Patent 6,824,791

The invention involves methods and products related to the micronization of hydrophobic drugs. A method of micronizing hydrophobic drugs using a set of solutions including an aqueous solution is provided. The invention also relates to products of micronized hydrophobic drugs and related methods of use.

Description of the Invention

FIELD OF THE INVENTION

The invention relates to methods of micronizing hydrophobic drugs and related products and methods of using the micronized hydrophobic drugs.

BACKGROUND OF THE INVENTION

Many hydrophobic agents, both active and non-active have utility in a variety of in vivo settings. Although techniques exist for preparing and formulating hydrophobic agents, these techniques are limited. Some methods of formulation cause a loss of bioactivity. Other methods produce large drug particles or particles of inconsistent sizes that lead to problems in drug delivery.

One method for formulating hydrophobic agents involves the generation of microparticles. Microparticles, microcapsules and microspheres (hereinafter "microparticles") have important applications in the pharmaceutical, agricultural, textile and cosmetics industry as delivery vehicles. In these fields of application, a drug, protein, hormone, peptide, fertilizer, pesticide, herbicide, dye, fragrance or other agent is encapsulated in a polymer matrix and delivered to a site either instantaneously or in a controlled manner in response to some external impetus (i.e., pH, heat, water, radiation, pressure, concentration gradients, etc.). Microparticle size can be an important factor in determining the release rate of the encapsulated material.

Many microencapsulation techniques exist which can produce a variety of particle types and sizes under various conditions. Methods typically involve solidifying emulsified liquid polymer droplets by changing temperature, evaporating solvent, or adding chemical cross-linking agents. Physical and chemical properties of the encapsulant and the material to be encapsulated can sometimes dictate the suitable methods of encapsulation, making only certain methodologies useful in certain circumstances. Factors such as hydrophobicity, molecular weight, chemical stability, and thermal stability affect encapsulation. Significant losses are frequently associated with multiple processing steps. These parameters can be particularly important in respect of encapsulating bioactive agents because losses in the bioactivity of the material due to the processing steps or low yields can be extremely undesirable.

SUMMARY OF THE INVENTION

In some aspects, the invention involves a method of micronizing hydrophobic drugs. The micronized drugs prepared by these methods have a variety of properties that are advantageous in the field of drug delivery. For instance, the methods of the invention allow for the formation of particles that have an average particle size of less than 1 micron. The micronized drugs also exhibit enhanced crystallinity and may be used to prepare particles which result in improved relative bioavailability when administered to a subject. Several of these surprising properties are demonstrated in the examples section below.

According to one aspect of the invention, a method for micronizing a hydrophobic agent is provided. A hydrophobic agent is dissolved in an effective amount of a first solvent that is free of polymer. The hydrophobic agent and the solvent form a mixture having a continuous phase. A second solvent and then an aqueous solution arc introduced into the mixture. The introduction of the aqueous solution causes precipitation of the hydrophobic agent and produces a composition of micronized hydrophobic agent having an average particle size of 1 micron or less.

According to another aspect of the invention, a method for micronizing a hydrophobic agent is provided. A hydrophobic agent is dissolved in an effective amount of a first solvent with a polymer. The hydrophobic agent and the first solvent form a mixture having a continuous phase. A second solvent and then an aqueous solution is introduced into the mixture. The introduction of the aqueous solution causes precipitation of the hydrophobic agent to produce a composition of micronized hydrophobic agent having an average particle size of 1 micron or less. In one embodiment, the final preparation contains less than 5% polymer. In yet another embodiment, the polymer is removed by the aqueous solution.

The hydrophobic agent may be dissolved in the first solvent in a variety of ways depending on the agent. Such methods include, but are not limited to, heating, sonicating, high shearing, or high stirring the hydrophobic agent in the first solvent.

The second solvent is optionally an alcohol selected from the group consisting of: methanol (methyl alcohol), ethanol, (ethyl alcohol), 1-propanol (n-propyl alcohol), 2-propanol (isopropyl alcohol), 1-butanol (n-butyl alcohol), 2-butanol (sec-butyl alcohol), 2-methyl-1-propanol (isobutyl alcohol), 2-methyl-2-propanol (t-butyl alcohol), 1-pentanol (n-pentyl alcohol), 3-methyl-1-butanol (isopentyl alcohol), 2,2-dimethyl-1-propanol (neopentyl alcohol), cyclopentanol (cyclopentyl alcohol), 1-hexanol (n-hexanol), cyclohexanol (cyclohexyl alcohol), 1-heptanol (n-heptyl alcohol), 1-octanol (n-octyl alcohol), 1-nonanol (n-nonyl alcohol), 1-decanol (n-decyl alcohol), 2-propen-1-ol (allyl alcohol), phenylmethanol (benzyl alcohol), diphenylmethanol (diphenylcarbinol), triphenylmethanol (triphenylcarbinol), glycerin, phenol, 2-methoxyethanol, 2-ethoxyethanol, 3-ethoxy-1,2-propanediol, Di(ethylene glycol) methyl ether, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 2,5-pentanediol, 3,4-, pentanediol, and 3,5-pentanediol. A preferred alcohol is isopropanol. The second solvent may also be a mixture of alcohols selected from the aforementioned group.

Microparticles of the micronized hydrophobic agent may be prepared by a variety of methods including, for example, spray drying, interfacial polymerization, hot melt encapsulation, phase separation encapsulation, spontaneous emulsion, solvent evaporation microencapsulation, solvent removal microencapsulation, coacervation, and low temperature microsphere formation. One preferred method of preparing microparticles of the hydrophobic agent is by performing phase inversion nanoencapsulation (PIN).

According to another aspect of the invention, microparticles are provided. The microparticles may be produced by the processes described above. The microencapsulated product may be composed of particles having various sizes. In one embodiment, more than 90% of the particles have a size less than 1 micron.

According to still another aspect of the invention, a composition comprising a preparation of micronized hydrophobic agent having an average particle size of less than 1 micron is provided. The preparation is composed of less than 5% polymer carrier and is free of surfactant. In one embodiment, the preparation is free of polymer carrier.

The invention, also provides in some aspects a composition comprising a preparation of micronized hydrophobic agent having an average particle size of less than 1 micron, wherein the preparation is free of polymer carrier and wherein the crystallinity of the micronized hydrophobic agent is at least 50% of the crystallinity of the non-micronized hydrophobic agent. In one embodiment, the crystallinity is at least 75%. In another embodiment, the crystallinity is greater than 90%.

The invention also encompasses methods for delivering a hydrophobic agent to a subject, by administering an encapsulated product including the agent, to the subject. The solid preparation of the micronized hydrophobic agent having an average particle size of less than 1 micron, composed of less than 5% polymer and free of surfactant is administered orally. In one embodiment, the bioactivity of the hydrophobic agent is retained. In another embodiment, the micronized hydrophobic agent has at least a 5% increase in relative bioavailability compared to the non-micronized hydrophobic agent. In some embodiments, the preparation is free of polymer.

In other aspects, the invention provides methods for delivering an agent to a subject by administering microparticles of a micronized hydrophobic agent encapsulated by phase inversion nanoencapsulation. The average microparticle size is less than 1 micron and the preparation, is composed of less than 5% polymer and is free of surfactant. The microencapsulated micronized hydrophobic agent may be administered orally. In one embodiment, the bioactivity of the hydrophobic agent is retained. In another embodiment, the micronized hydrophobic agent has at least a 5% increase in relative bioavailability compared to the non-micronized hydrophobic agent. In some embodiments, the preparation is free of polymer.

The invention also provides a method for achieving 100% bioactivity. The method comprises orally administering to the subject a solid preparation of micronized hydrophobic agent having an average particle size of less than 1 micron wherein 100% of the orally administered agent is bioactive. In an embodiment, the preparation is composed of less than 5% polymer and is free of surfactant. In some embodiments, the preparation is free of polymer.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in some aspects, on a new method for micronizing hydrophobic agents. It was discovered according to the invention that the method for micronizing hydrophobic agents results in a product having enhanced properties which lead to better plasma concentration in vivo administration. For instance, the micronized agent has reduced particle size, increased crystallinity and enhanced bioactivity and relative bioavailability when administered to a subject. The dramatic increases in bioactivity and relative bioavailability were unexpected.

The micronized agent prepared according to the methods of the invention may be used directly, e.g., administered to a subject, or it may be further manipulated into pharmaceutical compositions, such as microparticles. As shown in the Examples section, when the micronized agent was used to produce microparticles and those microparticles were delivered to animals the relative bioavailability increased dramatically compared to non-micronized formulations. In Example 4 it was demonstrated that these microparticles were capable of achieving close to and in some instances more than 100% relative bioavailability when orally administered (Table 4). Relative bioavailability as used herein refers to the amount of a drug available for detection in the systemic circulation as compared to an administered IV dose. The relative bioavailability of a drug administered by a route other than IV, is generally a function of the ability of the drug to permeate and penetrate into the systemic circulation. Relative bioavailability is affected by a variety of factors, most importantly permeability and solubility of the drug. Although absolute (100%) bioavailability might be achieved with IV administration, difficulties and limitations may be encountered in IV administration (such as drug precipitation/crystallinization in the blood) which render bioavailabity relative rather than absolute. If a drug administered by IV undergoes precipitation or crystallization, then the same drug administered by a different route may in some cases have a relative bioavailability greater than 100%.

The method in some aspects involves the formation of a continuous phase mixture or preparation of the hydrophobic agent to be micronized and a first solvent. This mixture or preparation is free of polymer. As used herein, a mixture or a preparation free of polymer refers to a mixture or preparation that has no detectable amount of polymer.

In some aspects of the invention, the mixture or preparation is substantially free of polymer. As used herein, substantially free of polymer is more than 97% free of polymer. In some embodiments the mixture or preparation is more than 98%, or more than 99%, or 100% free of polymer. In some embodiments, the mixture or preparation is absolutely free of polymer. As used herein absolutely free of polymer refers to a mixture or preparation that is 100% free of polymer.

The method may be accomplished by dissolving or dispersing the hydrophobic agent Is in an effective amount of the solvent. A second solvent that is immiscible with the original solvent and an aqueous solution are introduced into the mixture. The introduction of the aqueous solution causes the precipitation of the hydrophobic agent to produce a micronized drug.

The micronized hydrophobic agent may be used without further manipulation. For instance, it may be administered directly to a subject. The micronization procedure transforms the hydrophobic agent from a compound which when delivered directly to a subject has low relative bioavailability into one which, because of the micronized properties, has a much higher relative bioavailability when administered. Although it is not necessary, it is also possible to further process the micronized agent to produce microparticles or to incorporate the agent into other drug delivery devices. Microparticles of the micronized hydrophobic agent can be prepared by several common microencapsulation techniques. Suitable methods of encapsulation may be selected to produce the desired physical and chemical properties of the encapsulant and the material to be encapsulated. These optional methods are described in more detail below.

The methods are particularly useful for micronizing hydrophobic agents. The hydrophobic agent may be any type of hydrophobic compound including active agents and non-active agents. The hydrophobic agent is an agent that does not adsorb or absorb water. Hydrophobic active and non-active agents include, but are not limited to, adhesives, gases, pesticides, herbicides, fragrances, antifoulants, dies, salts, oils, inks, cosmetics, catalysts, detergents, curing agents, flavors, foods, fuels, metals, paints, photographic agents, biocides, pigments, plasticizers, propellants and the like. The active agent also may be a bioactive agent. The bioactive agent may be, for example, adrenergic agent; adrenocortical steroid; adrenocortical suppressant; aldosterone antagonist; amino acid; anabolic; analeptic; analgesic; anesthetic; anorectic; anti-acne agent; anti-adrenergic; anti-allergic; anti-amebic; anti-anemic; anti-anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholinergic; anticoagulant; anticonvulsant; antidepressant; antidiabetic; antidiarrheal; antidiuretic; anti-emetic; anti-epileptic; antifibrinolytic; antifungal; antihemorrhagic; antihistamine; antihyperlipidemia; antihypertensive; antihypotensive; anti-infective; anti-inflammatory; antimicrobial; antimigraine; antimitotic; antimycotic, antinauseant, antineoplastic, antineutropenic, antiparasitic; antiproliferative; antipsychotic; antirheumatic; antiseborrheic; antisecretory; antispasmodic; antithrombotic; anti-ulcerative; antiviral; appetite suppressant; blood glucose regulator; bone resorption inhibitor; bronchodilator; cardiovascular agent; cholinergic; depressant; diagnostic aid; diuretic; dopaminergic agent; estrogen receptor agonist; fibrinolytic; fluorescent agent; free oxygen radical scavenger; gastrointestinal motility effector; glucocorticoid; hair growth stimulant; hemostatic; histamine H₂ receptor antagonists; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive; imaging agent; immunizing agent; immunomodulator; immunoregulator; immunostimulant; immunosuppressant; keratolytic; LHRH agonist; mood regulator; mucolytic; mydriatic; nasal decongestant; neuromuscular blocking agent; neuroprotective; NMDA antagonist; non-hormonal sterol derivative; plasminogen activator; platelet activating factor antagonist; platelet aggregation inhibitor; psychotropic; radioactive agent; scabicide; sclerosing agent; sedative; sedative-hypnotic; selective adenosine Al antagonist; serotonin antagonist; serotonin inhibitor; serotonin receptor antagonist; steroid; thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer; amyotrophic lateral sclerosis agent; cerebral ischemia agent; Paget's disease agent; unstable angina agent; vasoconstrictor; vasodilator; wound healing agent; xanthine oxidase inhibitor; Anti-cancer, e.g. paclitaxel.

Bioactive agents also include immunological agents such as allergens (e.g., cat dander, birch pollen, house dust, mite, grass pollen, etc.) and antigens from pathogens such as viruses, bacteria, fungi and parasites. These antigens may be in the form of whole inactivated organisms, peptides, proteins, glycoproteins, carbohydrates or combinations thereof. Specific examples of pharmacological or immunological agents that fall within the above-mentioned categories and that have been approved for human use may be found in the published literature.

The hydrophobic agent is added to and dissolved in a first solvent. The first solvent is any suitable solvent that is capable of dissolving the hydrophobic agent. Typically the solvent will be a common organic solvent such as a halogenated aliphatic hydrocarbon such as methylene chloride, chloroform and the like; an alcohol; an aromatic hydrocarbon such as toluene; a halogenated aromatic hydrocarbon; an ether such as methyl t-butyl; a cyclic ether such as tetrahydrofuran; ethyl acetate; diethylcarbonate; acetone; or cyclohexane. The solvents may be used alone or in combination as the first solvent. It is desirable that the solvent be inert with respect to the agent being micronized. Those of skill in the art will be able to select an appropriate first solvent for the particular hydrophobic agent being micronized based on the guidance provided herein.

In some embodiments the hydrophobic agent is dissolved in an effective amount of the first solvent with a polymer. When a polymer is present in the first solvent, the agent is added to the solvent optionally after the polymer is dissolved in the solvent. If a polymer is used, the solvent chosen should be capable of dissolving the polymer, and it is desirable that the solvent be inert with respect to the polymer. It is preferred that the final product has a low concentration of polymer or no polymer at all. This could be achieved by choosing a polymer that is slightly soluble in water, or else a polymer that degrades in water. This way some of the polymer will disappear from the final formulation.

A "polymer" may be any suitable (micronizing) material consisting of repeating units including, but not limited to, nonbioerodible and bioerodible polymers. As used herein the term "polymer" includes polymer carriers and surfactants. A "polymer carrier" is a polymer that does not function as a surfactant. A surfactant is a surface-active agent that modifies the nature of surfaces, i.e., which may involve reducing the surface tension of water. Surfactants include but are not limited to wetting agents, detergents, emulsifiers, dispersing agents, penetrants, and antifoams. Some surfactants are polymers and others are non-polymeric. Thus only a subset of surfactants are polymeric surfactants.

Such polymers have been described in great detail in the prior art. They include, but are not limited to: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate), poly vinyl chloride polystyrene and polyvinylpryrrolidone.

Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of preferred biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters; polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide) and poly(lactide-co-caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The foregoing materials may be used alone, as physical mixtures (blends), or as co-polymers. Some preferred polymers are polyesters, polyanhydrides, polystyrenes and blends thereof.

Bioadhesive polymers are also useful. A bioadhesive polymer is one that binds to mucosal epithelium under normal physiological conditions. Bioadhesion in the gastrointestinal tract proceeds in two stages: (1) viscoelastic deformation at the point of contact of the synthetic material into the mucus substrate, and (2) formation of bonds between the adhesive synthetic material and the mucus or the epithelial cells. In general, adhesion of polymers to tissues may be achieved by (i) physical or mechanical bonds, (ii) primary or covalent chemical bonds, and/or (iii) secondary chemical bonds (i.e., ionic). These are particularly useful when the particles formed by the methods of the invention are delivered orally or to other mucosal tissue.

Physical or mechanical bonds can result from deposition and inclusion of the adhesive material in the crevices of the mucus or the folds of the mucosa. Secondary chemical bonds, contributing to bioadhesive properties, consist of dispersive interactions (i.e., Van der Waals interactions) and stronger specific interactions, which include hydrogen bonds. The hydrophilic functional groups primarily responsible for forming hydrogen bonds are the hydroxyl and the carboxylic groups. Numerous bioadhesive polymers are discussed in WO 93/21906. Representative bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules. 1993, 26:581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecl acrylate). A preferred polymer is poly(fumaric-co-sebacic)acid.

Polymers with enhanced bioadhesive properties can be provided wherein anhydride monomers or oligomers are incorporated into the polymer. The oligomer excipients can be blended or incorporated into a wide range of hydrophilic and hydrophobic polymers including proteins, polysaccharides and synthetic biocompatible polymers. Anhydride oligomers may be combined with metal oxide particles to improve bioadhesion even more than with the organic additives alone. Organic dyes because of their electronic charge and hydrophobicity/hydrophilicity can either increase or decrease the bioadhesive properties of polymers when incorporated into the polymers. The incorporation of oligomer compounds into a wide range of different polymers that are not normally bioadhesive dramatically increases their adherence to tissue surfaces such as mucosal membranes.

As used herein, the term "anhydride oligomer" refers to a diacid or polydiacids linked by anhydride bonds, and having carboxy end groups linked to a monoacid such as acetic acid by anhydride bonds. The anhydride oligomers have a molecular weight less than about 5000, typically between about 100 and 5000 daltons, or are defined as including between one to about 20 diacid units linked by anhydride bonds. In one embodiment, the diacids are those normally found in the Krebs glycolysis cycle. The anhydride oligomer compounds have high chemical reactivity.

The oligomers can be formed in a reflux reaction of the diacid with excess acetic anhydride. The excess acetic anhydride is evaporated under vacuum, and the resulting oligomer, which is a mixture of species which include between about one to twenty diacid units linked by anhydride bonds, is purified by recrystallizing, for example from toluene or other organic solvents. The oligomer is collected by filtration, and washed, for example, in ethers. The reaction produces anhydride oligomers of mono and poly acids with terminal carboxylic acid groups linked to each other by anhydride linkages.

The anhydride oligomer is hydrolytically labile. As analyzed by gel permeation chromatography, the molecular weight may be, for example, on the order of 200-400 for fumaric acid oligomer (FAPP) and 2000-4000 for sebacic acid oligomer (SAPP). The anhydride bonds can be detected by Fourier transform infrared spectroscopy by the characteristic double peak at 1750 cm^-1 and 1820 cm^-1, with a corresponding disappearance of the carboxylic acid peak normally at 1700 cm^-1.

In one embodiment, the oligomers may be made from diacids described for example in U.S. Pat. No. 4,757,128 to Domb et al., U.S. Pat. No. 4,997,904 to Domb, and U.S. Pat. No. 5,175,235 to Domb et al., the disclosures of which are incorporated herein by reference. For example, monomers such as sebacic acid, bis(p-carboxy-phenoxy)propane, isophathalic acid, fumaric acid, maleic acid, adipic acid or dodecanedioic acid may be used.

Organic dyes, because of their electronic charge and hydrophilicity/hydrophobicity, may alter the bioadhesive properties of a variety of polymers when incorporated into the polymer matrix or bound to the surface of the polymer. A partial listing of dyes that affect bioadhesive properties include, but are not limited to: acid fuchsin, alcian blue, alizarin red s, auramine o, azure a and b, Bismarck brown y, brilliant cresyl blue ald, brilliant green, carmine, cibacron blue 3GA, congo red, cresyl violet acetate, crystal violet, eosin b, eosin y, erythrosin b, fast green fcf, giemsa, hematoylin, indigo carmine, Janus green b, Jenner's stain, malachite green oxalate, methyl blue, methylene blue, methyl green, methyl violet 2b, neutral red, Nile blue a, orange II, orange G, orcein, paraosaniline chloride, phloxine b, pyronin b and y, reactive blue 4 and 72, reactive brown 10, reactive green 5 and 19, reactive red 120, reactive yellow 2, 3, 13 and 86, rose bengal, safranin o, Sudan III and IV, Sudan black B and toluidine blue.

The working molecular weight range for the polymer is on the order of 1 kDa-150,000 kDa, although the optimal range is 2 kDa-50 kDa. The working range of polymer concentration is 0.01-50% (weight/volume), depending primarily upon the molecular weight of the polymer and the resulting viscosity of the polymer solution. In general, the low molecular weight polymers permit usage of a higher concentration of polymer. A preferred concentration range according to the invention may be on the order of 0.0%-10% (weight/volume), while the optimal polymer concentration in the micronized product typically will be below 5% and preferably be close to or equal to 0%. It has been found that polymer concentrations on the order of 0-5% are particularly useful according to the methods of the invention.

The hydrophobic agent and the first solvent (with or without a polymer) form a continuous mixture. The hydrophobic agent is dissolved in the first solvent by any of a variety of methods, depending on the type of agent. Preferred methods include heating, sonicating, high shearing, or stirring the hydrophobic agent in the first solvent.

A second solution is then introduced into the mixture. The "second solvent" as used herein is a solvent that is immiscible with the original solvent. The second solvent includes, for example, any suitable alcohol or a combination of alcohols. Typically the solvent will be a common alcohol. Preferred alcohols are methanol (methyl alcohol), ethanol, (ethyl alcohol), 1-propanol (n-propyl alcohol), 2-propanol (isopropyl alcohol), 1-butanol (n-butyl alcohol), 2-butanol (sec-butyl alcohol), 2-methyl-1-propanol (isobutyl alcohol), 2-methyl-2-propanol (t-butyl alcohol), 1-pentanol (n-pentyl alcohol), 3-methyl-1-butanol (isopentyl alcohol), 2,2-dimethyl-1-propanol (neopentyl alcohol), cyclopentanol (cyclopentyl alcohol), 1-hexanol (n-hexanol), cyclohexanol (cyclohexyl alcohol), 1-heptanol (n-heptyl alcohol), 1-octanol (n-octyl alcohol), 1-nonanol (n-nonyl alcohol), 1-decanol (n-decyl alcohol), 2-propen-1-ol (allyl alcohol), phenylmethanol (benzyl alcohol), diphenylmethanol (diphenylcarbinol), triphenylmethanol (triphenylcarbinol), glycerin, phenol, 2-methoxyethanol, 2-ethoxyethanol, 3-ethoxy-1,2-propanediol, Di(ethylene glycol) methyl ether, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 2,5-pentanediol, 3,4-pentanediol, and 3,5-pentanediol. The most preferred alcohol is isopropanol. One or more of the alcohols can be used in combination.

An aqueous solution is then introduced into the above mixture. The addition of the aqueous solution causes precipitation of the hydrophobic agent and produces a composition of micronized agent. An "aqueous solution" as used herein is a solution in which water is the only or main component. The first and second solvents are selected such that when these two immiscible solvents are added to the aqueous solution, the three solutions become miscible.

The micronized hydrophobic agent may be used without further manipulation. For instance, it may be administered directly to a subject. Alternatively, the micronized hydrophobic agent may be further processed prior to use, e.g. it may be processed to produce microparticles. Microparticles of the micronized hydrophobic agent may be prepared using any one of several common microencapsulation techniques. Different microencapsulation techniques produce a variety of microparticles having different properties under various conditions. Suitable methods of encapsulation may be selected to produce the desired physical and chemical properties of the encapsulant and the material to be encapsulated. As used herein the terms "microparticle" and "microencapsulation" are used broadly to refer to particles, spheres or capsules that have sizes on the order of microns as well as nanometers. Thus, the terms "microparticle" "microsphere", "nanoparticle", "nanosphere", "nanocapsule" and "microcapsule" are used interchangeably.

Common microencapsulation techniques include but are not limited to spray drying, interfacial polymerization, hot melt encapsulation, phase separation encapsulation (solvent removal and solvent evaporation), spontaneous emulsion, solvent evaporation microencapsulation, solvent removal microencapsulation, coacervation, and low temperature microsphere formation and phase inversion nanoencapsulation (PIN). Each of these methods is well known in the art. A brief summary of the methods is presented below.

In spray drying, the core material to be encapsulated is dispersed or dissolved in a solution. Typically, the solution is aqueous and preferably the solution includes a polymer. The solution or dispersion is pumped through a micronizing nozzle driven by a flow of compressed gas, and the resulting aerosol is suspended in a heated cyclone of air, allowing the solvent to evaporate from the microdroplets. The solidified microparticles pass into a second chamber and are trapped in a collection flask.

Interfacial polycondensation is used to microencapsulate a core material in the following manner. One monomer and the core material are dissolved in a solvent. A second monomer is dissolved in a second solvent (typically aqueous) which is immiscible with the first. An emulsion is formed by suspending the first solution through stirring in the second solution. Once the emulsion is stabilized, an initiator is added to the aqueous phase causing interfacial polymerization at the interface of each droplet of emulsion.

In hot melt microencapsulation the core material (to be encapsulated) is added to molten polymer. This mixture is suspended as molten droplets in a nonsolvent for the polymer (often oil-based) which has been heated to approximately 10^oC. above the melting point of the polymer. The emulsion is maintained through vigorous stirring while the nonsolvent bath is quickly cooled below the glass transition of the polymer, causing the molten droplets to solidify and entrap the core material.

In solvent evaporation microencapsulation, the polymer is typically dissolved in a water immiscible organic solvent and the material to be encapsulated is added to the polymer solution as a suspension or solution in organic solvent. An emulsion is formed by adding this suspension or solution to a beaker of vigorously stirring water (often containing a surface active agent to stabilize the emulsion). The organic solvent is evaporated while continuing to stir. Evaporation results in precipitation of the polymer, forming solid microcapsules containing core material.

The solvent evaporation process is designed to entrap a liquid core material in PLA, PLA/PGA copolymer, or PLA/PCL copolymer microcapsules. The PLA or copolymer is dissolved in a miscible mixture of solvent and nonsolvent, at a nonsolvent concentration which is immediately below the concentration which would produce phase separation (i.e., cloud point). The liquid core material is added to the solution while agitating to form an emulsion and disperse the material as droplets. Solvent and nonsolvent are vaporized, with the solvent being vaporized at a faster rate, causing the PLA or copolymer to phase separate and migrate towards the surface of the core material droplets. This phase separated solution is then transferred into an agitated volume of nonsolvent, causing any remaining dissolved PLA or copolymer to precipitate and extracting any residual solvent from the formed membrane. The result is a microcapsule composed of PLA or copolymer shell with a core of liquid material.

In solvent removal microencapsulation, the polymer is typically dissolved in an oil miscible organic solvent and the material to be encapsulated is added to the polymer solution as a suspension or solution in organic solvent. An emulsion is formed by adding this suspension or solution to a beaker of vigorously stirring oil, in which the oil is a nonsolvent for the polymer and the polymer/solvent solution is immiscible in the oil. The organic solvent is removed by diffusion into the oil phase while continuing to stir. Solvent removal results in precipitation of the polymer, forming solid microcapsules containing core material.

In phase separation microencapsulation, the material to be encapsulated is dispersed in a polymer solution by stirring. While continuing to uniformly suspend the material through stirring, a nonsolvent for the polymer is slowly added to the solution to decrease the polymer's solubility. Depending on the solubility of the polymer in the solvent and nonsolvent, the polymer either precipitates or phase separates into a polymer rich and a polymer poor phase. Under proper conditions, the polymer in the polymer rich phase will migrate to the interface with the continuous phase, encapsulating the core material in a droplet with an outer polymer shell.

Spontaneous emulsification involves solidifying emulsified liquid polymer droplets by changing temperature, evaporating solvent, or adding chemical cross-linking agents. Physical and chemical properties of the encapsulant and the material to be encapsulated dictates the suitable methods of encapsulation. Factors such as hydrophobicity, molecular weight, chemical stability, and thermal stability affect encapsulation.

Some solvent evaporation processes are specifically designed to entrap a liquid core material in PLA, PLA/PGA copolymer, or PLA/PCL copolymer microcapsules. The PLA or copolymer is dissolved in a miscible mixture of solvent and nonsolvent, at a nonsolvent concentration which is immediately below the concentration which would produce phase separation (i.e., cloud point). The liquid core material is added to the solution while agitating to form an emulsion and disperse the material as droplets. Solvent and nonsolvent are vaporized, with the solvent being vaporized at a faster rate, causing the PLA or copolymer to phase separate and migrate towards the surface of the core material droplets. This phase separated solution is then transferred into an agitated volume of nonsolvent, causing any remaining dissolved PLA or copolymer to precipitate and extracting any residual solvent from the formed membrane. The result is a microcapsule composed of PLA or copolymer shell with a core of liquid material containing micronized hydrophobic agent.

In solvent removal microencapsulation, the polymer is typically dissolved in an oil miscible organic solvent and the material to be encapsulated is added to the polymer solution as a suspension or solution in organic solvent. An emulsion is formed by adding this suspension or solution to a beaker of oil with vigorous stirring, in which the oil is a nonsolvent for the polymer and the polymer/solvent solution is immiscible in the oil. The organic solvent is removed by diffusion into the oil phase while continuing to stir. Solvent removal results in precipitation of the polymer, forming solid microcapsules containing core material.

Encapsulation procedures for various substances using coacervation techniques have been described in the prior art, for example, in GB-B-929 406; GB-B-929 40 1; U.S. Pat. Nos. 3,266,987; 4,794,000 and 4,460,563. Coacervation is a process involving separation of colloidal solutions into two or more immiscible liquid layers (Ref. Dowben, R. General Physiology, Harper & Row, New York, 1969, pp. 142-143). Through the process of coacervation compositions comprised of two or more phases and known as coacervates may be produced. The ingredients that comprise the two phase coacervate system are present in both phases; however, the colloid rich phase has a greater concentration of the components than the colloid poor phase.

Components that may be used to formulate the coacervate system comprise anionic, cationic, amphoteric, and non-ionic surfactants. Anionic surfactants include di-(2 ethylhexyl) sodium sulfosuccinate; non-ionic surfactants include the fatty acids and the esters thereof; surfactants in the amphoteric group include (1) substances classified as simple, conjugated and derived proteins such as the albumins, gelatins, and glycoproteins, and (2) substances contained within the phospholipid classification, for example lecithin. The amine salts and the quaternary ammonium salts within the cationic group also comprise useful surfactants. Other surfactant compounds useful to form coacervates include compositions within the groups known as the polysaccharides and their derivatives, the mucopolysaccharides and the polysorbates and their derivatives. Synthetic polymers that may be used as surfactants include compositions such as polyethylene glycol and polypropylene glycol. Further examples of suitable compounds that may be utilized to prepare coacervate systems include glycoproteins, glycolipids, galactose, gelatins, modified fluid gelatins and galacturonic acid.

In addition, substances that are not intrinsically surface active may be used to prepare coacervates provided that they can be made so by chemical or other means. Fatty acids are not considered to be surface active compounds. However, when fatty acids are reacted with an alkaline chemical entity the resulting products will have surface-active properties.

Low temperature microsphere formation has been described, see, e.g., U.S. Pat. No. 5,019,400. The method is a process for preparing microspheres which involves the use of very cold temperatures to freeze polymer-biologically active agent mixtures into polymeric microspheres. The polymer is generally dissolved in a solvent together with an active agent that can be either dissolved in the solvent or dispersed in the solvent in the form of microparticles. The polymer/active agent mixture is atomized into a vessel containing a liquid non-solvent, alone or frozen and overlayed with a liquefied gas, at a temperature below the freezing point of the polymer/active agent solution. The cold liquefied gas or liquid immediately freezes the polymer droplets. As the droplets and non-solvent for the polymer is warmed, the solvent in the droplets thaws and is extracted into the non-solvent, resulting in hardened microspheres.

Phase separation microencapsulation proceeds more rapidly than the procedures described in the preceding paragraphs. A polymer is dissolved in the solvent. An agent to be encapsulated then is dissolved or dispersed in that solvent. The mixture then is combined with an excess of nonsolvent and is emulsified and stabilized, whereby the polymer solvent no longer is the continuous phase. Aggressive emulsification conditions are applied in order to produce microdroplets of the polymer solvent. After emulsification, the stable emulsion is introduced into a large volume of nonsolvent to extract the polymer solvent and form microparticles. The size of the microparticles is determined by the size of the microdroplets of polymer solvent.

One method for microencapsulating the micronized hydrophobic agent is by phase inversion nanoencapsulation (PIN). In PIN, a polymer is dissolved in an effective amount of a solvent. The agent to be encapsulated is also dissolved or dispersed in the effective amount of the solvent. The polymer, the agent and the solvent together form a mixture having a continuous phase, wherein the solvent is the continuous phase. The mixture is introduced into an effective amount of a nonsolvent to cause the spontaneous formation of the microencapsulated product, wherein the solvent and the nonsolvent are miscible. PIN has been described by Mathiowitz et al. in U.S. Pat. No. 6,131,211 and U.S. Pat. No. 6,235,224 that are incorporated herein by reference.

Thus, the invention also provides compositions of the micronized hydrophobic agent or microparticles produced using the micronized hydrophobic agent by the processes described above as well as other processes known in the art. The microencapsulated product or micronized hydrophobic agent consist of particles having various sizes. In some embodiments the particles have an average particle size of less than one micron. In other embodiments more than 90% of the particles have a size less than 1 micron.

The compositions of the invention have some properties which are advantageous over prior art products. For instance, the preparation of micronized hydrophobic agent has enhanced crystallinity over that of similar prior art preparations. The micronized hydrophobic agent of the invention may have at least 50% of the crystallinity of a non-micronized hydrophobic agent. In some embodiments it has at least 75%, 80%, 85%, 90%, or 95% of the crystallinity of a non-micronized hydrophobic agent. As used herein the term crystallinity refers to a property of a preparation having a body that is formed by the solidification of a chemical element, a compound, or a mixture and has a regularly repeating internal arrangement of its atoms and often external plane faces. The crystallinity of a preparation may be determined by methods known in the art. Some of the methods of measuring crystallinity are described in Introduction to Polymers, 2nd Edition; Young RJ, Lovell PA. 1991, Chapman and Hall Publishing, London, UK. One of the methods to measure crystallinity is thermal analysis. An example of this type of analysis is presented in Example 1 below. The magnitude of thermal change is proportional to the amount of crystalline component. A compound having enhanced crystallinity has improved release properties when administered in vivo.

As demonstrated in the Examples below, the compositions of the invention also allow for enhanced bioactivity and relative bioavailability of the hydrophobic agent. Many drug processing techniques result in a loss of bioactivity of the drug. The micronization process described herein is sufficient to allow the drug to retain its bioactivity. As a result, when the drug is delivered to a subject the drug is active. As used herein bioactivity refers to the normal function of a known drug. It refers to the presence or absence of a function and can be used to describe relative changes in levels of function rather than an absolute value.

The micronized hydrophobic agent also exhibits at least a 5% increase in relative bioavailability compared to the non-micronized hydrophobic agent. The dramatic difference in relative bioavailability of in vivo administered micronized agent versus non-micronized agent is demonstrated in the Examples. When administered through different routes, such as orally, the micronized hydrophobic agent exhibited dramatically increased relative bioavailability.

The compositions may include a physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer mixed with the micronized hydrophobic agent. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The micronized hydrophobic agent may be administered per se or in the form of a pharmaceutically acceptable salt. When used in medicine the salts may be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical formulations for parenteral administration include aqueous solutions of the micronized hydrophobic agent in water-soluble form. Additionally, suspensions of the micronized hydrophobic agent may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

The micronized hydrophobic agent may be administered by any ordinary route for administering medications. Depending upon the type of disorder to be treated, the micronized hydrophobic agent of the invention may be inhaled, ingested or administered by systemic routes. Systemic routes include oral and parenteral. For use in therapy, an effective amount of the micronized hydrophobic agent can be administered to a subject by any mode that delivers the nucleic acid to the organ or tissue being treated or monitored. Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, intratracheal, intrathecal, intravenous, inhalation, transdermal, intratracheobronchial (including intrapulmonary), ocular, vaginal, and rectal.

For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres, as described above, formulated for oral administration may also be used. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation. Inhaled medications are preferred in some embodiments because of the direct delivery to the lung. Several types of metered dose inhalers are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers. Techniques for preparing aerosol delivery systems are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the agent (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712; incorporated by reference).

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions are administered to a subject. A "subject" as used herein shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, or primate, e.g., monkey. The compositions are administered in effective amounts. An effective amount of a particular agent will depend on factors such as the type of agent, the purpose for administration, the severity of disease if a disease is being treated etc. Those of skill in the art will be able to determine effective amounts.

Claim 1 of 22 Claims

We claim:

1. A method for micronizing a hydrophobic agent, comprising:

dissolving a hydrophobic agent in an effective amount of a first solvent, with a polymer, wherein the hydrophobic agent and the first solvent form a mixture having a continuous phase, introducing a second solvent into the mixture, and introducing an aqueous solution into the mixture wherein the aqueous solution causes precipitation of the hydrophobic agent to produce a composition of micronized hydrophobic agent having an average particle size of 1 micron or less.

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