Nanoparticulate compositions having a peptide as a surface stabilizer

ES 2 366 646 T3

DESCRIPTION

Compositions in the form of nanoparticles that have a peptide as a surface stabilizer

Field of the invention

The present invention relates to nanoparticulate active agent compositions having a peptide adsorbed on the surface or associated with the surface of the active agent as a surface stabilizer, and to methods of preparation and use of such compositions.

Background of the invention

Nanoparticulate active agent compositions, first described in US Patent No. 5,145,684 ("the '684 patent"), are particles consisting of a sparingly soluble therapeutic or diagnostic agent that have adsorbed on their surface. or associated with its surface an uncrosslinked surface stabilizer. The '684 patent describes the use of a variety of surface stabilizers for nanoparticulate compositions. The use of a peptide as a surface stabilizer for nanoparticulate active agent compositions is not described in the '684 patent.

The '684 patent describes a method of screening active agents by identifying useful surface stabilizers that allow a nanoparticulate composition to be produced. Not all surface stabilizers can act to produce a stable, non-agglomerated nanoparticulate composition for all active agents. Furthermore, known surface stabilizers may be unable to produce a stable, non-agglomerated nanoparticulate composition for certain active agents. Thus, there is a need in the art to identify new surface stabilizers useful for producing nanoparticulate active agent compositions. Additionally, such new surface stabilizers may possess superior properties over previously known surface stabilizers.

Methods of preparing nanoparticulate active agent compositions are described, for example, in US Patent Nos. 5,518,187 and 5,862,999, both for "Method of Grinding Pharmaceutical Substances"; US Patent No. 5,718,388 for "Continuous Method of Grinding Pharmaceutical Substances"; and US Patent No. 5,510,118 for "Process for the Preparation of Therapeutic Compositions Containing Nanoparticles."

Nanoparticulate active agent compositions are also described, for example, in US Patent Nos. 5,298,262 for "Use of Ionic Cloud Point Modifiers to Prevent Particle Aggregation During Sterilization"; 5,302,401 for "Method for reducing particle size growth during lyophilization"; 5,318,767 for "X-ray Contrast Compositions Useful in Medical Imaging"; 5,326,552 for "New Formulation of Blood Blend Nanoparticulate X-Ray Contrast Agents Using High Molecular Weight Nonionic Surfactants", 5,328,404 for "X-Ray Optical Representation Method Using Iodinated Aromatic Propanedioates"; 5,336,507 for "Use of Charged Phospholipids to Reduce Nanoparticle Aggregation"; 5,340,564 for "Formulations comprising Olin 10-G to prevent particle aggregation and increase stability"; 5,346,702 for "Use of Nonionic Cloud Point Modifiers to Minimize Aggregation of Nanoparticles During Sterilization"; 5,349,957 for "Preparation and Magnetic Properties of Very Small Dextran-Magnetic Particles"; 5,352,459 for "Use of Purified Surface Modifiers to Prevent Particle Aggregation During Sterilization"; 5,399,363 and 5,494,683, both for "Surface Modified Anti-Cancer Compound Nanoparticles"; 5,401,492 for "Water Insoluble Non-Magnetic Manganese Particles as Magnetic Resonance Enhancing Agents"; 5,429,824 for "Use of Tyloxapol as a nanoparticle stabilizer"; 5,447,710 for "Method of Manufacturing Blood Blend X-ray Contrast Agents Using High Molecular Weight Nonionic Surfactants"; 5,451,393 for "X-ray Contrast Compositions Useful in Medical Imaging"; 5,466,440 for "Oral X-ray Contrast Agent Formulations for Gastrointestinal Diagnostics in Combination with Pharmaceutically Acceptable Clays"; 5,470,583 for "Method of preparing nanoparticulate compositions containing charged phospholipids to reduce aggregation"; 5,472,683 for "Nanoparticulate Diagnostic Mixed Carbamic Anhydrides for Optical Representations of Blood and Lymphatic System Combinations"; 5,500,204 for "Nanoparticulate Diagnostic Dimers as X-ray Contrast Agents for Optical Representations of Blood and Lymphatic System Combinations"; 5,518,738 for "Nanoparticulate Formulations of NSAIDs"; 5,521,218 for "Nanoparticulate Iodopamide Derivatives for Use as X-ray Contrast Agents"; 5,525,328 for "Nanoparticulate Diagnostic Diatrizoxy Ester X-ray Contrast Agents for Optical Representations of Blood and Lymphatic System Combinations"; 5,543,133 for "Process for the Preparation of X-ray Contrast Compositions Containing Nanoparticles"; 5,552,160 for "Surface Modified Nanoparticles of NSAIDs"; 5,560,931 for "Formulations of compounds in the form of nanoparticulate dispersions in digestible oils or fatty acids"; 5,565,188 for "Polyalkylene Block Copolymers as Nanoparticle Surface Modifiers"; 5,569,448 for "Sulfated Nonionic Block Copolymer Surfactant as Stabilizing Coatings

ES 2 366 646 T3

5,569,448 for "Sulphated Nonionic Block Copolymer Surfactant as Nanoparticle Stabilizing Coatings"; 5,571,536 for "Formulations of compounds in the form of nanoparticulate dispersions of digestible oils or fatty acids"; 5,573,749 for Nanoparticulate Diagnostic Mixed Carboxylic Anhydrides as X-ray Contrast Agents for Optical Representations of Blood and Lymphatic System Combinations "; 5,573,750 for "X-ray Contrast Agents for Diagnostic Optics"; 5,573,750 for "Diagnostic, Optical X-ray Contrast Agents"; 5,573,783 for "Redispersible Nanoparticulate Film Matrices with Protective Coatings"; 5,580,579 for Site Specific Adhesion within the Gl tract using high molecular weight linear poly (ethylene oxide) stabilized nanoparticles ”; 5,585,108 for "Formulations of oral gastrointestinal therapeutic agents in combination with pharmaceutically acceptable clays"; 5,587,143 for "Butylene Oxide-Ethylene Oxide Block Copolymer Surfactants as Stabilizing Coatings of Nanoparticulate Compositions"; 5,591,456 for "Naproxen milled with hydroxypropyl cellulose as dispersion stabilizer"; 5,593,657 for "New Barium Salt Formulations Stabilized with Nonionic and Anionic Stabilizers"; 5,622,938 for "Sugar-based surfactant for nanocrystals"; 5,628,981 for "Improved Formulations of Oral Gastrointestinal Diagnostic X-ray Contrast Agents and Oral Gastrointestinal Therapeutics"; 5,643,552 for "Nanoparticulate Diagnostic Mixed Carbonic Anhydrides as X-ray Contrast Agents for Optical Representations of Blood and Lymphatic System Combinations"; 5,718,388 for "Continuous method of grinding pharmaceutical substances": 5,718,919 for "Nanoparticles containing the R (-) enantiomer of ibuprofen"; 5,747-001 for "Aerosols Containing Beclomethasone Nanoparticle Dispersions"; 5,834,025 for "Reduction of Adverse Physiological Reactions Induced by Nanoparticulate Formulations Administered Intravenously"; 6,045,829 "Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors Using Cellulosic Surface Stabilizers"; 6,068,858 for "Methods of Preparation of Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors Using Cellulosic Surface Stabilizers"; 6,153,225 for "Nanoparticulate Naproxen Injectable Formulations"; 6,165,506 for "New solid dosage form of nanoparticulate naproxen"; 6,221,400 for "Methods of Treatment of Mammals Using Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors"; 6,264,922 for "Nebulized Aerosols Containing Nanoparticle Dispersions"; 6,267,989 for "Methods for preventing crystal growth and particle aggregation of nanoparticulate compositions"; 6,270,806 for "Use of PEG Derivatized Lipids as Surface Stabilizers of Nanoparticulate Compositions"; 6,316,029 for "Rapidly Disintegrating Solid Oral Dosage Form"; 6,375,986 for "Solid Nanoparticulate Pharmaceutical Compositions Comprising a Synergistic Combination of a Polymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate"; 6,428,814 for "Bioadhesive nanoparticulate compositions possessing cationic surface stabilizers"; 6,431,478 for "Small Scale Milling"; 6,432,381 for "Methods for targeting drug delivery to the lower and / or lower gastrointestinal tract"; Patent No. 6,582,285 for "Apparatus for sanitary wet grinding"; 6,592,903 for "Nanoparticulate Dispersions Comprising a Synergistic Combination of a Polymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate"; 6,742,734 for "System and Method of Grinding Materials"; and 6,745,962 for “Small-scale milling and its method”. Furthermore, US Patent Application No. 200 20012675 A1, published on January 31, 2002, for "Regulated release of nanoparticulate compositions" and WO 02/098565 for "Materials milling system and method", describe nanoparticulate compositions of active agents. None of these references describe nanoparticulate active agent compositions comprising a peptide surface stabilizer. "

Compositions of small amorphous particles are described, for example, in US Patent Nos. 4,783,484 for "Particulate Composition and Its Use as an Antimicrobial Agent"; 4,826,689 for "Method of manufacture of particles of uniform size starting from organic compounds insoluble in water"; 4,997,454 for "Method of manufacture of particles of uniform size starting from insoluble compounds"; 5,741,522 for “Undummed, ultra-small porous particles of uniform size to retain gaseous bubbles in their interior and methods; and 5,776,496 for "Ultra Small Porous Particles to Enhance Reverse Ultrasonic Scattering."

There is a need in the art for new surface stabilizers useful for preparing nanoparticulate active agent compositions. The present invention satisfies this need.

Summary of the invention

The present invention is directed to nanoparticulate compositions comprising at least one active agent and 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, adsorbed on the surface of the active agent or associated with its surface, in which the composition does not comprise nystatin or one of its salts.

Another aspect of the invention is directed to pharmaceutical compositions comprising a nanoparticulate composition of an active agent of the invention. The pharmaceutical compositions preferably comprise at least one active agent, 4: 1 poly (Lysine, Tryptophan) hydrobromide and a pharmaceutically acceptable carrier, as well as any desired excipients.

ES 2 366 646 T3

Still, in another embodiment, the invention is directed to bioadhesive nanoparticulate active agent compositions comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer. Such compositions can coat the gut, or the desired application site, and be retained for a period of time, thereby increasing the efficacy of the active agent as well as eliminating or decreasing the frequency of administration.

The invention further describes a method of preparing nanoparticulate active agent compositions having a peptide surface stabilizer adsorbed on the surface of the active agent or associated with its surface. Such a method comprises contacting an active agent with 4: 1 poly (Lysine, Tryptophan) hydrobromide for a period of time and under conditions sufficient to provide a nanoparticulate active agent / peptide composition. The peptide surface stabilizer can be contacted with the active agent either before, preferably during, or after the decrease in size of the active agent.

The present invention can be used in a method of treatment which comprises administering to a mammal a therapeutically effective amount of a nanoparticulate composition of an active agent, according to the invention.

Both the above general description and the detailed description that follow are exemplary and explanatory, and are intended to provide further explanation of the claimed invention. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the detailed description of the invention that follows.

Brief description of the drawings

Figure 1: Shows representative photomicrographs of nystatin crystals before (Fig. 1A) and after (Fig. 1B) grinding.

Figure 2: Shows the results of monitoring particle size stability over time, at 5 ° C (solid line), 25 ° C (dashed line), and 40 ° C (dotted line), of a composition nystatin nanoparticulate comprising the hydrobromide peptide of poly (Lysine, Tryptophan) 4: 1 as a surface stabilizer; and the

Figure 3: Shows representative photomicrographs of cells with anionic particles (Fig, 3A) and cationic particles (Fig. 3B).

Figures 1 to 3 have been retained for the sake of clarity, although the nystatin compound is not within the scope of the claims.

Detailed description of the invention

The present invention is directed to compositions comprising nanoparticulate active agents having at least 4: 1 poly (Lysine, Tryptophan) hydrobromide adsorbed on their surface or associated with their surface, and to methods of preparation and use of such nanoparticulate compositions, in which the composition does not comprise nystatin or one of its salts.

As the '684 patent teaches, not all surface stabilizer and active agent combinations can result in a stable nanoparticulate composition. The discovery of the present invention is surprising since peptides are biological compounds that possess secondary and tertiary structures that are critical for the activity of the peptide. It was surprising that 4: 1 poly (Lysine, Tryptophan) hydrobromide could be used successfully to stabilize a nanoparticulate active agent. Furthermore, it was even more surprising that grinding a peptide surface stabilizer did not change the activity and function of the peptide.

A "peptide" is defined as a compound consisting of two or more amino acids in which the carboxyl group in the alpha position of one of them is linked to the amino group in the alpha position of the other. A polypeptide is a long peptide chain. A protein is a large macromolecule made up of one or more polypeptide chains. In the context of the present invention "peptide" refers to a peptide or a polypeptide, but not to a protein.

A surprising feature of peptides is that they have well-defined three-dimensional structures. Peptides fold into compact structures with nominal binding lengths. The strong tendency of hydrophobic amino acid residues to flee from water drives the folding of soluble peptides.

An elongated or randomly arranged polypeptide chain is devoid of biological activity. This fact is due to the fact that the function of a peptide arises from its conformation, which is the three-dimensional arrangement of the atoms of a structure. See, for example, L. Stryer publication, Biochemistry, 3^to Edition, p. 1-41 (WH Freeman and Co., NY, 1988). The amino acid sequences are important because these sequences specify the conformation of the peptides. Id.

Peptides possess several different defined structures, including a primary, secondary, and tertiary structure. The primary structure of a peptide is generally the amino acid sequence of the peptide and the

ES 2 366 646 T3 disulfide position. See, for example, L. Stryer publication, Biochemistry, 3^to Edition, p. 31 (WH Freeman and Co., NY, 1988). Secondary structure refers to the spatial arrangement of amino acid residues that are close to each other in the linear sequence. Examples of these spherical relationships are structures known as alpha helix, beta folded sheet, and collagen helix. Id. Tertiary structure refers to the spatial arrangement of amino acid residues in a peptide or polypeptide that are widely separated in linear sequence.

Proteins, which comprise multiple polypeptide chains, also possess a quaternary structure, which refers to the spatial arrangement of the polypeptide subunits and the nature of their contacts. Id.

In addition to enabling the use of 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer for nanoparticulate active agents, this discovery is important given that the peptide surface stabilizer of the compositions of the invention may also possess therapeutic or anti-inflammatory properties. diagnosis. This fact is in contrast to prior art nanoparticulate active agent compositions, in which the surface stabilizer is generally a surfactant, lacking such therapeutic or diagnostic properties.

The nanoparticulate active agent compositions of the invention may also offer the following advantages compared to prior conventional non-nanoparticulate active agent compositions: (1) faster onset of action; (2) potential decrease in the frequency of administration; (3) smaller doses of active agents required to obtain the same pharmacological effect; (4) increased bioavailability; (5) increased dissolution rate; (6) improved performance characteristics for oral administration or by intravenous, subcutaneous or intramuscular injection, such as higher active agent dose loading and smaller volumes of tablets or liquid doses; (7) improved pharmacokinetic profiles, such as T profiles_max, C_maimproved x and AUC; (8) Pharmacokinetic profiles of the nanoparticulate compositions of substantially similar or bioequivalent active agents when administered in the ingested versus the fasted state: (9) bioadhesive compositions of active agents, which can coat the intestine or the desired application site and be retained for a prolonged period of time, thereby increasing the effectiveness of the active agent as well as eliminating or decreasing the frequency of administration; (10) high redispersion capacity of the nanoparticulate active agent particles present in the compositions of the invention after administration; (11) nanoparticulate active agent compositions can be formulated in a dry form that readily redisperses; (12) low viscosity nanoparticulate liquid dosage forms of active agents can be prepared; (13) for liquid nanoparticulate compositions of active agents that have low viscosity, better acceptance by the patient, due to the perception of a lighter formulation, easier to ingest and digest; (14) for liquid nanoparticulate compositions of active agents that possess low viscosity, ease of dispensing because a cup or syringe can be used; (15) nanoparticulate active agent compositions can be used in association with other active agents; (16) nanoparticulate active agent compositions can be sterilized by filtration; (17) nanoparticulate active agent compositions are suitable for parenteral administration; and (18) nanoparticulate active agent compositions do not require organic solvents or extreme pH.

The preferred administration form of the invention is a solid dosage form, although any pharmaceutically acceptable form of administration can be used. Exemplary pharmaceutical forms include, but are not limited to, tablets, capsules, sachets, troches, powders, pills, granules, liquid dispersions, oral suspensions, gels, aerosols (including nasal and pulmonary), ointments. and creams.

The dosage form of the invention may be, for example, a fast melt dosage form, a controlled release dosage form, a lyophilized dosage form, a delayed release dosage form, a long release dosage form, a release dosage form pulsatile, an immediate release and regulated release dosage form, mixed, or a combination thereof.

Furthermore, the compositions of the invention may be formulated for any suitable route of administration, such as oral, pulmonary, rectal, ophthalmic, colonic, parenteral, intracysteral, intravaginal, intraperitoneal, local, buccal, nasal or topical administration.

The present invention is described herein using various definitions, as set forth below and throughout the patent application.

As used herein, "about" will be understood by those skilled in the art and may vary to some extent in the context in which it is used. If there are uses of the term that are not clear to those skilled in the art given the context in which it is used, "about" means plus or minus 10% of the particular term.

ES 2 366 646 T3 "Conventional" or "non-nanoparticulate active agent" means an active agent that is solubilized or has an effective mean particle size greater than about 2 microns. The nanoparticulate active agents defined herein have an effective mean particle size of less than about 2 microns.

"Pharmaceutically acceptable", as used herein, refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of logical medical judgment, suitable for use in contact with the tissues of humans and of animals, without excessive toxicity, irritation, allergic response or other problem or complication, in proportion to a reasonable benefit / risk ratio.

"Pharmaceutically acceptable salts" as used herein refers to derivatives in which the parent compound has been modified by preparing acid or base salts thereof. Examples of acceptable pharmaceutical salts include, but are not limited to, mineral or organic acid salts of basic moieties such as amines; alkaline or organic salts of acidic moieties such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and salts prepared starting from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymax, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

"Poorly water soluble drugs", as used herein, means those that possess a solubility of less than about 30 mg / ml, preferably less than about 20 mg / ml, preferably less than 10 mg / ml. about, or, preferably, less than about 1 mg / ml. Such drugs tend to be eliminated from the gastrointestinal tract before being absorbed into the circulatory stream.

As used herein with reference to stable drug particles, "stable" includes, but is not limited to, one or more of the following parameters: (1) that the active agent particles do not flocculate or agglomerate appreciably due to to attractive forces between particles, nor, on the other hand, do they increase significantly in particle size over time; (2) that the physical structure of the active agent particles is not altered with time, as by transformation from an amorphous phase to a crystalline phase; (3) that the active agent particles are chemically stable; and / or (4) that the active agent has not been subjected to a heating step at or above the melting point temperature, when preparing the nanoparticles of the invention.

"Therapeutically effective amount", as used herein with respect to the dose of an active agent, means the dose that provides the specific pharmacological response for which the active agent is administered in a significant number of patients in need of such treatment. It is emphasized that "therapeutically effective amount" administered to a particular subject in a particular case will not always be effective in treating the diseases described herein, even though such doses are considered a "therapeutically effective amount" by those skilled in the art. . It is further to be understood that the doses of active agents are measured, in particular cases, as oral doses, or with reference to levels of active agent measured in the blood.

I. Preferred characteristics of the nanoparticulate active agent compositions of the invention

A. Increased bioavailability, dosing frequency and number of doses

The nanoparticulate active agent compositions of the invention, which have 4: 1 poly (Lysine, Tryptophan) hybrid bromide as a surface stabilizer, may preferably demonstrate increased bioavailability and require lower doses compared to non-nanoparticulate compositions of prior art, of the same active agent, administered at the same dose.

Any active agent can have adverse side effects. Therefore, lower doses of an active agent are desirable that can achieve the same or better therapeutic effects than those seen with higher doses of a non-nanoparticulate composition of the same active agent. Such lower doses can be achieved with the nanoparticulate compositions of active agents, of the invention, because the nanoparticulate compositions of active agents can manifest greater bioavailability compared to that of the non-nanoparticulate compositions of the same active agent, which means that, probably , lower doses of the active agent are needed to obtain the desired therapeutic effect.

The nanoparticulate compositions of active agents, of the invention, can be administered less frequently and in lower doses, compared to non-nanoparticulate compositions of the same active agent, in pharmaceutical forms such as liquid dispersions, powders, sprays, aerosols (pulmonary or nasal ), redispersible solid dosage forms, gels, ointments, creams, etc. from the people

ES 2 366 646 T3 nanoparticulate active. Lower doses can be administered because the small particle size of the active agent particles ensures greater absorption, and in the case of nanoparticulate compositions of bioadhesive active agents, the active agent is retained in the desired application site for a longer period. time compared to conventional non-nanoparticulate pharmaceutical forms of the same active agent.

In one embodiment of the invention, the therapeutically effective amount of the nanoparticulate active agent compositions is 1/6, 1/5, 1/4, 1/3, or 1/2 of the therapeutically effective amount of a non-nanoparticulate composition thereof. active agent.

Such lower doses are preferred as they can lessen or eliminate adverse effects of the active agent. Furthermore, such lower doses lower the cost of the dosage form and may increase patient acceptance.

B. Pharmacokinetic profiles of the nanoparticulate active agent compositions of the invention

The invention also preferably provides nanoparticulate active agent compositions having at least one peptide as a surface stabilizer and possessing a desirable pharmacokinetic profile when administered to mammals. The desirable pharmacokinetic profile of active agent compositions preferably includes, but is not limited to: (1) a T_max for an active agent, analyzed in the plasma of a mammal after administration which is preferably less than the T_max of a non-nanoparticulate composition of the same active agent, administered at the same dose; (2) a C_max of an active agent, analyzed in the plasma of a mammal after administration that is preferably greater than the C_max of a non-nanoparticulate composition of the same active agent, administered at the same dose, and / or (3) an AUC for an active agent, analyzed in the plasma of a mammal after administration that is preferably greater than the AUC of a non-nanoparticulate composition of the same active agent, administered at the same dose.

The desirable pharmacokinetic profile as used herein is the pharmacokinetic profile measured after the initial dose of the active agent. The compositions can be formulated in any manner as described herein and known to those of skill in the art.

A preferred active agent composition of the invention, which comprises at least one peptide as a surface stabilizer, shows in a comparative pharmacokinetic test with that of a non-nanoparticulate composition of the same active agent, administered at the same dose, a T_max not more than about 100%, not more than about 90%, not more than about 80%, not more than about 70%, not more than about 60%, not more than about 50%, not more than about 40%, no greater than about 30%, not greater than about 25%, not greater than about 20%, not greater than about 15%, not greater than about 10%, or not greater than about 5%, of the T_max evidenced by the non-nanoparticulate composition of the active agent. This T_max shorter translates into faster initiation of therapeutic activity.

A preferred active agent composition of the invention, which comprises at least one peptide as a surface stabilizer, shows in a comparative pharmacokinetic test with that of a composition

<td colspan="2">non-nanoparticulate</td><td colspan="4">same active agent, administered to</td><td>the</td><td>same</td><td colspan="2">dose, one C_max What is it</td><td>to the</td><td>less</td>

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non-nanoparticulate composition of the active agent.

A preferred active agent composition of the invention, which comprises at least one peptide as a surface stabilizer, shows in a comparative pharmacokinetic test with that of a non-nanoparticulate composition of the same active agent, administered in the same dose, an AUC that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200%, greater than the AUC revealed by the same non-nanoparticulate formulation of the active agent.

ES 2 366 646 T3

Any formulation that provides the desired pharmacokinetic profile is suitable for administration according to the present methods.

C. The pharmacokinetic profiles of the nanoparticulate active agent compositions of the invention are not substantially affected, preferably, by the ingested food or fasting state of the subject ingesting the compositions.

The invention encompasses nanoparticulate compositions of active agents, comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, in which, preferably, the pharmacokinetic profile of the active agent is not substantially affected by the state with ingested food or in fasting state of the subject ingesting the composition. This means that there is no substantial difference in the amount of active agent absorbed or in the rate of absorption of the active agent when nanoparticulate compositions of an active agent are administered in the fed state versus the fasted state. Thus, the nanoparticulate active agent compositions of the invention can preferably substantially eliminate the effect of food on the pharmacokinetic profiles of the active agent.

In another embodiment of the invention, the pharmacokinetic profile of active agent compositions of the invention comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, when administered to a mammal in the fasted state, is bioequivalent to the pharmacokinetic profile of the same nanoparticulate composition of the active agent administered at the same dose, when administered to a mammal in the fed state.

The "bioequivalence" is preferably established by a confidence interval (Cl) of 90%, between 0.80 and 1.25 for both, the C_max and AUC, under US Food and Drug Administration (USDFDA) regulatory guidelines, or a 90% Cl for an AUC of 0.80 to 1.25 and a 90% Cl for C_max from 0.70 to 1.43 under the regulatory guidelines of the European Medicines Evaluation Agency (EMEA) (the T_max not relevant for bioequivalence determinations under USFDA and EMEA guidelines).

Preferably, the difference in AUC (eg, absorption) of the nanoparticulate active agent composition of the invention comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, when administered in the ingested versus food state. to the fasted state, it is less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about , 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3%.

Furthermore, preferably, the differences of C_max of the nanoparticulate active agent composition of the invention, comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, when administered in the ingested food state versus the fasting state, it is less than approximately 100 %, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, smaller than, approximately 35%, less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, less than approximately 5%, or less than about 3%.

Finally, preferably, the difference in the T_max of the nanoparticulate compositions of active agents, of the invention, which comprise 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, when administered in the ingested food state versus the fasting state, it is less than approximately 100 %, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, smaller than, about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, or essentially no difference .

The benefits of a dosage form that substantially eliminates the effect of food include an increase in the suitability of the subject, thereby increasing the subject's complacency, since it is not necessary for the subject to ensure that they take a dose with or without food.

D. Redispersibility profiles of the nanoparticulate active agent compositions of the invention

An additional characteristic of the nanoparticulate active agent compositions of the invention, comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, is that the compositions redisperse in such a way that the effective average particle size of the redispersed active agent particles is less than about 2 microns. This fact is significant, since if after

ES 2 366 646 T3 administering the nanoparticulate active agent particles of the compositions of the invention, they will not be redispersed until obtaining a substantially nanoparticulate particle size, the pharmaceutical form could lose the benefits obtained when formulating the active agent with a nanoparticulate particle size .

This fact is due to the fact that the nanoparticulate active agent compositions of the invention benefit from the small particle size of the active agent; if the nanoparticulate active agent particles were not redispersed to obtain the small particle sizes after administration, "clumped" or agglomerated active agent particles would form. With the formation of such agglomerated particles, the bioavailability of the dosage form can fail.

Furthermore, the nanoparticulate active agent compositions of the invention demonstrate a dramatic redispersion of the active agent particles when administered to a mammal, such as a human or animal, as demonstrated by reconstitution in an aqueous medium. bio-relevant. Such aqueous bio-relevant media can be any aqueous media that bring out the desired ionic strength and the desired pH, which form the basis for the bio-relevance of the media. The desired pH and ionic strength are those that are representative of the physiological conditions found in the human body. Such bio-relevant aqueous media can be, for example, aqueous electrolytic solutions or aqueous solutions of a salt, an acid or a base, or one of their combinations, which reveal the desired pH and ionic strength.

Bio-relevant pH is well known in the art. For example, in the stomach, the pH ranges from slightly less than 2 (but typically greater than 1) to 4 or 5. In the small intestine the pH can range from 4 to 6, and in the colon it can range from 6 to 8. Bio-relevant ionic strength is also well known in the art. Gastric fluid in the fasted state has an ionic strength of approximately 0.1 M, while intestinal fluid has an ionic strength of approximately 0.14. See, for example, Lindahl et al., "Characterization of fluids from the stomach and proximal jejunum of men and women," Pharm. Res. 14 (4): 497-502 (1997).

The pH and ionic strength of the test solution are believed to be more critical than the specific chemical content. Accordingly, appropriate pH and ionic strength values ​​can be obtained by numerous combinations of strong acids, strong bases, salts, single or multiple conjugated acid-base pairs (i.e., weak acids and corresponding salts of such acids), electrolytes. monoprotic and polyprotic, etc.

Representative electrolyte solutions may be, but are not limited to, HCl solutions, ranging in concentration from about 0.001 to about 0.1 M, and NaCl solutions ranging in concentration from about 0.001 to about 0.1 M, and their mixtures. For example, electrolyte solutions may be, but not limited to, HCl about 0.1M or less, HCl about 0.01 M or less, HCl about 0.001 M or less, NaCl about 0.1M or less, NaCl about 0.01 M or less, NaCl about 0.001 M or less, and mixtures thereof. Of these electrolyte solutions, those of 0.01 M HCl and / or 0.1 M NaCl are the most representative of human physiological conditions in the fasting state, due to the pH and ionic strength conditions of the proximal gastrointestinal tract.

The electrolyte concentrations of 0.001 M HCl, 0.01 M HCl, and 0.1M HCl correspond, respectively, to pH 3, pH 2, and pH 1. Thus, a 0.01 M HCl solution simulates the typical acidic conditions found in stomach. A 0.1M NaCl solution provides a reasonable approximation of ionic strength conditions found throughout the body, including gastrointestinal fluids, although concentrations greater than 0.1M can be used to simulate conditions of the ingested food state. the interior of the human Gl tract.

Exemplary solutions of salts, acids, bases or their combinations that demonstrate the desired pH and ionic strength include, but are not limited to, phosphoric acid / phosphate salts + chloride salts. sodium, potassium and calcium, acetic acid / acetate salts + sodium, potassium and calcium chloride salts, carbonic acid / bicarbonate salts + sodium, potassium and calcium chloride salts, and citric acid / citrate salts + chloride salts sodium, potassium and calcium.

In other embodiments of the invention, the redispersed active agent particles of the invention (redispersed in a bio-relevant aqueous medium or any other suitable medium) have an effective mean particle size of less than about 1900 nm. less than about 1800 nm, less than about 1700 nm, less than about 1600 nm, less than about 1500 nm, less than about 1400 nm, less than about 1300 nm, less than about 1200 nm, less than about 1100 nm, less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than Approximately 500 nm, less than approximately 400 nm, less than approximately 300 nm, less than approximately 250 nm, less than approximately 200 nm, less than approximately 150 nm, less than approximately 100 nm, less than about 75 nm, or

ES 2 366 646 T3 less than about 50 nm, measured by light scattering, microscopy, or other appropriate methods.

Redispersibility can be tested using any suitable methods known in the art. See, for example, sections of US Patent No. 6,375,986 for "Solid Nanoparticulate Compositions Comprising a Synergistic Combination of a Polymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate."

E. Nanoparticulate compositions of bioadhesive active agents

The nanoparticulate bioadhesive active agent compositions of the invention comprise a cationic peptide surface stabilizer, 4: 1 poly (Lysine, Tryptophan) hydrobromide. Bioadhesive formulations of active agents demonstrate exceptional bioadhesion to biological surfaces, such as mucosa and skin.

Cationic surface stabilizers generally impart relatively large positive zeta potentials to the particles on which they adsorb or associate. To increase the bioadhesive properties of a nanoparticulate composition, two or more cationic surface stabilizers can be used.

In the case of nanoparticulate compositions of bioadhesive active agents, the term "bioadhesion" is used to describe the adhesion between the nanoparticulate compositions of active agents and a biological substrate (ie, gastrointestinal mucin, lung tissue, nasal mucosa, etc. .). See, for example, US Patent No. 6,428,814 for "Bioadhesive Nanoparticulate Compositions Possessing Cationic Surface Stabilizers."

There are basically two mechanisms that can be responsible for these bioadhesion phenomena: mechanical or physical interactions and chemical interactions. The first of them, the mechanical or physical mechanisms, involves the physical entanglement or interpenetration between a bioadhesive entity and the recipient tissue, which results from a good wetting of the bioadhesive surface, the swelling of the bioadhesive polymer, the penetration of the bioadhesive entity into a slit in the tissue surface, or the interpenetration of chains of the bioadhesive composition with those of the mucosa or other such related tissues. The second possible bioadhesion mechanism incorporates forces such as ionic attraction, dipole forces, Van der Waals interactions, and hydrogen bonds. It is this form of bioadhesion that is mainly responsible for the bioadhesive properties of the nanoparticulate active agent compositions of the invention. However, physical and mechanical interactions can also play a minor role in the bioadhesion of such nanoparticulate active agent compositions.

The bioadhesive active agent compositions of the invention are useful in any situation where it is desirable to apply the compositions to a biological surface. Bioadhesive active agent compositions preferably coat the target surface with a continuous, uniform film that is invisible to the naked eye to the human eye.

A nanoparticulate composition of a bioadhesive active agent slows the transit of the composition and some particles of the active agent could also most likely adhere to tissues other than mucosal cells and, therefore, provide prolonged exposure to the active agent. , thereby increasing the absorption and bioavailability of the administered doses.

The adhesion evidenced by the compositions of the invention means that the nanoparticulate active agent particles are not easily removed by washing, rubbing, or otherwise, from the biological surface over an extended period of time. The period of time in which a biological cell surface is replenished is the factor limiting the retention of bioadhesive nanoparticulate active agent particles on such a biological surface.

F. Low viscosity pharmaceutical forms of active agents

A liquid dosage form of a microcrystalline or non-nanoparticulate active agent composition was expected to be a relatively large volume, highly viscous substance that might not be well accepted by patient populations. Additionally, viscous solutions can be problematic for parenteral administration because those solutions require slow syringe flushing and can adhere to tubes. Furthermore, conventional formulations of poorly water soluble active agents tend to be unsafe for intravenous administration techniques, which are used primarily in association with highly water soluble substances.

The liquid dosage forms of the nanoparticulate active agent compositions of the invention, comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide, provide important advantages over a liquid dosage form of a conventional microcrystalline or solubilized composition of an agent. active. The low viscosity and silky texture of liquid dosage forms of nanoparticulate compositions of

Active agents of the invention result in advantages both in preparation and in use. These advantages include, for example: (1) better patient acceptance due to the perception of a lighter formulation that is easier to ingest and digest; (2) ease of dispensing because a cup or syringe can be used; (3) formulation potential of a higher concentration of active agent, which results in a lower dose volume and, therefore, less volume to be consumed by the subject; and (4) easier consequences of the global formulation.

Liquid dosage forms of active agents that are easier to consume are especially important when considering young patients, terminally ill patients, and older patients. Viscous or gritty formulations and those that require a relatively large dose volume are not well tolerated by these patient populations. Oral liquid dosage forms may be particularly preferable for patient populations who have difficulty swallowing tablets, such as children and the elderly.

The viscosities of liquid dosage forms of a nanoparticulate active agent according to the invention are preferably less than about 1/200, less than about 1/175, less than about 1/150, less than about 1 / 125, less than approximately 1/100, less than approximately 1/75, less than approximately 1/50, or less than approximately 1/25 that of an oral liquid pharmaceutical form of a non-nanoparticulate composition of the same active agent, in approximately the same concentration per ml of active agent.

The nanoparticulate pharmaceutical forms of active agents, typically liquid, of the invention, which comprise 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, have a viscosity in a shear rate of 0.1 (1 / s), measured at 20 ° C, from about 2000 mPa-s, to about 1 mPa-s, from about 1900 mPas to about 1 mPa-s, from about 1800 mPa-s to about 1 mPa-s, from about 1700 mPas to about 1 mPa-s, from about 1600 mPas to about 1 mPa-s, from about 1500 mPa-s to about 1 mPa-s, from about 1400 mPa-s to about 1 mPa-s, from about 1300 mPa s to about 1 mPa s, from about 1200 mPa s to about 1 mPa s, from about 1100 mPa s to about 1 mPa s, from about 1000 mPa s to about 1 mPa s, from about 900 mPa s to about 1 mPa s, from about 800 mPa s to about 1 mPa s, from about 700 mPa s to about 1 mPa s, from about 600 mPa s to about 1 mPas, from about 500 mPa s to about 1 mPa s, from about 400 mPa s to about 1 mPa s, from about 300 mPa s to about 1 mPa s, from about 200 mPa s to about 1 mPa s, from about 175 mPa s to about 1 mPa s, from about 150 mPa s to about 1 mPa s, from about 125 mPa s to about 1 mPa s, from about 100 mPa s to about 1 mPa s, from about 75 mPa s to about 1 mPas, from about 50 mPas to about 1 mPas, from about 25 mPas to about 1 mPas, from about 15 mPas to about 1 mPas, from about 10 mPas to about 1 mPas, or from about 5 mPas to about 1 mPas. Such viscosity is much more attractive for patient consumption and can lead to better overall patient acceptance.

Viscosity depends on concentration and temperature. Typically, a higher concentration results in a higher viscosity, while a higher temperature results in a lower viscosity. Viscosity, as defined above, refers to measurements taken at about 20 ° C. (The viscosity of water at 20 ° C is 1 mPa s.) The invention encompasses equivalent viscosities measured at different temperatures.

Another important aspect of the invention is that the nanoparticulate active agent compositions of the invention, formulated in a liquid pharmaceutical form, are not cloudy. "Haze", as used herein, refers to the property of particulate matter that can be seen with the naked eye or can be noted as "gritty". The nanoparticulate compositions of active agents of the invention, formulated in a liquid pharmaceutical form, can be poured or extracted from a container as easily as water, while it is expected that a liquid pharmaceutical form of a non-nanoparticulate or solubilized composition thereof active agent, bring out noticeably "heavier" characteristics.

The liquid formulations of this invention can be formulated for doses of any volume, but preferably volumes equivalent to or smaller than that of a liquid dosage form of a non-nanoparticulate composition of the same active agent.

G. Nanoparticulate compositions of active agents sterilized by filtration.

ES 2 366 646 T3

The nanoparticulate active agent compositions of the invention can be sterilized by filtration. This avoids the need for heat sterilization, which can damage or degrade an active agent, as well as result in crystal growth and aggregation of active agent particles.

Sterilizing filtration can be difficult due to the small particle size of the composition required. Filtration is an effective method of sterilizing homogeneous solutions when the pore size of the membrane filter is less than or equal to about 0.2 microns (200 nm) since a 0.2 micron filter is sufficient to essentially remove the all of the bacteria. Filter sterilization is not normally used to sterilize micrometer-sized active agent suspensions because the active agent particles are too large to pass through the pores of the membrane filter.

A sterile nanoparticulate dosage form of an active agent is especially useful for treating immunocompromised patients, children or young patients and the elderly, as these patients are the most susceptible to infections caused by a non-sterile liquid dosage form.

Since the nanoparticulate compositions of active agents, of the invention, comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer, formulated as a liquid pharmaceutical form, can be sterilized by filtration, and since the compositions can have With a very small effective average particle size of the active agent, the compositions are suitable for parenteral administration.

H. Compositions of combinations of pharmacokinetic profiles

In yet another embodiment of the invention, a first nanoparticulate composition of an active agent that provides a desired pharmacokinetic profile is co-administered, successively, or is combined with at least one other composition of another active agent that generates a different, desired pharmacokinetic profile. More than two active agent compositions can be administered together, successively, or in combination. While the first active agent composition has a nanoparticulate particle size, additional compositions of one or more active agents can be nanoparticulate, solubilized, or have a microparticulate particle size.

The second, third, fourth, etc. active agent compositions may differ from the first and from each other, for example; (1) in the identity of the active agent; (2) in the effective mean particle sizes of the active agent; or (3) in the dosage of the active agent. Such compositions in combination can reduce the required frequency of doses.

For example, a first active agent composition may have a nanoparticulate particle size that is communicated by a T_mashort x and typically a larger C_max This first composition of an active agent can be combined, co-administered or administered successively with a second composition comprising: (1) the same active agent having a larger particle size (but still nanoparticulate as defined herein), and that therefore manifests a slower absorption, a T_max longer and typically a C_max more low; or (2) a microparticulate or solubilized composition of the same active agent that exhibits a T_max longer and typically a C_max less.

If the second active agent composition has a nanoparticulate particle size, then preferably the active agent particles of the second composition have at least one surface stabilizer associated with the surface of the active agent particles. The surface stabilizer (s) may be the same or different from the surface stabilizer (s) present in the first active agent composition.

Preferably, when co-administration of a "fast acting" formulation and a "longer lasting" formulation is desired, the two formulations are combined into a single composition, eg, a double release composition.

I. Various benefits of the nanoparticulate active agent compositions of the invention

The nanoparticulate active agent compositions of the invention, comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide, preferably exhibit an increased dissolution rate compared to microcrystalline or non-nanoparticulate forms of the same agent. active. Furthermore, nanoparticulate active agent compositions preferably exhibit improved performance characteristics for oral administration or by intravenous, subcutaneous or intramuscular injection, such as administration of higher doses and smaller volumes of tablet or liquid doses. Furthermore, the nanoparticulate active agent compositions of the invention do not require organic solvents or extreme pHs.

ES 2 366 646 T3

II. Compositions

The compositions of the invention comprise a nanoparticulate active agent and 4: 1 poly (Lysine, Tryptophan) hydrobromide as a surface stabilizer adsorbed or associated with the surface of the active agent. In addition, the compositions may comprise one or more secondary surface stabilizers. The surface stabilizers useful herein physically adhere to the surface of the active agent or associate with the surface of the nanoparticulate active agent, but do not react chemically with the active agent itself. The individual molecules of the surface stabilizer are essentially devoid of intermolecular crosslinks.

The present invention also includes nanoparticulate active agent compositions having 4: 1 poly (Lysine, Tryptophan) hydrobromide as at least one surface stabilizer, formulated in compositions together with one or more non-toxic, pharmaceutically acceptable excipients, adjuvants or carriers, those collectively referred to as excipients.

A. Peptide surface stabilizer

The choice of a surface stabilizer is not a trivial matter and usually requires extensive experimentation to make the desired formulation. Accordingly, the present invention is directed towards the surprising discovery that 4: 1 poly (Llsin, Tryptophan) hydrobromide used as a nanoparticulate surface stabilizer provides stable nanoparticulate active agent compositions exhibiting low degrees of aggregation.

The compositions of the invention may comprise other peptides in addition to poly (Lysine, Tryptophan) 4 hydrobromide; 1, as surface stabilizers,

A "peptide" is defined as a compound consisting of two or more amino acids that are the basic structural units or "building blocks" of peptides. All peptides of all species, from bacteria to humans, are constructed from the same set of twenty commonly present genetically encoded amino acids, as shown in the table below.

Each amino acid contains an "amino" group (NH2), a "carboxyl" group (COOH), a hydrogen atom, and a distinctive R group, or side chains, attached to a carbon atom. Amino acids vary in their side chains, with variations in size, configuration, charge, hydrogen-binding capacity, and chemical reactivity. See, for example, L. Stryer publication, Biochemistry, 3^to Edition, 1-40 (WH Freeman and Co., NY, 1988).

<td>Amino acid</td><td>3 letter abbreviation</td><td>1 letter abbreviation</td>

<td>to the girl</td><td>TO</td><td>TO</td>

<td>asparagine</td><td>ASN</td><td>N</td>

<td>Aspartic acid</td><td>ASP</td><td>D</td>

<td>arginine</td><td>ARG</td><td>R</td>

<td>cysteine</td><td>CYS</td><td>C</td>

<td>glutamic acid</td><td>GLU</td><td>AND</td>

<td>glutamine</td><td>GLN</td><td>Q</td>

<td>glycine</td><td>GLY</td><td>G</td>

<td>histidine</td><td>HIS</td><td>H</td>

<td>isoleucine</td><td>ILE</td><td>I</td>

<td>leucine</td><td>LEU</td><td>L</td>

<td>lysine</td><td>LYS</td><td>K</td>

<td>methionine</td><td>MET</td><td>M</td>

<td>phenylalanine</td><td>PHE</td><td>F</td>

<td>proline</td><td>PRO</td><td>P</td>

ES 2 366 646 T3

<td>Amino acid</td><td>3 letter abbreviation</td><td>1 letter abbreviation</td>

<td>serine</td><td>TO BE</td><td>S</td>

<td>threonine</td><td>THR</td><td>T</td>

<td>tryptophan</td><td>TRP</td><td>w</td>

<td>tyrosine</td><td>TYR</td><td>Y</td>

<td>valine</td><td>VAL</td><td>V</td>

<td>aspartic acid or asparagine</td><td>ASX</td><td></td>

<td>glutamic acid or glutamine</td><td>GLX</td><td></td>

<td>Unknown or others</td><td>Xaa</td><td>X</td>

The peptides useful in the present invention may also comprise substituents other than amino acids. There are also naturally occurring chemical modifications of these twenty genetically encoded amino acids, such as the hydroxylation of proline, the addition of carbohydrates and lipids, and the phosphorylation of serine and tyrosine. In addition, D isomers of the amino acids have been synthesized into opposition to L isomers found in natural peptides and proteins.

The amino acids of a peptide are connected by an amido group, a covalent bond between the carboxyl group in the alpha position of one amino acid and the amino group in the alpha position of another amino acid. Many amino acids are joined by peptide bonds to form a polypeptide chain, which is unbranched. A polypeptide chain is a long peptide chain, consisting of a regularly repeating part, called the main chain, and a variable part, which comprises the distinctive side chains. Disulfide crosslinks can be formed in polypeptides by cysteine ​​residues of the polypeptides. Most natural polypeptide chains contain between 50 and 200 amino acid residues. The average molecular weight of an amino acid residue is approximately 110 daltons, and therefore the molecular weights of most polypeptide chains are between 5,500 and 220,000. See, for example, L. Stryer publication, Biochemistry. 3^to Edition, p. 22 (WH Freeman and Co., NY, 1988).

A protein is a large macromolecule made up of one or more polypeptide chains. In the context of the present invention, a "peptide" refers to a peptide or a polypeptide, but not a protein.

Preferably, the peptide surface stabilizers of the invention are. soluble in water. By "water soluble" it is meant that the peptide possesses a solubility in water greater than approximately 1 mg / ml, greater than approximately 10 mg / ml, greater than approximately 20 mg / ml, or greater than 30 mg / ml. , about. This is in contrast to prior art compositions that teach the use of a peptide as an active agent in a nanoparticulate composition of an active agent. See, for example, US Patent Nos. 6,270,806; 6,592,903; 6,428,814; and 6,375,986. In such prior art literature references, where a peptide is used as an active agent in a nanoparticulate composition, the peptide is poorly soluble in water.

There is an extensive catalog of commercially available peptides that can be used in the compositions of the invention. For example, the on-line peptide catalog http: // www. peptidecatalog.com/PC/Peptides provides a list of hundreds of commercially available peptides, along with their structures and molecular weights. In addition, for most commercially available peptides, the usual peptides can be prepared and used in the compositions of the invention.

B. Secondary or auxiliary surface stabilizers.

Compositions of the invention may also include one or more non-peptide surface stabilizers in addition to 4: 1 poly (Lysine, Tryptophan) hydrobromide as at least one peptide surface stabilizer.

The auxiliary surface stabilizers of the invention are preferably adsorbed on or associated with the surface of the active agent particles. Auxiliary surface stabilizers especially useful in this invention preferably do not react chemically with the active agent particles or with themselves. Preferably, the individual molecules of the auxiliary surface stabilizer are essentially free from intermolecular crosslinks.

Two or more auxiliary surface stabilizers may be employed in the compositions and methods of the invention.

ES 2 366 646 T3

Suitable surface stabilizers can be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products, and surfactants. Preferred auxiliary surface stabilizers include nonionic, anionic, cationic, zwitterionic, and ionic surfactants.

Representative examples of secondary surface stabilizers include gelatin, casein, lecithin (phosphatides), dextran, acacia, cholesterol, gum tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, ketostearyl alcohol, cetomacrogol emulsifying wax. , sorbitan esters, polyoxyethylene alkyl ethers (e.g. macrogol ethers such as ketomacrogol 1000), polyoxyethylene castor oil derivatives, polyoxyethylenated sorbitan fatty acid esters (for example, commercially available Tweens® such as, for example, Tween 20® and Tween 80® (ICI Specialty Chemicals)); polyethylene glycols (eg, Carbowax 3550® and 934® (Union Carbide)); Polyoxyethylene stearates, colloidal silicon dioxide, sodium dodecyl sulfate phosphates, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl celluloses (for example, HPC, HPC-SL and HPC-L (HPC-L), hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polymer of 4- (1,1,3,3-tetramethylbutyl) phenol with ethylene oxide and formaldehyde (also known as tyloxapol, superione and triton), poloxamers (for example, Pluronics F68® and F108® which are block copolymers of oxide ethylene and propylene oxide), poloxamines (for example, Tetronic 908® also known as Poloxamine 908®, which is a tetrafunctional block copolymer derived from the successive addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, NJ)); Tetronic 1508® (T-1508) (BASF Wyandotte Corporation), dialkyl esters of the sodium salt of sulfosuccinic acid (for example, Aerosol OT®, which is a dioctyl ester of the sodium salt of sulfosuccinic acid (DOSS) (American Cyanamid) ); Duponol P®, which is a sodium lauryl sulfate (DuPont); Tritons X-200®, which is an alkyl aryl polyether sulfonate (Rohm and Haas); Crodestas F-110®, which is a mixture of sucrose stearate and sucrose distearate (Croda Inc.); p-isononylphenoxypoly (glycidol), also known as Olin-IOG® or Surfactant 10-G® (Olin Chemicals, Stamford, CT); Crodestas SL-40® (Croda, Inc); and SA9OHCO, which is Ci₈H37CH2C (O) N (CH3) -CH2 (CHOH) 4 (CH2OH)₂ (Eastman Kodak Co.): Decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside; n-decyl ^ -D-maltopyranoside; n-dodecyl ^ -D-glucopyranoside; n-dodecyl-βD-maltoside; heptanoyl-N-methylglucamine; n-heptyl ^ -D-glucopyranoside; n-heptyl ^ -D-thioglucoside; n-hexyl ^ -Dglucopyranoside; nonanoyl-N-methylglucamide; n-nonanoyl ^ -D-glucopyranoside; octanoyl-N-methylglucamide; η-οοίϋ-β-ϋglucopyranoside; octyl ^ -D-thioglucopyranoside; lysozyme; PEG-derivatized phospholipid, PEG-derivatized cholesterol, PEG-derivatized cholesterol, PEG-derivatized vitamin A, PEG-derivatized vitamin E, random copolymers of vinylpyrrolidone and vinyl acetate, and the like.

Examples of useful cationic surface stabilizers include, but are not limited to, polymers, biopolymers, polysaccharides, cellulosic compounds, alginates, phospholipids, and non-polymeric compounds, such as zwitterionic stabilizers, poly-n-methylpyridinium, anthriulpyridinium chloride, cationic phospholipids, charged phospholipids such as dimyristoylphosphatidylglycerol, chitosan, polysine, polyvinylimidazole, polybrene, poly (methyl methacrylate) trimethylammonium bromide (PMMTMABr), hexyldesyltrimethylammonium bromide (HDMAB) and polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethylsulfate.

Other useful cationic stabilizers include, but are not limited to, cationic lipids composed of sulfonium, phosphonium, and quaternary ammonium, such as stearyltrimethylammonium chloride, benzyl-di (2-chloroethyl) ethylammonium bromide, coccotrimethylammonium chloride or bromide, chloride or bromide. of cocomethyldihydroxyethylammonium; dodecyltrimethylammonium bromide decyltriethylammonium chloride, decyldimethylhydroxyethylammonium chloride or bromide, Ci chloride or bromide₂.i5dimethylhydroxyethylammonium, cocodimethylhydroxyethylammonium chloride or bromide, myristyltrimethylammonium methylsulfate, lauryldimethylbenzylammonium chloride or bromide, lauryldimethyl (ethenoxy) 4 ammonium chloride or bromide, N-alkylchloride₂.i8) dimethylbenzylammonium, N-tetradecyldimethylbenzylammonium chloride monohydrate, dimethyldidecylammonium chloride, N-alkyl chloride and (Ci₂_i4) dimethyl 1-naphthylmethylammonium, trimethylammonium halide, alkyltrimethylammonium salts and dialkyldimethylammonium salts, lauryltrimethylammonium chloride, ethoxylated salt of alkylamidoalkyldialkylammonium and / or ethoxylated salt of trialkyldimethylammonium-chloro dimethyl ammonium dimethyl ammonium, tetrakyldimethylammonium-chloro-di-alkylbeniozenediodide, ethoxylated salt of trialkyldimethylammonium-chloro dimethyl ammonium dimethyl ammonium, tetrakyldimethylammonio-chloro-di-ethoxylated salt, tetrakyl dimethyl ammonioidemoniodide , monohydrate, N-alkyl chloride (Ci₂_i4) dimethyl-1-naphthylmethylammonium and dodecyldimethylbenzylammonium chloride, dialkylbenzenealkylammonium chloride, lauryltrimethylammonium chloride, alkylbenzylmethylammonium chloride, Ci bromide₂, Ci5Ci7-trimethylammonium, dodecylbenzyltriethylammonium chloride, poly-diallyldimethylammonium chloride (DADMAC), dimethylammonium chlorides, alkyldimethylammonium halides, tricetylmethylammonium chloride, decyltrimethylammonium bromide ™ (DADMAC), Dimethylammonium chlorideAT6, alkyldimethylammonium halides, tricethylmethylammonium chloride, decyltrimethylammonium bromide ™ (DADMAC), 33 decyl trimethylammonium trimethyl ammonium bromide, 33-decyl trimethylammonium trimethyl ammonium bromide, AL , POLYQUAT 10 ™, tetrabutylammonium bromide, benzyltrimethylammonium bromide, choline esters (such as fatty acid choline esters), benzalkonium chloride, stearalkonium chloride compounds (such as stearyltrimonium chloride and di-stearyldimonium chloride), cetylpyridinium bromide or chloride, quaternized polyoxyethylalkylamines halide salts, MIRAPOL ™ and ALKAQUAT ™ (Alkaril Chemical Company), alkylpyridinium salts; amines, such as alkylamines, dialkylamines, alkanolamines, polyethylenepolyamines, N, N-dialkylaminoalkyl acrylates, and vinylpyridine, amine salts, such as laurylamine acetate, stearylamine acetate, alkylpyridinium salt and alkylimidazolium salt, and amine oxides, you come out of

ES 2 366 646 T3 imidazolinium, protonated quaternary acrylamides, methylated quaternary polymers, such as poly (diallyldimethylammonium chloride) and poly (N-methylvinylpyridinium chloride); and cationic guar gum.

Such exemplary cationic surface stabilizers and other useful cationic surface stabilizers have been described by: J. Cross and E. Singer in Cationic Surfactants: Analytical and Biological Evaluation (Marcel Dekker, 1994); P and D. Rubingh (Compiler), Cationic Surfactants Physical Chemistry (Marcel Dekker, 1991); and J. Richmond, Cationic Surfactants Organic Chemistry (Marcel Dekker, 1990).

Particularly preferred non-polymeric primary stabilizers are any non-polymeric compound, such as benzalkonium chloride, carbonium compounds, phosphonium compounds, oxonium compounds, halonium compounds, cationic organometallic compounds, quaternary phosphorous compounds, pyridinium compounds, anilinium compound , immonium compounds, hydroxyammonium compounds, primary ammonium compounds, secondary ammonium compounds, tertiary ammonium compounds and quaternary ammonium compounds of the formula NR- ^ RsR © For compounds of the formula NR1R2R3RZ (i) none of R1-R4 are CH3;

(ii) one of R1-R4 is CH₂;

(iii) three of R1-R4 are CH3;

(iv) all of R1-R4 are CH3;

(v) two of R1-R4 are CH3, one of R1-R4 is CeHsCl-h, and one of R1-R4 is an alkyl chain of seven carbon atoms or less;

(vi) two of R1-R4 are CH3, one of R1-R4 is CeHsCI-h, and one of R1-R4 is an alkyl chain of nineteen carbon atoms or more;

(vii) two of R1-R4 are CH3 and one of R1-R4 is the group CeH5 (CH₂)_n, in whose formula n&gt; 1;

(viii) two of R1-R4 are CH3, one of R1-R4 is CeHsCHz and one of R1-R4 comprises at least one heteroatom;

(ix) two of R1-R4 are CH3, one of R1-R4 is CeHsCI-h and one of R1-R4 comprises at least one halogen;

(x) two of R1-R4 are CH3, one of R1-R4 is CeHsCI-h and one of R1-R4 comprises at least one cyclic fragment;

(xi) two of R1-R4 are CH3, and one of R1-R4 is a phenyl ring; or (xii) two of R1-R4 are CH3, and two of R1-R4 are simply aliphatic fragments.

Such compounds include, but are not limited to, behenalkonium chloride, benzethonium chloride, cetylpyridinium chloride, behentrimonium chloride, lauralkonium chloride, ketalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylamine hydrofluoride. Chloralylmethenamine Chloride (Quaternium-15), Distearyldimonium Chloride (Quaternium-5), Dodecyldimethylethylbenzylammonium Chloride (Quaternium-14), Quaternium-22, Quaternium-26, Quaternium-18 Hectorite, Dimethylaminoethyl Cichloride Hydrochloride, Dimethylaminoethyl Chloride Hydrochloride diethanolammonium POE (10) olethyl ether, diethanolammonium POE (3) oleyl ether phosphate, tallowalkonium chloride, dimethyldioctadecylammonium bentonite, stearalkonium chloride, domiphene bromide, Denatonium benzoate, myristalkonium chloride, laurtrimonium chloride, ethylenediamine dihydrochloride, guanidine hydrochloride, pyridoxine hydrochloride, iofetamine hydrochloride, meglumine hydrochloride, methylbenzethonium chloride, polyethenyl trimonium hydrochloride, 1-olemonium trimonium chloride, polyethenyltrimonium bromide, myrtrimonium hydrochloride procaine, cocobetaine, stearalkonium bentonite, stearalkonium hectonite, stearyltrihydroxyethylpropylenediamine dihydrofluoride, sebotrimonium chloride, and hexadecyltrimethylammonium bromide.

Most of these surface stabilizers are known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, jointly published by the American Pharmaceutical Association and The Pharmaceutical Society of Great Britain (The Pharmaceutical Press, 1986). Surface stabilizers are commercially available and / or can be prepared by procedures known in the art.

C. Active agents

The nanoparticles of the invention comprise at least one active, therapeutic or diagnostic agent, collectively referred to as "drug". A therapeutic agent can be a pharmaceutical agent, including biological compounds such as proteins, peptides, and nucleotides, or a diagnostic agent, such as a contrast agent, including X-ray contrast agents.

ES 2 366 646 T3

The active agent exists as a crystalline phase, an amorphous phase, a semi-morpho phase, a semi-crystalline phase, or mixtures thereof. The crystalline phase differs from a non-crystalline or amorphous phase, which results from precipitation techniques, such as those described in EP Patent No. 275,796.

The invention can be practiced with a wide variety of active agents. The active agent is preferably present in essentially pure form, is poorly soluble and is dispersible in at least one liquid dispersion medium. By "sparingly soluble" is meant that the active agent possesses a solubility in a liquid dispersion medium of less than about 30 mg / ml, less than about 20 mg / ml, less than about 10 mg / ml, or less than 1 mg / ml, approximately. Liquid dispersion media include, but are not limited to, water, aqueous saline solutions, safflower oil, and solvents such as ethanol, t-butanol, hexane, and glycol. A preferred liquid dispersion medium is water.

Two or more active agents can be used in combination.

1. Active agents in general

The active agent can be selected from a variety of known classes of drugs, including, for example, nutraceuticals, COX-2 inhibitors, retinoids. anticancer agents, NSAIDS, proteins, peptides, nucleotides, anti-obesity drugs, dietary supplements, carotenoids, corticosteroids, elastase inhibitors, antifungal products, cancer therapy products, antiemetics, analgesics, cardiovascular agents, anti-inflammatory agents, anthelmintics, antiarrhythmic agents antibiotics (including penicillins), anticoagulants, antidepressants, antidiabetic agents, antiepileptics, antihistamines, antihypertensive agents, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, immunosuppressants, antithyroid agents, antiviral agents, anxiolytics, sedatives (hypnotics and neuroleptics), astringents, beta-adrenergic blocking agents, inotropic agents and blood substitutes, cardiac agents contrast, corticosteroids, cough suppressants (expectorants and mucolytics), diagnostic agents, optic diagnostic agents, diuretics, dopaminergic (antiparkinsonian agents), hemostats, immunological agents, lipid regulating agents, muscle relaxants, parasympathomimetics, calcitonin and parathyroid bisphosphonates, prostaglandins, radiopharmaceuticals, sex hormones (including spheroids) antiallergic, stimulant and anoretic agents, sympathomimetics, thyroid agents, vasodilators and xanthines.

Examples of representative active agents useful in the invention include, but are not limited to, acyclovir, alprazolam, altretamine, amiloride, amiodarone, benzotropine mesylate, bupropion, cabergoline, candesartan, cerivastatin, chlorpromazine, ciprofloxacin, cisapride, clarithomycin, clonidine clopidogrel, cyclobenzaprine, cyproheptadine, delavirdine, desmopressin, diltiazem, dipyridamole, dolasetron, enalapril maleate, enalaprilat, famotidine, felodipine, Furazolidone, Glipizide, Irbesartan, Ketoconazole, Lansoprazole, Loratadine, Loxapine, Mebendazole, Mercaptopurine, Milrinone Lactate, Minocycline, Mitoxantrone, Nelfinavir Mesylate, Nimodipine, Norfloxacin, Olanzapine, Penzifloprazolimide, Taxifloprazolimide, Pinozifloprazolimid, rifampin, risperidone, rizatriptan, saquinavir, sertraline, sildenafil, acetylsulfisoxazole, temazepam, thiabendazole, thioguanine, trandolapril, triamterene, trimetrexate, troglitazone, trovafloxacin, verapamil, vinblastine sulfate, mycophenolate, atovaquone, proguanil, ceftazidime, cefuroxime, etoposide, terbinafine, thalidomide, fluconazole, amsacrine, dacarbazine, teniposide, and acetylsalicylate.

Exemplary nutraceutical products and dietary supplements are described, for example, in Roberts et al., Nutraceuticals: The Complete Encyclopedia of Supplements, Herbs, Vitamins, and Healing Foods (American Nutraceutical Association, 2001). A nutraceutical agent or dietary supplement, also known as a phytochemical agent or functional food, is, in general, any one of a class of dietary supplements, vitamins, minerals, herbs, or healing foods that have medicinal or pharmaceutical effects on the body. Exemplary nutraceutical products or dietary supplements include, but are not limited to, lutein, folic acid, fatty acids (eg, DHA and ARA), fruit and plant extracts, vitamin and mineral supplements, phosphatidylserine, acid lipoic, melatonin, glucosamine / chondroitin, Aloe Vera, Guggul, glutamine, amino acids (for example, isoleucine, leucine, lysine, methionine, phenylanine, threonine, tryptophan, and valine), green tea, lycopene, whole foods, food additives, herbs, phytonutrients, antioxidants, fruit flavonoid constituents, evening primrose oil (EPO), flax seeds, fish and marine animal oils, and probiotic agents. Nutraceutical products and dietary supplements also include bio-transformed foods, genetically transformed to have desired properties, also known as "pharmaceuticals"

Active agents for administration in an aerosol formulation are preferably selected from the group consisting of proteins, peptides, bronchodilators, corticosteroids, elastase inhibitors, analgesics, antifungals, agents for cystic fibrosis therapies, asthma therapies, therapies of emphysema, respiratory failure syndrome therapies, chronic bronchitis therapies, obstructive, chronic pulmonary disease therapies, transplant rejection therapies, therapies for tuberculosis and other infections of the

ES 2 366 646 T3 lung, fungal infection therapies, respiratory failure therapies associated with acquired immunodeficiency syndrome, cancer drugs, antiemetics, analgesics and cardiovascular agents.

2. Active anticancer agents

Useful anticancer agents are preferably selected from alkylating agents, antimetabolites, natural products, hormones and antagonists, and various agents, such as radiosensitizers.

Examples of alkylating agents include; (1) alkylating agents possessing the bis- (2-chloroethyl) -amino group such as, for example, chlormethine, chlorambucil, melphalan, uramustine, manomustine, extramustine phosphate, mechlorethamine oxide, cyclophosphamide, ifosphamide, and triphosphamide; (2) alkylating agents possessing a substituted aziridine group such as, for example, tretamine, thiotepa, triaziquone, and mitomycin; (3) Alkylating agents of the alkyl sulfonate type, such as, for example, busulfan, piposulfan and piposulfam; (4) alkylating derivatives of N-alkyl-N-nitrosourea such as, for example, carmustine, lomustine, semustine, or streptozotocin; and (5) alkylating agents such as mitobronitol, dacarbazine, and procarbazine.

Examples of antimetabolites include: (1) folic acid analogs such as, for example, methotrexate; (2) pyrimidine analogs such as, for example, fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine, and flucytosine; and (3) purine derivatives such as, for example, mercaptopurine, thioguanine, azathioprine, thiamiprine, vidarabine, pentostatin, and puromycin.

Examples of natural products include: vinca alkaloids such as, for example, vinblastine and vincristine; (2) epipodophyllotoxins such as, for example, etoposide and teniposide; (3) antibiotics such as, for example, adriamycin, daunomycin, doctinomycin, daunorubicin, doxorubicin, mithramycin, bleomycin, and mitomycin; (4) enzymes such as, for example, L-asparaginase; (5) biological response modifiers such as, for example, alpha-interferon; (6) camptothecin; (7) taxol; and (8) retinoids, such as retinoic acid.

Examples of hormones and antagonists include: (1) adrenocorticosteroids such as, for example, prednisone; (2) progestins such as, for example, hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; (3) estrogens such as, for example, diethylstilbestrol and ethinyl estradiol; (4) anti-estrogens such as, for example, tamoxifen; (5) androgens such as, for example, testosterone propionate and fluoxymesterone; (6) antiandrogens such as, for example, flutamide; and (7) gonadotropin-releasing hormone analogs such as, for example, leuprolide.

Examples of various agents include: (1) radiosensitizers such as, for example, 1,2,4-benzotriazin-3-amino-1,4-dioxide (SR 4889) and 1,2,4-benzotriazine-7-amino-1, 4-dioxide (WIN 59075); (2) platinum coordination complexes such as cisplatin and carboplatin; (3) anthracenediones such as, for example, mitoxantrone; (4) substituted ureas such as, for example, hydroxyurea; and (5) adrenocortical suppressors such as, for example, mitotane and aminoglutethimide.

In addition, the anticancer agent can be an immunosuppressive drug such as, for example, cyclosporine, azathioprine, sulfasalzan, methoxsalcen, and thalidomide.

The anticancer agent can also be a COX-2 inhibitor.

3. Active analgesic agents

An analgesic can be, for example, an NSAID or a COX-2 inhibitor

Exemplary NSAIDs that may be formulated into compositions of the invention include, but are not limited to, non-acid compounds and suitable acidic compounds. Suitable non-acidic compounds include, for example, nabumetone, thiaramide, proquazone, bufexamac, flumizole, epirazole, tinoridine, timegadine, and dapsone. Suitable acidic compounds include, for example, carboxylic acids and enolic acids. Suitable carboxylic acid type NSAIDs include, for example: (1) salicylic acids and their esters, such as aspirin, diflunisal, benorylate, and phosphosalt; (2) acetic acids such as phenylacetic acids, including diclofenac, alclofenac, and fenclofenac; (3) carbocyclic and heterocyclic acetic acids such as etodolac, indomethacin, sulindac, tolmetin, fenthiazac, and tilomisole; (4) propionic acids such as carprofen, fenbufen, flurbiprofen, ketoprofen, oxaprozin, suprofen, tiaprofenic acid, ibuprofen, naproxen, fenoprofen, indoprofen, and pyrprofen; and (5) Phenamic acids such as flufenamic, mefenamic, meclofenamic, and niflumic. Suitable enolic acid type NSAIDs include, for example: (1) pyrazolones such as oxyphenbutazone, phenylbutazone, apazone and feprazone; and (2) oxicams such as piroxicam, sudoxicam, isoxicam, and tenoxicam.

Exemplary COX-2 inhibitors that may be formulated in combination with the nanoparticulate nimesulide composition of the invention include, but are not limited to, celecoxib (SC-58635, CELEBREX®, Pharmacia / Searle and Co.), rofecoxib (MK-966, L-748731, VIOXX®, Merck and Co.), meloxicam

ES 2 366 646 T3 (MOBIC®, jointly marketed by Abbott Laboratories, Chicago, IL, and Boehringer Ingelheim Pharmaceuticals), valdecoxib (BEXTRA®, GD Searle and Co.), parecoxib (GD Searle and Co.), etoricoxib (MK663; Merck), SC-236 (chemical name for 4- [5- (4-chlorophenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl)] benzenesulfonamide; GD Searle and Co., Skokie, IL); NS-398 (N- (2-cyclohexyloxy-4-nitrophenyl) methane sulfonamide; Taisho Pharmaceutical Co., Ltd., Japan), SC-58125 (methylsulfone-spiro (2,4) hept-5-ene I; Pharmacia / Searle and Co.); SC-57666 (Pharmacia / Searle and Co.); SC-558 (Pharmacia / Searle and Co.); SC-560 (Pharmacia / Searle and Co.); etodolac (Lodine®, Wyeth-Ayerst Laboratories Inc.); DFU (5,5-dimethyl-3- (3-fluorophenyl) -4- (4-methylsulfonyl) phenyl 2 (5H) -furanone); monteleukast (MK-476), L-745337 ((5-methanesulfonamide-6- (2,4-difluorothiophenyl) -1-indanone), L-761066, L-761000, L-748780 (all from Merck and Co.) ; DUP-697 (5-bromo-2- (4-fluorophenyl) -3- (4- (methylsulfonyl) phenyl; DuPont Merck Pharmacuetical Co.); PGV 20229 (1- (7-tert-butyl-2,3- dihydro-3,3-dimethylbenzo (b) furan-5-yl) -4-cyclopropylbutan-1-one; Procter and Gamble Pharmaceuticals); iguratimod (T-614; 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H -1benzopyran-4-one; Toyama Corp., Japan); BF 389 (Biofor, USA); CL 1004 (PD 136095), PD 136005, PD 142893, PD 138387, and PD 145065 (all from Parke-Davis / Warner Lambed Co.); flurbiprofen (ANSAID®; Pharmacia and Upjohn); nabumetone (FELAFEN®; SmithKIine Beecham, pie); flosulide (CGP 28238; Novartis / Ciba Geigy); piroxicam (FELDANE®; Pfizer); diclofenac (VOLTAREN® and CATAFLAM®, Novartis); lumiracoxib (COX-189; Novartis); D 1367 (Celltech Chiroscience, footnote); R 807 (3-benzoyldifluoromethane sulfonanilide, diflumidone); JTE-522 (Japan Tobáceo, Japan); FK-3311 (4'-acetyl-2 '- (2,4-difluorophenoxy) methanesulfonanilide), FK 867; FR 140423, and FR 115068 (all from Fujisawa, Japan); GR 253035 (Glaxo Wellcome); RWJ 63556 (Johnson and Johnson); RWJ 20485 (Johnson and Johnson); ZK 38997 (Schering); S 2474 ((E) - (5) - (3,5-di-tert-butyl-4-hydroxybenzylidene) -2-ethyl-1,2-isothiazolidine-1,1 dioxide indomethacin (Shionogi and Co., Ltd, Japan); Zomepirac analogs such as RS 57067 and RS 104897 (Hoffmann La Roche) RS 104894 (Hoffmann La Roche); SC 41930 (Monsanto); pranlukast (SB 205312, Ono-1078, ONON®, ULTAIR®; SmithKIine Beecham); SB 209670 (SmithKIine Beecham); and APHS (heptynyl sulfide).

D. Particle size of the nanoparticulate active agent

Compositions of the invention contain nanoparticulate active agent particles having an effective mean particle size of less than about 2000 nm (ie, 2 microns). In other embodiments of the invention, the active agent particles are smaller than about 1900 nm, less than about 1800 nm, less than about 1700 nm, less than about 1600 nm, less than about 1500 nm, less than about 1400 nm. , less than approximately 1,300 nm, less than approximately 1,200 nm, less than approximately 1,100 nm, less than approximately 1,000 nm, less than approximately 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than About 150 nm, less than about 100 nm, less than about 75 nm, or less than about 50 nm, as measured by light scattering, microscopy, or other appropriate methods.

By "an effective mean particle size less than about 2000 nm" is meant that at least 50% by weight of the active agent particles have a smaller particle size than the effective mean size, that is, less than about 2000nm, 1900nm, 1800nm, etc., when measured by the aforementioned techniques. In other embodiments of the invention, at least about 70%, at least about 90%, at least about 95%, or at least about 99% of the active agent particles have a size of particle smaller than effective mean size, ie smaller than about 2000 nm, 1900 nm, 1800 nm, etc.

If the nanoparticulate active agent composition is combined with a conventional active agent composition, then such composition is either solubilized or possesses an effective mean particle size greater than about 2 microns. By "an effective mean particle size greater than about 2 microns" is meant that at least 50% of the microparticulate active agent particles have a particle size greater than about 2 microns by weight, measured using the aforementioned techniques. In other embodiments of the invention, at least about 70%, at least about 90%, at least about 95%, or at least about 99%, by weight, of the microparticulate active agent particles have a particle size greater than 2 microns. , about.

In the present invention, the D50 value of a nanoparticulate composition of an active agent is the particle size below which 50% by weight of the active agent particles fall. Similarly, D90 and D99 are the particle sizes below which 90% and 99% by weight, respectively, of the active agent particles fall.

5. Concentration of the nanoparticulate active agent and of the poly (L¡sina, Tryptophan) hydrobromide 4: 1.

The relative amounts of active agent and 4: 1 poly (Lysine, Tryptophan) hydrobromide, and optionally one or more secondary surface stabilizers, can vary widely. The optimal amount of components

Individual ES 2 366 646 T3 depends, for example, on the particular active agent selected, the hydrophilic lipophilic balance (HLB), the melting point and the surface tension of the aqueous solutions of the stabilizer, etc.

The concentration of the peptide surface stabilizer can range from about 0.5% to about 99.999%, from about 5.0% to about 99.9%, or from about 10% to about 99.5%, by weight, based on the Total combined dry weight of the at least one active agent and the poly (Lysine, Tryptophan) hydrobromide 4: 1, excluding other excipients.

The concentration of the at least one active agent may range from about 99.5% to about 0.001%, from about 95% to about 0.1%, or from about 90% to about 0.5%, by weight, based on the total combined dry weight of the active agent and poly (Lysine, Tryptophan) hydrobromide 4: 1, excluding other excipients.

B. Methods for preparing nanoparticulate formulations of active agents

Nanoparticulate active agent compositions of the invention comprising 4: 1 poly (Lysine, Tryptophan) hydrobromide can be prepared, for example, using milling, homogenization or precipitation techniques. Exemplary methods of preparing nanoparticulate compositions are described in the '684 patent. Methods of preparing nanoparticulate active agent compositions are also described in US Patent No. 5,518,187 for "Method of grinding pharmaceutical substances"; US Patent No. 5,718,388 for "Continuous Method of Grinding Pharmaceutical Substances", US Patent No. 5,862,999 for "Method of Grinding Pharmaceutical Substances"; US Patent No. 5,665,331 for "Co-Microprecipitation of Nanoparticulate Pharmaceutical Agents with Crystal Growth Modifiers"; US Patent No. 5,662,883 for "Co-Microprecipitation of Nanoparticulate Pharmaceutical Agents with Crystal Growth Modifiers"; US Patent No. 5,560,932 for "Microprecipitation of Nanoparticulate Pharmaceutical Agents"; US Patent No. 5,543,133 for "Process for the Preparation of X-ray Contrast Compositions Containing Nanoparticles"; US Patent No. 5,534,270 for "Method of Preparation of Stable Drug Nanoparticles"; US Pat. No. 5,510,118 for "Process for the preparation of therapeutic compositions containing nanoparticles"; and US Patent No. 5,470,583 for "Method of preparing nanoparticulate compositions containing charged phospholipids to reduce aggregation."

The resulting nanoparticulate active agent compositions can be used in any desired dosage form.

1. Grinding to obtain nanoparticulate dispersions of active agents

The milling of the active agent to obtain a nanoparticulate dispersion comprises dispersing particles of the active agent in a liquid dispersion medium in which the active agent is sparingly soluble, followed by the application of mechanical means, in the presence of milling media to reduce the size. active agent particle size to the desired effective average particle size. The dispersion media can be, for example, water, safflower oil, ethanol, t-butanol, glycerin, polyethylene glycol (PEG), hexane or glycol. Water is the preferred dispersion medium.

The active agent particles are preferably reduced in size in the presence of 4: 1 poly (Lysine, Tryptophan) hydrobromide. Alternatively, the active agent particles can be contacted with 4: 1 poly (Lysine, Tryptophan) hydrobromide, either during or after attrition. One or more secondary stabilizers can also be added before, during or after wear. Other compounds, such as a diluent, can be added to the peptide surface stabilizer / active agent composition before, during, or after the size reduction process. Dispersions can be prepared continuously or in batches.

2. Precipitation to obtain nanoparticulate compositions of active agents

Another method of forming the desired nanoparticulate active agent composition is by microprecipitation. This is a method of preparing stable dispersions of poorly soluble active agents in the presence of one or more peptide surface stabilizers including 4: 1 poly (Lysine, Tryptophan) hydrobromide, and one or more colloidal surface active agents that enhance the stability, free from traces of toxic solvents or solubilized heavy metal impurities. Such a method comprises, for example: (1) dissolving the poorly soluble active agent in a suitable solvent; (2) adding the formulation from step (1) to a solution comprising at least poly (Lysine, Tryptophan) 4; 1 hydrobromide as a peptide surface stabilizer and, optionally, one or more secondary surface stabilizers, to form a solution clear; and (3) precipitating the formulation from step (2) using an appropriate non-solvent. The method can be followed by removing any salt formed, if present, by dialysis or diafiltration and concentrating the dispersion by conventional methods.

3. Homogenization to obtain nanoparticulate compositions of active agents

ES 2 366 646 T3

Exemplary homogenization methods for preparing nanoparticulate active agent compositions are described in US Patent No. 5,510,118 for "Method of Preparation of Therapeutic Compositions Containing Nanoparticles."

Such a method comprises dispersing the active agent particles in a liquid dispersion medium in which the active agent is poorly soluble, followed by subjecting the dispersion to homogenization to reduce the active agent particle size to the effective average particle size. wanted. The active agent particles can be reduced in size in the presence of 4: 1 poly (Lysine, Tryptophan) hydrobromide as at least one peptide surface stabilizer and, if desired, one or more additional surface stabilizers. Alternatively, the active agent particles can be contacted with 4: 1 poly (Lysine, Tryptophan) hydrobromide and, if desired, one or more additional surface stabilizers, or during or after attrition. Other compounds, such as a diluent, can be added to the active agent / peptide surface stabilizer composition either before, during or after the size reduction process. Dispersions can be made continuously or in batches.

C. Methods of use of nanoparticulate formulations of active agents

The nanoparticulate active agent compositions of the present invention can be administered to humans and animals by any conventional means including, but not limited to, oral, rectal, ocular, parenteral (intravenous, intramuscular, or intravenous) routes. subcutaneous), intracisternal, pulmonary, intravaginal, intraperitoneal, local (powders, ointments or drops), or as a buccal or nasal spray.

Compositions suitable for parenteral injection may comprise sterile, physiologically acceptable aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution to obtain sterile injectable solutions or dispersions. Examples of suitable aqueous or non-aqueous excipients, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerin, and the like), suitable mixtures thereof, vegetable oils (such as olive oil), and esters. injectable organics such as ethyl oleate. A proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Nanoparticulate active agent compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. The prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be achieved through the use of agents that delay absorption, such as aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one of the following: (a) one or more inert excipients (or carriers), such as sodium citrate or dicalcium phosphate; (b) solid fillers or diluents, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (c) binders, such as carboxymethylcellulose, alginates, polyvinylpyrrolidone, sucrose, and acacia; (d) humectants, such as glycerin; (e) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (f) dissolution retarders, such as paraffin; (g) absorption accelerators, such as quaternary ammonium compounds; (h) wetting agents, such as cetyl alcohol and glyceryl monostearate; (i) adsorbents, such as kaolin and bentonite; and (j) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. For capsules, tablets and pills, the dosage forms may also comprise buffering agents. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent, liquid dosage forms may comprise inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents. Exemplary emulsifying agents are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils such as cottonseed oil, peanut oil. , corn germ oil, olive oil, castor oil, and sesame oil, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols, sorbitan fatty acid esters, or mixtures of these substances, and the like.

In addition to such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and flavoring agents.

Current levels of active agent doses in the nanoparticulate compositions of the invention can be varied to obtain an amount of active agent effective to achieve the desired therapeutic response for a particular composition and method of administration. The dose level selected depends, for

ES 2 366 646 T3 consequently of the desired therapeutic effect, the route of administration, the potency of the active agent administered, the desired duration of treatment, and other factors.

Administration unit compositions may contain amounts of such submultiples thereof, which can be used to obtain daily doses. It is to be understood, however, that the specific dose level for a particular patient will depend on a variety of factors including weight, general health, sex, diet, time and route of administration, potency of the active agent administered, the rates of absorption and excretion, the combination with other active agents, and the severity of the particular disease being treated.

The examples that follow, with the exception of Examples 1 to 3, are provided to illustrate the present invention. However, it is to be understood that the invention is not to be limited to the specific conditions or details that are described in these examples.

Examples 1 to 3 have been retained for the sake of clarity and for a more complete understanding of the invention.

The formulations listed in the examples below were also investigated using an optical microscope. Here, "stable" nanoparticulate dispersions (uniform Brownian motion) could be easily distinguished from "aggregate" dispersions (relatively large, non-uniform particles, no motion).

Example 1

The purpose of this example was to prepare a nanoparticulate nystatin composition containing 4: 1 poly (Lysine, Tryptophan) hydrobromide as a peptide surface stabilizer.

Nystatin is a poorly water soluble antifungal polyene antibiotic obtained from Streptomyces nourset. It is an antifungal agent indicated for the treatment of oral, gastrointestinal and vaginal candidiasis. Oral candidiasis, in particular, is a common disease of immunocompromised patients. Nystatin is indicated for the therapeutic treatment of all infections caused by susceptible microorganisms, in those patients in whom candidal infections (monilial) are probably complicating therapy.

A suspension of nystatin (Sigma-Aldrich Co.) at 2% (w / w) and poly (Lysine, Tryptophan) hydrobromide 4: 1 ("Poly (Lys, Trp))" (Sigma; St. Louis, MO) 1% (w / w), which is a random cationic co-polyamino acid having a molecular weight of 38,000, in water, it was ground for 1 day using low energy techniques (ball mill) in the presence of milling media YTZ ceramics.

The mean nystatin particle size after grinding was 149 nm, with a D90 of 270 nm, determined by static light scattering using a Horiba LA-910 light scattering particle size analyzer (Horiba Instruments, Irvine, CA). The composition had a zeta potential (electrokinetic) of 47.7 mV, measured by electrophoresis in 5x10 'NaCl⁴ M (Malvern ZetaSizer). The dispersion capacity was verified by phase contrast microscopy.

Figure 1 shows representative photomicrographs of nystatin crystals before (Fig. 1A) and after (Fig. 1B) grinding.

The stability of the particle size, under regulated conditions, was verified over time. Figure 2 shows the results of the verification of nystatin particle size stability over time, at 5 ° C (solid line), 25 ° C (dashed line) and 40 ° C (dotted line). ), for the nanoparticulate nystatin / peptide composition.

These results demonstrate that a peptide surface stabilizer can be used successfully to stabilize an active agent at a nanoparticulate particle size. Furthermore, such a peptide surface stabilizer can impart additional therapeutic benefits to the final formulation. For example, the peptide surface stabilizer, Poly (Lys, Trp) is cationic and therefore nanoparticulate active agent compositions using this surface stabilizer can be bioadhesive

The resulting composition exhibited a mean particle size of 149 nm and was free of agglomeration. Furthermore, the nanoparticulate nystatin / peptide composition exhibited virtually no particle size growth at all three temperatures tested.

Example 2

The purpose of this example was to determine whether a cationic surface charge, such as that obtained with the use of a cationic peptide surface stabilizer, enhances the adhesion of small particles to cells.

ES 2 366 646 T3

Cell binding experiments were carried out with polystyrene latex microspheres as a model. A positive surface charge was expected to enhance the interaction of particles with cell surface macromolecules, which possess a net negative charge.

Cationic microspheres with a mean zeta potential (51.5 mV), comparable to that of the nystatin / peptide nanoparticulate composition of Example 1, were tested against anionic microspheres (mean zeta potential = -50.9 mV). Microspheres were incubated with NIH / 3T3 fibroblasts, washed thoroughly, fixed, and subjected to SEM analysis.

Figure 3 shows representative photomicrographs of cells with anionic particles (Fig. 3A) and cationic particles (Fig. 3B).

The results indicate that positively charged particles interact more strongly with the cell surface than negatively charged particles, and it is thought that nanoparticulate active agent compositions having a cationic peptide as a surface stabilizer, with comparable zeta potentials, may follow the same trend. .

Example 3

The purpose of this example was to determine whether grinding an active agent, such as nystatin, which has a peptide surface stabilizer, affects the activity of the active agent.

The minimum inhibitory concentration (MIC) of a ground nystatin composition, which had Poly (Lys, Trp) as a peptide surface stabilizer, was compared with the MIC of two unground nystatin compositions. Nystatin for the ground nanoparticulate composition was obtained from Sigma-Aldrich Co. and the two unground nystatin compositions were obtained from Sigma-Aldrich Co. and Paddock Laboratories, Inc. Details for ground and unground nystatin compositions are provided in Table 1 below, including particle size of ground nanoparticulate nystatin / Poly (Lys, Trp) composition, and potency (U / ml, USP) and the MIC of each nystatin composition.

<td colspan="5">Table 1</td>

<td>Nystatin concentration</td><td>Surface stabilizer and concentration</td><td>Average particle size (nm)</td><td>Potency (U / ml, USP)</td><td>MIC</td>

<td>2% (Sigma)</td><td>Poly (Lys, Trp) 1%^Ί</td><td> 129</td><td> 101.200</td><td> 1:10.000</td>

<td>5% (Sigma)</td><td>N / A - unground</td><td>N / A</td><td> 253.000</td><td> 1:10.000</td>

<td>4% (Paddock)</td><td>N / A - unground</td><td>N / A</td><td> 253.000</td><td> 1:100.000</td>

<td colspan="5">'Poly (Lysine, Tryptophan) is a random cationic co-polyamino acid</td>

The nanoparticulate sample was ball milled for 26 hours with YTZ ceramic grinding media.

The minimum inhibitory concentration (MIC) of the ground nystatin / peptide composition and that of the two unground samples were determined in C. albicans cultures. The MIC indicated here is the maximum dilution of the formulation in the culture broth that inhibits the growth of C. albicans. As Table 1 shows, the ground nystatin / peptide composition exhibited no significant difference in MIC and, surprisingly, was more active than at least one of the unground nystatin samples. These results confirm that the milling process does not decrease nystatin activity.

Example 4

The purpose of this example was to prepare a nanoparticulate composition of a diuretic, Compound A, using a peptide surface stabilizer. Diuretics can be used to reduce bloating and fluid retention caused by various medical problems, including heart or liver disease. Diuretics are also used to treat high blood pressure.

A suspension of Compound A at 2% (w / w) and of poly (Lysine, Tryptophan) hydrobromide 4: 1 at 1% (w / w) as a peptide surface stabilizer, in water, was ground for 3 days in a medium aqueous environment in a low energy mill, in the presence of yttrium stabilized 0.8 mm ceramic media.

Particle size analysis of the resulting Compound A dispersion was carried out by laser light diffraction using the Horiba LA 910 particle size analyzer (Horiba Instruments, Irvine, CA) and water

ES 2 366 646 T3 as diluent, The mean particle size of the dispersion of Compound A was 99 nm, with a D90 of 138 nm. The composition was stable.

Example 5

The purpose of this example was to prepare a nanoparticulate composition of paclitaxel using a peptide surface stabilizer. Paclitaxel belongs to a group of drugs called antineoplastics. This drug is used to treat cancer of the ovaries, breast, certain types of lung cancer, and a cancer of the skin and mucous membranes most commonly found in patients with acquired immunodeficiency syndrome (AIDS). It can also be used to treat other types of cancer.

Paclitaxel has the following chemical structure:

<img file="ES2366646T3_D0001.tif" />

A suspension of paclitaxel at 2% (w / w) and of poly (Lysine, Tryptophan) hydrobromide 4: 1 at 1% (w / w) as a peptide surface stabilizer, in water, was ground for 3 days in an environment aqueous in a low energy mill, in the presence of yttrium stabilized 0.8 mm ceramic media.

Particle size analysis of the resulting paclitaxel dispersion was carried out by laser light diffraction using the Horiba LA910 particle size analyzer (Horiba Instruments, Irvine, CA) and water as a diluent. The mean particle size of the ground paclitaxel dispersion was 139 nm with a D90 of 185 nm. The composition was stable.

Example 6

The purpose of this example was to prepare a nanoparticulate amphotericin B composition using a peptide surface stabilizer. Amphotericin B is a poorly water soluble antifungal agent. It is used topically to treat yeast skin infections; IV is used to treat a variety of life-threatening fungal infections.

Amphotericin B has the following chemical structure:

<img file="ES2366646T3_D0002.tif" />

In this experiment, amphotericin B was milled with 4: 1 poly (Lys, Trp) hydrobromide as a peptide surface stabilizer. A 2% (w / w) aqueous suspension of amphotericin B (Sigma) was prepared, with 1% (w / w) poly (Lys, Trp) (Sigma). The composition was ball milled for 24 hours with YTZ 0.8mm ceramic media. The

ES 2 366 646 T3 particle size of the resulting amphotericin B dispersion was characterized by static laser light scattering on a Horiba LA-910 particle size distribution analyzer. The results are set forth in Table 2 below.

<td colspan="5">TABLE 2</td>

<td>Drug and Concentration</td><td>Surface Stabilizer and Concentration</td><td>Average particle size (nm)</td><td>D50 (nm)</td><td>D90 (nm)</td>

<td>Amphotericin B 2%</td><td>Poly (Lis, Trp) 1%</td><td> 121</td><td> 96</td><td> 230</td>

These results demonstrate that amphotericin B dispersions can be successfully stabilized by a peptide surface stabilizer, such as the 4: 1 poly (Lys, Trp) hydrobromide random co-polypeptide.

It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present invention. Therefore, the present invention is intended to cover the modifications and variations of this invention provided, included within the scope of the claims and their added equivalents.