 2.4; logP = 6.17), cinnamic acid, phenylacetic acid, (±) -alcamphor-10sulfonic acid, dodecylbenzenesulfonic acid (pK_to = -1.8; logP = 6.6), or their combinations. Other non-limiting examples of hydrophobic acids include dodecylsulfuric acid (pK_to -0.09; logP = 4.5), dioctyl sulfosuccinic acid (i.e. docusate acid) (pK_to =

-0.8; logP = 5.2), dioleoyl phosphatidic acid (pK_to = ~ 2), or Vitamin D₃-sulfate (pK_to = -1.5).

In some embodiments, the hydrophobic acid can be a bile acid. Non-limiting examples of bile acids include chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid (pK_to = 4.65; logP = 3.79), hicolic acid, beta-muricolic acid, cholic acid (pK_to = ~ 4.5; logP = 2.48), taurocholic acid, collesteryl sulfate acid (pK_to = -1.4), lithocholic acid, an amino acid-conjugated bile acid, or combinations thereof. An amino acid-conjugated bile acid can be conjugated to any suitable amino acid. In some embodiments, the amino acid-conjugated bile acid is a glycine-conjugated bile acid or a taurine-conjugated bile acid.

In certain cases, the hydrophobic acid can be a polyelectrolyte. For example, the polyelectrolyte can be a polysulfonic acid (eg, polystyrene sulfonic acid or dextran sulfate) or a polycarboxylic acid (eg, polyacrylic acid or polymethacrylic acid).

In some cases, a contemplated hydrophobic acid may have a molecular weight of less than about 1000 Da, in some embodiments, less than about 500 Da, in some embodiments, less than about 400 Da, in some forms of embodiments, less than about 300 Da, in some embodiments, less than about 250 Da, in some embodiments, less than about 200 Da, and in some embodiments, less than about 150 Da. In some cases, the hydrophobic acid may have a molecular weight between about 100 Da and about 1000 Da, in some embodiments, between about 200 Da and about 800 Da, in some embodiments, between about 200 Da and about 600 Da, in some embodiments, between about 100 Da and about 300 Da, in some embodiments, between about 200 Da and about 400 Da, in some embodiments, between around 300 Da and around 500 Da, and in some embodiments, between around 300 Da and around 1000 Da. In certain embodiments, a contemplated acid may have a molecular weight greater than about 200 kDa, in some embodiments, greater than about 300 Da, in some embodiments, greater than about 400 Da, and in some embodiments, greater than about 500 Da. In certain embodiments, the rate of release of a therapeutic agent from a nanoparticle can be decreased by increasing the molecular weight of the hydrophobic acid used in the nanoparticle formulation.

In some cases, a contemplated hydrophobic acid may have a molecular weight of less than 1000 Da, in some embodiments less than 500 Da, in some embodiments less than 400 Da, in some embodiments less than 300 It gives, in some embodiments, less than 250 Da, in some embodiments, less than 200 Da, and in some embodiments, less than 150 Da. In some cases, the hydrophobic acid may have a molecular weight between 100 Da and 1000 Da, in some embodiments, between 200 Da and 800 Da, in some embodiments, between 200 Da and 600 Da, in some forms of implementation, between 100 Da and 300 Da, in some embodiments, between 200 Da and 400 Da, in some embodiments, between 300 Da and 500 Da, and in some embodiments, between 300 Da and 1000 Da. In certain embodiments, a contemplated acid may have a molecular weight greater than 200 kDa, in some embodiments, greater than 300 Da, in some embodiments, greater than 400 Da, and in some embodiments, greater than 500 Da.

In some embodiments, a hydrophobic acid can be selected, at least in part, based on the concentration of the acid. For example, hydrophobic acid may have an acid dissociation constant in water (pK_to) from about -5 to about 7, in some embodiments, about -3 to about 5, in some embodiments, about -3 to about 4, in some embodiments, about - 3 to about 3.5, in some embodiments, about -3 to about 3, in some embodiments, about -3 to about 2, in some embodiments, about -3 to about 1 , in some embodiments, about -3 to about 0.5, in some embodiments, about -1.0 to about 5.0, in some embodiments, about -0.5 to about 0.5, in some embodiments, about 1 to about from 7, in some embodiments, about 2 to about 7, in some embodiments, about 2.0 to about 5.0, in some embodiments, about 3 to about 7, in some embodiments , about 4 to about 6, in some embodiments, about 4 to about 5.5, in some embodiments, about 4 to about 5, and in some embodiments, about 4.5 to about 5 , determined at 25 ° C. In some embodiments, the acid may have a pK_to less than about 7, less than about 5, less than about 3.5, less than about 3, less than about 2, less than about 1, or less than about 0, determined at 25 ° C.

In some embodiments, the hydrophobic acid may have an acid dissociation constant in water (pK_to) -5 to 7, in some embodiments, -3 to 5, in some embodiments, -3 to 4, in some embodiments, -3 to 3.5, in some embodiments, -3 to 3 , in some embodiments, -3 to 2, in some embodiments, -3 to 1, in some embodiments, -3 to 0.5, in some embodiments, -1.0 to 5.0, in some embodiments , -0.5 to 0.5, in some embodiments, 1 to 7, in some embodiments, 2 to 7, in some embodiments, 2.0 to 5.0, in some embodiments, 3 to 7, in some embodiments, 4 to 6, in some embodiments, 4 to 5.5, in some embodiments, 4 to 5, and in some embodiments, 4.5 to 5, determined at 25 ° C. In some embodiments, the acid may have a pK_to less than 7, less than 5, less than 3.5, less than 3, less than 2, less than 1, or less than 0, determined at 25 ° C.

In certain embodiments, the hydrophobic acid may be selected, at least in part, on the basis of the difference between the pK_to hydrophobic acid and pK_to of the protonated therapeutic agent. For example, in some cases, the difference between the pK_to hydrophobic acid and pK_to of the protonated therapeutic agent can be between about 1 pK unit_to and about 15 pK units_to, in some embodiments, enter about 1 pK unit_to and about 10 pK units_to, in some embodiments, enter about 1 pK unit_to and about 5 pK units_to, in some embodiments, enter about 1 pK unit_to and about 3 pK units_to, in some embodiments, enter about 1 pK unit_to and about 2 pK units_to, in some embodiments, between about 2 pK units_to and about 15 pK units_to, in some embodiments, between about 2 pK units_to and about 10 pK units_to, in some embodiments, between about 2 pK units_to and about 5 pK units_to, in some embodiments, between about 2 pKa units and about 3 pKa units, in some embodiments, between about 3 pKa units and about 15 pK_tounits, in some embodiments, between about 3 pKa units and about 10 pKa units, in some embodiments, between about 3 pKa units and about 5 pKa units, in some embodiments, between about 4 pKa units and about 15 pKa units, in some embodiments, between about 4 pKa units and about 10 pKa units, in some embodiments, between about 4 pKa units and about 6 pKa units, in some embodiments, between about 5 pKa units and about 15 pKa units, in some embodiments, between about 5 pKa units and about 10 pKa units , in some embodiments, between about 5 pKa units and about 7 pKa units, in some embodiments, between about 7 pKa units and about 15 pKa units, in some embodiments, between about 7 pKa units and about 9 pKa units, in some embodiments, between about 9 pKa units and about 15 pKa units, in some embodiments, between about 9 pKa units and about 11 pKa units , in some embodiments, between about 11 pKa units and about 13 pKa units, and in some embodiments, between about 13 pKa units and about 15 pKa units, determined at 25 ° C.

In certain embodiments, the difference between the pK_to hydrophobic acid and pK_to of the protonated therapeutic agent can be between 1 pKa unit and 15 pKa units, in some embodiments, between 1 pKa unit and 10 pKa units, in some embodiments, between 1 pKa unit and 5 pKa units, in some embodiments , between 1 pKa unit and 3 pKa units, in some embodiments, between 1 pKa unit and 2 pKa units, in some embodiments, between 2 pKa units and 15 pKa units, in some embodiments, between 2 pKa units and 10 pKa units, in some embodiments, between 2 pKa units and 5 pKa units, in some embodiments, between 2 pKa units and 3 pKa units, in some embodiments, between 3 pKa units and 15 pKa units, in some embodiments, between 3 pKa units and 10 pKa units, in some embodiments, between 3 pKa units and 5 pKa units, in some embodiments, between 4 pKa units and 15 pKa units, in some embodiments, between 4 pKa units and 10 pKa units, in some embodiments, between 4 pKa units and 6 pKa units, in some embodiments, between 5 pKa units and 15 pKa units, in some embodiments, between 5 pKa units and 10 pKa units, in some embodiments, between 5 pKa units and 7 pKa units, in some embodiments, between 7 pKa units and 15 pKa units, in some embodiments, between 7 pKa units and 9 pKa units, in some embodiments, between 9 pKa units and 15 pKa units, in some embodiments, between 9 pKa units and 11 pKa units, in some embodiments, between 11 pKa units and 13 units pKa, and in some embodiments, between 13 pKa units and 15 pKa units, determined at 25 ° C.

In some cases, the difference between the pK_to hydrophobic acid and pK_to of the protonated therapeutic agent can be at least about 1 pKa unit, in some embodiments, at least about 2 pKa units, in some embodiments, at least about 3 pKa units, in some embodiments , at least about 4 pKa units, in some embodiments, at least about 5 pKa units, in some embodiments, at least about 6 pKa units, in some embodiments, at least about 7 pKa units, in some embodiments at least about 8 pKa units, in some embodiments at least about 9 pKa units, in some embodiments at least about 10 pKa units , and in some embodiments, at least about 15 pKa units, determined at 25 ° C.

In some embodiments, the difference between the pK_to hydrophobic acid and pK_to of the protonated therapeutic agent may be at least 1 pKa unit, in some embodiments at least 2 pKa units, in some embodiments at least 3 pKa units, in some embodiments at least 4 units pKa, in some embodiments, at least 5 pKa units, in some embodiments, at least 6 pKa units, in some embodiments, at least 7 pKa units, in some embodiments, at least 8 pKa units, in some embodiments at least 9 pKa units, in some embodiments at least 10 pKa units, and in some embodiments at least 15 pKa units, determined to 25 ° C.

In some embodiments, the hydrophobic acid may have a logP of between about 2 and about 15, in some embodiments, between about 5 and about 15, in some embodiments, between about 5 and about of 10, in some embodiments, between about 2 and about 8, in some embodiments, between about 4 and about 8, in some embodiments, between about 2 and about 7, or in some embodiments, between about 4 and about 7. In some cases, the hydrophobic acid may have a logP greater than about 2, greater than about 4, greater than about 5, or greater than about 6.

In some embodiments, the hydrophobic acid may have a logP of between 2 and 15, in some embodiments, between 5 and 15, in some embodiments, between 5 and 10, in some embodiments, between 2 and 8, in some embodiments, between 4 and 8, in some embodiments, between 2 and 7, or in some embodiments, between 4 and 7. In some cases, the hydrophobic acid may have a logP greater than 2 , greater than 4, greater than 5, or greater than 6.

In some embodiments, a contemplated hydrophobic acid may have a convenient phase transition temperature, for example, to enhance the properties of therapeutic nanoparticles. For example, hydrophobic acid may have a melting point of less than about 350 ° C, in some cases less than 300 ° C, in some cases less than about 100 ° C, and in some cases less than around 50 ° C. In certain embodiments, the hydrophobic acid can have a melting point between about 5 ° C and about 25 ° C, in some cases, between about 15 ° C and about 50 ° C, in some cases, between around 30 ° C and around 100 ° C, in some cases between around 75 ° C and around 150 ° C, in some cases between around 125 ° C and around 200 ° C in some cases , between around 150 ° C and around 250 ° C, in some cases, between around 200 ° C and around 300 ° C, and in some cases between around 250 ° C and around 350 ° C. In some cases, the hydrophobic acid may have a melting point of less than about 15 ° C, in some cases, less than about 10 ° C, or in some cases, less than about 0 ° C. In certain embodiments, the hydrophobic acid can have a melting point of between about -30 ° C and about 0 ° C or in some cases, between about -20 ° C and about -10 ° C.

In some embodiments, the hydrophobic acid may have a melting point of less than 350 ° C, in some cases less than 300 ° C, in some cases less than 100 ° C, and in some cases less than 50 ° C. In certain embodiments, the hydrophobic acid may have a melting point between 5 ° C and 25 ° C, in some cases between 15 ° C and 50 ° C, in some cases between 30 ° C and 100 ° C , in some cases between 75 ° C and 150 ° C, in some cases between 125 ° C and 200 ° C, in some cases between 150 ° C and 250 ° C, in some cases between 200 ° C and 300 ° C, and in some cases, between 250 ° C and 350 ° C. In some cases, the hydrophobic acid may have a melting point of less than 15 ° C, in some cases, less than 10 ° C, or in some cases, less than 0 ° C. In certain embodiments, the hydrophobic acid can have a melting point between -30 ° C and 0 ° C, or in some cases, between -20 ° C and -10 ° C.

For example, the hydrophobic acid for use in the methods and nanoparticles disclosed in this application can be selected, at least in part, on the basis of the solubility of the therapeutic agent in a solvent comprising the acid. For example, in some embodiments, depending on the solvent, the therapeutic agent dissolved in a solvent comprising the acid may have a solubility of between about 15 mg / ml and about 200 mg / ml, between about 20 mg / ml and around 200mg / ml, between around 25mg / ml and around 200mg / ml, between around 50mg / ml and around 200mg / ml, between around 75mg / ml and around 200 mg / ml, between around 100 mg / ml and around 200 mg / ml, between about 125mg / ml and about 175mg / ml, between about 15mg / ml and about 50mg / ml, between about 25mg / ml and about 75mg / ml. In some embodiments, the therapeutic agent dissolved in a solvent containing the hydrophobic acid may have a solubility of greater than about 10 mg / ml, greater than about 50 mg / ml, or greater than about 100 mg / ml. In some embodiments, the therapeutic agent dissolved in a solvent containing the hydrophobic acid (eg, a first solution consisting of the therapeutic agent, solvent, and hydrophobic acid) can have a solubility of at least about 2 times greater. , in some embodiments, at least about 5 times greater, in some embodiments, at least about 10 times greater, in some embodiments, at least about 20 times greater, in some embodiments, about 2 times to about 20 times greater, or in some embodiments, about 10 times to about 20 times greater, than when the therapeutic agent is dissolved in a solvent that does not contain the hydrophobic acid (for example, a second solution consisting of the therapeutic agent and the solvent).

In some embodiments, depending on the solvent, the therapeutic agent dissolved in a solvent comprising the acid can have a solubility of between 15 mg / ml and 200 mg / ml, between 20 mg / ml and 200 mg / ml, between 25 mg / ml and 200 mg / ml, between 50 mg / ml and 200 mg / ml, between 75 mg / ml and 200 mg / ml, between 100 mg / ml and 200 mg / ml, between 125 mg / ml and 175 mg / ml, between 15 mg / ml and 50 mg / ml, between 25 mg / ml and 75 mg / ml. In some embodiments, the therapeutic agent dissolved in a solvent containing the hydrophobic acid may have a solubility of greater than 10 mg / ml, greater than 50 mg / ml, or greater than 100 mg / ml. In some embodiments, the therapeutic agent dissolved in a solvent containing the hydrophobic acid (for example, a first solution consisting of the therapeutic agent, solvent, and hydrophobic acid) may have at least 2-fold solubility, in some embodiments, at least 5 times greater, in some embodiments, at least 10 times greater, in some embodiments, at least 20 times greater, in some embodiments, 2 times to 20 times greater, or in some embodiments, 10 times to 20 times greater, than when the therapeutic agent is dissolved in a solvent that does not contain the hydrophobic acid (for example, a second solution consisting of the therapeutic agent and the solvent).

In some cases, the concentration of hydrophobic acid in a drug solution (i.e., the therapeutic agent solution) can range from about 1 weight percent to about 30 weight percent, in some embodiments, from about 2 percent by weight to about 30 percent by weight, in some embodiments, from about 3 percent by weight to about 30 percent by weight, in some embodiments, from about 4 percent by weight to about 30 percent by weight, in some embodiments, from about 5 percent by weight to about 30 percent by weight, in some embodiments, from about 6 weight percent to about 30 weight percent, in some embodiments, from about 8 weight percent to about 30 weight percent, in some embodiments, from about 10 weight percent to about 30 weight percent, in some embodiments, from about 12 weight percent to about 30 weight percent, in some embodiments, about 14 weight percent to about 30 weight percent, in some embodiments, from about 16 weight percent to about 30 weight percent, in some embodiments, from about 1 percent by weight to about 5 percent by weight, in some embodiments, from about 3 percent by weight to about 9 percent by weight, in some embodiments, from about 6 weight percent to about 12 weight percent, in some embodiments, from about 9 weight percent to about 15 weight percent, in some embodiments, from about 12 percent by weight to about 18 percent by weight, and in some embodiments, from about 15 percent by weight to about 21 percent by weight. In certain embodiments, the concentration of hydrophobic acid in a drug solution may be about 1 percent by weight or greater, in some embodiments, about 2 percent by weight or greater, in some embodiments, about 3 weight percent or greater, in some embodiments, about 5 weight percent or greater, in some embodiments, about 10 weight percent or greater, in some embodiments, about 15 percent by weight or greater, and in some embodiments, about 20 percent by weight or greater.

In some cases, the concentration of hydrophobic acid in a drug solution (i.e., the therapeutic agent solution) can range from 1 percent by weight to 30 percent by weight, in some embodiments, from 2 percent by weight. weight to 30 percent by weight, in some embodiments, from 3 percent by weight to 30 percent by weight, in some embodiments, from 4 percent by weight to 30 percent by weight, in some forms realization, 5 percent by weight to 30 percent by weight, in some embodiments, 6 percent by weight to 30 percent by weight, in some embodiments, 8 percent by weight to 30 percent by weight , in some embodiments, from 10 weight percent to 30 weight percent, in some embodiments, from 12 weight percent to 30 weight percent, in some embodiments, from 14 weight percent. weight to 30 weight percent, in some embodiments, from 16 percent by weight to 30 percent by weight, in some embodiments, from 1 percent by weight to 5 percent by weight, in some embodiments, from 3 percent by weight to 9 percent by weight , in some embodiments, from 6 percent by weight to 12 percent by weight, in some embodiments, from 9 percent by weight to 15 percent by weight, in some embodiments, from 12 percent by weight. weight to 18 weight percent, and in some embodiments, from 15 percent by weight to 21 percent by weight. In certain embodiments, the concentration of hydrophobic acid in a drug solution may be 1 percent by weight or greater, in some embodiments 2 percent by weight or greater, in some embodiments 3 percent. by weight or greater, in some embodiments, 5 percent by weight or greater, in some embodiments, 10 percent by weight or greater, in some embodiments, 15 percent by weight or greater, and in some embodiments, twenty percent by weight or greater.

In certain embodiments, the molar ratio of hydrophobic acid to therapeutic agent (for example, initially during formulation of the nanoparticles and / or on the nanoparticles) may range from about 0.25: 1 to about 6: 1, in about from 5: 1, to about 4: 1, to about 3: 1, to about 2: 1, in some embodiments, some embodiments, some embodiments, some embodiments, some forms of realization, from around 0.25: 1 to from around 0.25: 1 to from around 0.25: 1 to from around 0.25: 1 to from around 0.25: 1 to around 1.5: 1, in some embodiments, from around 0.25: 1 to about 1: 1, in some embodiments, from about 0.25: 1 to about 0.5: 1, in some embodiments, from about 0.5: 1 to about about about

6: 1, in some ways

5: 1, in some ways

4: 1, in some embodiments, of embodiment, of embodiment, from about 0.5: 1 to about 0.5: 1 to about 0.5: 1 to about 3: 1, in some embodiments, from about 0.5: 1 to about 2: 1, in some embodiments, from about 0.5: 1 to about 1.5: 1, in some embodiments, from about 0.5: 1 to about 1: 1, in some embodiments, from around 0.5: 1 to around 0.75: 1, in some embodiments, from about 0.75: 1 to about 2: 1, in some embodiments, from about 0.75: 1 to about 1.5: 1, in some embodiments, from about 0.75: 1 to about 1.25: 1, in some embodiments, from about 0.9: 1 to about 1.1: 1, in some embodiments, from about 0.95: 1 to about 1.05: 1, in some embodiments , around 1: 1, in some embodiments, from about 0.75: 1 to about 1: 1, in some embodiments, from about 1: 1 to about 6: 1, in some embodiments, from about 1: 1 to about 5: 1 , in some embodiments, from about 1: 1 to about 4: 1, in some embodiments, from about 1: 1 to about 3: 1, in some embodiments, from about 1: 1 to about 2: 1, in some embodiments, from about 1: 1 to about 1.5: 1, in some embodiments, from about 1.5: 1 to about 6: 1, in some embodiments, from about 1.5: 1 to about 5: 1, in some embodiments, from about 1.5: 1 to about 4: 1, in some embodiments, from about 1.5: 1 to about 3: 1, in some embodiments, from about 2: 1 to about 6: 1, in some embodiments , from about 2: 1 to about 4: 1, in some embodiments, from about 3: 1 to about 6: 1, in some embodiments, from about 3: 1 to about 5: 1, and in some embodiments, from about 4: 1 to about 6: 1.

In certain embodiments, the molar ratio of hydrophobic acid to therapeutic agent (eg, initially during formulation of nanoparticles and / or nanoparticles) can range from 0.25: 1 to 6: 1, in some embodiments, 0.25: 1 to 5: 1, in some embodiments 0.25: 1 to 4: 1, in some embodiments 0.25: 1 to 3: 1, in some embodiments 0.25: 1 to 2: 1, in some embodiments 0.25: 1 to 1.5: 1, in some embodiments, from 0.25: 1 to 1: 1, in some embodiments, from 0.25: 1 to 0.5: 1, in some embodiments, from 0.5: 1 to 6: 1, in some embodiments , 0.5: 1 to 5: 1, in some embodiments, 0.5: 1 to 4: 1, in some embodiments, 0.5: 1 to 3: 1, in some embodiments, 0.5: 1 to 2: 1, in some embodiments, from 0.5: 1 to 1.5: 1, in some embodiments, from 0.5: 1 to 1: 1, in some embodiments, 0.5: 1 to 0.75: 1, in some embodiments 0.75: 1 to 2: 1, in some embodiments 0.75: 1 to 1.5: 1, in some embodiments 0.75: 1 to 1.25: 1, in some embodiments, 0.9: 1 to 1.1: 1, in some embodiments, 0.95: 1 to 1.05: 1, in some embodiments, 1: 1, in some embodiments, 0.75: 1 to 1: 1, in some embodiments, 1: 1 to 6: 1, in some embodiments, 1: 1 to 5: 1, in some embodiments, from 1: 1 to 4: 1, in some embodiments, from 1: 1 to 3: 1, in some embodiments, from 1: 1 to 2: 1, in some embodiments , 1: 1 to 1.5: 1, in some embodiments, 1.5: 1 to 6: 1, in some embodiments, 1.5: 1 to 5: 1, in some embodiments, 1.5: 1 to 4: 1, in some embodiments, from 1.5: 1 to 3: 1, in some embodiments, from 2: 1 to 6: 1, in some embodiments, from 2: 1 to 4: 1, in some embodiments, from 3: 1 to 6: 1, in some embodiments, from 3: 1 to 5: 1, and in some embodiments, from 4: 1 to 6: 1.

In some cases, the initial molar ratio of hydrophobic acid to a therapeutic agent (i.e., during formulation of the nanoparticles) may be different from the molar ratio of hydrophobic acid to a therapeutic agent in the nanoparticles (i.e., after removal of the non-encapsulated hydrophobic acid and therapeutic agent). In other cases, the initial molar ratio of hydrophobic acid to a therapeutic agent (i.e., during formulation of the nanoparticles) may be essentially equal to the molar ratio of hydrophobic acid to a therapeutic agent in the nanoparticles (i.e., after removal of non-encapsulated hydrophobic acid and therapeutic agent).

In one embodiment, when the nanoparticle contains the hydrophobic acid, the nanoparticle comprising the therapeutic agent, 1— (4 - {[4— (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-11-1,3,5-triazin-2-yl) phenyl] urea, can form a salt with the hydrophobic acid. In other words, the hydrophobic acid associated with the therapeutic agent is the pharmaceutically acceptable salt. Therefore, in one embodiment, the present invention relates to a therapeutic nanoparticle comprising therapeutic agent or a pharmaceutically acceptable salt thereof and a polymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer. or a poly (lactic acid-coglycolic acid) -poly (ethylene) glycol diblock copolymer and combinations thereof, where the therapeutic agent is 1— (4 - [[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — II— 1,3,5 —Triazin-2-yl) phen i I] urea.

In some cases, a solution containing the therapeutic agent can be prepared, separately from a solution containing the polymer, and the two solutions can then be combined prior to formulation of the nanoparticle. For example, in one embodiment, a first solution contains the therapeutic agent and the hydrophobic acid, and a second solution contains the polymer and, optionally, the hydrophobic acid. Formulations where the second solution does not contain the hydrophobic acid may be suitable, for example, to minimize the amount of hydrophobic acid used in a process, or in some cases, to minimize the contact time between the hydrophobic acid and, for example, a polymer that can degrade in the presence of the hydrophobic acid. In other cases, a single solution can be prepared containing the therapeutic agent, polymer, and hydrophobic acid.

In some embodiments, the hydrophobic ion pair can be formed prior to formulation of the nanoparticles. For example, a solution containing the hydrophobic ion pair can be prepared prior to formulation of the contemplated nanoparticles (eg, by preparing a solution containing appropriate amounts of the therapeutic agent and hydrophobic acid). In other embodiments, the hydrophobic ion pair can be formed during formulation of the nanoparticles. For example, a first solution containing the therapeutic agent and a second solution containing the hydrophobic acid can be combined during a process step for the preparation of the nanoparticles (for example, before emulsion formation and / or during the formation of emulsion). In certain embodiments, the hydrophobic ion pair can be formed prior to encapsulation of the therapeutic agent and hydrophobic acid in a contemplated nanoparticle. In other embodiments, the hydrophobic ion pair can be formed on the nanoparticle, for example, after encapsulation of the therapeutic agent and hydrophobic acid.

In certain embodiments, the hydrophobic acid may have a solubility of less than about 2 g per 100 ml of water or less, in some embodiments, about 1 g per 100 ml of water or less;

in some embodiments, about 100mg per 100ml of water or less, in some embodiments, about 10mg per 100ml of water or less, and in some embodiments, about 1mg per 100ml water or less, determined at 25 ° C. In other embodiments, the hydrophobic acid can have a solubility ranging from about 1 mg per 100 ml of water to about 2 g per 100 ml of water; in some embodiments, from about 1mg per 100ml of water to about 1g per 100ml of water, in some embodiments, from about 1mg per 100ml of water to about 500mg per 100 ml of water, and in some embodiments, from about 1 mg per 100 ml of water to about 100 mg per 100 ml of water, determined at 25 ° C. In some embodiments, the hydrophobic acid can be essentially insoluble in water at 25 ° C.

In certain embodiments, the hydrophobic acid may have a solubility of less than 2 g per 100 ml of water or less, in some embodiments, 1 g per 100 ml of water or less; in some embodiments, 100 mg per 100 ml of water or less, in some embodiments, 10 mg per 100 ml of water or less, and in some embodiments, 1 mg per 100 ml of water or less, determined at 25 ° C. In other embodiments, the hydrophobic acid can have a solubility ranging from 1 mg per 100 ml of water to 2 g per 100 ml of water; in some embodiments, from 1 mg per 100 ml of water to 1 g per 100 ml of water, in some embodiments, from 1 mg per 100 ml of water to 500 mg per 100 ml of water, and in some forms of realization, from 1 mg per 100 ml of water to 100 mg per 100 ml of water, determined at 25 ° C. In some embodiments, the hydrophobic acid can be essentially insoluble in water at 25 ° C.

In some embodiments, the disclosed nanoparticles may be essentially free of the hydrophobic acid used during the preparation of the nanoparticles. In other embodiments, the disclosed nanoparticles may comprise the hydrophobic acid. For example, in some embodiments, the acid content in the disclosed nanoparticles can range from about 0.05 percent by weight to about 35 percent by weight, in some embodiments, from about 0.05 percent by weight. to about 30 weight percent, in some embodiments, from about 0.05 weight percent to about 20 weight percent, in some embodiments, from about 0.5 percent by weight to about 30 percent by weight, in some embodiments, from about 1 percent by weight to about 30 percent by weight, in some embodiments, from about 2 weight percent to about 30 weight percent, in some embodiments, from about 3 weight percent to about 30 weight percent, in some embodiments, from about 5 percent by weight to about 30 percent by weight, in some embodiments, from about 7 percent by weight to about 30 percent by weight, in some embodiments, from about 10 weight percent to about 30 weight percent, in some embodiments, from about 15 weight percent to about 25 weight percent, in some embodiments, from about 15 percent by weight to about 30 percent by weight, in some embodiments, from about 20 percent by weight to about 30 percent by weight, in some embodiments, from about 0.05 weight percent to about 0.5 weight percent, in some embodiments, from about 0.05 weight percent to about 5 weight percent, in some embodiments, from about 1 percent by weight to about 5 percent by weight, in some embodiments, from about 3 percent by weight to about 10 percent by weight, in some embodiments, from about 1 weight percent to about 10 weight percent, in some embodiments, from about 5 weight percent to about 10 weight percent, in some embodiments, from about 5 weight percent to about 15 weight percent, and in some embodiments, from about 10 weight percent to about 20 weight percent.

In some embodiments, the acid content in the disclosed nanoparticles may range from 0.05 weight percent to 35 weight percent, in some embodiments, from 0.05 weight percent to 30 weight percent, in some embodiments, from 0.05 weight percent to 20 weight percent, in some embodiments, 0.5 weight percent to 30 weight percent, in some embodiments, 1 weight percent to 30 percent by weight, in some embodiments, from 2 percent by weight to 30 percent by weight, in some embodiments, from 3 percent by weight to 30 percent by weight, in some embodiments, from 5 percent by weight to 30 percent by weight, in some embodiments, from 7 percent by weight to 30 percent by weight, in some embodiments, from 10 percent by weight to 30 percent by weight, in some embodiments , from 15 percent by weight to 25 percent by weight, in some embodiments, from 15 weight percent to 30 weight percent, in some embodiments, from 20 weight percent to 30 weight percent, in some embodiments, from 0.05 weight percent to 0.5 percent by weight, in some embodiments, from 0.05 percent by weight to 5 percent by weight, in some embodiments, from 1 percent by weight to 5 percent by weight, in some embodiments , from 3 percent by weight to 10 percent by weight, in some embodiments, from about 1 percent by weight to about 10 percent by weight, in some embodiments, from about 5 percent by weight to about 10 percent by weight, in some embodiments, from 5 percent by weight to 15 percent by weight, and in some embodiments, from 10 percent by weight to 20 percent by weight.

In some embodiments, the disclosed nanoparticles release substantially immediately (eg, about 1 minute to about 30 minutes, about 1 minute to about 25 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 1 hour, about 1 hour, or about 24 hours). In other cases, the release profile is slower: about 2% or less is released; about 5% or less; about 10% or less; about 15% or less; about 20% or less; about 25% or, about 30% or less, about 40% or less of the therapeutic agent, by weight, for example, when placed in a phosphate buffer solution, for example, a buffer comprising phosphate buffer monobasic and dibasic (such as sodium chloride buffer, 0.138 M, potassium chloride, 0.0027 M, monobasic sodium or potassium phosphate, about 0.02 M, and dibasic sodium or potassium phosphate, about 0.01 M, dissolved in 1 liter of water, for example, RODI water), at room temperature (for example, 25 ° C) and / or at 37 ° C. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above), e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to about

0.01 to about 50%, in some embodiments, about 0.01 to about 25%, in some embodiments, about 0.01 to about 15%, in some embodiments, about 0.01 to about 10 %, in some embodiments, about 1 to about 40%, in some embodiments, about 5 to about 40%, and in some embodiments, about 10 to about 40% of the therapeutic agent released, by weight, for about 1 hour. In some embodiments, the nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (eg, a phosphate buffer solution), for example, at 25 ° C and / or at 37 ° C. , at a rate substantially corresponding to about 10 to about 70%, in some embodiments, about 10 to about 45%, in some embodiments, about 10 to about 35%, or in some embodiments, about 10 to about 25%, therapeutic agent released by weight over about 4 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above), e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to about 0.01 to about 50%, in some embodiments, about 0.01 to about 25%, in some embodiments, about 0.01 to about 15%, in some embodiments, about 0.01 to about 10%, in some embodiments, about 0.01 to about 5%, and in some embodiments, about 0.01 to about 3%. % therapeutic agent released by weight over about 4 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to about 0.01 to about 60%, in some embodiments, about 0.01 to about 25%, in some embodiments, about 0.01 to about 15%, in some embodiments, about 0.01 to about 10%, in some embodiments, about 0.01 to about 5%, and in some embodiments, about 0.01 to about 3% of the therapeutic agent released by weight over about 10 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to about 0.01 to about 70%, in some embodiments, about 0.01 to about 50%, in some embodiments, about 0.01 to about 25%, in some embodiments, about 0.01 to about 15%, in some embodiments, about 0.01 to about 10%, in some embodiments, about 0.01 to about of 5%, and in some embodiments, about 0.01 to about 3% of the therapeutic agent released by weight over about 20 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to about 1 to about 80%, in some embodiments, about 1 to about 50%, in some embodiments, about 1 to about 30%, in some embodiments, about 1 to about 25%, in some embodiments, about 1 to about 15%, in some embodiments, about 1 to about about 10%, and in some embodiments, about 1 to about 5% of the therapeutic agent released by weight over about 40 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to about 10 to about 100%, in some embodiments, about 10 to about 80%, in some embodiments, about 10 to about 70%, in some embodiments, about 10 to about 60%, in some embodiments, about 10 to about 50%, in some embodiments, about 10 to about 40% , in some embodiments, about 10 to about 30%, in some embodiments, about 10 to about 20% of the therapeutic agent released by weight over about 100 hours.

In some embodiments, the disclosed nanoparticles release substantially immediately (eg, 1 minute to 30 minutes, 1 minute to 25 minutes, 5 minutes to 30 minutes, 5 minutes to 1 hour, 1 hour, or 24 hours) . In other cases, the release profile is slower: 2% or less is released; 5% or less; 10% or less; 15% or less; 20% or less; 25%, 30% or less 40% or less of the therapeutic agent, by weight, for example, when placed in a phosphate buffer solution, e.g., a buffer comprising monobasic and dibasic phosphate buffer (such as phosphate buffer). sodium chloride, 0.138 M, potassium chloride, 0.0027 M, monobasic sodium or potassium phosphate, about 0.02 M, and dibasic sodium or potassium phosphate, about 0.01 M, dissolved in 1 liter of water, for example, water RODI), at room temperature (eg 25 ° C) and / or at 37 ° C. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to 0.01 to 50%, in some embodiments, 0.01 to 25%, in some embodiments, 0.01 to 15%, in some embodiments, 0.01 to 10%, in some embodiments, 1 to 40%, in some embodiments, 5 to 40%, and in some embodiments, 10 to 40% of the therapeutic agent released by weight over 1 hour. In some embodiments, the nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (eg, a phosphate buffer solution), eg, at 25 ° C and / or at 37 ° C. , at a rate substantially corresponding to 10 to 70%, in some embodiments 10 to 45%, in some embodiments 10 to 35%, or in some embodiments 10 to 25%, of therapeutic agent, released by weight over 4 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to 0.01 to 50%, in some embodiments, 0.01 to 25%, in some embodiments, 0.01 to 15%, in some embodiments, 0.01 to 10%, in some embodiments 0.01 to 5%, and in some embodiments 0.01 to 3% of the therapeutic agent released by weight over 4 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to 0.01 to 60%, in some embodiments, 0.01 to 25%, in some embodiments, 0.01 to 15%, in some embodiments, 0.01 to 10%, in some embodiments 0.01 to 5%, and in some embodiments 0.01 to 3% of the therapeutic agent released by weight over 10 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to 0.01 to 70%, in some embodiments, 0.01 to 50%, in some embodiments, 0.01 to 25%, in some embodiments, 0.01 to 15%, in some embodiments 0.01 to 10%, in some embodiments 0.01 to 5%, and in some embodiments 0.01 to 3% of the therapeutic agent released by weight over 20 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to 1 to 80%, in some embodiments, 1 to 50%, in some embodiments, 1 to 30%, in some embodiments, 1 to 25 %, in some embodiments, 1 to 15%, in some embodiments, 1 to 10%, and in some embodiments, 1 to 5% of the therapeutic agent released by weight over 40 hours. In certain embodiments, nanoparticles comprising the therapeutic agent can release the therapeutic agent when placed in an aqueous solution (e.g., a phosphate buffer solution, as described in the present application above) e.g., at 25 ° C and / or 37 ° C, at a rate substantially corresponding to 10 to 100%, in some embodiments, 10 to 80%, in some embodiments, 10 to 70%, in some embodiments, 10 to 60%, in some embodiments 10 to 50%, in some embodiments 10 to 40%, in some embodiments 10 to 30%, in some embodiments 10 to 20% of the therapeutic agent released by weight during 100 hours.

In some embodiments, the disclosed nanoparticles can substantially retain the therapeutic agent, for example, for at least about 1 minute, at least about 1 hour or more, when placed in a phosphate buffer solution at 37 ° C.

In some embodiments, the disclosed nanoparticles can substantially retain the therapeutic agent, eg, for at least 1 minute, at least 1 hour or more, when placed in a 37 ° C phosphate buffer solution.

In one embodiment, the disclosed therapeutic nanoparticles may include a targeting ligand, eg, a low molecular weight ligand. In certain embodiments, the low molecular weight ligand is conjugated to a polymer, and the nanoparticle comprises a certain ratio of ligand-conjugated polymer (eg, PLA-PEG Ligand) to non-functionalized polymer (eg, PLA-PEG or PLGA-PEG).

The nanoparticle can have an effective ratio of these two polymers, such that an effective amount of ligand is associated with the nanoparticle for the treatment of a disease or disorder, such as cancer. For example, higher ligand density can increase binding to the target (cell binding / target uptake), thus making the nanoparticle "target specific". Alternatively, a certain concentration of non-functionalized polymer (eg, non-functionalized PLGA-PEG copolymer) in the nanoparticle can control inflammation and / or immunogenicity (i.e., the ability to elicit an immune response), and allow the nanoparticle have adequate half-life for the treatment of a disease or disorder. For example, in one embodiment, the molar ratio of non-functionalized polymer to ligand-conjugated polymer ranges from about 0.01 to about 0.1, and in another embodiment, from about 0.01 to about 0.05, such as, for example, around 0.025. Additionally, the non-functionalized polymer, in some embodiments, can decrease the clearance rate from the circulatory system via the reticuloendothelial system (RES). Therefore, the non-functionalized polymer can provide the nanoparticle with characteristics that can allow travel of the particle through the body upon administration. In some embodiments, a non-functionalized polymer can balance an otherwise high concentration of ligands, which may otherwise accelerate clearance from the subject, thereby producing less delivery to target cells.

In another embodiment, the molar ratio of non-functionalized polymer to ligand-conjugated polymer ranges from 0.01 to 0.1, and in another embodiment, from 0.01 to 0.05, such as, for example, 0.025.

In some embodiments, the nanoparticles disclosed in this application may include ligand-conjugated functionalized polymers constituting a range of about 0.1 to about 50, eg, about 0.1 to about 30, eg, about 0.1 to about 20, for example, about 0.1 to about 10 mole percent of the entire polymer composition of the nanoparticle (i.e. functionalized polymer + non-functionalized polymer). Further disclosed in this application are, in another embodiment, nanoparticles that include a polymer conjugated (for example, covalently (i.e. through a linker (eg, an alkylene linker)) or a bond) with one or plus low molecular weight ligands, where the weight percentage of low molecular weight ligand relative to total polymer ranges from about 0.001 to about 5, for example, from about 0.001 to about 2, for example, from about 0.001 to about 1.

In some embodiments, the nanoparticles disclosed in this application may include ligand-conjugated functionalized polymers constituting a range of 0.1 - 50, eg 0.1 - 30, eg 0.1 20, eg 0.1 - 10 percent. in moles of the entire polymer composition of the nanoparticle (ie, functionalized polymer + non-functionalized polymer). In addition, nanoparticles that include a polymer conjugated with one or more low molecular weight ligands are disclosed in this application, where the weight percentage of low molecular weight ligand with respect to the total polymer ranges from 0.001 to 5, for example, from 0.001 to 2, for example 0.001 to 1.

In general, a "nanoparticle" refers to any particle that has a diameter of less than 1000 nm, for example, about 10 nm to about 200 nm. The disclosed therapeutic nanoparticles may include nanoparticles having a diameter ranging from about 60 to about 120 nm, or from about 70 to about 120 nm, or from about 80 to about 120 nm, or from about 90 at around 120 nm, or around 100 to around 120 nm, or around 60 to around 130 nm, or around 70 to around 130 nm, or around 80 to around 130 nm, or around 90 to around 130 nm, or about 100 to about 130 nm, or about 110 to about 130 nm, or about 60 to about 140 nm, or about 70 to about 140 nm, or about 80 to around 140 nm, or around 90 to around 140 nm, or around 100 to around 140 nm, or around 110 to around 140 nm, or around 60 to around 150 nm, or around 70 to around 150 nm, or around 80 to around 150 nm, or about 90 to about 150 nm, or about 100 to about 150 nm, or about 110 to about 150 nm, or about 120 to about 150 nm.

The disclosed therapeutic nanoparticles can include nanoparticles having a diameter ranging from 60 to 120 nm, or from 70 to 120 nm, or from 80 to 120 nm, or from 90 to 120 nm, or from 100 to 120 nm, or from 60 at 130 nm, or from 70 to 130 nm, or from 80 to 130 nm, or from 90 to 130 nm, or from 100 to 130 nm, or from 110 to 130 nm, or from 60 to 140 nm, or from 70 to 140 nm, or 80 to 140 nm, or 90 to 140 nm, or 100 to 140 nm, or 110 to 140 nm, or 60 to 150 nm, or 70 to 150 nm, or 80 to 150 nm, or from 90 to 150 nm, or 100 to 150 nm, or 110 to 150 nm, or 120 to 150 nm.

Polymers

In some embodiments, the nanoparticles can comprise a mixture of polymers and the therapeutic agent. In some embodiments, the therapeutic agent and / or targeting moiety (ie, a low molecular weight ligand) may be associated with at least part of the polymeric matrix. For example, in some embodiments, a targeting moiety (eg, ligand) can be covalently associated with the surface of a polymeric matrix. In some embodiments, the covalent association is mediated by a linker. The therapeutic agent can be associated with the surface of the polymeric matrix, or encapsulated within the polymeric matrix, or surrounded by the polymeric matrix, and / or dispersed throughout the polymeric matrix.

A wide variety of polymers and methods for the formation of particles from them are known in the art of drug delivery. In some embodiments, the disclosure is directed to nanoparticles with at least two macromolecules, where the first macromolecule comprises a first polymer linked to a low molecular weight ligand (for example , targeting moiety); and the second macromolecule comprises a second polymer that is not linked to a targeting moiety. The nanoparticle may optionally include one or more additional, non-functionalized polymers.

Any suitable polymer can be used in the disclosed nanoparticles. Polymers can be natural or unnatural (synthetic). The polymers can be homopolymers or copolymers comprising two or more monomers. In terms of sequence, the copolymers can be random, blocky, or comprise a combination of random and block sequences. The polymers are usually organic polymers.

The term "polymer", as used in the present application, is given its common meaning as used in the art, that is, a molecular structure comprising one or more repeating units (monomers), connected by covalent bonds. The repeating units may all be identical, or in some cases, there may be more than one type of repeating unit present within the polymer. In some cases, the polymer can be biologically derived, that is, a biopolymer. Non-limiting examples include peptides or proteins. In some cases, additional moieties may also be present on the polymer, for example biological moieties such as those described below. If more than one type of repeating unit occurs within the polymer, then the polymer is said to be a "copolymer". It is understood that in any embodiment employing a polymer, the polymer employed may be a copolymer, in some cases. The repeating units that make up the copolymer can be arranged in any way. For example, the repeating units can be arranged in a random order, in an alternating order, or as a block copolymer, that is, comprising one or more regions where each comprises a first repeating unit (for example, a first block), and one or more regions each comprising a second repeating unit (eg, a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of different blocks.

The disclosed particles may include copolymers, which, in some embodiments, describe two or more polymers (such as those described in this application) that have been associated with each other, usually by covalently bonding the two or more polymers to each other. yes. Therefore, a copolymer can comprise a first polymer and a second polymer, which have been conjugated to each other to form a block copolymer where the first polymer can be a first block of the block copolymer, and the second polymer can be a second. block of the block copolymer.

Naturally, those skilled in the art will understand that a block copolymer, in some cases, may contain multiple polymer blocks, and that a block copolymer, as used in the present application, is not limited to only one block copolymers having a single first block and a single second block. For example, a block copolymer can comprise a first block comprising a first polymer, a second block comprising a second polymer, and a third block comprising a third polymer or the first polymer, etc. In some cases, the block copolymers can contain any number of first blocks of a first polymer and second blocks of a second polymer (and in certain cases, third blocks, fourth blocks, etc.). Furthermore, it should be noted that block copolymers can also, in some cases, be formed from other block copolymers. For example, a first block copolymer can be conjugated with another polymer (which can be a homopolymer, a biopolymer, another block copolymer, etc.), to form a new block copolymer containing multiple types of blocks, and / or with other moieties (eg with non-polymeric moieties).

In some embodiments, the polymer (eg, copolymer, eg, block copolymer) may be amphiphilic, that is, it has a hydrophilic portion and a hydrophobic portion, or a relatively hydrophilic portion and a relatively hydrophobic portion. A hydrophilic polymer can be one that generally attracts water, and a hydrophobic polymer can be one that generally repels water. A hydrophilic or hydrophobic polymer can be identified, for example, by preparing a sample of the polymer and measuring its contact angle with water (typically, the polymer will have a contact angle of less than 60 °, whereas a hydrophobic polymer will have a contact angle greater than about 60 °). In some cases, the hydrophilicity of two or more polymers can be measured relative to each other, that is, a first polymer can be more hydrophilic than a second polymer. For example, the first polymer may have a lower contact angle than the second polymer.

In one group of embodiments, a polymer (eg, copolymer, eg, block copolymer) contemplated in this application includes a biocompatible polymer, that is, the polymer that does not ordinarily induce an adverse response when inserted or injected. in a living subject, for example, without significant inflammation and / or acute rejection of the polymer by the immune system, for example, by means of a T cell response. Consequently, the therapeutic particles contemplated herein may be non-immunogenic. The term non-immunogenic, as used in the present application, refers to endogenous growth factor in its native state, which, normally, does not produce circulating antibodies, T cells, or reactive immune cells, or produces only minimal levels of them, and that it does not normally produce an immune response against itself in the individual.

Biocompatibility usually refers to the acute rejection of material by at least a portion of the immune system; that is, a non-biocompatible material implanted in a subject elicits an immune response in the subject, which may be severe enough that rejection of the material by the immune system cannot be adequately controlled, and is often of a degree of such that the material must be removed from the subject. A simple test to determine biocompatibility can consist of exposing a polymer to cells in vitro; biocompatible polymers are polymers that will not typically cause significant cell death at moderate concentrations, for example, at concentrations of 50 micrograms / 10⁶ cells. For example, a biocompatible polymer can produce less than about 20% cell death when exposed to cells such as fibroblasts or epithelial cells, even when it is phagocytosed or otherwise taken up by such cells. Non-limiting examples of biocompatible polymers that may be useful in various embodiments include polydioxanone (PDO), polyhydroxyalkanoate, polyhydroxybutyrate, poly (glycerol sebacate), polyglycolide (i.e., poly (glycolic acid)) (PGA), polylactide (i.e. , poly (lactic acid)) (PLA), poly (lactic acid) -co-poly (glycolic acid) (PLGA), polycaprolactone, or copolymers or derivatives that include these and / or other polymers.

In certain embodiments, the contemplated biocompatible polymers may be biodegradable, that is, the polymer is capable of degrading, chemically and / or biologically, within a physiological environment, such as within the body. As used in the present application, biodegradable polymers are those that, when introduced into cells, are broken down by cellular machinery (biologically degradable) and / or by a chemical process, such as hydrolysis (chemically degradable) into components that the cells can either be reused or disposed of without significant toxic effect on the cells. In one embodiment, the biodegradable polymer and its degradation by-products can be biocompatible.

The particles disclosed in this application may or may not contain PEG. Furthermore, certain embodiments may be targeted to a poly (ester-ether) containing copolymers is, for example, polymers having repeating units linked by ester bonds (for example, R bonds¹⁰⁰-C (O) -OR¹) and ether bonds (for example, R bonds¹-OR¹ where R¹⁰⁰ and R¹ are independently hydrocarbyl moieties which may be optionally substituted and which may be the same or different). In some embodiments, a biodegradable polymer, such as a hydrolyzable polymer, containing carboxylic acid groups, can be conjugated to poly (ethylene glycol) repeating units to form a poly (ester-ether). A polymer (eg, copolymer, eg, block copolymer) containing poly (ethylene glycol) repeating units may further be referred to as a PEGylated polymer.

For example, a contemplated polymer may be one that hydrolyzes spontaneously on exposure to water (eg, within a subject), or the polymer may degrade on exposure to heat (eg, at temperatures around 37 ° C). . The degradation of a polymer can occur at various rates, depending on the polymer or copolymer used. For example, the half-life of the polymer (the time at which 50% of the polymer can degrade into monomers and / or other non-polymeric moieties) can be on the order of days, weeks, months, or years, depending on the polymer. Polymers can be biologically degraded, eg, by enzymatic activity or cellular machinery, in some cases, eg, through exposure to a lysozyme (eg, having a relatively low pH). In some cases, polymers can break down into monomers and / or other non-polymeric moieties that cells can either reuse or dispose of without significant toxic effect on cells (for example, polylactide can be hydrolyzed to form lactic acid; polyglycolide can be hydrolyzed to form glycolic acid, etc.).

In some embodiments, the polymers can be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly (lactic acid-co-glycolic acid) and poly (lactide-co-glycolide), collectively referred to in this application as PLGA; and homopolymers comprising glycolic acid units, referred to herein as PGA, and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D, L-lactic acid, poly-L-lactide , poly-D-lactide and poly-D, L-lactide, collectively referred to herein as PI_A. In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEGylated polymers and copolymers of lactide and glycolide (eg, PEGylated PI_A, PEGylated PGA, PEGylated PLGA, and their derivatives). In some embodiments, polyesters include, for example, polyanhydrides, PEGylated poly (ortho ester), poly (ortho ester), poly (caprolactone), PEGylated poly (caprolactone), polylysine, PEGylated polylysine, poly (ethyleneimine), poly ( PEGylated ethyleneimine), poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L-proline ester), poly [a- (4-amino-butyl) - L-glycolic acid], and its derivatives.

In some embodiments, a polymer can be PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and the various forms of PLGA can be characterized by the ratio of lactic acid: glycolic acid. The lactic acid can be L-lactic acid, Lactic acid, or D, L-lactic acid. The degradation rate of PLGA can be adjusted by alternating the lactic acid-glycolic acid ratio. In some embodiments, PLGA can be characterized by a lactic acid: glycolic acid molar ratio of about 85:15, about 75:25, about 60:40, about 50:50, about 40:60, about 25:75, or about 15:85. In some embodiments, the molar ratio of lactic acid monomers to glycolic acid in the particle polymer (eg, pLGA block copolymer or PLGA-PEG block copolymer), can be selected to optimize the various parameters such as water absorption, therapeutic agent release, and / or polymer degradation kinetics.

In some embodiments, the polymers can be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, copolymers of acrylic acid and methacrylic acid, copolymers of methyl methacrylate, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, acrylic polyphacid), methacrylic polyphacid), acid copolymer methacrylic alkylamide, polymethyl methacrylate), methacrylic polyacrylamide polyacrylamide, amino alkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates and blends comprising one or more of the above polymers. The acrylic polymer may comprise fully polymerized copolymers of acrylic and methacrylic acid esters, with a low content of quaternary ammonium groups.

In some embodiments, the polymers can be cationic polymers. In general, cationic polymers are capable of condensing and / or protecting negatively charged strands of nucleic acids (eg, DNA, RNA, or derivatives thereof). Amine-containing polymers such as poly (lysine), polyethyleneimine (PEI), and poly (amidoamine) dendrimers are contemplated for use, in some embodiments, in a disclosed particle.

In some embodiments, the polymers can be degradable polyesters bearing cationic side chains. Examples of these polyesters include poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L-proline ester).

It is contemplated that PEG may be terminated, and include an end group, for example, when PEG is not conjugated to a ligand. For example, PEG can terminate in a hydroxyl, a methoxy or other alkoxy group, a methyl or other alkyl group, an aryl group, a carboxylic acid, an amine, an amide, an acetyl group, a guanidino group, or an imidazole. Other contemplated end groups include azide, alkyl, maleimide, aldehyde, hydrazide, hydroxylamine, alkoxyamine or otiol moieties.

Methods and techniques for the PEGylation of a polymer will be known to those skilled in the art, for example, using EDC (I-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to react a polymer. with an amine-terminated PEG group, by ring opening polymerization (ROMP) techniques, or the like.

In one embodiment, the molecular weight (or, for example, the molecular weight ratio of, for example, different blocks of a copolymer) of the polymers can be optimized for effective processing as disclosed in this application. For example, the molecular weight of a polymer can affect the rate of particle degradation (such as when the molecular weight of a biodegradable polymer can be adjusted), solubility, water absorption, and drug release kinetics. For example, the molecular weight of the polymer (or, for example, the ratio of molecular weights of, for example, different blocks of a copolymer) can be adjusted such that the particle biodegrades in the treated subject within a reasonable period of time. (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.).

A disclosed particle may comprise, for example, a diblock copolymer of PEG and PL (G) A, where, for example, the pEG portion may have a number average molecular weight of about 1000-20,000, for example, about 2000-20,000, for example about 2 to about 10,000, and the pL (G) A portion may have a number average molecular weight of from about 5000 to about 20,000, or about 5000

1,000,000, for example, about 20,000-70,000, for example, about 15,000-50,000.

For example, an exemplary therapeutic nanoparticle is disclosed herein that includes from about 10 to about 99 percent by weight of poly (lactic acid) -poly (ethylene) glycol copolymer or poly (lactic acid) -co-acid copolymer. poly (glycol) - poly (ethylene) glycol, or about 50 to about 99.75 weight percent, about 20 to about 80 weight percent, about 40 to about 80 weight percent , or from about 30 to about 50 percent by weight, or from about 70 to about 90 percent by weight, from about 70 to about 99.75 percent by weight, from about 80 to about 99.75 percent weight percent, about 70 to about 80 weight percent, or about 85 to about 95 weight percent, poly (lactic acid) -poly (ethylene) glycol copolymer or poly (acid) copolymer. (lactic) -co-poly (glycolic acid) -poly (ethylene) glycol. In some embodiments, a therapeutic nanoparticle comprises about 50 percent by weight, about 55 percent by weight, about 60 percent by weight, about 65 percent by weight, about 70 percent by weight, about 75 percent by weight, about 80 percent by weight, about 85 percent by weight, about 90 weight percent or about 95 weight percent poly (lactic acid) -poly (ethylene) glycol copolymer or poly (lactic) -co-poly (glycolic acid) -poly (ethylene) copolymer glycol. Exemplary poly (lactic acid) -poly (ethylene) glycol copolymers may include a number average molecular weight ranging from about 15 to about 20 kDa, or from about 10 to about 25 kDa of poly (lactic acid). ), and a number average molecular weight of from about 4 kDa to about 6 kDa, from about 4 kDa to about 10 kDa, from about 6 kDa to about 10 kDa, or from about 2 kDa to about 10 kDa of poly (ethylene) glycol.

In another example, an exemplary therapeutic nanoparticle is disclosed herein that includes 10 to 99 percent by weight of poly (lactic acid) -poly (ethylene) glycol copolymer or poly (lactic) -co-poly (glycolic acid) copolymer. ) - poly (ethylene) glycol, or 50 to 99.75 weight percent, 20 to 80 weight percent, 40 to 80 weight percent, or 30 to 50 weight percent, or 70 to 90 weight percent, 70 to 99.75 weight percent, 80 to 99.75 weight percent, 70 to 80 weight percent, or 85 to 95 weight percent, poly (lactic) acid-poly (ethylene) glycol copolymer or poly (lactic) -co-acid poly ( glycolic) - poly (ethylene) glycol. In some embodiments, a therapeutic nanoparticle comprises 50 percent by weight, 55 percent by weight, 60 percent by weight, 65 percent by weight, 70 percent by weight, 75 percent by weight, 80 percent by weight. weight, 85 weight percent, 90 weight percent, or 95 weight percent of poly (lactic) acid -poly (ethylene) glycol copolymer or poly (lactic) -co-poly (glycolic acid) copolymer —Poly (ethylene) glycol. Exemplary poly (lactic acid) -poly (ethylene) glycol copolymers can include a number average molecular weight ranging from 15 to 20 kDa, or from 10 to 25 kDa of poly (lactic acid) and a number average molecular weight 4 kDa to 6 kDa, 4 kDa to 10 kDa, 6 kDa to 10 kDa, or 2 kDa to 10 kDa of poly (ethylene) glycol.

In some embodiments, the poly (lactic acid) -poly (ethylene) glycol copolymer may have a poly (lactic acid) number average molecular weight fraction of from about 0.6 to about 0.95, in some embodiments , from about 0.7 to about 0.9, in some embodiments, from about 0.6 to about 0.8, in some embodiments, from about 0.7 to about 0.8, in some embodiments, from about 0.75 to about 0.85, in some embodiments, from about 0.8 to about 0.9, and in some embodiments, from about 0.85 to about 0.95. It should be understood that the number average molecular weight fraction of poly (lactic acid) can be calculated by dividing the number average molecular weight of the poly (lactic acid) component of the copolymer by the sum of the number average molecular weight of the poly (lactic acid) component. (lactic) and the number average molecular weight of the poly (ethylene) glycol component.

In some embodiments, the poly (lactic acid) poly (ethylene) glycol copolymer may have a poly (lactic acid) number average molecular weight fraction of 0.6 to 0.95, in some embodiments, 0.7 to 0.9 , in some embodiments 0.6 to 0.8, in some embodiments 0.7 to 0.8, in some embodiments 0.75 to 0.85, in some embodiments 0.8 to 0.9, and in some embodiments realization, from 0.85 to 0.95.

In certain embodiments, the therapeutic nanoparticle comprises 1- (4 - {[4— (dimethylamino) piperidine-1 -yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl— 1 , 3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 7. In certain embodiments, the therapeutic nanoparticle comprises 1- (4 - {[4- (dimethylamino) piperidin-1-yl] carboniIJphenyl) -3— [4— (4,6-dimorpholin-4-yl-1,3 , 5-triazin-2-yl) phenyl] urea and PLAPEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 4. In certain embodiments, the therapeutic nanoparticle comprises 1- (4 - {[4— (dimethylamine) piperidin-1 -yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl— 1,3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1:14 (therapeutic agent: PLA-PEG). In certain embodiments, the therapeutic nanoparticle comprises 1- (4 {[4— (dimethylamino) piperidine-1 —¡l] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — ¡I— 1,3,5-triazin-2-11) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 3.

In certain embodiments, the therapeutic nanoparticle comprises 1— (4 - {[4— (dimethylamino) piperidine — 1 —yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — ¡I— 1,3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of 1: 7. In certain embodiments, the therapeutic nanoparticle comprises' l— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4— 1-1,3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of 1: 4. In certain embodiments, the therapeutic nanoparticle comprises 1— (4 - ([4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl-1 , 3,5-triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1:14 (therapeutic agent: PLA-PEG). In certain embodiments, the therapeutic nanoparticle comprises 1- (4 {[4— (dimethylamino) piperidine-1 —l] carbonl} phenl) —3— [4— (4,6 — dimorpholin — 4 —I — 1,3,5— triazin-2-yl) phenyl] urea and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of 1: 3.

The disclosed nanoparticles can optionally include about 1 to about 50 weight percent poly (lactic acid) or poly (lactic acid) -co-poly (glycolic acid) (which does not include PEG), or they can optionally include from about 1 to about 50 percent by weight, or from about 10 to about 50 percent by weight or from about 30 to about 50 percent by weight of poly (lactic acid) or poly (lactic) - co-poly (glycolic) acid. For example, poly (lactic) acid or poly (lactic) -co-poly (glycolic) acid can have a number average molecular weight ranging from about 5 to about 15 kDa, or from about 5 to about 12 kDa. Exemplary PLA can have a number average molecular weight ranging from about 5 to about 10 kDa. Exemplary PLGA can have a number average molecular weight ranging from about 8 to about 12 kDa.

The disclosed nanoparticles may optionally include 1 to 50 percent by weight of poly (lactic acid) or poly (lactic acid) -co-poly (glycolic acid) (not including PEG), or may optionally include 1 to 50 percent by weight. by weight, or 10 to 50 weight percent or 30 to 50 weight percent of poly (lactic acid) or poly (lactic acid) -co-poly (glycolic acid). For example, poly (lactic) acid or poly (lactic) -co-poly (glycolic) acid can have a number average molecular weight ranging from 5 to 15 kDa, or from 5 to 12 kDa. Exemplary PLA can have a number average molecular weight ranging from 5 to 10 kDa. Exemplary PLGA can have a number average molecular weight ranging from 8 to 12 kDa.

A therapeutic nanoparticle, in some embodiments, may contain from about 10 to about 30 percent by weight, from about 10 to about 25 percent by weight, from about 10 to about 20 percent by weight. , from about 10 to about 15 percent by weight, from about 15 to about 20 percent by weight, from about 15 to about 25 percent by weight, from about 20 to about 25 percent in weigh, from about 20 to about 30 percent by weight, or from about 25 to about 30 percent by weight, of poly (ethylene) glycol, where the poly (ethylene) glycol may be present as an acid copolymer poly (lactic) -poly (ethylene) glycol, poly (lactic acid) -co-poly (glycolic acid) -poly (ethylene) glycol copolymer, or poly (ethylene) glycol homopolymer. In certain embodiments, the polymers of the nanoparticles can be conjugated to a lipid. The polymer can be, for example, a lipid terminated PEG.

A therapeutic nanoparticle, in some embodiments, may contain 10 to 30 percent by weight, 10 to 25 percent by weight, 10 to 20 percent by weight, 10 to 15 percent by weight, 15 to 20 percent by weight, 15 to 25 percent by weight, 20 to 25 percent by weight, 20 to 30 percent by weight, or 25 to 30 percent by weight of poly (ethylene) glycol, where the poly (ethylene) glycol may be present as a copolymer of poly (lactic acid) - poly (ethylene) glycol, poly (lactic acid) -co-poly (glycolic acid) -poly (ethylene) glycol copolymer or poly (ethylene) glycol homopolymer.

In certain embodiments, the therapeutic nanoparticle comprises the PLA-PEG polymer, and the PLA-PEG molar ratio is around 5: 1. In other embodiments, the therapeutic nanoparticle comprises the PLA-PEG polymer, and the molar ratio of PLA-PEG is 5: 1.

Addressing remains.

Provided in this application, in some embodiments, nanoparticles that may include an optional targeting moiety, that is, a moiety capable of different binding or association, with a biological entity, for example, a membrane component, a receptor cell surface, an antigen, or the like. A targeting moiety present on the surface of the particle may allow the particle to be localized to a particular targeting site, eg, a tumor, a disease site, a tissue, an organ, a cell type, etc. In this way, the nanoparticle can then be "target specific". The drug or other cargo can then, in some cases, be released from the particle, and interact locally with the particular targeting site.

In one embodiment, a disclosed nanoparticle includes a targeting moiety that is a low molecular weight ligand. The term "binding" or "binding", as used in the present application, refers to the interaction between a corresponding pair of molecules or their portions that exhibit mutual affinity or binding capacity, usually due to specific interaction or binding. or non-specific, including, without limitation, biochemical, physiological, and / or chemical interactions. "Biological bonding" defines a type of interaction that occurs between pairs of molecules that include proteins, nucleic acids, glycoproteins, carbohydrates, hormones, or the like. The term "binding partner" refers to a molecule that can undergo binding with a particular molecule. "Specific binding" refers to molecules, such as polynucleotides, that are capable of binding or recognizing a binding partner (or a limited number of binding partners) to a substantially higher degree than other similar biological entities. In one group of embodiments, the targeting moiety has an affinity (as measured by a dissociation constant) of less than about 1 micromolar, at least about 10 micromolar, or at least about 100 micromolar.

In some embodiments, the targeting moiety has an affinity (as measured by a dissociation constant) of less than 1 micromolar, at least 10 micromolar, or at least 100 micromolar.

For example, a targeting portion can cause the particles to be localized to a tumor (eg, a solid tumor), a disease site, a tissue, an organ, a cell type, etc., within the body of a subject, according to the rest of the addressing used. For example, a low molecular weight ligand can be localized to a solid tumor, eg, breast or prostate tumors, or cancer cells. The subject can be a human or non-human animal. Examples of subjects include, without limitation, a mammal such as a dog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat, a mouse, a guinea pig, a hamster. , a primate, a human or the like.

Contemplated targeting moieties can include small molecules. In certain embodiments, the term "small molecule" refers to organic, natural or artificially created (eg, by chemical synthesis) compounds that have relatively low molecular weight and are not proteins, polypeptides, or nucleic acids. Small molecules usually have multiple carbon-carbon bonds. In certain embodiments, the small molecules are about 2000 g / mol or less in size. In some embodiments, the small molecules are around 1500 g / mol or less, or around 1000 g / mol or less. In some embodiments, the small molecules are around 800 g / mol or less, 500 g / mol or less, for example, from around 100 g / mol to around 600 g / mol, or around 200 g / mol to about 500 g / mol.

In certain embodiments, the small molecules are 2000 g / mole or less in size. In some embodiments, the small molecules are 1500 g / mol or less, or 1000 g / mol or less. In some embodiments, the small molecules are 800 g / mol or less, 500 g / mol or less, for example, 100 g / mol to 600 g / mol, or 200 g / mol to 500 g / mol. .

In some embodiments, the low molecular weight ligand is of Formulas I, II, III, or IV:

<img file="CU24488B1_D0003.tif" />

II III IV and their enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates;

where m and n are independently 0, 1, 2 or 3; p is 0 or 1;

R¹, R², R⁴ and R⁵ independently are selected from the group consisting of substituted or unsubstituted alkyl (e.g., C- | _10-alkyl, C1-6-alkyl, or C1-4-alkyl), substituted or unsubstituted aryl (e.g., phenyl or pyridinyl), and any combination thereof; and R³ is H or Ci_6-alkyl (eg CH3).

For compounds of Formulas I, II, III, and IV, R¹, R², R⁴ or R⁵they comprise nanoparticle attachment sites, eg, a polymer attachment site that is part of a disclosed nanoparticle, eg, PEG. The point of attachment can be formed by a covalent bond, ionic bond, hydrogen bond, a bond formed by adsorption that includes chemical adsorption and physical adsorption, a bond formed from van der Waals bonds, or forces of dispersion. For example, if R¹, R², R⁴, or R⁵ are defined as an aniline or a Ci-₆-alkyl-NH₂, any hydrogen (eg, an amino hydrogen) from these functional groups could be removed such that the low molecular weight ligand is covalently bound to the polymer matrix (eg, the pEG block of the polymer matrix) of the nanoparticle. As used in the present application, the term "covalent bond" refers to a bond between two atoms formed by sharing at least one pair of electrons.

In particular embodiments of Formulas I, II, III or IV, R¹, R², R⁴ and R⁵ are independently Ci-6-alkyl or phenyl, or any combination of Ci ~ 6-alkyl or phenyl, which are independently substituted one or more times with OH, SH, NH2 or CO2H, and where the alkyl group may be interrupted by N (H), S, or O. In another embodiment, R¹, R², R⁴ and R⁵ are, independently, CH2-Ph, (CH₂)₂-SH, CH₂-SH, (CH₂) 2C (H) (NH2) CO₂H, CH₂C (H) (NH₂)CO₂H, CH (NH₂) CH₂CO₂H, (CH₂)₂C (H) (SH) CO₂H, CH₂-N (H) -Ph, O-CH₂-Ph, or O- (CH₂)₂-Ph, where Ph is phenyl, and where each Ph can be independently substituted one or more times with OH, NH₂, CO₂H or SH. For these formulas, the NH groups₂, OH or 5 SH serve as the covalent point of attachment to the nanoparticle (for example,

-N (H) -PEG, -O-PEG or -S-PEG).

Examples of ligands include:

<img file="CU24488B1_D0004.tif" />

<img file="CU24488B1_D0005.tif" />

<img file="CU24488B1_D0006.tif" />

<img file="CU24488B1_D0007.tif" />

and their enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates, where the NH groups₂, OH or SH serve as the covalent point of attachment to the nanoparticle (e.g., -N (H) -PEG, -OPEG, oS-PEG) or

A indicates the point of attachment to the nanoparticle, where n is 1, 2, 3, 4, 5, or 6, and where R is independently selected from the group consisting of NH₂, SH, OH, CO₂H, C ^ -e-alkyl substituted with NH₂, SH, OH, or CO₂H, and phenyl substituted with NH₂, SH, OH, or CO₂H, and where R serves as the covalent point of attachment to the nanoparticle (e.g., -N (H) -PEG, -S-PEG, -O-PEG, or CO₂-PEG). These compounds can be additionally substituted with NH₂, SH, OH, CO₂H, Ci ~ 6-alkyl which is substituted with NH₂, SH, OH, or CO₂H, or phenyl which is substituted with NH₂, SH, OH or CO₂H, where these functional groups can further serve as the covalent point of attachment to the nanoparticle.

In some embodiments, small molecule targeting moieties that can be used to target cells associated with solid tumors such as prostate or breast cancer tumors include PSMA peptidase inhibitors such as 2-PMPA, GPI5232, VA- 033, phenylalkylphosphonamidates and / or their analogues and their derivatives. In some embodiments, small molecule targeting moieties that can be used to target cells associated with prostate cancer tumors include derivatives of thiol and indole thiol, such as derivatives of 2MPPA and 3- (2-mercaptoethyl) -1 / - / - Indole-2-carboxylic acid. In some embodiments, small molecule targeting moieties that can be used to target cells associated with prostate cancer tumors include hydroxamate derivatives. In some embodiments, small molecule targeting moieties that can be used to target cells associated with prostate cancer tumors include PBDA and urea-based inhibitors, such as ZJ 43, ZJ 11, ZJ 17, ZJ 38, and / or their analogues and their derivatives, androgen receptor targeting agents (ARTAs), polyamines, such as putrescine, spermine and spermidine, glutamate carboxylase II (GCPII) inhibitors, also known as NAAG Peptidase or NAALADase.

In another embodiment, the targeting moiety can be a ligand that targets the Her2 receptor, EGFR, folate, or toll receptors. In another embodiment, the targeting moiety is folate, folic acid, or an EGFR binding molecule.

For example, contemplated targeting moieties can include a nucleic acid, polypeptide, a glycoprotein, a carbohydrate, or a lipid. For example, a targeting moiety can be a nucleic acid targeting moiety (eg, an aptamer, eg, the A10 aptamer) that binds to a cell type specific marker. In general, an aptamer is an oligonucleotide (eg, DNA, RNA, or an analog or derivative) that binds to a particular target, such as a polypeptide. In some embodiments, a targeting moiety can be a natural or synthetic ligand for a cell surface receptor, for example, a growth factor, a hormone, LDL (low density lipid), transferrin, etc. A targeting moiety can be an antibody, where such term is intended to include antibody fragments. Characteristic portions of the antibodies, single chain targeting residues, can be identified, for example, using procedures such as phage display.

The targeting moieties disclosed in this application may, in some embodiments, be conjugated to a disclosed polymer or copolymer (eg, PLA-PEG), and said polymer conjugate may be part of a disclosed nanoparticle.

In certain embodiments, the therapeutic nanoparticle has an additionally present targeting ligand, and the ligand is PLAPEG-GL, where GL has the following structure:

»NH

<img file="CU24488B1_D0008.tif" />

Η Η Η H

In some embodiments, a therapeutic nanoparticle can include a polymer-drug conjugate. For example, a drug can be conjugated to a disclosed polymer or copolymer (eg, PLA-PEG), and said polymer-drug conjugate can be part of a disclosed nanoparticle. For example, a disclosed therapeutic nanoparticle can optionally include from about 0.2 to about 30 percent by weight of a PLA-PEG or PLGA-PEG, where the pEG is functionalized with a drug (eg, PLA-PEG-drug). .

In another example, a disclosed therapeutic nanoparticle may optionally include 0.2 to 30 weight percent of a PLA-PEG or PLGAPEG, where the pEG is functionalized with a drug (eg, PLAPEG-drug).

A disclosed polymeric conjugate (eg, a polymer-ligand conjugate) can be formed using any suitable conjugation technique. For example, two compounds such as a drug or targeting moiety and a biocompatible polymer (eg, a biocompatible polymer and a poly (ethylene glycol)) can be conjugated to each other using techniques such as EDC-NHS (I-ethyl-3 - (3— dimethylaminopropyl) carbodiimide hydrochloride and / V-hydroxysuccinimide) or a reaction involving a maleimide or a carboxylic acid, which can be conjugated to one end of a thiol, an amine or a similarly functionalized polyether. Conjugation of a targeting moiety or drug and a polymer to form a polymer-targeting moiety conjugate or a polymer-drug conjugate can be performed in an organic solvent, such as, without limitation, dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, acetone, or the like. Specific reaction conditions can be determined by those of skill in the art using no more than routine experimentation.

In another group of embodiments, a conjugation reaction can be performed by reacting a polymer comprising a carboxylic acid functional group (eg, a poly (ester-ether compound)) with a polymer or other moiety (such as a targeting moiety or therapeutic drug) having an amine functionality thereon. For example, a targeting moiety, such as a low molecular weight ligand, or the therapeutic agent can be reacted with an amine to form an amine-containing moiety, which can then be conjugated to the carboxylic acid of the polymer. Said reaction can take place as a one-step reaction, that is, the conjugation is carried out without the use of intermediates such as A / -hydroxysuccinimide or a maleimide. In some embodiments, the therapeutic agent can be reacted with an amine-containing linker to form an amine-containing drug, which can then be conjugated to the carboxylic acid of the polymer as described above. The conjugation reaction between the amine-containing moiety and the carboxylic acid terminated polymer (such as a poly (ester-ether) compound) can be accomplished, in a group of embodiments, by adding the amine-containing moiety. , solubilized in an organic solvent such as (without limitation) dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines, dioxane, or dimethylsulfoxide, to a solution containing the carboxylic acid terminated polymer. The carboxylic acid terminated polymer can be contained within an organic solvent such as, without limitation, dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, or acetone. The reaction between the amine-containing moiety and the carboxylic acid terminated polymer can occur spontaneously, in some cases. Unconjugated reagents can be washed off after such reactions, and the polymer can be precipitated from solvents such as, for example, ethyl ether, hexane, methanol, or ethanol. In certain embodiments, a conjugate between an alcohol-containing moiety and carboxylic acid functional group of a polymer can be formed, which can be achieved in a manner similar to that described above for amine-carboxylic acid conjugates.

Preparation of nanoparticles.

Another aspect of this disclosure is directed to systems and methods for the manufacture of the disclosed nanoparticles. In some embodiments, using two or more different polymers (eg, copolymers, eg, block copolymers) in different ratios and producing particles from the polymers (eg, copolymers, eg, block copolymers), the properties of the particles can be controlled. For example, one polymer (eg, copolymer, eg, block copolymer) may include a low molecular weight ligand, while another polymer (eg, copolymer, eg, block copolymer) may

100 be selected for its biocompatibility and / or its ability to control the immunogenicity of the resulting particle.

In some embodiments, a solvent used in a nanoparticle preparation process (eg, a nanoprecipitation process or a nanoemulsion process as described below) can include a hydrophobic acid, which can confer desirable properties on the prepared nanoparticles. using the process. As discussed above, in some cases, hydrophobic acid can enhance the drug loading of the disclosed nanoparticles. Additionally, in some cases, the controlled release properties of the disclosed nanoparticles can be enhanced by the use of hydrophobic acid. In some cases, the hydrophobic acid can be included in, for example, an organic solution or an aqueous solution used in the process. In one embodiment, the therapeutic agent is combined with an organic solution and the hydrophobic acid and optionally one or more polymers. The concentration of hydrophobic acid in a solution used to dissolve the therapeutic agent is discussed above, and can range, for example, from about 1 percent by weight to about 30 percent by weight or from 1 percent by weight to 30 percent by weight, etc.

In one group of embodiments, the particles are formed by providing a solution comprising one or more polymers, and contacting the solution with a non-solvent polymer to produce the particle. The solution can be miscible or immiscible with the non-solvent polymer. For example, a water-miscible liquid such as acetonitrile can contain the polymers, and the particles are formed as the acetonitrile comes into contact with water, a non-solvent polymer, for example, by pouring the acetonitrile into water. at a controlled rate. The polymer contained within

101 From the solution, on contact with the non-solvent polymer, it can then precipitate to form particles such as nanoparticles. Two liquids are said to be "immiscible" or immiscible with each other when one is not soluble in the other at a level of at least 10% by weight at ambient temperature and pressure. Usually an organic solution (for example, dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines, dioxane, dimethylsulfoxide, etc.) and an aqueous liquid (for example, water, or water containing dissolved salts or other species, cellular or biological media, ethanol, etc.) are immiscible with each other. For example, the first solution can be dumped into the second solution (at a suitable rate or speed). In some cases, particles such as nanoparticles can form as the first solution contacts the second immiscible liquid; For example, precipitation of the polymer on contact causes the polymer to form nanoparticles while the first solution is poured into the second liquid, and in some cases, for example, when the rate of introduction is carefully controlled and kept at a relatively low rate. slow, nanoparticles can form. Control of such particle formation can be optimized without difficulty by one of skill in the art using only routine experimentation.

Properties such as surface functionality, surface charge, size, zeta (*) potential, hydrophobicity, ability to control immunogenicity, and the like can be highly controlled using a disclosed process. For example, a library of particles can be synthesized and screened to identify particles that have a particular polymer ratio that allows the particles to have a specific residue density (e.g., low molecular weight ligands ) present on the surface of the particle. This allows

102 preparation of particles having one or more specific properties, eg, a specific size and specific surface residue density, without undue degree of stress. Therefore, certain embodiments target screening techniques using such libraries, as well as any particles identified using such libraries. Furthermore, identification can occur by any suitable method. For example, identification can be direct or indirect, or proceed quantitatively or qualitatively.

In some embodiments, the already formed nanoparticles are functionalized with a targeting moiety using procedures analogous to those described for the production of ligand-functionalized polymeric conjugates. For example, a first copolymer (PLGA-PEG, poly (lactide-co-glycolide) and poly (ethylene glycol)) is mixed with the protonatable nitrogen-containing therapeutic agent to form particles. The particles then associate with a low molecular weight ligand to form nanoparticles that can be used to treat cancer. The particles can be associated with various amounts of low molecular weight ligands in order to control the surface density of the ligand of the nanoparticle, so as to alter the therapeutic characteristics of the nanoparticle. Additionally, for example, by controlling parameters such as molecular weight, molecular weight of PEG, and surface charge of the nanoparticle, particles with very precise control can be obtained.

In another embodiment, a nanoemulsion process is provided, such as the process depicted in Figures 1, 2A and 2B. For example, a therapeutic agent, a hydrophobic acid, a first polymer (for example, a diblock copolymer such as PLA-PEG or PLGA-PEG,

103 any of which may be optionally linked to a ligand) and an optional second polymer (eg (PL (G) A-PEG or PLA), may combine with an organic solution to form a first organic phase. Said first phase may include about 1 to about 50% by weight solids, about 5 to about 50% by weight solids, about 5 to about 40% by weight solids, about 1 to about 15% by weight solids, or about 10 to about 30% by weight solids. The first organic phase can be combined with a first aqueous solution to form a second phase. The organic solution can include, for example, toluene, methyl ethyl ketone, acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate, dimethylformamide, methylene chloride, dichloromethane, chloroform, acetone, benzyl alcohol, Tween ™ 80, Span 80, or the like, and their combinations. In one embodiment, the organic phase can include benzyl alcohol, ethyl acetate, and combinations thereof. The second phase can range from about 0.1 to 50% by weight, or from about 1 to 50% by weight, or from about 5 to 40% by weight, or from about 1 to 15% by weight, of solids. . The aqueous solution can be water, optionally, in combination with one or more of sodium cholate, ethyl acetate, polyvinyl acetate, and benzyl alcohol. In some embodiments, the pH of the aqueous phase can be selected based on the pK_to protonated basic therapeutic agent and / or pK_to of hydrophobic acid. For example, in certain embodiments, the therapeutic agent, when protonated, may have a first pK_to, the hydrophobic acid may have a second pK_to, and the aqueous phase can have a pH equal to one pK unit_to between the first pK_to and the second pK_to. In a particular embodiment, the pH of the aqueous phase can be equal to one pK unit._to which is approximately equidistant between the first pK_to and the second pK_to.

104

In another embodiment, the first phase may include 1 to 50% by weight of solids, 5 to 50% by weight of solids, 5 to 40% by weight of solids, 1 to 15% by weight of solids, or 10 to 30% by weight of solids. In one embodiment, the second phase can range from 0.1 to 50% by weight, or from 1 to 50% by weight, or from 5 to 40% by weight, or from 1 to 15% by weight of solids. In a particular embodiment, the pH of the aqueous phase can be equal to one unit of pK_to which is equidistant between the first pK_to and the second pK_to.

For example, the organic or oil phase can use a solvent that is only partially miscible with the non-solvent (water). Therefore, when mixed at a sufficiently low ratio and / or when presaturated water is used with the organic solvents, the oil phase remains liquid. The oil phase can be emulsified in an aqueous solution, and as liquid droplets, cut into nanoparticles using, for example, high energy dispersion systems, such as homogenizers or sonicators. The aqueous portion of the emulsion, otherwise known as the "water phase", can be a surfactant solution consisting of sodium cholate and presaturated with ethyl acetate and benzyl alcohol. In some cases, the organic phase (eg, first organic phase) may include the basic therapeutic agent. Additionally, in certain embodiments, the aqueous solution (eg, first aqueous solution) can include the substantially hydrophobic acid. In other embodiments, both the basic therapeutic agent and the substantially hydrophobic acid can be dissolved in the organic phase.

Emulsifying the second stage to form an emulsion stage can be carried out, for example, in one or two emulsion stages. For example, a primary emulsion can be prepared, and then emulsified to form a fine emulsion. The primary emulsion can be formed, for example,

105 using simple mixing, a high pressure homogenizer, probe sonicator, stir bar, or rotor stator homogenizer. The primary emulsion can be formed as a fine emulsion through the use of, for example, a probe sonicator or a high pressure homogenizer, for example, using 1, 2, 3 or more passes through a homogenizer. For example, when using a high pressure homogenizer, the pressure used can be around 2.06 to around 4.13 bar (around 30 to around 60 psi), around 2.75 to around 3.44 bar (around 40 to around 50 psi), around 68.9 to around 551.5 bar (around 1000 to around 8000 psi), around 137.8 to around 275.7 bar (around 2000 to around 4000 psi), around 275.7 to around 551.5 bar (around 4000 to around 8000 psi), or around 275.7 to around 344.7 bar (around 4000 to around 5000 psi), for example around 137.8, 172.3, 275.7 or 344.7 bar (around 2000, 2500, 4000 or 5000 psi).

In another example, when using a high pressure homogenizer, the pressure used can be 2.06 to 4.13 bar (30 to 60 psi), 2.75 to 3.44 bar (40 to 50 psi), 68.9 to 551.5 bar (1000 to 8000 psi ), 137.8 to 275.7 bar (2000 to 4000 psi), 275.7 to 551.5 bar (4000 to 8000 psi), or 275.7 to 344.7 bar (4000 to 5000 psi), for example 137.8, 172.3, 275.7 or 344.7 bar (2000, 2500, 4000 or 5000 Psi).

In some cases, fine emulsion conditions, which can be characterized by a very high surface-to-volume ratio of the droplets in the emulsion, can be selected so as to maximize the solubility of the therapeutic agent and hydrophobic acid and form the desired HIP. In certain embodiments, under fine emulsion conditions, the equilibrium of dissolved components can occur very rapidly, i.e.

106 that is, faster than the solidification of the nanoparticles. Consequently, the selection of a HIP based on, for example, the difference of pK_to between the protonated form of the therapeutic agent and the hydrophobic acid, or adjusting other parameters such as the pH of the fine emulsion and / or the pH of the quench solution, can have a significant effect on drug loading and properties release of the nanoparticles by dictating, for example, the formation of a HIP in the nanoparticle, in contrast to diffusion of the therapeutic agent and / or hydrophobic acid out of the nanoparticle.

In some embodiments, the therapeutic agent and the substantially hydrophobic acid can be combined in the second phase prior to emulsifying the second phase. In some cases, the therapeutic agent and the substantially hydrophobic acid may form a hydrophobic ion pair prior to emulsifying the second phase. In other embodiments, the therapeutic agent and the substantially hydrophobic acid can form a hydrophobic ion pair during second phase emulsification. For example, the therapeutic agent and the substantially hydrophobic acid can be combined in the second phase substantially concurrently with the emulsion of the second phase, for example, the therapeutic agent and the substantially hydrophobic acid can be dissolved in separate solutions (for example, two substantially immiscible solutions), which are then combined during emulsification. In another example, the therapeutic agent and the substantially hydrophobic acid can be dissolved in separate miscible solutions which are then fed into the second phase during emulsification.

Either dilution or solvent evaporation may be required to complete solvent extraction and solidify the particles. For better control over extraction kinetics and a more scalable process,

107 A solvent dilution can be used by aqueous quenching. For example, the emulsion can be diluted in cold water to a concentration sufficient to dissolve all of the organic solvent to form a quenched phase. In some embodiments, quenching can be done at least partially at a temperature of about 5 ° C or less. For example, the water used in the shutdown may be at a temperature lower than room temperature (for example, around 0 to around 10 ° C, or around 0 to around 5 ° C). In certain embodiments, a quench may be selected having a suitable pH for quenching the emulsion phase, for example, by improving the properties of the nanoparticles, such as the release profile, or by improving a parameter of the nanoparticles. , such as drug loading. The pH of the slake can be adjusted by titration of acid or base, for example, or by appropriate selection of a regulator. In some embodiments, the pH of the quench can be selected based on the pK_to protonated basic therapeutic agent and / or pK_to of hydrophobic acid. For example, in certain embodiments, the basic therapeutic agent, when protonated, may have a first pK_to, the hydrophobic acid may have a second pK_to, and the emulsion phase can be quenched with an aqueous solution having a pH equal to one pK unit_to between the first pK_to and the second pK_to. In some embodiments, the resulting quenched phase may further have a pH equal to one unit of pK._to between the first pK_to and the second pK_to. In a particular embodiment, the pH can be equal to one unit of pK_to which is approximately equidistant between the first pK_to and the second pK_to.

In certain embodiments, HIP formation can take place during or after emulsion, for example, as a result of equilibrium conditions in the fine emulsion. No desire to limit yourself to any

108 In theory, it is believed that soluble organic counterions (ie, hydrophobic acid) can facilitate diffusion of the therapeutic agent into a nanoparticle of an emulsion as a consequence of HIP formation. Without wishing to be bound by any theory, the HIP can remain in the nanoparticle before solidification of the nanoparticle, since the solubility of HIP in the nanoparticle is greater than the solubility of HIP in the aqueous phase of the emulsion and / or in the shutdown. For example, when selecting a pH for shutdown that is between the pK_to of the basic therapeutic agent and the pK_toof hydrophobic acid, the formation of ionized therapeutic agent and hydrophobic acid can be optimized. However, selecting a pH that is too high may tend to cause the hydrophobic acid to diffuse out of the nanoparticle, while selecting a pH that is too low may tend to cause the therapeutic agent to diffuse out of the nanoparticle. .

In some embodiments, the pH of an aqueous solution used in a nanoparticle formulation process (e.g., including, without limitation, the aqueous phase, the emulsion phase, the quench, and the quenched phase) can be independently selected and it can range from about 1 to about 3, in some embodiments, from about 2 to about 4, in some embodiments, from about 3 to about 5, in some embodiments, from about 4 to about 6, in some embodiments, from about 5 to about 7, in some embodiments, from about 6 to about 8, in some embodiments, from about 7 to about 9, and in some embodiments, from about 8 to about 10. In certain embodiments, the pH of an aqueous solution used in a nanoparticle formulation process can range from about 3 to about 4, in some

109 embodiments, from about 4 to about 5, in some embodiments, from about 5 to about 6, in some embodiments, from about 6 to about 7, in some embodiments, from about 7 to about 8, and in some embodiments, from about 8 to about 9.

In some embodiments, the pH of an aqueous solution used in a nanoparticle formulation process (eg, including, without limitation, the aqueous phase, the emulsion phase, the quench, and the quenched phase) can be independently selected. and can range from 1 to 3, in some embodiments, from 2 to 4, in some embodiments, from 3 to 5, in some embodiments, from 4 to 6, in some embodiments, from 5 to 7, in some embodiments, from 6 to 8, in some embodiments, from 7 to 9, and in some embodiments, from 8 to 10. In certain embodiments, the pH of an aqueous solution used in a nanoparticle formulation process can vary from 3 to 4, in some embodiments, from 4 to 5, in some embodiments, from 5 to 6, in some embodiments, from 6 to 7, in some embodiments, from 7 to 8, and in some embodiments, 8 to 9.

In some embodiments, not all of the therapeutic agent is encapsulated in the particles at this stage, and a drug solubilizer is added to the quenched phase to form a solubilized phase. The drug solubilizer can be, for example, polysorbate 80 (Tween ™ 80), Tween ™ 20, polyvinyl pyrrolidone, cyclodextran, sodium dodecyl sulfate, sodium cholate, diethylnitrosamine, sodium acetate, urea, glycerol, propylene glycol, glycofurol, poly (ethylene) glycol, bris (polyoxyethylene glycol) dodecyl ether, sodium benzoate, sodium salicylate, polyoxyethylene (100) stearyl ether, or combinations thereof. For example, Tween ™ 80 can be added to the suspension.

110 nanoparticles quenched, in order to solubilize the free drug and prevent the formation of drug crystals. In some embodiments, a ratio of drug solubilizer to protonatable nitrogen-containing therapeutic agent is from about 200: 1 to about 10: 1, or in some embodiments, from about 100: 1 to about 10: 1.

In some embodiments, a ratio of drug solubilizer to protonatable nitrogen-containing therapeutic agent is 200: 1 to 10: 1, or in some embodiments, 100: 1 to 10: 1.

The solubilized phase can be filtered in order to recover the nanoparticles. For example, ultrafiltration membranes can be used to concentrate the nanoparticle suspension and substantially remove organic solvent, free drug (ie, unencapsulated therapeutic agent), drug solubilizer, and other processing aids (surfactants). Exemplary filtration can be accomplished using a tangential flow filtration system. For example, by using a membrane with a pore size suitable to retain nanoparticles while allowing solutes, micelles and organic solvent to pass through, the nanoparticles can be selectively separated. Exemplary membranes with molecular weight cutoffs ranging from about 300 to about 500 kDa (~ about 5 to about 25 nm) can be used. Exemplary membranes with molecular weight cutoffs ranging from 300 to 500 kDa (~ 5 to 25 nm) can be used.

Diafiltration can be performed using a constant volume approach, which means that the diafiltrate (cold deionized water, for example, at around 0 to around 5 ° C, or 0 to around 10 ° C) can be added to the suspension. Feed at the same rate as the filtrate is removed from the suspension. In some embodiments, the filtration may include a first filtration using a first temperature of about 0 to

111 around 5 ° C, or 0 to around 10 ° C, and a second temperature of around 20 to around 30 ° C, or 15 to around 35 ° C. In some embodiments, the filtration may include processing from about 1 to about 30, in some cases, about 1 to about 15, or in some cases, 1 to about 6 diavolumes. For example, filtration may include processing of about 1 to about 30, or in some cases, about 1 to about 6 diavolumes, at about 0 to about 5 ° C, and processing of at least one diavolume (eg, about 1 to about 15, about 1 to about 3, or about 1 to about 2 diavolumes) at about 20 to about 30 ° C. In some embodiments, the filtration comprises processing different diavolumes at different distinctive temperatures.

In some embodiments, the filtration may include a first filtration using a first temperature of 0 to 5 ° C, or 0 to 10 ° C, and a second temperature of 20 to 30 ° C, or 15 to 35 ° C. In some embodiments, the filtration may include processing 1 to 30, in some cases 1 to 15, or in some cases 1 to 6 diavolumes. For example, filtration may include processing 1 to 30, or in some cases 1 to 6 diavolumes, at 0 to 5 ° C, and processing at least one diavolume (for example, 1 to 15, 1 to 3, or 1 to 2 diavolumes) at 20 to 30 ° C.

After purification and concentration of the nanoparticle suspension, the particles can be passed through one, two or more sterilization and / or depth filters, for example, using a ~ 0.2 pm depth prefilter. For example, a sterilization filtration step may involve filtration of therapeutic nanoparticles using a train

112 filtration at a controlled rate. In some embodiments, the filter train may include a depth filter and a sterilization filter.

In another embodiment of the preparation of the nanoparticles, an organic phase composed of a mixture of the therapeutic agent and polymer (homopolymer, copolymer and copolymer with ligand) is formed. The organic phase is mixed with an aqueous phase at a ratio of approximately 1: 5 (oil phase: aqueous phase) where the aqueous phase is composed of a surfactant and some dissolved solvent. The primary emulsion is formed by combining two phases under simple mixing or through the use of a rotor stator homogenizer. The primary emulsion is then formed into a fine emulsion through the use of a high pressure homogenizer. The fine emulsion is then quenched by addition to deionized water with mixing. In some embodiments, the quench: emulsion ratio can be from about 2: 1 to about 40: 1, or in some embodiments, from about 5: 1 to about 15: 1. In some embodiments, the quench: emulsion ratio is approximately 8.5: 1. In some embodiments, the quench: emulsion ratio can be from 2: 1 to 40: 1, or in some embodiments, 5: 1 to 15: 1. In some embodiments, the quench: emulsion ratio is 8.5: 1. A Tween ™ solution (eg, Tween ™ 80) is then added to the quench to achieve approximately 2% Tween ™ in total. This serves to dissolve free unencapsulated therapeutic agent. The nanoparticles are then isolated through centrifugation or ultrafiltration / diafiltration.

It will be appreciated that the amounts of polymer, therapeutic agent, and hydrophobic acid that are used in preparing the formulation may differ from those in a final formulation. For example, part of the therapeutic agent may not be fully incorporated into a nanoparticle, and such agent

113 Free therapeutic can, for example, be filtered. For example, in one embodiment, a first organic solution containing about 11 percent by weight of theoretical loading of therapeutic agent in a first organic solution containing about 9% of a first hydrophobic acid (eg, an acid fatty), a second organic solution containing about 89 percent by weight of polymer (for example, the polymer can include about 2.5 mole percent of a polymer-conjugated targeting moiety and about 97.5 mole percent PLA-PEG), and an aqueous solution containing about 0.12% of a second hydrophobic acid ( e.g. a bile acid) can be used in the preparation of a formulation that produces, for example, a final nanoparticle comprising about 2 percent by weight of therapeutic agent, about 97.5 weight percent polymer (where the polymer can include about 1.25 mole percent of a targeting moiety conjugated to a polymer and about 98.75 mole percent PLAPEG), and about 0.5% total hydrophobic acid. Such processes can provide final nanoparticles suitable for administration to a subject, including about 1 to about 20 weight percent therapeutic agent, eg, about 1, about 2, about 3, about 4, about 5, about 8, about 10, or about 15 percent therapeutic agent by weight.

In another embodiment, a first organic solution containing 11 percent by weight of therapeutic agent theoretical loading in a first organic solution containing 9% of a first hydrophobic acid (eg, a fatty acid), a second organic solution containing 89 weight percent polymer (for example, the polymer may include 2.5 mole percent of a targeting moiety conjugated to a polymer and 97.5 percent

114 mole percent PLA-PEG), and an aqueous solution containing 0.12% of a second hydrophobic acid (for example, a bile acid) can be used in the preparation of a formulation that achieves, for example, a final nanoparticle comprising 2 weight percent therapeutic agent, 97.5 weight percent polymer (where the polymer may include 1.25 mole percent of a targeting moiety conjugated to a polymer and 98.75 mole percent PLA-PEG) and 0.5% total hydrophobic acid. Such processes can provide final nanoparticles suitable for administration to a subject, including 1 to 20 percent by weight therapeutic agent, eg, 1, 2, 3, 4, 5, 8, 10, or 15 percent agent. therapeutic weight.

In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1— (4 - {[4— (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4— I — 1,3,5 — triazin — 2 — yl) phenl] urea and pamoic acid in a weight ratio of therapeutic agent to pamoic acid of about 0.1: 1, about about

0.5: 1, about 1: 1, about 1.1: 1, about 1.2: 1,

1.3: 1, about 1.4: 1, about 1.5: 1, about 1.6: 1, about

1.7: 1, around

1.8: 1, around

1.9: 1, around

2: 1, around

2.5: 1, around

3: 1, around

3.5: 1, around

4: 1, around

4.5: 1, around

5: 1, around

5.5: 1, around

6: 1, around

6.5: 1, around

7: 1, around

7.5: 1, around

8: 1, around 8.5: 1, around 9: 1, around 9.5: 1, or around 10: 1.

In some embodiments, the therapeutic nanoparticle comprises PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 0.5: 1, about 1: 1, about 1: 2, around 1: 3, around 1: 4, around 1: 5, around 1: 6, around 1: 7, around 1: 8, around 1: 9, around 1: 10, around 1:15, or around 1:20. In certain embodiments, the

115 Therapeutic nanoparticle comprises the therapeutic agent 1- (4-¿[4 (dimethylamino) piperidin-1 -¡l] carbonyl} phenyl) -3- [4- (4,6-dimorpholin-4- ¡1-1.3, 5— triazin-2-l) phenyl] urea, pamoic acid, in a weight ratio of therapeutic agent to pamoic acid of about 1.8: 1, PLA-PEG (in a molar ratio of 16: 5) in a ratio by weight of therapeutic agent to PLA-PEG of about 1: 3, and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL of about 44: 1. In other embodiments, the therapeutic nanoparticle further comprises a solubilizer. In certain such embodiments, the solubilizer is polyoxyethylene (100) stearyl ether. In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1- (4 - {[4- (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin— 4-yl-1,3,5-triazin-2-yl) phenyl] urea and pamoic acid in a weight ratio of therapeutic agent to acidic pamoic acid of 0.1: 1, 0.5: 1, 1: 1, 1.1: 1 , 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4 : 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, or 10: 1. In some embodiments, the therapeutic nanoparticle comprises PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of 0.5: 1, 1: 1, 1: 2, 1: 3 , 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1:10, 1:15, or 1:20. In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1- (4 - {[4- (dimethylamino) piper¡d¡n1-¡l] carbonyl} phenyl) —3— [4— (4,6-dimorpholin) —4-yl-1,3,5-triazin-2-yl) phenyl] urea, pamoic acid, at a weight ratio of therapeutic agent to pamoic acid of 1.8: 1, PLA-PEG (at a molar ratio of 16 : 5) in a weight ratio of therapeutic agent to PLA-PEG of 1: 3, and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL of 44: 1. In other embodiments, the therapeutic nanoparticle further comprises a solubilizer. In

116 In certain such embodiments, the solubilizer is polyoxyethylene (100) stearyl ether.

In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1- (4 - {[4- (dimethylamino) piperidin-1-¡l] carbonyl} phenyl) -3- [4 (4,6-dimorpholin) 4-yl-1,3,5-triazin-2-yl) phenyl] urea and oleic acid in a weight ratio of therapeutic agent to oleic acid of 0.1: 1, about 0.5: 1, about 1: 1, about 1.1: 1, about 1.2: 1, about 1.3: 1, about 1.4: 1, about 1.5: 1, about 1.6: 1, about 1.7: 1, about 1.8: 1, about 1.9: 1, about 2: 1, about 2.5: 1, about 3: 1, about 3.5: 1, about 4: 1, about 4.5: 1, about 5: 1, about 5.5: 1, around 6: 1, around 6.5: 1, around 7: 1, around 7.5: 1, around 8: 1, around 8.5: 1, around 9: 1, around 9.5: 1, or about 10: 1. In some embodiments, the therapeutic nanoparticle comprises PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLAPEG of about 0.5: 1, about 1: 1, about 1: 2, around 1: 3, around 1: 4, around 1: 5, around 1: 6, around 1: 7, around 1: 8, around 1: 9, around 1:10, around 1:11, around 1:12, around 1:13, around 1:14, around 1:15, around 1:20, around 1:25, or around 1:30. In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1- (4 {[4— (dimethylamino) piperidine-1 -yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4 — ¡ I — 1,3,5— triazin-2-yl) phenyl] urea, oleic acid, in a weight ratio of therapeutic agent to oleic acid of about 1: 1.6, PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 1: 7, and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL of about 46: 1. In some embodiments, the

117 Therapeutic nanoparticle further comprises cholic acid. In other embodiments, the therapeutic nanoparticle further comprises a solubilizer. In certain such embodiments, the solubilizer is polysorbate 80. In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1- (4 - {[4- (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4— yl-1,3,5-triazin-2-yl) phenyl] urea and oleic acid in a weight ratio of therapeutic agent to oleic acid of 0.1: 1, 0.5: 1, 1: 1, 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, or 10: 1. In some embodiments, the therapeutic nanoparticle comprises PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of 0.5: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:20, 1:25, or 1:30. In certain embodiments, the therapeutic nanoparticle comprises the therapeutic agent 1- (4 {[4— (dimethylamino) piperidin-1 -yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl —1,3,5— triazin-2-yl) phenyl] urea, oleic acid in a 6: 1 weight ratio of therapeutic agent to oleic acid, PLA-PEG (in a 16: 5 molar ratio) weight ratio of therapeutic agent to PLA-PEG of 1: 7, and PLA-PEGGL in a weight ratio of PLA-PEG to PLA-PEG-GL of 46: 1. In some embodiments, the therapeutic nanoparticle additionally comprises cholic acid. In other embodiments, the therapeutic nanoparticle further comprises a solubilizer. In certain such embodiments, the solubilizer is polysorbate 80.

In some embodiments, the therapeutic nanoparticle is a nanoparticle prepared by emulsifying a first organic phase comprising a first polymer, a therapeutic agent, and a substantially hydrophobic acid, so as to form an emulsion phase; he

118 quenching the emulsion phase so as to form a quenched phase; and filtration of the quenched phase in order to recover the therapeutic nanoparticles, where the therapeutic agent is 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4 , 6-dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

In other embodiments, the therapeutic nanoparticle is a nanoparticle prepared by the process of combining a first organic phase with a first aqueous solution to form a second phase; emulsifying the second phase to form an emulsion phase, wherein the emulsion phase comprises a first polymer, therapeutic agent, and a substantially hydrophobic acid; quenching the emulsion phase so as to form a quenched phase; and filtration of the quenched phase in order to recover the therapeutic nanoparticles, where the therapeutic agent is 1 (4 - {[4— (dimethylamino) pipendin-1 — ¡l] carbonyl} phen¡l) —3— [4— (4,6 — dimorpholin — 4 — ¡I— 1,3,5 — triazin — 2 — ¡l) phenyl] urea, the first organic phase comprises the therapeutic agent and pamoic acid in a weight ratio of therapeutic agent to pamoic acid of around 11: 1 and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 1: 3 in an organic solvent comprising benzyl alcohol and ethyl acetate in a weight ratio of benzyl alcohol to ethyl acetate of about 1.25, and the first aqueous solution comprises a polyoxyethylene (100) stearyl ether dissolved in benzyl alcohol in a weight ratio of 0.005: 1, and the combination of the first organic phase and the first aqueous phase in a weight ratio of about 1: 5 to form a second phase; the emulsion of the second phase formed therein and the quenching of the emulsion phase with citric acid, 0.1 M, in aqueous solution at pH 4.5; and the concentration of the resulting product.

119

The therapeutic agent can include alternative forms such as pharmaceutically acceptable salt forms, free base forms, hydrates, isomers, and their prodrugs.

An effective amount, when used in conjunction with a compound of this invention, is an amount effective to inhibit mTOR or PI3K in a subject.

The therapeutic agent of the present invention exhibits an inhibitory activity of mTOR (target of rapamycin in mammalian cells), and therefore, the therapeutic nanoparticle prepared from the therapeutic agent can be used to inhibit the abnormal growth of cells in which mTOR plays a role. Consequently, the therapeutic nanoparticle of the present invention is effective in treating disorders with which abnormal cell growth actions of mTOR are associated, such as restenosis, atherosclerosis, bone disorders, arthritis, diabetic retinopathy, psoriasis, benign prosthetic hypertrophy, atherosclerosis, inflammation, angiogenesis, immune disorders, pancreatitis, kidney disease, cancer, etc. In particular, the compounds of the present invention possess excellent cancer cell growth inhibitory effects, and are effective in the treatment of cancers, preferably all solid cancers and malignant lymphomas, and especially leukemia, cancer of skin, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, gastric cancer, brain tumor, advanced renal cell carcinoma, acute lymphoblastic leukemia, malignant melanoma, bone or soft tissue sarcoma, etc.

The therapeutic agent of the present invention exhibits PI3 kinase inhibitory activity, and therefore, the therapeutic nanoparticle prepared from the therapeutic agent can be used to inhibit growth.

120 abnormal cell in which PI3 kinases play a role. Consequently, the therapeutic agent of the present invention is effective in the treatment of disorders with which actions of abnormal cell growth are associated, of PI3 kinases, such as restenosis, atherosclerosis, bone disorders, arthritis, diabetic retinopathy, psoriasis, prosthetic hypertrophy. benign, atherosclerosis, inflammation, angiogenesis, immune disorders, pancreatitis, kidney disease, cancer, etc. In particular, the therapeutic nanoparticle of the present invention possesses excellent cancer cell growth inhibitory effects, and is effective in treating cancer types, preferably, all types of solid cancer and malignant lymphomas, and especially, leukemia, cancer. skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, gastric cancer, brain tumor, head and neck cancer, for example, cancer of the following regions: oral cavity, pharynx, larynx, sinuses and nasal cavity, or salivary glands), advanced renal cell carcinoma, acute lymphoblastic leukemia, malignant melanoma soft tissue or bone sarcoma, etc.

The therapeutic agent is also useful in the treatment of a type of cancer associated with PTEN deficiency. The phosphatase-tensin homologue deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase, and functions as a protein phosphatase by dephosphorylating protein substrates at serine, threonine, and tyrosine residues. PTEN also functions as a lipid phosphatase through dephosphorylation of phosphoinosital 3,4,5-triphosphate (PIP3), a key signaling component of phosphoinositol-3 kinase (PI3-kinase). PTEN is a known tumor suppressor that has been implicated in cellular processes including mediation of the MAP kinase signaling pathway, centromeric maintenance, and is implicated

121 in DNA repair pathways through mediation of Rad51 gene expression. Tumor suppressors play roles in maintaining genome stability, and loss of function of these tumor suppressors is known to lead to genomic instability. Genetic instability represents an unavoidable consequence of the loss of tumor suppressors. In fact, frequent occurrence of PTEN mutation and genetic instability is found in a wide range of PTEN-deficient cancers. Also, several tumor cell lines are known to be deficient in PTEN. Similarly, several tumor cell lines are known to be deficient in PTEN. Embryonic stem cells devoid of PTEN were shown to exhibit DNA repair checkpoint defects in response to ionizing radiation, resulting in accumulation of unrepaired chromosomes with DNA double-stranded breaks and gaps. Other mechanistic studies revealed that the observed G2 checkpoint defects may result from the functional alteration of the checkpoint protein, CHK1, due to the lack of PTEN. PTEN deficiency directly elevates AKT kinase activity, which triggers phosphorylation of CHK1. Phosphorylated CHK1 undergoes ubiquitination, preventing its entry into the nucleus. CHK1 sequestration in the cytoplasm disrupts its normal function in initiating a DNA repair checkpoint. Furthermore, the inactivation of CHK1 in PTEN-deficient cells leads to the accumulation of DNA double-strand breaks. Examination of the localization of CHK1 in a large panel of primary human breast carcinomas indicates an increased cytoplasmic level of CHK1 in tumor cells with lower expression of PTEN and high phosphorylation of AKT. Additionally, aneuploidy was frequently observed both in human breast carcinomas with low PTEN expression and in mouse prostatic intraepithelial neoplasia.

122

Pten.up. + / -. Such in vitro and in vivo observations indicate that PTEN deficiencies are involved in the initiation of an oncogenic signaling process by causing dysfunction of important checkpoint proteins. The cytoplasm has been considered the primary site of PTEN for the production of its tumor suppressor function, and the ability of PTEN to block the pl3 kinase pathway through its phosphatase activity has been considered the key mechanism by which PTEN suppresses the carcinogenesis. Although the cellular distribution of PTEN varies in different tissues, endogenous PTEN in neurons, gliomas, and thyroid cells, pancreas, and skin is most commonly found in the nuclear compartment. Increasing evidence indicates that malignant diseases may be accompanied by translocation of PTEN from the nucleus to the cytoplasm. Inactivation of PTEN, either by mutations, deletions or promoter hypermethylation, has been identified in a wide variety of tumors. The therapeutic agent of the present invention is a method of treating a type of cancer associated with a PTEN deficiency, such as endometrial carcinoma, glioblastoma (glioblastoma multiforme / anaplastic astrocytoma), prostate cancer, kidney cancer, cell lung carcinoma. small, meningioma, head and neck cancer, thyroid cancer, bladder cancer, colorectal cancer, breast cancer, melanoma.

Pharmaceutical formulations.

The nanoparticles disclosed in this application can be combined with pharmaceutically acceptable carriers to form a pharmaceutical composition, according to another aspect. As one skilled in the art will appreciate, carriers can be selected on the basis of route of

123 administration, as described below, the location of the target tissue, the drug delivered, the time course of drug delivery, etc.

The pharmaceutical compositions can be administered to a patient or subject by any means known in the art, including oral and parenteral routes. The terms "patient" or "subject", as used in the present application, are interchangeable, and refer to humans, as well as non-humans, including, for example, mammals, birds, reptiles, amphibians, and fish. For example, non-humans can be mammals (eg, rodent, rat, mouse, rabbit, monkey, dog, cat, primate, or pig). In certain embodiments, parenteral routes are desirable, as they avoid contact with digestive enzymes found in the alimentary canal. According to said embodiments, the compositions of the invention can be administered by injection (for example, intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments or drops) , or by inhalation (for example, by sprays).

In a particular embodiment, the nanoparticles are administered to a subject in need, systemically, for example, by injection or IV infusion.

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable wetting or dispersing agents. The sterile injectable preparation may further be a sterile injectable solution, suspension, or emulsion in a non-toxic, parenterally acceptable solvent or diluent, for example, as a solution in 1,3-butanediol. Among the acceptable solvents and vehicles that may be employed are water, compound sodium chloride solution, USP (Pharmacopoeia

124 of the United States) and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed, as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Furthermore, fatty acids such as oleic acid are used in the preparation of injectable products. In one embodiment, the conjugate of the invention is suspended in a carrier liquid comprising 1% (w / v) of sodium carboxymethyl cellulose and 0.1% (v / v) of Tween ™ 80. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retentive filter, or by incorporation of sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to delivery. use.

Solid dosage forms for oral administration include capsules, tablets, lozenges, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated conjugate is mixed with at least one inert, pharmaceutically acceptable carrier or excipient, such as sodium citrate or dicalcium phosphate, and / or (a) fillers or extenders such as starches, lactose , sucrose, glucose, mannitol and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates and sodium carbonate, (e) solution retarding agents, such as paraffin, (f) accelerators of the absorption, such as quaternary ammonium compounds, (g) wetting agents such as, for example, 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,

125 sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and lozenges, the dosage form may further comprise pH regulating agents.

It will be appreciated that the exact dosage of a nanoparticle containing the therapeutic agent is selected by the individual physician in view of the patient to be treated; in general, the dosage and administration are adjusted so as to provide an effective amount of the therapeutic agent nanoparticle to the treated patient. As used in the present application, the effective amount of a nanoparticle containing a protonatable nitrogen-containing therapeutic agent refers to the amount necessary to produce the desired biological response. As will be appreciated by those skilled in the art, the effective amount of a nanoparticle containing the therapeutic agent can vary according to factors such as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration. , etc. For example, the effective amount of a nanoparticle containing the therapeutic agent could be the amount that achieves a reduction in tumor size by a desired amount over a desired period of time. Additional factors that can be considered include the severity of the disease; the age, weight and gender of the treated patient; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance / response to therapy.

Nanoparticles can be formulated in unit dosage form, for ease of administration and uniformity of dosage. The term "unit dosage form", as used in the present application, refers to a physically separate unit of nanoparticle appropriate for the patient to be treated. However, it will be understood that the total daily use of the

126 Compositions will be decided by the treating physician, within the scope of sound medical judgment. For any nanoparticle, the therapeutically effective dose can be estimated initially either in cell culture assays, or in animal models, usually mice, rabbits, dogs or pigs. The animal model is further used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in humans. The therapeutic efficacy and toxicity of nanoparticles can be determined by means of conventional pharmaceutical procedures, in cell cultures or experimental animals, for example, ED₅₀ (the dose that is therapeutically effective in 50% of the population) and the LD₅₀ (the lethal dose for 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Pharmaceutical compositions that exhibit high therapeutic indices may be useful in some embodiments. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.

In one embodiment, the compositions disclosed in this application may include about 10 ppm palladium or less, about 8 ppm palladium or less, or about 6 ppm palladium or less. For example, a composition is provided herein that includes nanoparticles having a polymeric conjugate where the composition has less than about 10 ppm palladium or less.

In one embodiment, the compositions disclosed in this application may include 10 ppm palladium or less, 8 ppm palladium or less, or 6 ppm palladium or less. For example, a

127 composition including nanoparticles having a polymeric conjugate where the composition has less than 10 ppm palladium or less.

In some embodiments, a composition suitable for freezing is contemplated, including nanoparticles disclosed in this application and a solution suitable for freezing, for example, a sugar such as a mono, di, or polysaccharide, for example, sucrose and / or a trehalose, and / or a salt and / or a cyclodextrin solution that is added to the nanoparticle suspension. Sugar (eg sucrose or trehalose) can function, for example, as a cryoprotectant in order to prevent aggregation of the particles upon freezing. For example, provided in this application is a nanoparticle formulation comprising a plurality of the disclosed nanoparticles, sucrose, an ion halide, and water; where nanoparticles / sucrose / water / ionic halide are around 340% / 10-40% / 20-95% / 0.1-10% (w / w / w / w) or around 5-10% / 10-15 % / 8090% / 1—10% (p / p / p / p). For example, said solution can include nanoparticles as disclosed in this application, about 5% to about 20% by weight of sucrose and an ionic halide such as sodium chloride, in a concentration of about 10-100 mM. In another example, provided in the present application is a nanoparticle formulation comprising a plurality of the disclosed nanoparticles, trehalose, cyclodextrin, and water; where nanoparticles / trehalose / water / cyclodextrin are around 340% / 1-25% / 20—95% / 1—25% (w / w / w / w) or around 5-10% / 1 -25% / 8090% / 10-15% (p / p / p / p).

In another example, the present application provides a nanoparticle formulation comprising a plurality of the disclosed nanoparticles, sucrose, an ionic halide and water; where nanoparticles / sucrose / water / ion halide are 3-40% / 10-40% / 20-95% / 0.1

128

10% (p / p / p / p) or 5-10% / 10-15% / 80-90% / 1-10% (p / p / p / p). For example, said solution can include nanoparticles as disclosed in this application, 5% to 20% by weight of sucrose and an ionic halide such as sodium chloride, in a concentration of 10-100 mM. In another example, provided in the present application is a nanoparticle formulation comprising a plurality of the disclosed nanoparticles, trehalose, cyclodextrin, and water; where nanoparticles / trehalose / water / cyclodextrin are 3-40% / 1-25% / 2095% / 1-25% (w / w / w / w) or 5-10% / 1-25% / 80-90 % / 10-15% (p / p / p / p).

For example, a contemplated solution may include nanoparticles as disclosed in this application, about 1% to about 25% by weight of a disaccharide such as trehalose or sucrose (for example, about 5% to about 25% trehalose. or sucrose, for example about 10% trehalose or sucrose, or about 15% trehalose or sucrose, for example about 5% sucrose) by weight) and a cyclodextrin such as β-cyclodextrin, in a concentration of from about 1% to about 25% by weight (for example about 5% to about 20%, for example 10% or about 20% by weight, or about 15% to about 20% by weight of cyclodextrin). Contemplated formulations may include a plurality of the disclosed nanoparticles (eg, nanoparticles having PLAPEG and an active ingredient), and about 2% to about 15% by weight (or about 4% to about 6% by weight , for example, about 5% by weight) sucrose and about 5% by weight to about 20% (for example, about 7% by weight to about 12% by weight, for example, about 10% by weight) of a cyclodextrin, for example, HPbCD).

In another example, a contemplated solution may include nanoparticles as disclosed in this application, 1% to 25% by weight of a disaccharide such as trehalose or sucrose (eg, 5% to 25% trehalose or sucrose,

129 for example, 10% trehalose or sucrose, or 15% trehalose or sucrose, for example 5% sucrose) by weight) and a cyclodextrin such as β-cyclodextrin, in a concentration of 1% to 25% by weight (for eg 5% to 20%, eg 10% or 20% by weight, or 15% to 20% by weight of cyclodextrin). Contemplated formulations can include a plurality of the disclosed nanoparticles (eg, nanoparticles having PLA-PEG and an active ingredient), and 2% to 15% by weight (or 4% to 6% by weight, eg 5% by weight) of sucrose and 5% by weight to 20% (for example, 7% by weight to 12% by weight, for example 10% by weight) of a cyclodextrin, for example HPbCD).

The present disclosure relates, in part, to lyophilized pharmaceutical compositions which, when reconstituted, have a minimal amount of large aggregates. Such large aggregates can be about 0.5 pm or larger, about 1 pm or larger, about 10 pm or larger, and may be undesirable in a reconstituted solution. Aggregate sizes can be measured using a variety of techniques including those set forth in the United States Pharmacopeia ("USP") at &lt;788&gt;, incorporated into this application by reference. These tests reviewed in USP &lt;788&gt; include a light obscuration particle count test, microscopic particle count test, laser diffraction, and single particle optical detection. In one embodiment, the particle size in a given sample is measured using laser diffraction and / or single particle optical detection.

USP &lt;788&gt; for a light obscuration particle count test establishes guidelines for sampling the particle sizes in a suspension. For solutions with an amount less than or equal to 100 ml, the preparation meets the test if the number

130 Average number of particles present does not exceed 6000 per container, which are&gt; 10 pm, and 600 per container, which are&gt; 25 pm.

As outlined in USP &lt;788&gt;, the microscopic particle count test establishes guidelines for the determination of particle quantities using a binocular microscope set to 100 ± 10x magnification having an eyepiece micrometer. An ocular micrometer is a circular diameter graticule consisting of a circle divided into quadrants with black reference circles denoting 10 pm and 25 pm when viewed at 100x magnification. A linear scale is provided below the graticule. The number of particles with reference to 10 pm and 25 pm is visually adjusted. For solutions with an amount less than or equal to 100 ml, the preparation complies with the test if the average number of particles present does not exceed 3000 per container that are&gt; 10 pm, and 300 per container, that are&gt; 25 pm.

In some embodiments, a 10 ml aqueous sample of a disclosed composition, upon reconstitution, comprises fewer than 600 particles per ml that are greater than or equal to 10 microns in size; and / or less than 60 particles per mi that are greater than or equal to 25 microns in size.

Dynamic light scattering (DLS) can be used for particle size measurement, although it is based on Brownian motion so the technique may miss some larger particles. Laser diffraction is based on the differences in refractive index between the particle and the suspension medium. The technique is capable of detecting particles in the submicron to millimeter range. Relatively small amounts (eg, about 1-5% by weight) of larger particles can be determined in suspensions of

131 nanoparticles. Single Particle Optical Detection (SPOS) uses light dimming of dilute suspensions to quantify individual particles around 0.5 pm. By knowing the particle concentration of the measured sample, the weight percentage of aggregates or the aggregate concentration (particles / ml) can be calculated.

Aggregate formation can take place during lyophilization due to dehydration of the surface of the particles. This dehydration can be avoided by using lyoprotectants, such as disaccharides, in the suspension, prior to lyophilization. Suitable disaccharides include sucrose, lactulose, lactose, maltose, trehalose, or cellobiose, and / or mixtures thereof. Other contemplated disaccharides include kojibiose, nigerosa, isomaltose, β, β-trehalose, α, β-trehalose, soforose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiase, melibiose, melibiulose, rutylinose, and x-ray. Reconstitution shows equivalent size distributions of DLS compared to the initial suspension. However, laser diffraction can detect particles&gt; 10 pm in size in some reconstituted solutions. Additionally, SPOS can also detect particles&gt; 10 pm in size at a concentration higher than that of the FDA [United States Food and Drug Administration] guidelines (10⁴-10⁵ particles / ml for particles&gt; 10 pm).

In some embodiments, one or more ionic halide salts can be used as an additional lyoprotectant to a sugar, such as sucrose, trehalose, or mixtures thereof. Sugars can include disaccharides, monosaccharides, trisaccharides and / or polysaccharides, and can include other excipients, for example, glycerol and / or surfactants. Optionally, a cyclodextrin can be included as an additional lyoprotectant. Cyclodextrin

132 it can be added in place of the ion halide salt. Alternatively, the cyclodextrin can be added in addition to the ionic halide salt.

Suitable ionic halide salts can include sodium chloride, calcium chloride, zinc chloride, or mixtures thereof. Additional suitable salts of ionic halide include potassium chloride, magnesium chloride, ammonium chloride, sodium bromide, calcium bromide, zinc bromide, potassium bromide, magnesium bromide, ammonium bromide, sodium iodide, iodide. of calcium, zinc iodide, potassium iodide, magnesium iodide or ammonium iodide, and / or their mixtures. In one embodiment, about 1 to about 15 weight percent sucrose can be used with an ion halide salt. In one embodiment, 1 to 15 weight percent sucrose can be used with an ionic halide salt. In one embodiment, the lyophilized pharmaceutical composition may comprise from about 10 to about 100 mM sodium chloride. In one embodiment, the lyophilized pharmaceutical composition may comprise 10 to 100 mM sodium chloride. In another embodiment, the lyophilized pharmaceutical composition may comprise about 100 to about 500 mM of divalent ionic chloride salt, such as calcium chloride or zinc chloride. In another embodiment, the lyophilized pharmaceutical composition may comprise 100 to 500 mM of divalent ionic chloride salt, such as calcium chloride or zinc chloride. In still another embodiment, the suspension to be lyophilized may further comprise a cyclodextrin, for example, about 1 to about 25 weight percent cyclodextrin may be used. In still another embodiment, the suspension to be lyophilized may additionally comprise a cyclodextrin, for example 1 to 25 weight percent cyclodextrin may be used.

133

A suitable cyclodextrin can include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or mixtures thereof. Examples of cyclodextrins contemplated for use in the compositions disclosed in this application include hydroxypropyl-p-cyclodextrin (HPbCD), hydroxyethyl-β-cyclodextrin, sulfobutyl ether-p-cyclodextrin, methyl-P-cyclodextrin, dimethyl-P-cyclodextrin, carboxymethyl -p-cyclodextrin, carboxymethyl ethyl

-β-cyclodextrin, diethyl-β-cyclodextrin, tri-O-alkyl-β-cyclodextrin, glocosyl-p-cyclodextrin and maltosyl-P-cyclodextrin. In one embodiment, about 1 to about 25 weight percent trehalose (eg, about 10% to about 15%, eg, 5 to about 20% by weight) with cyclodextrin. In one embodiment, the lyophilized pharmaceutical composition may comprise from about 1 to about 25 weight percent β-cyclodextrin. An exemplary composition may comprise nanoparticles comprising PLA-PEG, an active ingredient / therapeutic agent, about 4% to about 6% (eg, about 5% by weight) of sucrose and about 8 to about 12%. percent by weight (eg about 10 percent by weight) HPbCD. In one embodiment, 1 to 25 percent by weight of trehalose (eg, 10% to 15%, eg, 5 to 20% by weight) can be used with cyclodextrin. In one embodiment, the lyophilized pharmaceutical composition may comprise 1 to 25 percent by weight of β-cyclodextrin. An exemplary composition can comprise nanoparticles comprising PLA-PEG, an active ingredient / therapeutic agent, 4% to 6% (eg 5% by weight) sucrose, and 8 to 12 percent by weight (eg 10 percent by weight). by weight) of HPbCD.

In one aspect, a lyophilized pharmaceutical composition is provided comprising the disclosed nanoparticles, where, upon reconstitution of the

134 lyophilized pharmaceutical composition in a nanoparticle concentration of about 50 mg / ml, in a lower amount or about 100 ml of an aqueous medium, the reconstituted composition suitable for parenteral administration comprises less than 6000, such as less than 3000, microparticles with a size greater than or equal to 10 microns; and / or less than 600, such as less than 300, microparticles greater than or equal to 25 microns.

The amount of microparticles can be determined by means known to those skilled in the art, such as those described in USP &lt;788&gt;; by means of a light obscuration particle count test, such as that described in USP &lt;788&gt;; by the test of microscopic particle count, laser diffraction and single particle optical detection.

In one aspect, a pharmaceutical composition suitable for parenteral use with reconstitution is provided, which comprises a plurality of therapeutic particles comprising a copolymer having a hydrophobic polymer segment and a hydrophilic polymer segment; an active ingredient; a sugar; and a cyclodextrin.

For example, the copolymer can be poly (lactic acid) -poly (ethylene) glycol block copolymer. Upon reconstitution, 100 ml of aqueous sample can comprise fewer than 6000 particles that are greater than or equal to 10 microns in size; and fewer than 600 particles that are greater than or equal to 25 microns in size.

The step of adding a disaccharide and an ionic halide salt may comprise the addition of about 5 to about 15 percent by weight of sucrose or about 5 to about 20 percent by weight of trehalose (for example, about 10 to about 20 percent by weight of

135 trehalose), and about 10 to about 500 mM ion halide salt. The ion halide salt can be selected from sodium chloride, calcium chloride and zinc chloride, or their mixtures. In one embodiment, about 1 to about 25 weight percent cyclodextrin is further added.

In another embodiment, the step of adding a disaccharide and an ionic halide salt may comprise the addition of 5 to 15 percent by weight of sucrose or 5 to 20 percent by weight of trehalose (eg, 10 to 20 weight percent trehalose), and 10 to 500 mM ion halide salt. In one embodiment, 1 to 25 weight percent cyclodextrin is further added.

In another embodiment, the step of adding a disaccharide and a cyclodextrin may comprise the addition of about 5 to about 15 percent by weight of sucrose or about 5 to about 20 percent by weight of trehalose (for example, about 10 to about 20 weight percent trehalose), and about 1 to about 25 weight percent cyclodextrin. In one embodiment, about 10 to about 15 weight percent cyclodextrin is added. The cyclodextrin can be selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or mixtures thereof.

In another embodiment, the step of adding a disaccharide and a cyclodextrin may comprise the addition of 5 to 15 percent by weight of sucrose or 5 to 20 percent by weight of trehalose (e.g., 10 to 20 percent in trehalose), and 1 to 25 weight percent cyclodextrin. In one embodiment, 10 to 15 percent by weight of cyclodextrin is added.

136

In another aspect, a method of preventing the substantial aggregation of particles in a pharmaceutical nanoparticle composition is provided, which comprises adding a sugar and a salt to the lyophilized formulation, in order to avoid aggregation of the nanoparticles upon reconstitution. . In one embodiment, a cyclodextrin is further added to the lyophilized formulation. In still another aspect, a method of preventing substantial particle aggregation in a pharmaceutical nanoparticle composition is provided, which comprises adding a sugar and a cyclodextrin to the lyophilized formulation, in order to avoid aggregation of the nanoparticles with the reconstitution.

A contemplated lyophilized composition may have a therapeutic particle concentration of greater than about 40 mg / ml. The formulation suitable for parenteral administration may have less than about 600 particles that are greater than 10 microns in size in a 10 ml dose. Lyophilization may comprise freezing the composition to a temperature greater than about -40 ° C, or for example, less than about -30 ° C, to form a frozen composition; and drying the frozen composition to form the lyophilized composition. The drying step can take place at about 0.066 mbar (50 mTorr) at a temperature of about -25 to about -34 ° C, or about -30 to about -34 ° C.

A contemplated lyophilized composition may have a therapeutic particle concentration of greater than 40 mg / ml. The formulation suitable for parenteral administration may have fewer than 600 particles having a size greater than 10 microns in a 10 ml dose. Freeze-drying may comprise freezing the composition at a temperature above 40 ° C, or for example, below -30 ° C, to form a composition.

137 frozen; and drying the frozen composition to form the lyophilized composition. The drying step can take place at 0.066 mbar (50 mTorr) at a temperature of -25 to -34 ° C, or -30 to -34 ° C.

Treatment methods.

In some embodiments, the targeted nanoparticles can be used for the purpose of treatment. As used in the present application, the terms "treat" or "treatment" mean relief, amelioration, palliation, delayed onset, inhibition of progression, reduction of severity, and / or reduction of incidence of one or more symptoms or traits of a disease, disorder, and / or condition. In some embodiments, the targeted nanoparticles can be used for the treatment of solid tumors, eg, cancer and / or cancer cells. In certain embodiments, the targeted nanoparticles or pharmaceutical compositions comprising the nanoparticles can be used for the treatment of any type of cancer where the prosthetic specific membrane antigen (PSMA) is expressed on the surface of cancer cells or in tumor neovasculature. in a subject in need, which includes neovasculature of the prostate or non-prosthetic solid tumors. Examples of the pSMA-related indication include, without limitation, prostate cancer, breast cancer, non-small cell lung cancer, colorectal carcinoma, and glioblastoma.

In some embodiments, the targeted nanoparticles or pharmaceutical compositions comprising the nanoparticles can be used for the preparation of a medicament for the relief, amelioration, amelioration, delay of onset, inhibition of advancement, reduction of

138 severity, and / or reduction in the incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, the targeted nanoparticles or pharmaceutical compositions comprising the nanoparticles can be used for the preparation of a medicament for the treatment of any type of cancer where the prosthetic specific membrane antigen (PSMA) is expressed on the surface of the cells. cancer cells, or in tumor neovasculature in a subject in need thereof, including neovasculature of the prostate or solid non-prostatic tumors.

The term "cancer" includes premalignant as well as malignant types of cancer. Cancer types include, without limitation, cancer of the blood (eg, chronic myelogenous leukemia, chronic myelomonocytic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia, mantle cell lymphoma), prostate, gastric cancer, colorectal cancer , skin cancer, for example, melanomas or basal cell carcinomas, lung cancer (for example, non-small cell lung cancer), breast cancer, head and neck cancer, bronchial cancer, pancreatic cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, oral cavity or pharyngeal cancer, liver cancer (for example, hepatocellular carcinoma), kidney cancer ( eg, renal cell carcinoma), testicular cancer, cancer of the biliary tract, cancer of the small intestine or appendix, gastrointestinal stromal tumor, cancer of the salivary glands, cancer of the thyroid gland, cancer of the adrenal gland, osteosarcoma, chondrosarcoma, cancer of the hematological tissues, cancer of the head or neck, and the like. Cancer cells can present in the form of a tumor (ie, a solid tumor), they can present themselves within

139 from a subject (eg, leukemia cells), or they can be cell lines derived from a type of cancer.

Cancer can be associated with a variety of physical symptoms. Cancer symptoms generally depend on the type and location of the tumor. For example, lung cancer can cause coughing, shortness of breath, and chest pain, while colon cancer often causes diarrhea, constipation, and blood in the stool. However, to give just a few examples, the following symptoms are often generally associated with many types of cancer: fever, chills, night sweats, cough, dyspnea, weight loss, loss of appetite, anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly, hemoptysis, fatigue, malaise, cognitive dysfunction, depression, hormonal disturbances, neutropenia, pain , sores without healing, enlarged lymph nodes, peripheral neuropathy and sexual dysfunction.

In one aspect, a method for treating cancer (eg, leukemia) is provided. In some embodiments, the treatment of cancer comprises the administration of a therapeutically effective amount of the targeted particles of the invention to a subject in need thereof, in amounts and for as long as necessary to achieve the desired result. In certain embodiments, a "therapeutically effective amount" of a targeted particle of the invention is that amount effective to treat, alleviate, ameliorate, alleviate, delay onset, inhibit progression, reduce severity, and / or reduce the incidence of one or more symptoms or features of cancer.

In one aspect, a method is provided for administering the compositions of the invention to a subject suffering from cancer (eg, leukemia). In some embodiments, the particles can be administered to a subject in amounts and for as long as

140 necessary to achieve the desired result (i.e., cancer treatment). In certain embodiments, a "therapeutically effective amount" of a targeted particle of the invention is that amount effective to treat, alleviate, ameliorate, alleviate, delay onset, inhibit progression, reduce severity, and / or reduce the incidence of one or more symptoms or features of cancer.

The therapeutic protocols of the invention involve the administration of a therapeutically effective amount of a targeted particle of the invention to a healthy individual (ie, a subject who does not exhibit any symptoms of cancer and / or who has not been diagnosed with cancer). For example, healthy individuals can be "immunized" with a targeted particle of the invention, prior to the development of cancer and / or the onset of cancer symptoms; individuals at risk (eg, patients who have a family history of cancer; patients who carry one or more genetic mutations associated with the development of cancer; patients who have a genetic polymorphism associated with the development of cancer; patients infected with a virus associated with the development of cancer; patients with habits and / or lifestyles associated with the development of cancer; etc.) can be treated substantially contemporaneously with (eg, within 48 hours, within 24 hours, or within 12 hours) the onset of cancer symptoms. Of course, individuals known to have cancer can receive the treatment of the invention at any time.

In other embodiments, the disclosed nanoparticles can be used to inhibit the growth of cancer cells, eg, myelogenous leukemia cancer cells. As used in the present application, the term "inhibits the growth of cancer cells" or "inhibits the growth of cancer cells" refers to any decrease in the

141 speed of proliferation and / or migration of cancer cells, arrest of proliferation and / or migration of cancer cells, or death of cancer cells, such that the growth rate of cancer cells is reduced compared to that observed or predicted rate of growth of an untreated control cancer cell. The term "inhibits growth" can further refer to a reduction in the size or disappearance of a cancer cell or tumor, as well as a reduction in its metastatic potential. Preferably, such inhibition at the cellular level can reduce size, arrest growth, reduce aggression, or prevent or inhibit metastasis of a cancer in a patient. Those skilled in the art can determine without difficulty, by any of a variety of suitable cues, whether the growth of cancer cells is inhibited.

Inhibition of cancer cell growth can be evidenced, for example, by arrest of cancer cells in a particular phase of the cell cycle, eg, arrest in the G2 / M phase of the cell cycle. Inhibition of cancer cell growth can further be evidenced by direct or indirect measurement of tumor or cancer cell size. In human cancer patients, such measurements are generally performed using well-known imaging methods, such as magnetic resonance imaging, computerized axial tomography, and X-rays. Cancer cell growth can further be determined indirectly, such as by determining levels of circulating carcinoembryonic antigen, prostate specific antigen, or other cancer specific antigens that correlate with cancer cell growth. Inhibition of

142 Cancer growth is also generally correlated with prolonged survival and / or greater health and well-being of the subject.

Also provided in this application are methods of administration to a patient, of the nanoparticles disclosed in this application that include an active agent, where, upon administration to a patient, said nanoparticles substantially reduce the volume of distribution and / or substantially reduce the C_mfree ax, compared to administration of the agent alone (ie, not as a disclosed nanoparticle).

In some embodiments, the therapeutic nanoparticle is administered with a compound selected from the group consisting of a topoisomerase I inhibitor, a MEK 1/2 inhibitor, an HSP90 inhibitor, procarbazine, dacarbazine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campatechins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5-fluorouracil, docetaxel, paclitaxel, leucovorin, levamisole, nitrogenous, irinotecan, BCNuustin, carobustin, erynotecan vinblastine, vincristine, vinorelbine, oxaliplatin, imatinib mesylate, bevacizumab, hexamethylmelamine, topotecan, tyrosine kinase inhibitors, thyphostines, herbimycin A, genistein, erbstatin, hydroxyzine, glatiramer acetate, interferon beta-1a, interferon beta-1b, natalizumab, and lavendustine A; and a pharmaceutically acceptable carrier.

In another aspect, a therapeutic nanoparticle is provided as described in the present application for use as a medicament in a subject.

143

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in producing an antiproliferative effect in a subject.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in a subject as an anti-invasive agent in the containment and / or treatment of solid tumor disease.

In yet another aspect, there is provided the use of a therapeutic nanoparticle as described in the present application in the prevention or treatment of cancer in a subject.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in the prevention or treatment of cancer in a subject.

In yet another aspect, the use of a therapeutic nanoparticle as described in the present application is provided in the manufacture of a medicament for the prevention or treatment of cancer in a subject.

In still another aspect, the use of a therapeutic nanoparticle as described in the present application for the production of an antiproliferative effect in a subject is provided.

In still another aspect, the use of a therapeutic nanoparticle as described in the present application is provided in the manufacture of a medicament for use in producing an antiproliferative effect in a subject.

In still another aspect, the use of a therapeutic nanoparticle as described in the present application is provided in the manufacture of a medicament for use in a subject as an anti-invasive agent in the containment and / or treatment of a solid tumor disease. .

144

In yet another aspect, there is provided a method for producing an antiproliferative effect in a subject in need of such treatment, comprising administering to said subject an effective amount of a therapeutic nanoparticle as described in the present application.

In still another aspect, there is provided a method for producing an anti-invasive effect by containing and / or treating a solid tumor disease in a subject in need of such treatment, comprising administering, to said subject, an amount effectiveness of a therapeutic nanoparticle as described in the present application.

In yet another aspect, a therapeutic nanoparticle is provided as described in the present application for use in the prevention or treatment of a solid tumor disease in a subject.

In yet another aspect, there is provided the use of a therapeutic nanoparticle as described in the present application in the manufacture of a medicament for use in the prevention or treatment of a solid tumor disease in a subject.

In yet another aspect, there is provided a method for the prevention or treatment of a solid tumor disease in a subject in need of such treatment, comprising administering, to said subject, an effective amount of a therapeutic nanoparticle as described in the present application.

US Patent No. 8,206,747, issued June 26, 2012, entitled "Drug-loaded polymeric nanoparticles, and methods for their manufacture and use" is incorporated into this application by reference, in its entirety.

FORMS OF REALIZATION

145

Some embodiments of this invention are as follows.

1. A therapeutic nanoparticle comprising:

about 0.05 to about 30 percent by weight of a substantially hydrophobic acid;

about 0.2 to about 25 percent by weight of a therapeutic agent; where the pK_to of the protonated therapeutic agent is by or less about 1.0 pKa units greater than the pK_to hydrophobic acid; and about 50 to about 99.75 weight percent of a polymer selected from a poly (lactic acid) diblock copolymer - poly (ethylene) glycol or a poly (lactic acid-co-glycolic acid) diblock copolymer - poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol, where the therapeutic agent is 1- (4 - {[4 (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3- [4- (4,6-dimorpholin-4-yl-1,3,5— triazin -2-l) phenyl] urea or a pharmaceutically acceptable salt thereof.

2. The therapeutic nanoparticle of embodiment 1, where the amount of the therapeutic agent is about 0.2 to about 20 percent by weight.

3. The therapeutic nanoparticle according to embodiment 1 or 2, comprising:

- (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6— dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea;

and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 7 of 1— (4 — ¿[4— (dimethylamine) piper¡din — 1 — yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-l—1,3,5-triazin-2-yl) phenyl] urea: PLA-PEG.

Four. The therapeutic nanoparticle according to embodiment 1 or 2, comprising:

146 - (4 - {[4— (dimethylamino) pipe ridin-1 -yl] carbonyl} phenyl) -3— [4— (4,6dimorpholin-4-¡I-1,3,5-triazin-2-yl) phenyl] urea;

and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1:14 of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3 - [4— (4,6-dimorpholin — 4-11—1,3,5-triazin-2-yl) phenyl] urea: PLA-PEG.

5. The therapeutic nanoparticle according to embodiment 1 or 2, comprising:

- (4 - {[4— (dimethylamino) piperidin-1 -yl] carbonyl} phenyl) -3— [4— (4,6— dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea;

and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of about 1: 5 of 1— (4 - {[4— (dimethylamino) piperidin — 1 —yl] carbonyl} phenyl) —3 - [4— (4,6-dimo rf or I in-4-i 1-1,3,5-tri azi n-2-yl) phen i I] u rea: P LA-P EG.

6. A therapeutic nanoparticle comprising:

about 0.2 to about 25 percent by weight of a therapeutic agent;

a substantially hydrophobic acid, where the molar ratio of the substantially hydrophobic acid to the therapeutic agent ranges from about 0.25: 1 to about 2: 1 and where the pKa of the protonated therapeutic agent is at least about 1.0 pKa units greater than the pK_to hydrophobic acid; and about 50 to about 99.75 weight percent of a polymer selected from a poly (lactic acid) diblock copolymer - poly (ethylene) glycol or a poly (lactic acid-co-glycolic acid) diblock copolymer - poly (ethylene) glycol and combinations thereof, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene) glycol, where the therapeutic agent is 1- (4 - {[4 (dimethylamino) piperidin — 1 —iljcarbonyljphenyl) —3— [4— (4,6 — dimorpholin — 4 — ¡1—1,3,5— triazin — 2— yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

147

7. The therapeutic nanoparticle of embodiment 6, wherein the amount of the therapeutic agent is about 0.2 to about 20 weight percent.

8. A therapeutic nanoparticle comprising:

a substantially hydrophobic acid;

a therapeutic agent; where the pK_to of the protonated therapeutic agent is at least about 1.0 pKa units greater than the pK_to hydrophobic acid; and a polymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer or a poly (lactic acid-co-glycolic acid) -poly (ethylene) glycol diblock copolymer and combinations thereof, where the therapeutic agent is 1— (4 - {[4— (dimethylamine) piper¡din-1-yl] carbon¡l} phenyl) —3— [4— (4,6— dimorpholin — 4 — ¡1—1 , 3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

9. A therapeutic nanoparticle comprising:

a therapeutic agent;

a substantially hydrophobic acid, where the molar ratio of the substantially hydrophobic acid to the therapeutic agent ranges from about 0.25: 1 to about 2: 1 and where the pKa of the protonated therapeutic agent is at least about 1.0 pKa units greater than the pK_to hydrophobic acid; and a polymer selected from poly (lactic acid) -poly (ethylene) glycol diblock copolymer or a poly (lactic acid-co-glycolic acid) -poly (ethylene) glycol diblock copolymer and combinations thereof, and, where the therapeutic agent is 1 - (4 - {[4— (dimethylamino) piperidine-1 -yl] carbonyl} phenyl) —3— [4— (4,6— dimorpholin-4-yl — 1,3,5-triazin— 2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

10. The therapeutic nanoparticle of embodiment 6 or 9,

148 where the molar ratio of the substantially hydrophobic acid to the therapeutic agent is from about 0.5: 1 to about 1.5: 1.

eleven. The therapeutic nanoparticle of embodiment 6 or 9, where the molar ratio of substantially hydrophobic acid to therapeutic agent is from about 0.25: 1 to about 1: 1.

12. The therapeutic nanoparticle of embodiment 6 or 9, where the molar ratio of substantially hydrophobic acid to therapeutic agent is from about 0.75: 1 to about 1.25: 1.

13. The therapeutic nanoparticle of any of embodiments 1-12, where the pK_to of the protonated therapeutic agent is at least about 2.0 pKa units greater than the pK_to of hydrophobic acid.

14. The therapeutic nanoparticle of any of embodiments 1-12, where the pK_to of the protonated therapeutic agent is at least about 4.0 pKa units greater than the pK_to of hydrophobic acid.

fifteen. A therapeutic nanoparticle comprising:

a hydrophobic ion pair comprising a hydrophobic acid and therapeutic agent; where the difference between the pKa of the protonated therapeutic agent and the hydrophobic acid is at least about 1.0 pKa units or greater; and about 50 to about 99.75 weight percent of a poly (lactic) acid-poly (ethylene) glycol diblock copolymer, where the poly (lactic acid) -poly (ethylene) glycol copolymer has an average molecular weight in number from about 15 kDa to about 20 kDa of poly (lactic acid) and a number average molecular weight of about 4 kDa to about 6 kDa of poly (ethylene) glycol, where the therapeutic agent is 1— (4 - {[4— (dimethylamino) piperidin-1 -yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl— 1,3,5— triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

16. The therapeutic nanoparticle of embodiment 15, where

149 the difference between the pKa of the protonated therapeutic agent and the hydrophobic acid is at least about 2.0 pKa units.

17. The therapeutic nanoparticle of embodiment 15, where the difference between the pKa of the protonated therapeutic agent and the hydrophobic acid is at least about 4.0 pKa units.

18. The therapeutic nanoparticle of any of embodiments 1-5, 8, or 13-17, comprising about 0.05 to about 20 weight percent of the hydrophobic acid.

19. The therapeutic nanoparticle of any of embodiments 1-18, where the substantially hydrophobic acid has an iogP ranging from about 2 to about 7.

twenty. The therapeutic nanoparticle of any of embodiments 1-18, where the substantially hydrophobic acid has an IogP ranging from about 4 to about 8.

twenty-one. The therapeutic nanoparticle of any of embodiments 1-20, where the substantially hydrophobic acid has a pK_to in water from about -1.0 to about 5.0.

22. The therapeutic nanoparticle of any of embodiments 1-20, where the substantially hydrophobic acid has a pK_to in water from about 2.0 to about 5.0.

22. The therapeutic nanoparticle of any of embodiments 1-22, where the substantially hydrophobic acid and the therapeutic agent form a hydrophobic ion pair on the therapeutic nanoparticle.

24. The therapeutic nanoparticle of any of embodiments 1-23, where the hydrophobic acid is a fatty acid.

25. The therapeutic nanoparticle of embodiment 24, wherein the fatty acid is a saturated fatty acid selected from the group consisting

150 in: caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosilic acid, behenic acid , tricosilic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melisic acid, henatriacontyl acid, lacceroic acid, psilic acid, gedic acid, ceroplastic acid, hexatriacontyl acid, and their combinations.

26. The therapeutic nanoparticle of embodiment 24, wherein the fatty acid is an omega-3 fatty acid selected from the group consisting of: hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid , docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, and combinations thereof.

27. The therapeutic nanoparticle of embodiment 24, wherein the fatty acid is an omega-6 fatty acid selected from the group consisting of: linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, and their combinations.

28. The therapeutic nanoparticle of embodiment 24, wherein the fatty acid is an omega-9 fatty acid selected from the group consisting of: oleic acid, eicosenoic acid, Mead acid, erucic acid, nervonic acid, and combinations thereof.

29. The therapeutic nanoparticle of embodiment 28, where the fatty acid is oleic acid.

30. The therapeutic nanoparticle of embodiment 29, where

151 the weight ratio of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4- (4,6-dimorpholin-4-II-1,3,5 -triazin-2-yl) phenyl] urea an oleic acid is about 6: 1.

31. The therapeutic nanoparticle of embodiment 24, wherein the fatty acid is a polyunsaturated fatty acid selected from the group consisting of: rumen acid, α-calendic acid, β-calendic acid, jacaric acid, α-eleostearic acid, β- eleostearic, cathalpic acid, punicic acid, rumelenic acid, α-parinaric acid, β-parinaric acid, bosseo pentaenoic acid, pinolenic acid, podocarpic acid, and their combinations.

32. The therapeutic nanoparticle of any of embodiments 1-24, wherein the hydrophobic acid is a bile acid.

33. The therapeutic nanoparticle of embodiment 32, wherein the bile acid is selected from the group consisting of chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, hicolic acid, beta-muricolic acid, cholic acid, lithocholic acid, an amino acid conjugated bile acid , and their combinations.

3. 4. The therapeutic nanoparticle of embodiment 33, where the bile acid is cholic acid.

35. The therapeutic nanoparticle of embodiment 33, wherein the amino acid-conjugated bile acid is a glycine-conjugated bile acid or a taurine-conjugated bile acid.

36. The therapeutic nanoparticle of any of embodiments 1-23, where the hydrophobic acid is selected from the group consisting of dioctyl sulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid, naphthalene-1,5-disulfonic acid, Naphthalene-2-sulfonic acid, pamoic acid, undecanoic acid, and combinations thereof.

152

37. The therapeutic nanoparticle of embodiment 36, wherein the hydrophobic acid is pamoic acid.

38. The therapeutic nanoparticle of embodiment 37, where the weight ratio of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin —4-II — 1,3,5-triazin-2-yl) phenyl] urea to pamoic acid is about 1.8: 1.

39. The therapeutic nanoparticle of any of embodiments 1-38, comprising from about 1 to about 20 percent by weight of the therapeutic agent.

40. The therapeutic nanoparticle of any of embodiments 1-38, comprising from about 2 to about 20 percent by weight of the therapeutic agent.

41. The therapeutic nanoparticle of any of embodiments 1-38, comprising from about 4 to about 20 percent by weight of the therapeutic agent.

42. The therapeutic nanoparticle of any of embodiments 1-38, comprising about 5 to about 20 weight percent of the therapeutic agent.

43. The therapeutic nanoparticle of any of embodiments 1-38, where the hydrophobic acid has a molecular weight of between about 200 Da and about 800 Da.

44. The therapeutic nanoparticle of any of embodiments 1-43, wherein the therapeutic nanoparticle substantially retains the therapeutic agent for at least 1 minute when placed in a 37 ° C phosphate buffer solution.

Four. Five. The therapeutic nanoparticle of any of embodiments 1-43, wherein the therapeutic nanoparticle substantially releases

153 Immediately less than about 30% of the therapeutic agent when placed in a phosphate buffer solution at 37 ° C.

46. The therapeutic nanoparticle of any of embodiments 1-43, where the therapeutic nanoparticle releases about 10 to about 45% of the therapeutic agent over about 1 hour when placed in a phosphate buffer solution at 37 ° C.

47. The therapeutic nanoparticle of any of the embodiments 1-43, where the therapeutic nanoparticle releases about 0.01 to about 15% of the therapeutic agent over about 4 hours when placed in a phosphate buffer solution at 37 ° C.

48. The therapeutic nanoparticle of any one of embodiments 1-43, wherein the therapeutic nanoparticle releases about 0.01 to about 15% of the therapeutic agent over about 10 hours when placed in a phosphate buffer solution at 37 ° C.

49. The therapeutic nanoparticle of any of embodiments 1-43, where the therapeutic nanoparticle releases about 0.01 to about 25% of the therapeutic agent over about 20 hours when placed in a phosphate buffer solution at 37 ° C.

fifty. The therapeutic nanoparticle of any of embodiments 1-43, wherein the therapeutic nanoparticle releases about 1 to about 40% of the therapeutic agent over about 40 hours when placed in a phosphate buffer solution at 37 ° C.

51. The therapeutic nanoparticle of any of embodiments 1-43, wherein the therapeutic nanoparticle has a release profile that is substantially the same as a release profile for a control nanoparticle that is substantially the same as the therapeutic nanoparticle except that it does not contain a fatty acid or bile acid.

154

52. The therapeutic nanoparticle of any of embodiments 1-51, wherein the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.6 to about of 0.95.

53. The therapeutic nanoparticle of any of embodiments 1-51, wherein the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.6 to about of 0.8.

54. The therapeutic nanoparticle of any of embodiments 1-51, wherein the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.75 to about of 0.85.

55. The therapeutic nanoparticle of any of embodiments 1-51, wherein the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight fraction of poly (lactic acid) of from about 0.7 to about of 0.9.

56. The therapeutic nanoparticle of any one of embodiments 1-55, wherein the therapeutic nanoparticle comprises about 10 to about 25 weight percent poly (ethylene) glycol.

57. The therapeutic nanoparticle of any of embodiments 1-55, wherein the therapeutic nanoparticle comprises about 10 to about 20 weight percent poly (ethylene) glycol.

58. The therapeutic nanoparticle of any one of embodiments 1-55, wherein the therapeutic nanoparticle comprises about 15 to about 25 weight percent poly (ethylene) glycol.

59. The therapeutic nanoparticle of any of embodiments 1-55, wherein the therapeutic nanoparticle comprises about

155 to about 30 weight percent poly (ethylene) glycol.

60. The therapeutic nanoparticle of any of embodiments 1-59, where the poly (lactic acid) -poly (ethylene) glycol copolymer has a number average molecular weight of about 15 kDa to about 20 kDa of poly (acid). lactic acid) and a number average molecular weight of about 4 kDa to about 6 kDa of poly (ethylene) glycol.

61. The therapeutic nanoparticle of any of embodiments 1-60, further comprising about 0.2 to about 30 weight percent poly (lactic acid) -poly (ethylene) glycol copolymer functionalized with a targeting ligand.

62. The therapeutic nanoparticle of any of embodiments 1-60, further comprising about 0.2 to about 30 weight percent poly (lactic acid) -acid copolymer poly (glycolic) poly (ethylene) glycol coi functionalized with a targeting ligand.

63. The therapeutic nanoparticle of embodiment 61 or 62, where the targeting ligand is covalently bound to the poly (ethylene) glycol

64. The therapeutic nanoparticle of any of embodiments 1-63, wherein the hydrophobic acid is a polyelectrolyte.

65. The therapeutic nanoparticle of embodiment 64, wherein the polyelectrolyte is selected from the group consisting of a poly (styrene sulfonic acid), polyacrylic acid, and polymethacrylic acid.

66. The therapeutic nanoparticle of any of embodiments 1-65, where the substantially hydrophobic acid is a mixture of two or more substantially hydrophobic acids.

67. The therapeutic nanoparticle of embodiment 66, comprising a mixture of two substantially hydrophobic acids.

156

68. The therapeutic nanoparticle of embodiment 67, where the two substantially hydrophobic acids are oleic acid and cholic acid.

69. The therapeutic nanoparticle of embodiment 66, comprising a mixture of three substantially hydrophobic acids.

70. The therapeutic nanoparticle of embodiment 66, comprising a mixture of four substantially hydrophobic acids.

71. The therapeutic nanoparticle of embodiment 66, comprising a mixture of five substantially hydrophobic acids.

72. A therapeutic nanoparticle prepared by a process comprising the steps of:

emulsifying a first organic phase comprising a first polymer, a therapeutic agent, and a substantially hydrophobic acid, so as to form an emulsion phase;

quenching the emulsion phase so as to form a quenched phase; and filtration of the quenched phase in order to recover the therapeutic nanoparticles, where the therapeutic agent is 1- (4 - {[4- (dimethylamino) piper¡din1 —il] carbon¡IJphenyl) —3— [4— (4 , 6-dimorpholin-4-II-1,3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

73. The therapeutic nanoparticle of embodiment 72, where the hydrophobic acid is a fatty acid.

74. The therapeutic nanoparticle of embodiment 73, wherein the fatty acid is a saturated fatty acid selected from the group consisting of: caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosilic acid, behenic acid, acid

157 tricosilic, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, meisic acid, henatriacontylic acid, lacceroic acid, psilic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, and their combinations.

75. The therapeutic nanoparticle of embodiment 73, wherein the fatty acid is an omega-3 fatty acid selected from the group consisting of: hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eiccosapentaenoic acid, heneicosapentaenoic acid , docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, and combinations thereof.

76. The therapeutic nanoparticle of embodiment 73, wherein the fatty acid is an omega-6 fatty acid selected from the group consisting of: linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, and their combinations.

77. The therapeutic nanoparticle of embodiment 73, wherein the fatty acid is an omega-9 fatty acid selected from the group consisting of: oleic acid, eicosenoic acid, Mead acid, erucic acid, nervonic acid, and combinations thereof.

78. The therapeutic nanoparticle of embodiment 77, where the fatty acid is oleic acid.

79. The therapeutic nanoparticle of embodiment 73, where the fatty acid is a polyunsaturated fatty acid selected from the group consisting of: rumen acid, α-calendic acid, β-calendic acid, jacaric acid, α-eleostearic acid, β- acid eleostearic acid, cathalpic acid, punicic acid, rumelenic acid, α-parinaric acid, β-parinaric acid, acid

158 bosseo pentaenoic, pinolenic acid, podocarpic acid, and their combinations.

80. The therapeutic nanoparticle of any of embodiments 72, wherein the hydrophobic acid is a bile acid.

81. The therapeutic nanoparticle of embodiment 80, where the bile acid is selected from the group consisting of chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, hicolic acid, beta-muricolic acid, cholic acid, lithocholic acid, an amino acid conjugated bile acid , and their combinations.

82. The therapeutic nanoparticle of embodiment 81, where the bile acid is cholic acid.

83. The therapeutic nanoparticle of embodiment 81, where the amino acid-conjugated bile acid is a glycine-conjugated bile acid or a taurine-conjugated bile acid.

84. The therapeutic nanoparticle of any of embodiments 72, wherein the hydrophobic acid is selected from the group consisting of dioctyl sulfosuccinic acid, 1-hydroxy-2-naphthoic acid, dodecylsulfuric acid, naphthalene-1,5-disulfonic acid, naphthalene acid -2-sulfonic, pamoic acid, undecanoic acid and their combinations.

85. The therapeutic nanoparticle of embodiment 84, where the hydrophobic acid is pamoic acid.

86. The therapeutic nanoparticle of any of embodiments 72-85, where the hydrophobic acid has a molecular weight of between about 200 Da and about 800 Da.

87. The therapeutic nanoparticle of any of embodiments 72-86, wherein the therapeutic nanoparticle substantially retains the therapeutic agent for at least 1 minute when placed in a

159 phosphate buffer solution at 37 ° C.

88. The therapeutic nanoparticle of any of embodiments 72-86, wherein the therapeutic nanoparticle immediately releases substantially less than about 30% of the therapeutic agent when placed in a phosphate buffer solution at 37 ° C.

89. The therapeutic nanoparticle of any of embodiments 72-86, where the therapeutic nanoparticle releases about 10 to about 45% of the therapeutic agent over about 1 hour when placed in a phosphate buffer solution at 37 ° C.

90. The therapeutic nanoparticle of any of embodiments 72-86, where the therapeutic nanoparticle releases about 0.01 to about 15% of the therapeutic agent over about 4 hours when placed in a phosphate buffer solution at 37 ° C.

91. The therapeutic nanoparticle of any of embodiments 72-86, where the therapeutic nanoparticle releases about 0.01 to about 15% of the therapeutic agent over about 10 hours when placed in a phosphate buffer solution at 37 ° C.

92. The therapeutic nanoparticle of any of embodiments 72-86, where the therapeutic nanoparticle releases about 0.01 to about 25% of the therapeutic agent over about 20 hours when placed in a phosphate buffer solution at 37 ° C.

93. The therapeutic nanoparticle of any of embodiments 72-86, wherein the therapeutic nanoparticle releases about 1 to about 40% of the therapeutic agent over about 40 hours when placed in a phosphate buffer solution at 37 ° C.

94. The therapeutic nanoparticle of any of embodiments 72-86, wherein the therapeutic nanoparticle has a profile of

160 release that is substantially the same as a release profile for a control nanoparticle that is substantially the same as the therapeutic nanoparticle except that it does not contain a fatty acid or bile acid.

95. The therapeutic nanoparticle of any of embodiments 72-94, wherein the first polymer is poly (lactic acid) -poly (ethylene) glycol copolymer.

96. The therapeutic nanoparticle of any of embodiments 72-94, wherein the first polymer is poly (lactic acid) -co-poly (glycolic acid) -poly (ethylene) glycol copolymer.

97. The therapeutic nanoparticle of any of embodiments 72-96, where the substantially hydrophobic acid is a mixture of two or more substantially hydrophobic acids.

98. The therapeutic nanoparticle of embodiment 97, comprising a mixture of two substantially hydrophobic acids.

99. The therapeutic nanoparticle of embodiment 97, comprising a mixture of three substantially hydrophobic acids.

100. The therapeutic nanoparticle of embodiment 97, comprising a mixture of four substantially hydrophobic acids.

101. The therapeutic nanoparticle of embodiment 97, comprising a mixture of five substantially hydrophobic acids.

102. The therapeutic nanoparticle of any of embodiments 1, 5-95, or 97-101, where the polymer is PLA-PEG and the molar ratio of PLA-PEG is 5: 1.

103. A therapeutic nanoparticle prepared by the process comprising the steps of:

combining a first organic phase with a first aqueous solution to form a second phase;

161 emulsifying the second phase to form an emulsion phase, wherein the emulsion phase comprises a first polymer, therapeutic agent, and a substantially hydrophobic acid;

quenching the emulsion phase so as to form a quenched phase; and filtration of the quenched phase in order to recover the therapeutic nanoparticles, where the therapeutic agent is 1- (4 - {[4- (dimethylamino) piperidin1-yl] carbonyl} phenyl) —3— [4— (4,6 —Dimorpholin —- 4 — ¡1—1,3,5-triazin — 2 — ¡l) phenyl] urea, the first organic phase comprises the therapeutic agent and pamoic acid in a weight ratio of therapeutic agent to pamoic acid of about of 11: 1, and PLA-PEG (in a molar ratio of 16: 5) in a weight ratio of therapeutic agent to PLA-PEG of about 1: 3, in an organic solvent comprising benzyl alcohol and ethyl acetate in a weight ratio of benzyl alcohol to ethyl acetate of about 1.25, and the first aqueous solution comprises a polyoxyethylene (100) stearyl ether dissolved in benzyl alcohol in a weight ratio of 0.005: 1; and combining the first organic phase and the first aqueous phase in a weight ratio of about 1: 5 to form a second phase; emulsion of the second phase formed therein and quenching of the emulsion phase with citric acid, 0.1 M, in aqueous solution at pH 4.5; and the concentration of the resulting product.

104. A therapeutic nanoparticle of 1- (4 - {[4 (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — ¡1—1,3,5— triazin -2-yl) phenyl] urea or its pharmaceutically acceptable salt.

105. A therapeutic nanoparticle comprising a therapeutic agent or one of its pharmaceutically acceptable salts and a polymer selected from a poly (lactic acid) -poly (ethylene) glycol diblock copolymer or a poly (lactic acid-co-glycolic acid diblock copolymer ) -

162 poly (ethylene) glycol and its combinations, where the therapeutic agent is 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4 —Il-1,3,5-triazin-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

106. The therapeutic nanoparticle of any of the embodiments 1-71, 104, or 105, where a targeting ligand is additionally presented, which is PLA-PEG-GL, where GL has the following structure:

NH

<img file="CU24488B1_D0009.tif" />

107. The therapeutic nanoparticle according to any of embodiments 1-71 or 104-106, further comprising a solubilizer.

108. The therapeutic nanoparticle according to embodiment 107, where the solubilizer is polysorbate 80.

109. The therapeutic nanoparticle of embodiment 107, where the solubilizer is polyoxyethylene (100) stearyl ether.

110. The therapeutic nanoparticle of any of embodiments 1-109, where the therapeutic agent is 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4, 6-dimorpholin-4-yl-1,3,5-triazi n-2-yl) phenyl] urea.

111. A pharmaceutical composition comprising a therapeutic nanoparticle of any of Embodiments 1-110 and a pharmaceutically acceptable excipient.

112. The pharmaceutical composition of embodiment 111 comprising a plurality of therapeutic nanoparticles.

113. The pharmaceutical composition of embodiment 111 or

163

112, which further comprises a saccharide.

114. The pharmaceutical composition of any of embodiments 111-113, further comprising a cyclodextrin.

115. The pharmaceutical composition of embodiments 113 or 114, wherein the saccharide is a disaccharide selected from the group consisting of sucrose, trehalose, and a mixture thereof.

116. A method of treating cancer in a subject in need, which comprises administering to the subject a therapeutically effective amount of a therapeutic nanoparticle of any of embodiments 1-110 or a pharmaceutical composition of any of the forms of realization 111-115.

117. The method of embodiment 116, where the cancer is chronic myelogenous leukemia.

118. The method of embodiment 116, where the cancer is gastrointestinal stromal tumor.

119. The method of embodiment 116, wherein the cancer is selected from the group consisting of chronic myelomonocytic leukemia, hypereosinophilic syndrome, renal cell carcinoma, hepatocellular carcinoma, Philadelphia chromosome positive acute lymphoblastic leukemia, non-cell lung cancer. small, pancreatic cancer, breast cancer, a solid tumor, head and neck cancer, and mantle cell lymphoma.

120. The method of embodiment 119, where the cancer is breast cancer.

121. A process for the preparation of a therapeutic nanoparticle, comprising the steps of:

the combination of a first organic phase with a first solution

164 aqueous to form a second phase;

emulsifying the second phase to form an emulsion phase, wherein the emulsion phase comprises a first polymer, therapeutic agent, and a substantially hydrophobic acid;

quenching the emulsion phase so as to form a quenched phase; and filtration of the quenched phase in order to recover the therapeutic nanoparticles, where the therapeutic agent is 1- (4 - {[4- (dimethylamino) piperidin1 —i I] carbonyl} phenyl I) —3— [4— ( 4,6-dimorpholin-4-yl-1,3,5-triazi n-2-yl) phenyl] urea or a pharmaceutically acceptable salt thereof.

122. The process of embodiment 121, further comprising combining the therapeutic agent and the substantially hydrophobic acid in the second phase prior to emulsifying the second phase.

123. The process of embodiment 122, where the therapeutic agent and the substantially hydrophobic acid form a hydrophobic ion pair prior to emulsifying the second phase.

124. The process of embodiment 122, where the therapeutic agent and the substantially hydrophobic acid form a hydrophobic ion pair prior to or during second phase emulsification.

125. The process of embodiment 121, further comprising combining the therapeutic agent and the substantially hydrophobic acid in the second phase substantially concurrently with the emulsion in the second phase.

126. The process of embodiment 125, wherein the first organic phase comprises the therapeutic agent, and the first aqueous solution comprises the substantially hydrophobic acid.

127. The process of any of embodiments 121-126,

165 where the therapeutic agent, when protonated, has a first pK_to, the substantially hydrophobic acid has a second pK_to, and the emulsion phase is quenched with an aqueous solution having a pH equal to one pKa unit between the first pK_to and the second pK_to.

128. The process of embodiment 127, where the quenched phase has a pH equal to one pKa unit between the first pK_to and the second pK_to.

129. The process of any of embodiments 121-128, where the therapeutic agent, when protonated, has a first pK_to, the substantially hydrophobic acid has a second pK_to, and the first aqueous solution has a pH equal to one pKa unit between the first pK_to and the second pK_to.

130. The process of any of embodiments 127-129, where the pH equals one pKa unit that is approximately equidistant between the first pK_to and the second pK_to.

131. The process of any one of embodiments 121-130, where the therapeutic agent is 1- (4 - {[4- (dimethylamino) piperidin-1yl] carbonyl} phenyl) —3— [4— (4,6 —Dimorpholin-4-II-1,3,5-triazin-2-yl) phenyl] urea.

EXAMPLES

Having described the invention in general terms, it will be better understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments, and which are not intended to limit the invention in any way.

EXAMPLE 1 - PREPARATION OF FORMULATION A WITH A THERAPEUTIC AGENT.

166 (a) Preparation of organic phase loading: Benzyl alcohol (8932.5 mg) was dissolved in 67.5 mg of RODI water (deionized by reverse osmosis) with mixing. The therapeutic agent, 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — II— 1,3,5— Triazi n-2-yl) phenyl] urea, (150 mg), was added to the solution, and then the solution was sonicated until dissolution of the drug. PLA-PEG-GL (19.2 mg) and PLA-PEG were added in a ratio of 16 mol / 5 mol (830.8 mg) and vortexed until dissolution.

(b) Preparation of aqueous phase loading: Sodium cholate (2.75 g) was dissolved in RODI water (955.5 g) on ​​a stir plate. Benzyl alcohol (40 g) was added to the sodium cholate / water solution, and the mixture was stirred on a stir plate until dissolved.

(c) Emulsion formation: The weight ratio of aqueous phase to organic phase was 5: 1. The organic phase, weighing 10 g, was poured into 50 g of the aqueous phase which was cooled in a water bath, and the mixture was homogenized using a hand-held homogenizer for 15 seconds. The coarse emulsion was fed through a high pressure homogenizer with the pressure set at 722.9 bar (10485 psi) on the gauge for 1 pass to form a nanoemulsion (fine emulsion).

(d) Nanoparticle formation: the nanoemulsion was poured into 600 g of cold RODI water (less than 2 ° C) while stirring on a stir plate to form a quenched phase (the quench to emulsion weight ratio was 10: 1). 64.3 grams of a polysorbate 80 solution (350 grams dissolved in 650 g of RODI water) were added to the quenched phase with mixing.

(e) Concentration of nanoparticles through tangential flow filtration (TFF): The quenched phase was concentrated using TFF with Pali 300 kDa cassette (2 membranes) to form a nanoparticle concentrate of

167 approximately 200 ml. The nanoparticle concentrate was diafiltered with approximately 20 diavolumes of cold RODI water at less than 2 ° C. The volume of the diafiltered nanoparticle concentrate was reduced to a minimum volume.

Therefore, this formulation contained 1- (4 - {[4- (dimethylamino) piperidin1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl-1, 3,5-triazin-2-yl) phenyl] urea, and the polymers PLA-PEG (in a molar ratio of 16: 5) and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG- GL of about 43: 1 and a therapeutic agent weight ratio, 1- (4 - {[4- (dimethylamine) piperdin-1-yl] carbonyl} phenyl) —3— [4— (4.6 —Dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea, to polymers, 15:85. There was no counter ion or hydrophobic acid in this formulation. The particle size of a nanoparticle so formed as described in the present application above was around 116 nm.

EXAMPLE 2 - PREPARATION OF FORMULATION B WITH THERAPEUTIC AGENT.

(a) Preparation of organic phase loading: Oleic acid (900 mg), trifluoroacetic acid (TFA) (273 mg) were dissolved in benzyl alcohol (8827 mg). The therapeutic agent, 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl I) —3— [4— (4,6-di morpholin-4-yl-1,3, 5-triazi n-2-yl) phenyl] urea (120 mg), was mixed with the oleic acid / TFA / benzyl alcohol solution, and the mixture was heated to 80 ° C for 10 minutes in order to dissolve the agent there therapeutic. Once the 1- (4 - {[4- (dimethylamino) piperidine-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl-1,3,5-triazin-2 —Yl) phenyl] urea, the solution was allowed to cool to room temperature. This solution was thoroughly mixed with a polymer solution of PLA-PEG in a ratio of 16 moles / 5 moles (860 mg), PLA-PEG-GL (18.9 mg) and ethyl acetate (4549 mg) to form a solution.

168 (b) Preparation of aqueous phase loading: Sodium cholate (4.5 g) was dissolved in RODI water (955.5 g) on ​​a stir plate. Benzyl alcohol (40 g) was added to the sodium cholate / water solution, and the mixture was stirred on a stir plate until dissolved.

(c) Emulsion formation: The weight ratio of aqueous phase to organic phase was 5: 1. The organic phase was poured into 33.4 g of the aqueous phase which was cooled in an ice water bath, and the mixture was homogenized using a manual homogenizer for 15 seconds. The coarse emulsion was fed through a high pressure homogenizer with the pressure set at 722.9 bar (10485 psi) on the gauge for 1 pass, to form a nanoemulsion (fine emulsion).

(d) Nanoparticle formation: The nanoemulsion was poured into 401.2 g of cold RODI water (less than 2 ° C) while stirring on a stir plate to form a quenched phase. To the quenched phase 51.4 grams of a polysorbate 80 solution (350 g dissolved in 650 g RODI water) were added with mixing.

(e) Concentration of nanoparticles through tangential flow filtration (TFF): The quenched phase was concentrated using TFF with Pali 300 kDa cassette (2 membranes) to form a nanoparticle concentrate of approximately 200 ml. The nanoparticle concentrate was diafiltered with approximately 20 diavolumes of cold RODI water at less than 2 ° C. The volume of the diafiltered nanoparticle concentrate was reduced to a minimum volume.

Consequently, this formulation contained 1- (4 - {[4 (dimethylamino) piperidi n-1 -¡l] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl-1,3 , 5-triazin-2-yl) phenyl] urea, and the polymers PLA-PEG (in a molar ratio of 16: 5) and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL

169 of about 46: 1 and a therapeutic agent weight ratio, 1- (4 - {[4 (dimethylamino) piperidine-1 — l] carbonyl} phenyl) —3— [4— (4,6-dimorpholin— 4-I-1,3,5-triazin-2-yl) phenyl] urea, to polymers, from 12:88. It contained about 5.7% by weight of 1 - (4 - {[4— (dimethylamino) piperidine-1 -yl] carbonyl} phenyl) -3— [4— (4,6— dimorpholin-4-yl-1.3 , 5-triazin-2-yl) phenyl] urea and about 9% by weight of oleic acid in 3% of trifluoroacetic acid. The particle size of a nanoparticle thus formed as described in the present application above was around 74 nm.

EXAMPLE 3. PREPARATION OF FORMULATION C WITH A THERAPEUTIC AGENT.

(a) Preparation of organic phase load: Trifluoroacetic acid (1600 mg), benzyl alcohol (8827 mg) and RODI water (1500 mg) were mixed, and if necessary, heated to form a solution. To this solution was added the therapeutic agent, 1- (4 - {[4- (dimethylamino) piperidine-1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — ¡1—1.3 , 5-triazin-2-yl) phenyl] urea (1468.8 mg), and the resulting mixture was sonicated to form a solution. After the therapeutic agent dissolved, the solution was allowed to cool to room temperature. This solution was added to a solution of pamoic acid (136.5 mg) and DMSO (331.2 mg). This solution was thoroughly mixed with a PLA-PEG polymer solution in a ratio of 16 mol / 5 mol (643.5 mg) of PLAPEG-GL (14.5 mg) and ethyl acetate (7200 mg).

(b) Preparation of aqueous phase charge: A surfactant, Brij S 100 (polyoxyethylene (100) stearyl ether) (200 mg) was dissolved in benzyl alcohol (40.0 g), with stirring, and cold RODI water (959.8 g ) and mixed on ice until the solution cleared. The aqueous phase charge was cooled to less than 2 ° C with stirring.

170 (c) Emulsion formation: The weight ratio of aqueous phase to organic phase was 5: 1. The organic phase was poured into 50.07 g of the aqueous phase which was cooled in an ice water bath, and the mixture was homogenized using a manual homogenizer for 15 seconds. The coarse emulsion was fed through a high pressure homogenizer with the pressure set at 722.9 bar (10485 psi) on the gauge for 1 pass, to form a nanoemulsion (fine emulsion).

(d) Nanoparticle formation: The nanoemulsion was poured into a cold RODI water quench solution (1000 g) cooled to less than 2 ° C, and was stirred on a stir plate. To the quenched solution a cooled solution (less than 2 ° C) of polysorbate 80 (350 g) dissolved in RODI water (650 g) was added, with mixing.

(e) Concentration of nanoparticles through tangential flow filtration (TFF): The quenched phase was concentrated using TFF with Pali 300 kDa cassette (2 membranes) to form a nanoparticle concentrate of approximately 200 ml. The nanoparticle concentrate was diafiltered with approximately 20 diavolumes of cold RODI water at less than 2 ° C. The volume of the diafiltered nanoparticle concentrate was reduced to a minimum volume.

Consequently, this formulation contained 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl — 1,3,5 - triazin-2-yl) phenyl] urea, and the polymers PLA-PEG (in a molar ratio of 16: 5) and PLA-PEG-GL in a weight ratio of PLA-PEG to PLA-PEG-GL of about of 44: 1 and a therapeutic agent weight ratio, 1— (4 - {[4 (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl —1,3,5— triazin-2-yl) phenyl] urea, to polymers, from 22:64. It contained about 60% by weight of pamoic acid at 1— (4 - {[4— (dimethylamino) piperidine-1 — ¡l] carbonyl} phenyl) -

171

3— [4— (4,6-dimorpholin — 4 — 1—1,3,5-triazin-2-yl) phenyl!] Urea. Consequently, the formulation contained about 5% by weight of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4— yl-1,3,5-triazin-2-yl) phenyl] urea and 3.2% by weight of pamoic acid. The particle size of a nanoparticle thus formed as described in the present application above was around 92 nm.

EXAMPLE 4: FORMULATION D WITH THERAPEUTIC AGENT.

(a) Preparation of organic loading solution: A heated 7% by weight solution of xinaphoic acid in benzyl alcohol combined with PLA-PEG in a molar ratio of 16: 5 with ethyl acetate was vortexed until dissolution. The therapeutic agent, 1- (4 - {[4- (dimethylamino) piperidin1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl-1,3,5-triazin) was added —2-yl) phenyl] urea, to achieve a final concentration of 15% by weight.

(b) Preparation of aqueous phase charge: Sodium cholate (2.75 g) was dissolved in RODI water (955.5 g) with stirring. Benzyl alcohol (40 g) was added to the aqueous sodium cholate solution, and the mixture was stirred until dissolved.

(c) Emulsion formation: The weight ratio of aqueous phase to organic phase was 5: 1. The organic phase was poured into the aqueous phase, which was cooled in an ice water bath, and the mixture was homogenized using a hand-held homogenizer for 15 seconds. The coarse emulsion was fed through a high pressure homogenizer with the pressure set at 722.9 bar (10485 psi) on the gauge for 1 pass, to form a nanoemulsion (fine emulsion).

(d) Nanoparticle formation: The nanoemulsion was poured into a quench buffer solution consisting of anhydrous citric acid (19.2 g) in cold RODI water (1000 g) cooled to less than 2 ° C, and brought to pH 4.5 with

172

N sodium hydroxide, and the resulting solution was stirred on a stir plate. To the quenched solution, a cooled (less than 2 ° C) solution of polysorbate 80 (350 g) dissolved in RODI water (650 g) was added with mixing.

(e) The nanoparticles were concentrated through tangential flow filtration according to the procedure of Example 1.

Consequently, this formulation contained the counterion xinaphoic acid. It contained 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6— dimorpholin-4-yl — 1,3,5-triazin — 2— yl) phenyl] urea. The particle size of a nanoparticle thus formed as described in the present application above was around 109 nm.

COMPARATIVE EXAMPLE 1, CONTROL SOLUTION, (a) Preparation of organic loading solution: A 7.5% by weight solution of benzyl alcohol, prepared by dissolving benzyl alcohol in RODI water, was combined with PLA-PEG in a mixture that had a 16: 5 molar ratio with ethyl acetate, and was vortexed until dissolution.

(b) Preparation of aqueous phase charge: Sodium cholate (2.75 g) was dissolved in RODI water (955.5 g) with stirring. Benzyl alcohol (40 g) was added to the sodium cholate / water solution, and the mixture was stirred until dissolved.

(c) Emulsion formation: The weight ratio of aqueous phase to organic phase was 5: 1. The organic phase was poured into the aqueous phase, which was cooled in an ice water bath, and the mixture was homogenized using a manual homogenizer for 15 seconds. The coarse emulsion was fed through a high pressure homogenizer with the pressure set at 722.9 bar (10485 psi) on the gauge for 1 pass, to form a nanoemulsion (fine emulsion).

(d) Nanoparticle formation: The nanoemulsion was poured into 600 g of cold RODI water (less than 2 ° C) while stirring on a plate of

173 stirring to form a quenched phase (the weight ratio of quench to emulsion was 10: 1). 64.3 g of a polysorbate 80 solution (350 g dissolved in 650 g of RODI water) were added to the quenched phase, with mixing.

(e) Concentration of nanoparticles through tangential flow filtration (TFF): The quenched phase was concentrated using TFF with Pali 300 kDa cassette (2 membranes) to form a nanoparticle concentrate of approximately 200 ml. The nanoparticle concentrate was diafiltered with approximately 20 diavolumes of cold RODI water at less than 2 ° C. The volume of the diafiltered nanoparticle concentrate was reduced to a minimum volume.

COMPARATIVE EXAMPLE 2, SOLUTION B.

(a) Preparation of organic phase organic charge: Oleic acid (900 mg), trifluoroacetic acid (TFA) (273 mg) were dissolved in benzyl alcohol (8827 mg). The therapeutic agent, 1— (4 - {[4— (dimethylamine) piperidin— 1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl) was mixed —1,3,5-triazin-2-yl) phenyl] urea (120 mg) with the oleic acid / TFA / benzyl alcohol solution, and the mixture was heated to 80 ° C for 10 minutes in order to dissolve the therapeutic agent. Once the 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6— dimorpholin-4-yl — 1,3,5-triazin —2-l) phen l] urea, the solution was allowed to cool to room temperature. This solution was thoroughly mixed with a polymer solution of PLA-PEG in a ratio of 16 moles / 5 moles (860 mg), PLAPEG-GL (18.9 mg), and ethyl acetate (4549 mg) to form a solution.

(b) Preparation of aqueous phase loading: Sodium cholate (4.5 g) was dissolved in RODI water (955.5 g) on ​​a stir plate. Benzyl alcohol (40 g) was added to the sodium cholate / water solution, and the mixture was stirred on a stir plate until dissolved.

174 (c) Emulsion formation: The weight ratio of aqueous phase to organic phase was 5: 1. The organic phase was poured into 33.4 g of the aqueous phase, which was cooled in an ice water bath, and the mixture was homogenized using a hand-held homogenizer for 15 seconds. The coarse emulsion was fed through a high pressure homogenizer with a pressure set at 722.9 bar (10485 psi) on the gauge, for 1 pass to form a nanoemulsion (fine emulsion).

(d) Nanoparticle formation: The nanoemulsion was poured into 401.2 g of cold RODI water (less than 2 ° C) while stirring on a stir plate, to form a quenched phase. To the quenched phase, 51.4 grams of a polysorbate 80 solution (350 g dissolved in 650 g of RODI water) were added, with mixing.

(e) Concentration of nanoparticles through tangential flow filtration (TFF): The quenched phase was concentrated using TFF with a 300 kDa Pali cassette (2 membranes) to form a nanoparticle concentration of approximately 200 ml. The nanoparticle concentrate was diafiltered with approximately 20 diavolumes of cold RODI water at less than 2 ° C. The volume of the diafiltered nanoparticle concentrate was reduced to a minimum volume.

Consequently, this formulation contained 1— (4 - {[4— (dimethylamino) piperidine-1 —yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4 — ¡1—1.3, 5-triazin-2-yl) phenyl] urea and PLA-PEG polymers (in a ratio of about 16: 5) in a weight ratio of therapeutic agent to polymers of about 1: 14.7. It contained about 6.0% by weight of 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin — 4 —Il-1,3,5-triazin-2-yl) phenyl] urea, about 5.4% cholic acid and about 1.1% by weight oleic acid.

175

EXAMPLE 5: FORMULATION E WITH THERAPEUTIC AGENT,

The procedure of Example 1 was repeated, except that there was no presence of PLA-PEG-GL polymer. The pLA-PEG-GL polymer was replaced with 19.2 mg of PLA-PEG in a ratio of 16 mol / 5 mol, so that the total amount of PLA-PEG present was 850 mg.

EXAMPLE 6: FORMULATION F WITH THERAPEUTIC AGENT.

The procedure of Example 2 was repeated, except that there was no PLA-PEG-GL polymer present. The pLA-PEG-GL polymer was replaced with 20 mg of PLA-PEG in a ratio of 16 mol / 5 mol so that the total amount of PLA-PEG present was 860 mg.

EXAMPLE 7: FORMULATION F WITH THERAPEUTIC AGENT.

The procedure of Example 3 was repeated except that there was no PLA-PEG-GL polymer present. The polymer pLA-PEG-GL was replaced with 14.5 mg of PLA-PEG in a ratio of 16 mol / 5 mol so that the total amount of PLA-PEG present was 658 mg.

EXAMPLE 8: FORMULATION RELEASE PROFILE.

Each formulation was prepared on a scale sufficient to deliver 200 mg of therapeutic agent, 1— (4 - {[4— (dimethylamino) piper¡din — 1— ¡l] carbon¡l} phen¡I) —3— [4 - (4,6-dimorpholin-4-I-1,3,5-triazin-2-yl) phenyl-urea, at a concentration of&gt; 2.5 mg / ml (FORMULATION A = 25 g, FORMULATION B = 20 g, FORMULATION C = 10 g). Nanoparticle formulations with 30% sucrose by weight were prepared and poured into vials in aliquots of&gt; 11 mg of therapeutic agent. Table 1 summarizes the attributes of the nanoparticles prepared for this study.

176

Table 1: Synthesis of FORMULATION A, B, C.

<td>Formulation</td><td>Lot No.</td><td>Therapeutic agent loading</td><td>Particle size (nm)</td><td>API released within 24 h</td>

<td>TO</td><td> 237-46</td><td> 4%</td><td> 130</td><td> 60%</td>

<td>B</td><td> 237-45</td><td> 5%</td><td> 95</td><td> 22%</td>

<td>C</td><td> 237-44</td><td> 16%</td><td> 100</td><td> 2%</td>

All three batches satisfied the criteria for particle size and therapeutic agent release (90-150 nm, &lt;50% therapeutic agent released at&gt; 2 h). Except for the nanoparticles of FORMULATION A, the batches also met the criteria for therapeutic agent loading of&gt; 5%. Historically, the therapeutic agent loading for FORMULATION A has been at the lower limit or below the target loading threshold, so this result was not unexpected.

Figure 3 shows the in vitro release curves for each batch. The in vitro release method used determines the release profiles of these nanoparticles under conditions at 37 ° C using the centrifugal system. The samples were centrifuged at 264,000 xg for 30 minutes, and the supernatant was assayed to establish the concentration of therapeutic agent. The cumulative percent release was determined by comparing the supernatant concentration with the total therapeutic agent concentration prior to centrifugation.

The in vitro release profile in Figure 3 shows that the therapeutic agent release rate was quantifiably different for each of the formulations. Figure 4 depicts the pharmacokinetics of the therapeutic agent nanoparticles in Wistar Han rats.

The protocol was as follows: Male Wistar Han rats (approximately six weeks old; n = 4 / group) were dosed with resident cannulas

177 in the jugular vein, intravenously, with a 1 mg / kg bolus of nanoparticles of FORMULATION A, B and C, or nanoparticles of Formulation A, B and C diluted in 0.9% saline solution. At various times after dosing, serial blood collections were made from jugular vein cannulas, and plasma concentrations of therapeutic agent were qualified by LC-MS / MS. Figure 4 (a) shows nanoparticle pharmacokinetics versus free therapeutic agent, while (b) shows the same information with omitted free therapeutic agent.

Figure 4 (a) indicates that the three formulations A, B and C evaluated exhibited substantially longer retention times in the bloodstream, over free API. This corresponds to higher AUC and ti / 2 values, summarized in Table 2 (TA = therapeutic agent).

Table 2: Synthesis of AUC_totai and you data /₂ for FORMULATION A, B &amp; C nanoparticles evaluated.

<td>Parameter</td><td>TA</td><td>Formulation TO</td><td>60% EA</td><td>7% Xinafoic acid</td><td>Formulation C</td><td>Formulation B</td>

<td>AUC_total] (h-ng / ml)</td><td> 919.5</td><td> 312,972</td><td> 485,188</td><td> 653,749</td><td> 601,768</td><td> 550,539</td>

<td>tl / 2 (h)</td><td> 13.2</td><td> 17.7</td><td> 23.8</td><td> 25.1</td><td> 23.8</td><td> 23.7</td>

EXAMPLE 9: RELEASE PROFILE FOR FORMULATION C.

FORMULATION C was again prepared as in Example 3, using the traditional batch process on the 2 g and 5 g scale. An additional 2 g batch of FORMULATION C was prepared using an acid buffer.

178 citrus, 50 mM, titrated at pH 4.5 with sodium hydroxide, in order to favor potential ion pairing. This pH was selected because it was between the pK_to of pamoic acid (~ 2.5) and the first pK_to of 1— (4 - {[4— (dimethylamino) piperidine — 1 —yl] carboni!} phenyl) —3— [4— (4,6 — dimorpholin — 4 — 1—1,3,5— triazin— 2-yl) phenyl] urea (~ 6.7). Table 3 summarizes the particle attributes for these small-scale batches.

Table 3: Effect of using citric acid regulator, pH 4.5, 50 mM, for the quenching medium in Formulation C.

<td>Lot</td><td>Lot No.</td><td>Therapeutic agent loading</td><td>Size (nm)</td>

<td>2 g RODI off</td><td> 237-34-1</td><td> 4.48%</td><td> 101</td>

<td>5 g RODI off</td><td> 237-34-2</td><td> 2.43%</td><td> 98</td>

<td>2 g Regulated shutdown</td><td> 237-34-3</td><td> 13.18%</td><td> 92</td>

Longer emulsion processing time from a 2g batch to a 5g batch achieved a substantial drop in therapeutic agent loading. However, it was shown that using a quench regulated at pH 4.5 an almost three-fold increase in the loading of the therapeutic agent was achieved, 1— (4 - {[4— (dimethylamine) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-11-1,3,5-triazin-2-yl) phenyl] urea.

The in vitro release method was used to determine the release profiles of these nanoparticles under conditions at 37 ° C using the centrifugal system. The samples were centrifuged at 264,000 xg for 30 minutes, and the supernatant was assayed to establish the concentration of therapeutic agent. The cumulative percent release was determined by comparing the supernatant concentration with the total therapeutic agent concentration prior to centrifugation. Figure 5 shows that the in vitro release profile was not affected by the use of a regulated quench.

179

EXAMPLE 10: DETERMINATION OF PARTICLE ATTRIBUTES FOR FORMULATION C.

Two 10 g batches of FORMULATION C with quench of citric acid regulator, 100 mM, titrated to pH 4.5, each were prepared by assembling five batches of 2 g in order to avoid the effects of processing time on the drug loading of the therapeutic agent, 1- (4 - {[4 (dimethylamino) piperidin-1-yl] carbonll} phenyl) -3- [4- (4,6-dimorpholin-4-1-1 3,5-triazin-2-l) phenyl] urea. Table 4 summarizes the particle attributes for these batches.

Table 4. Particle attributes for batches of FORMULATION C using citric acid buffer quench, pH 4.5.

<td>Lot No.</td><td>Description</td><td>White API load</td><td>Solid organic phase</td><td>Formulation C charge</td><td>Particle size (nm)</td><td>Surfactan</td>

<td> 237-34-3</td><td>8 wt% TFA 7.5 wt% water in BA, 1: 1 pamoic to therapeutic agent, 20:80 (BA + DMSO): EA, 50 mM quenched citric acid pH 4.5</td><td> 22%</td><td> 10%</td><td> 13.18%</td><td> 92</td><td>Brij, 0.02% by weight</td>

<td> 237-36</td><td>8% by weight TFA 7.5% by weight water in BA, 1: 1 pamoic to therapeutic agent, 20:80 (BA + DMSO): EA, 100 mM quenched citric acid, pH 4.5</td><td> 22%</td><td> 10%</td><td> 18.25%</td><td> 100</td><td>Brij, 0.02% by weight</td>

<td> 237-44</td><td>8 wt% TFA 7.5 wt% water in BA, 1: 1 pamoic to therapeutic agent, 20:80 (BA + DMSO): EA, 100 mM quenched citric acid, pH 4.5, targeted GL</td><td> 22%</td><td> 10%</td><td> 16.30%</td><td> 100</td><td>Brij, 0.02% by weight</td>

BA = benzyl alcohol EA = ethyl alcohol

The in vitro release profiles were conducted as follows:

the in vitro release method was used to determine release profiles

180 of these nanoparticles under conditions at 37 ° C using the centrifuge system. The samples were centrifuged at 264,000 xg for 30 minutes, and the supernatant was assayed to establish the concentration of therapeutic agent. The percent cumulative release was determined by comparing the supernatant concentration with the total therapeutic agent concentration prior to centrifugation. The results are shown in Figure 6.

From the investigations, it was determined that the maximum therapeutic agent loading in formulation C was achieved at pH 4.5. Without wishing to be limiting, it is believed that this can be attributed to the fact that ion pairing between the therapeutic agent and the counter ion is encouraged when the pH of the solution is below pK._to of protonated therapeutic agent drug and higher than pK_to of the acid molecule (pamoic acid). This effect is believed to be theoretically maximized when the larger fraction of both species is in their ionized state.

EXAMPLE 11: PROGRAMMING STUDY OF MDAMB361 XENOGRAFT OF NANOPARTICLES OF 1- (4- (14 (DIMETHYLAMINE) PIPERIDIN-1-illCARBONYL) PHENYL) -3-i4- (4,6-DIMORPHOLIN-4-ÍI-1,3,5- TRIAZIN-2-yl) PHENYLLUREA Q4D VERSUS Q8D.

Female SCID / bg mice, about 6 weeks old, were obtained from Charles River Laboratories (Wilmington, MA). Animals were kept under clean room conditions in lidded cages filtered by Alpha-Dri bed sterilization and housed in ventilated HEPA filtered feeders. Animals received sterile rodent chow and water ad libitum. All procedures were conducted in accordance with the Institute for Laboratory Animal Research Guidance, for the

Care and Use of Laboratory Animals [Institute for Laboratory Animal

Research Guide for the Care and Use of Laboratory Animals] and with the guidelines

181 from Pfizer's Animal Care and Use Committee,

Three to four days prior to tumor cell inoculation, animals were implanted with a 0.36 mg minisphere, 60 d release, 17P-estradiol (Innovative Research of America). The MDA-MB-361 cells were harvested at 80-90% confluence, and viability over 80-90% (NS) was supplemented with 50% Matrigel (BD Bioscíences, San Jose CA) in order to facilitate the taking of the tumor. The cells (5 x 106 in 200 μΙ) were implanted subcutaneously (S. C.) in the hind flank region of the mouse and grown to the designated size prior to compound administration for each experiment. Tumor size was determined by measurement with an electronic caliper, and tumor volume was calculated as the product of its length x width.² x 0.5. When tumor volumes reached an average of 250 mm³, the mice were randomized by treatment groups that included a vehicle control group with intravenous (iv) injections of the corresponding drug, in a dose of 10 ml / kg of volume with a program of every four days (Q4D) or every eight days (Q8D). The animals were treated with 5 or 10 mg / kg of 1- (4 - [[4— (dimethylamino) piperidine — 1 —yl] carbonyl} phenyl) —3— [4— (4,6 — dimorpholin — 4 — ¡ 1—1,3,5— triazin — 2 — i |) phenyl] urea or 25 mg / kg of the nanoparticle of Formulation B in each injection.

Figure 7 shows that Formulation B nanoparticles dosed every 8 days have similar efficacy compared to once every 4 days, and that Formulation B nanoparticles can achieve a dosing frequency of 2 weeks in the clinic.

EXAMPLE 12: STUDY OF DELAY OF TUMOR GROWTH AND INHIBITION OF TUMOR GROWTH MDAMB361.

182

Female SCID / bg mice, about 6 weeks old, were obtained from Charles River Laboratories (Wilmington, MA). Animals were kept under clean room conditions in lidded cages filtered by Alpha-Dri bed sterilization and housed in ventilated HEPA filtered feeders. Animals received sterile rodent chow and water ad libitum. All procedures were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and the guidelines of the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. Pfizer of the Animal Care and Use Committee,

Three to four days prior to tumor cell inoculation, animals were implanted with a 0.36 mg minisphere, 60 d release, 17P-estradiol (Innovative Research of America). MDA-MB-361 cells were harvested at 80-90% confluence, and viability over 80-90% (NS) was supplemented with 50% Matrigel (BD Biosciences, San Jose CA) in order to facilitate tumor uptake. . Cells (5x106in 200 µΙ) were implanted subcutaneously (SC) into the hind flank region of the mouse and allowed to grow to the designated size prior to compound administration for each experiment. Tumor size was determined by measuring with an electronic caliper, and tumor volume was calculated as the product of its length x width.² x 0.5. When tumor volumes reached an average of 250 mm³, the mice were randomized by treatment groups that included a vehicle control group with intravenous (iv) injections of the corresponding drug, in a dose of 10 ml / kg of volume with a program of every four days (Q4D) for 4 doses. After the 4th. dose, the animals were further monitored in order to establish the

183 delay of tumor growth. Animals were treated with 10 mg / kg of 1 (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3— [4— (4,6-dimorpholin-4-yl— 1, 3,5-triazin-2-yl) phenyl] urea; 2, 10, or 25 mg / kg of the nanoparticles of Formulation A or B; or 10 or 25 mg / kg of Formulation C nanoparticles in each injection.

Figures 8A, 8B and 8C show that Formulation B nanoparticles and Formulation C nanoparticles inhibit tumor growth with improved efficacy, compared to 1- (4 - {[4— (dimethylamino) piperidin — 1— yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4 — ii — 1,3,5— triazin-2-yl) phenyl] urea (naked API) and the nanoparticles of Formulation A.

EXAMPLE 13: MODEL WM266-4 TUMOR GROWTH INHIBITION STUDY.

Female nu / nu mice, about 8 weeks old, were obtained from Charles River Laboratories (Wilmington, MA). Animals were kept in clean room conditions in lidded cages filtered by Alpha-Dri bed sterilization and housed in ventilated HEPA filtered feeders. Animals received sterile rodent chow and water ad libitum. All procedures were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and the guidelines of the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals Pfizer of the Animal Care and Use Committee.

WM266-4 cells were harvested at 80-90% confluence, and viability over 80-90% (NS) was supplemented with 50% Matrigel (BD Biosciences, San Jose CA) in order to facilitate tumor uptake. Cells (2 x 10⁶in 200 μΙ) were implanted subcutaneously (SC) in the flank region

184 mouse hindquarters, and grown to designated size prior to compound administration for each experiment. Tumor size was determined by measuring with an electronic caliper, and tumor volume was calculated as the product of its length x width.² x 0.5. When tumor volumes reached an average of 400 mm³, the mice were randomized by treatment groups that included vehicle control group with daily oral doses (QD (every day)) of PF-0192513-00-0004 (PD-901) and / or intravenous (iv) injections of the nanoparticle drugs, B and C, in a volume of 10 ml / kg, with a program of every four days (Q4D) for 4 doses. Dosage and drug are described in the legends to the Figures. Animals were treated with 10 mg / kg of 1- (4 {[4- (dimethylamino) piperidin-1-yl] carbonyl} phenyl) -3- [4- (4,6-dimorpholin-4-II-1 3,5-triazin-2-yl) phenyl] urea or 10, 25 or 50 mg / kg of the nanoparticles of Formulation B or C in each injection.

Figure 9 illustrates that Formulation C Nanoparticles produce greater tolerance and efficacy than Formulation B nanoparticles or 1 (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4 - (4,6-dimorpholin-4-II- 1,3,5-triazin-2-yl) phenyl] urea (naked API).

EXAMPLE 14: IN VIVO TARGET MODULATION STUDIES WITH NANOPARTICLES.

In vivo target modulation studies were conducted to determine the effects of treatment with nanoparticles of Formulation A, B and C on phosphorylation of S6 on S235 / S236 and AKT on S473 and T308 by ELISA. Fresh resected tumors were ground to fine powder, using a Martor metal mortar and a granite track, under liquid nitrogen. Tumor powder was stored -80 ° C until preparation of Tumor Uses for ELISA. Briefly, an aliquot (50 mg) was placed

185 of tumor powder in a 2 ml Martor tube previously cooled, 500 μΙ of cold lysis regulator [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0 mM Na₂ EDTA, 1'mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, I mM β-glycerophosphate, 1 mM NasVCU, 1 pg / ml leupeptin, 1 mM PMSF, 1x inhibitor cocktail protease / phosphatase]; The tube was embedded in wet ice, and the samples were homogenized at a speed of 6 for 30 seconds, using a tissue homogenizer. Samples were collected, and snap frozen on dry ice and thawed on wet ice. The freeze-thaw cycle was repeated, then the samples were centrifuged in a cold Eppendorf refrigerated centrifuge at 13,000 rpm for 10 minutes. The supernatant was collected, and centrifuged again. Total protein and phosphoAKT levels (S473 and T308) and total protein and phosphoS6 levels in tumor lysates were determined by ELISA. The extent of phosphorylation in resected tumors from treated animals was compared to that in resected tumors from vehicle-treated animals, at the same point.

Figures 8A, 8B and 8C show that Formulation B nanoparticles and Formulation C nanoparticles inhibit pS6 with improved efficacy compared to 1- (4 - {[4- (dimethylamino) piperidine-1yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea (naked API) and nanoparticles of Formulation A, and demonstrate persistent target modulation observed until day 7 after dosing.

EXAMPLE 15: ANALYSIS OF GLUCOSE AND INSULIN LEVELS AFTER TREATMENT WITH NANOPARTICLES.

Glucose: In mouse or rat studies, approximately 100 μΙ of plasma (EDTA as an anticoagulant) was used for evaluation of glucose content based on an enzymatic assay.

186 published by Slein (Bergmeyer HU, ed. Slein MW. Methods of Enzymatic Analysis. New York, NY: Academic Press; 1974: 1196-1201.), using the enzymes hexokinase and glucose-6-phosphate dehydrogenase. Plasma glucose was measured with the Advia® 120 Glucose Hexokinase_3 (GLUH_3) system with the automated hematology analysis instrument (Siemens Healthcare Diagnostics Inc., Tarrytown, New York). The Advia Chemistry Glucose Hexokinase_3 (GLUH_3) assay used a two-component reagent. The plasma sample was added to Reagent 1, which contained the regulator, ATP, and NAD. Sample absorbance readings were taken in Reagent 1 and used to correct for interfering substances in the sample. Reagent 2 (the regulator, ATP, NAD, Hexokinase, and G6PD) was added, which initiated glucose conversion and absorbance development at 340/410 nm. The difference between the absorbance in Reagent 1 and Reagent 2 was proportional to the glucose concentration.

Insulin'. In the mouse or rat studies, approximately 20 µΙ of plasma (EDTA as an anticoagulant) was used for the assessment of insulin content. The insulin assay was an intercalation enzyme-linked immunosorbent assay (ELISA) based on a rat / mouse insulin ELISA kit purchased from EMD Míllipore Corporation (St. Charles, Missouri). . The test procedure was as follows: 1) capture of insulin molecules from plasma samples into the wells of a microtiter plate coated with a previously titrated amount of a monoclonal antibody to mouse anti-mouse insulin, and the binding of biotinylated polyclonal antibodies to captured insulin, 2) washing of unbound materials from samples, 3) binding of horseradish peroxidase to immobilized biotinylated antibodies, 4) washing of

187 free enzyme conjugates, and 5) the quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the 3,3 ', 5,5'tetramethylbenzidine substrate. Enzyme activity was measured spectrophotometrically by the highest absorbance at 450 nm, which was directly proportional to the amount of insulin captured in the plasma sample. Plasma insulin concentration was calculated by interpolation from a reference curve generated in the same assay with reference models of known rat or mouse insulin concentrations.

Figure 10 illustrates that Formulation B and C nanoparticles may have an improved safety profile over 1— (4 - {[4— (dimethylamino) piperidin-1-yl] carbonyl} phenyl) —3— [4— (4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl) phenyl] urea (naked API).

EQUIVALENTS

Those skilled in the art will recognize, or be able to evaluate using no more than routine experimentation, many equivalents of the specific embodiments of the invention, as described in this application. Such equivalents are intended to be contemplated by the following claims.