Title of Invention	"METHOD OF PREPARING A CONTROLLED RELEASE MICROPARTICLE COMPOSITIONS"
Abstract	The compositions disclosed herein are for use as controlled release therapeutics for the treatment of a wide variety of diseases. In particular, the compositions provide water soluble bioactive agents, organic ions and polymers where the bioactive agent is efficiently released over time with minimal degradation products. The resulting controlled release composition is capable of administration in a decreased dose volume due to the high drug content and predominance of non-degraded bioactive agent after release. Additionally, the compositions, of the present invention are capable of long term, sustained releases.

Title of Invention

"METHOD OF PREPARING A CONTROLLED RELEASE MICROPARTICLE COMPOSITIONS"

Abstract

The compositions disclosed herein are for use as controlled release therapeutics for the treatment of a wide variety of diseases. In particular, the compositions provide water soluble bioactive agents, organic ions and polymers where the bioactive agent is efficiently released over time with minimal degradation products. The resulting controlled release composition is capable of administration in a decreased dose volume due to the high drug content and predominance of non-degraded bioactive agent after release. Additionally, the compositions, of the present invention are capable of long term, sustained releases.

Full Text	CONTROLLED RELEASE COMPOSITIONS Field of the Invention The present invention relates to controlled release compositions including a water soluble bioactive agent, an organic ion and a polymer, wherein the content of said bioactive agent is about two to four fold higher than in the absence of organic ion. BACKGROUND OF THE INVENTION Currently there are numerous controlled release formulations on the market that contain various bioactive agents, such as GnRH analogs, human growth hormone, risperidone, and somatostatin analogs of which octreotide acetate is an example. Such formulations are preferred by physicians and veterinarians and by their patients because they reduce the need for multiple injections. Unfortunately, there are many problems with the formulations for controlled release compositions such as the fact that many popular bioactive agents are not good candidates for controlled release compositions due to their substantial water solubility. The use of highly soluble bioactive agents may result in tow drug content and an undesirable "burst" of bioactive agent upon contact with an aqueous solution, such as by administration to a patient or introduction to a physiological medium, which often limits the useable content of bioactive agent achievable in practice. Various methods of solving the water solubility problem have been devised with some success. One such effort is disclosed in US Patent No. 5,776,885 by Orsolini et al. Orsolini converted water soluble peptides to their water insoluble addition salts with pamoic, tannic or stearic acid prior to their encapsulation in sustained release compositions. Encapsulation within a polymer matrix is then performed by dispersing the water insoluble peptide in a polymer solution and forming controlled release compositions via either extrusion or a cumbersome coacervation method. One disadvantage of this approach is the requirement to obtain the water insoluble addition salt prior to encapsulation within a polymer matrix. Furthermore, it was discovered in the present work that when pre-formed addition salts were employed in an emulsion process to form microparticles the result was release of substantial amounts of modified or degraded peptide from the composition when placed in an aqueous physiological buffer. Modification was in the form of undesirable acylation of bioactive agent. Controlled release composition with a high drug load, low burst effect upon administration, and minimum degradation of the bioactive agent are greatly needed to realize the benefits of these types of composition as human or veterinary therapeutics. STATEMENT OF THE INVENTION: Accordingly, the present invention provides a method of preparing a controlled release composition, said method comprises the steps of: a) combining a bioactive agent and a polymer with an organic solvent to obtain a homogeneous organic phase, wherein the bioactive agent is selected from the group comprising of proteins, peptides, LHRH agonists and synthetic analogs thereof, leuprolide, oxytocin, somatostatin and synthetic analogs thereof and hormones; b) combining an organic ion and water to obtain an aqueous phase, wherein the organic ion is selected from the group consisting of pamoate, trifluoromethyl-p-toluate, cholate, 2-napthalene sulfonate, 2,3-napthalene dicarboxylate, I-hydroxy-2-naphthoate, 3-hydroxy-2-naphthoate, 2-.na phthoate, and salicylsalicylate; c) contacting the homogenous organic phase and the aqueous phase through the use of an emulsion process so that the molar stoichiometry of the bioactive agent relative to the organic ion ranges' from 0.5 to 2.0; and d) recovering said controlled release composition in a manner known per se having diameter between 20.0 nm to 1.0 mm. SUMMARY OF THE INVENTION: It has now been discovered that controlled release compositions capable of administration in a concentrated, low dose volume form which release active agent for a prolonged period of time can originate from water soluble bioactive agentswithout first converting them to a water insoluble form. This finding is in direct contrast to previous work in this field that suggested that water soluble bioactive agents would need to be manipulated in some way, for example 1:0 render them water insoluble, prior to forming a sustained release composition. Additionally, the conversion process required in previous compositions, resulted in a high percentage of degradation of thtbioactive agent, whereas the compositions of the present invention are prepared with an understanding of how to reduce degradation. The presence of the bioactive agent in a physiologically relevant form creates an increased core load with regard to previously prepared compositions, which are susceptible to significant degradation. Reduced degradation translates into increased release of unmodified bioactive agent, thus allowing for a smaller injection volume to deliver the same dose of bioactive agent. The compositions of the present invention offer a vast improvement over previous inventions in that they provide a way to deliver a relatively undegraded bioactive agent over a prolonged period of time using a reduced dose volume. In one embodiment, the controlled release compositions are micro particles and nanoparticles. In a particular embodiment, the microparticles and nanoparticles are biodegradable. In another particular embodiment, the polymer is selected from the group consisting of, but not limited to, poly (lactide)s, poly (glycolide)s, poly (Iactide-co-glycolide)s, poly (lactic acid)s, poly (glycolic acid)s, poly (lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly (amino acids), polyorthoesters, polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of polyethylene glycol and poly(lactide)s or poly(lactide-co-glycolide)s, biodegradable polyurethanes, blends and copolymers thereof. In a particular embodiment, the microparticle is made of poly(d,l-lactide-co-glycolide) (PLGA). PLGA degrades when exposed to physiological pH and hydrolyzes to form lactic acid and glycolic acid, which are normal byproducts of cellular metabolism. The disintegration rate of PLGA polymers will vary depending on the polymer molecular weight, ratio of lactide to glycolide monomers in the polymer chain, and stereoregularity of the monomer subunits. Mixtures of L and D stereoisomers that disrupt the polymer crystallinity will increase polymer disintegration rates. In addition, microspheres may contain blends of two or more biodegradable polymers, of different molecular weight and/or monomer ratio. In other alternative embodiments, derivatized biodegradable polymers, including hydrophilic polymers attached to PLGA, can be used to form microspheres. In particular embodiments, the hydrophilic polymer is selected from the group consisting of, but not limited to, poly(ethylene glycol), poly(propylene glycol) and copolymers of poly(ethylene glycol) and polypropylene glycol). Biodegradable Nanoparticles In certain embodiments, the controlled release composition is a nanoparticle. In certain embodiments, the bioactive agent, with or without a hydrophilic polymer attached, is associated with biodegradable submicron particles for controlled release of the bioactive agent. A nanoparticle has a diameter ranging from 20.0 nanometers to about 2.0 microns and is typically between 100.0 nanometers and 1.0 micron. Nanoparticles can be created in the same manner as microparticles, except that high-speed mixing or homogenization is used to reduce the size of the polymer/bioactive agent emulsions to less than 2.0 microns and typically below 1.0 micron. Alternative methods for nanoparticle production are known in the art and may be employed for the present invention. Production of Controlled Release Compositions Controlled release compositions of the present invention may be made by any emulsion process known in the art. In one embodiment an organic phase, containing one or more solvents, a bioactive agent and a polymer, is contacted with an aqueous phase, containing an organic ion. In a particular embodiment, the organic phase additionally includes a cosolvent. In another particular embodiment, the aqueous phase additionally includes an emulsifying agent. In a particular embodiment, the organic phase is contacted with the aqueous phase to form an emulsion wherein the emulsion comprises droplets of the organic phase dispersed in the aqueous phase. Solvent is subsequently removed from the emulsion droplets to form hardened microparticles. The hardened microparticles may then be recovered from the aqueous phase and dried. In one embodiment, the organic phase may contain solvents including but not limited to, methylene chloride, ethyl acetate, benzyl alcohol, acetone, acetic acid, propylene carbonate and other solvents in which the biodegradable polymer is soluble. In a particular embodiment, the solvent of the organic phase may be selected from the group consisting of ethyl acetate and methylene chloride. In one embodiment, the aqueous phase may contain solvents including but not limited to, methanol, benzyl alcohol, isopropyl alcohol, water, dimethylsulfoxide, N,N-dimethylformamide, methylene chloride and other solvents in which the bioactive agent is soluble. In another embodiment, cosolvents may be added to the organic phase. They are optionally used to promote solubility of the bioactive agent in the organic phase. In a particular embodiment, they are selected from the group consisting of, but not limited to, methyl alcohol, ethyl alcohol, isopropyl alcohol, N-methyl pyrrolidinone, dimethyl sulfoxide, N,N-dimethyl formamide, PEG 200, PEG 400 and benzyl alcohol. In another particular embodiment, the cosolvent may be present between about 0 and 90% w/w of the solvent of the organic phase. In another particular embodiment, the cosolvent is present between about 0 and 50% w/w of the solvent of the organic phase. The bioactive agent may be dissolved first in an appropriate volume of the cosolvent which is then added to the solvent of the organic phase, preferably having the biodegradable polymer dissolved therein, so as to form a solution of all the components of the organic phase. A person of ordinary skill can adjust the volumes and order of addition to achieve the desired solution of bioactive agent and biodegradable polymer. In a certain embodiment, the bioactive agent will be present in the organic phase at a concentration of about 1 - 20% w/w. In a particular embodiment, the biodegradable polymer will be present in the organic phase at a concentration of about 2-40% w/w. In another particular embodiment, the biodegradable polymer will be present in the organic phase at a concentration of about 5-20% w/w. Organic ions are dissolved in the aqueous phase. In a certain embodiment, they are dissolved at a concentration of between about 0.1 and 1000 mM. In a particular embodiment, they are dissolved at a concentration of between about 1 to 100 mM. The concentration may be adjusted for each particular organic ion agent and bioactive agent to achieve the desired drug loading and encapsulation efficiency. One or more emulsifying agents may be added to the aqueous phase to stabilize the emulsion. Emulsifying agents may be selected from the group consisting of, but not limited to, poly(vinyl alcohol), albumin, lecithin, vitamin E TPGS and polysorbates. The emulsifying agents are present at a concentration in the aqueous phase between 0 and 10% (w/w). In a particular embodiment, they are present at a concentration between 0.5 to 5% w/w. Pharmaceutical Formulations In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other alternative methods of administration of the present invention may also be used, including but not limited to intradermal administration, pulmonary administration, buccal administration, transdermal and transmucosal administration. Transmucosal administration may include, but is not limited to, ophthalmic, vaginal, rectal and intranasal. All such methods of administration are well known in the art. In a particular embodiment, the controlled release composition of the present invention may be administered intranasally, such as with nasal solutions or sprays, aerosols or inhalants. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. Antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in any of the formulations. Preservatives and other additives may be selected from the group consisting of, but not limited to, antimicrobials, anti-oxidants, chelating agents, inert gases and the like. Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines and are used for asthma prophylaxis. In another embodiment, controlled release compositions of the present invention are applied topically. Such controlled release compositions include, but are not limited to, lotions, ointments, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Excipients, Carriers and Diluents Controlled release compositions of the present invention can be formulated in any excipient the biological system or entity can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, polyethylene glycol and injectable organic esters such as ethyl oleate may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Iris buffer, while examples of preservatives include thimerosol, cresols, formalin and benzyl alcohol. Pharmaceutical carriers for controlled release compositions of the present invention are known to those skilled in the art. Those most typically utilized are likely to be standard carriers for administration to humans including solutions such as sterile water, saline and buffered solutions at physiological pH. The controlled release compositions of the present invention may be suspended in any aqueous solution or other diluent for injection in a human or animal patient in need of treatment. Aqueous diluent solutions may further include a viscosity enhancer selected from the group consisting of sodium carboxymethylcellulose, sucrose, mannitol, dextrose, trehalose and other biocompatible viscosity enhancing agents. The viscosity may be adjusted to a value between 2 centipoise (cp) and 100 cp, preferably between 4 and 40 cp. In a particular embodiment, a surfactant may be included in the diluent to enhance suspendability of the controlled release composition. Surfactants may be selected from the group consisting of, but not limited to, polysorbates and other biocompatible surfactants. Surfactants are used at a concentration of between 0 and 5% (w/w), preferably between 0.1 and 1% w/w. A composition of the present invention comprising a bioactive agent, an organic ion and a polymer is found to show surprising properties. The composition therefore is found to be synergistic. EXAMPLES The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 Conventional Preparation of Octreotide Acetate Encapsulated in Poly(lactide-co-glycolide) (PLGA) Microparticles Using Co-Solvents According to Previously Used Methods. Octreotide acetate microparticle formulations were prepared to investigate the effect of different co-solvents in the organic phase. Formulations A-F, prepared using an oil-in-water emulsion/solvent extraction technique, are summarized in Table 1. PLGA polymer (50:50 lactide/glycolide, MW 24,000,180 mg) was dissolved in ethyl acetate (EtOAc, 900 µL), and octreotide acetate (20 mg) dissolved in a co-solvent (Table 1) was added to the polymer solution. The resulting homogeneous organic phase was added to an aqueous phase (2 ml) containing 1% poly(vinyl alcohol) (PVA) and the mixture was vortexed for 15-30 seconds. The emulsion was poured into a solvent extraction solution (10 mM sodium phosphate, pH 8.0,150 ml) and stirred for four hours to extract EtOAc. Particles were isolated by filtration, washed with water and air dried overnight. The formulations were characterized by particle size, scanning electron microscopy (SEM), morphology, octreotide core load and in vitro release profiles. Formulation D was repeated using an emulsifying device, such as that disclosed in PCT Application Serial No. PCT/US04/11485, that combined a homogeneous organic phase (2 ml) consisting of octreotide acetate (20 mg), MeOH (100 µL), PLGA polymer (50:50 lactide/glycolide, MW 24,000,180 mg) and EtOAc (1.9 mL) with a 1 % PVA aqueous phase (4 mL). The emulsion was then added to a solvent extraction solution and stirred for four hours to extract EtOAc. This process produced formulation D2 (Table 1). The co-solvents investigated had a small influence on particle size and core load. Particle sizes were larger with the higher viscosity poly(ethylene glycol) (PEG) co-solvents. In contrast, core loads were similar for the methanol (MeOH) and PEG co-solvents (formulations A-C). The highest core loads were obtained for the MeOH cosolvent with a pH 8 buffered emulsion step (formulation D2) and for the dimethyl sulfoxide (DMSO) cosolvent (formulation F). In vitro release kinetics were measured in either phosphate buffered saline (PBS, pH 7.2, 37°C) or 100 mM sodium acetate (NaOAc, pH 4.0, 37°C). An example is shown in Table 2 (Formulation D2). The PEG co-solvent systems showed the highest initial peptide burst (8-10%), while the remaining formulations had an initial burst in the range of 2-3%. All the formulations released peptide for at least 6 weeks. There was a decrease in the relative release rates for formulations prepared with polar aprotic solvents (formulations E - F) resulting in lower total release of peptide relative to the other formulations. Octreotide acetate as the free peptide was measured to be 95% intact by high-pressure liquid chromatography (HPLC) following incubation in the release medium (PBS, pH 7.2, 37°C) after 49 days. In contrast, incubation of octreotide acetate PLGA microparticle formulations produced 55% of modified peptide species after 70 days in the release medium (PBS, pH 7.2, 37°C, Table 2). HPLC analysis showed that the new peptide entities were more hydrophobic than native octreotide acetate. HPLC/MS analysis revealed masses consistent with acylation of the parent peptide by PLGA polymer. The masses found were consistent with random acylation, for example, peptide plus one or two glycolic or lactic acid monomers in any combination. It may be that acylation products arise from attack on PLGA fragments or the polymer backbone by nucleophilic moieties in octreotide. At a lower pH these moieties likely would be protonated reducing their nucleophilicity and consequently the amount of acylated product. It was observed that formation of acylated byproducts for octreotide acetate PLGA microparticles incubated in 100 mM sodium acetate (NaOAc, pH 4.0) buffer was reduced to 1.25% at 49 days, in marked contrast to the results for PBS buffer (55%). Table 1. Octreotide Acetate Encapsulation in PLGA Microparticles. (Table Removed) Table 2. In vitro release of formulations D2 and AG. NaOAc buffer contains 100 mM NaOAc (pH 4.0), 0.02% Tween-20 and 0.05% NaN3. PBS is phosphate buffered saline (pH 7.2) containing 0.02% Tween-20 and 0.05% NaN3. Samples were incubated in a shaking (150 Hz) water bath incubator at 37C. Peptide and acylated peptide release values are listed as cumulative percent released. (Table Removed) Example 2 Production of Water Insoluble Organic Acid Salts (Complexes) of Octreotide and Encapsulation in PL6A Microparticles According to Previously Used Methods. Organic ion agents were investigated where the organic ion was initially complexed with octreotide acetate to form a water insoluble salt followed by encapsulation in PLGA microparticles. Sodium Dodecvlsulfate (SDS). An octreotide-SDS complex was prepared by dissolving octreotide acetate (100 mg) in H2O (500 µL). SDS (1.5 equiv, 43.2 mg) dissolved in H2O (500 µL) was added drop wise to the octreotide acetate solution with vortexing at room temperature. A precipitate immediately formed. The sample was centrifuged at 10,000 rpm for 1 minute and the supernatant removed by pipette. The precipitate was washed with cold water and lyophilized providing an octreotide-SDS complex (95.3 mg). RP-HPLC analysis showed a pronounced broadening of the octreotide peak indicating formation of the octreotide/SDS complex. Formulations G-l were prepared using an oil-in-water emulsion/solvent extraction technique. PLGA polymer (MW 24,000,180 mg) was dissolved in EtOAc (900 µL). Octreotide/SDS complex was dissolved in MeOH (100 µL) and added to the polymer solution. This resulted in a heterogeneous organic phase. In the case of formulation I (Table 3) an additional aliquot of MeOH (100 µL) was added to produce a homogeneous organic phase. The resulting organic phase was added to an aqueous phase (2 ml) containing 1% PVA and the mixture was vortexed for 15 - 30 seconds. The emulsion was poured into a solvent extraction solution (10 mM sodium phosphate, pH 8.0,150 mL) and stirred for four hours to extract EtOAc. Particles were isolated by filtration, washed with water and air dried overnight. The formulations were characterized by particle size, SEM morphology, octreotide core load and in vitro release profiles. The measured core load for formulations G-l prepared from the octreotide-SDS complex were relatively low, between 0.6-2.6% (Table 3). Also the median particle size was reduced by approximately 40% relative to formulations (A-F) prepared with octreotide acetate. The in vitro release profiles of formulations G-l in PBS were quite similar. Each had an initial burst of approximately 20% followed by three weeks of 1.5% release/week. After three weeks the rate of release increased to approximately 7.0 % release/week, culminating in approximately 80% total peptide release at 9 weeks. The in vitro PBS release assay with these formulations resulted in the release of similar amounts of acylated (40-55%) and total peptide compared to octreotide acetate (formulations A-F). Table 3. Octreotide-SDS Complex in the Organic Phase. (Table Removed) Benzoic Acid. Formulations (J-M) were prepared using one to ten equivalents of benzoic acid co-dissolved in the organic phase with PLGA. PLGA polymer (MW 24,000,180 mg) and benzoic acid (2.4 - 24 mg) were dissolved in EtOAc (900 µL). Octreotide acetate was dissolved in MeOH (100 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was added to an aqueous phase (2 ml) containing 1% PVA and the mixture was vortexed for 15 - 30 seconds. The emulsion was poured into a solvent extraction solution (10 mM sodium phosphate, pH 8.0,150 ml) and stirred for four hours to extract EtOAc. Particles were isolated by filtration, washed with water and air dried overnight. The core loads measured between 0.88-1.67% over the range of 1-10 added equivalents of benzoic acid per equivalent of octreotide acetate (Table 4). PamoicAcid. An octreotide-pamoate complex was prepared by dissolving pamoic acid (19.4 mg, 0.05 mmol) in 0.2 N NaOH (500 µL) to provide the sodium pamoate salt. Octreotide acetate (100 mg, 0.10 mmol) was dissolved in deionized water (100 µL) and added drop wise with gentle vortexing to the sodium pamoate salt solution. This produced a flocculent light yellow precipitate. The precipitate was pelleted by centrifugation, and the supernatant removed by pipette. The pellet was washed with water (1.0 ml), re-suspended in water and lyophilized to a light yellow powder (113 mg). The octreotide/pamoate ratio of this preparation was 1.71 as measured by RP-HPLC. A second octreotide-pamoate complex was prepared by dissolving pamoic acid (19.4 mg, 0.05 mmol) in 0.4 N NaOH (250 µL) and dioxane (250 µL) to provide a solution of sodium pamoate in dioxane/water (1:1). Octreotide acetate (50 mg, 0.05 mmol) was dissolved in dioxane/water (1:1, 200 µL). The octreotide acetate solution was added drop wise to the sodium pamoate with mixing to provide a light yellow, homogenous solution. This material was lyophilized to dryness providing a light yellow powder (65 mg). The octreotide/pamoate ratio of this preparation was 1.02 as measured by RP-HPLC. These two preparations were used to prepare new PLGA microparticle formulations. Table 4. Benzoic Acid and Octreotide Acetate in Organic Phase. (Table Removed) Microparticle formulations (Table 5, Q-W) were prepared by an oil-in-water emulsion/ solvent extraction method. PLGA polymer (MW 24,000,180 mg) was dissolved in EtOAc (1000 µL). Octreotide pamoate (20 or 40 mg) was dissolved in benzyl alcohol (BnOH, 1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1 % PVA aqueous phase in a ratio of 1:2 to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. Formulation characterization (Table 5) revealed that the initial octreotide/pamoate ratio of 1.7 had little effect on the encapsulation efficiency and core load relative to the formulations prepared with the octreotide/pamoate ratio of 1.02. In contrast, changing the co-solvent to benzyl alcohol increased the encapsulation efficiency by approximately 60% relative to methanol (e.g. Formulation S compared to T). The in vitro release profiles of these formulations in PBS demonstrated total peptide released (79-92%, Table 5 Q-T) is comparable to PLGA octreotide acetate microparticles made by conventional methods (formulations D, F, Table 1) while the amount of acylated peptide released (28-40%, Table 5 Q-T) is decreased slightly relative to conventional formulations (44-55%, Table 1, A-D). Formulations prepared using the octreotide/pamoate ratio of 1:1 did not show as strong a dependence of encapsulation efficiency and core load on the nature of the co-solvent as the 1.7 ratio formulations above. The differences in solubility for the complexes with different octreotide/pamoate ratios in the co-solvent is proposed as an explanation for this observation. The higher octreotide/pamoate ratio material had an increased solubility in benzyl alcohol relative to methanol resulting in higher encapsulation efficiency. In contrast, it was found that there was no significant difference in solubility in methanol versus benzyl alcohol for the 1:1 octreotide/pamoate complex. This resulted in similar encapsulation efficiencies and core loads independent of the co-solvents. The in vitro release profiles of these 1:1 formulations (U-W) reveal similar trends as discussed above, namely, that the total percent of peptide released (85 -110%, Table 5 U-W) is again comparable to conventional formulations (Example 1) (ca. 85%, Table 2) while the amount of acylated product released (35 - 44% Table 5 U-W) is decreased somewhat relative to conventional formulations (44-55%, Table 1, A-D2). Analysis of the final octreotide/pamoate molar ratio showed a wide variation among the formulations tested (Table 5) with a range from 2.1:1 (formulation W) to over 200:1 (formulation R). In all cases the ratio is more than twice the octreotide/pamoate ratio of the starting peptide salt complex. Thus the use of a preformed pamoate salt of the peptide octreotide yielded highly variable octreotide/pamoate molar ratios in the final sustained release formulation. Table 5. Octreotide-Pamoate Microparticles Prepared Using Pre-Formed (Table Removed) Example 3 Octreotide Acetate Encapsulation in PLGA Microspheres Using Organic Acid Salts in the Aqueous Emulsion Phase According to the Present Invention. Surprisingly it was discovered that the use of an organic acid salt in the aqueous phase of the emulsification process allowed use of a water soluble peptide and eliminated the need to prepare complexed species in an independent step prior to preparing the formulation. The present invention provided added benefits such as increased drug coreload, consistent octreotide/organic ion ratio, and decreased peptide degradation during in vitro release. Microparticle formulations were prepared by an oil-in-water emulsion/solvent extraction method. PLGA polymer (MW 24,000,140 -180 mg) was dissolved in EtOAc (1000 µL). Octreotide acetate (20 -60 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogenous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10-50 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This resulted in a final octreotide/pamoate ratio of approximately 1-1.5 in the microparticle formulation measured by RP-HPLC (Table 6). The effects of various experimental parameters on core load were investigated including organic to aqueous phase ratio, nature of co-solvent, and volume of co-solvent. BnOH was found to be a more suitable co-solvent than MeOH. It was possible to use BnOH in larger volumes than MeOH, as MeOH induced polymer precipitation in the organic phase. BnOH also led to a small increase in core load versus MeOH (Formulation Y, AB, Table 6). However, the use of BnOH without the organic ion in the aqueous phase did not provide high core loads or encapsulation efficiencies (Al, Table 6). It was also found that decreasing the organic to aqueous phase ratio increased the encapsulation efficiency slightly when BnOH was used as the co-solvent (Formulations AE, AF, Table 6). In all cases the molar ratio of octreotide to pamoate was tightly grouped between about 1.0 and 1.5, in contrast to the formulations of Example 2 (Table 5) where the use of a preformed octreotide/pamoate complex led to wide variations of the final octreotide/pamoate ratio from 2.1 to over 200. Significantly, product with predictable and elevated drug core loads ranging from 5-17.5% could be formed with the method of the present invention, (formulations AD, AG, AH, Table 6), in contrast to the prior art methods of Examples 1 and 2 where the maximum drug core load achieved was about 8% (Table 5 - S) with averages ranging from 2-6% (Tables 1-5). In addition, the compositions of the present invention have consistent stoichiometry for the molar ratio of bioactive agent to organic ion (Table 6). This is in contrast to the compositions made using previous methods (Table 5). Furthermore, the relative production of acylated peptide is lower for microparticles made with the organic ion in the aqueous phase (Table 6) than for microparticles made with the use of preformed octreotide-pamoate (Table 5) or octreotide acetate (Table 2). Table 6. Octreotide-Pamoate Complex Microparticles by an in situ Process. (Table Removed) The effect of organic acid concentration in the aqueous phase was explored to determine the optimal manufacturing parameters. PLGA polymer (MW 24,000,160 mg) was dissolved in EtOAc (1000 µL). Octreotide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogenous organic phase. The resulting organic phase was combined with a 1 % PVA aqueous phase containing 20 or 50 mM sodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. Formulations AJ - AL show that 20 or 50 mM disodium pamoate had no effect on core load relative to 10 mM disodium pamote (Table 7). However, the disodium pamoate concentration in the aqueous phase did have a measurable effect on the "day one" in vitro PBS release. The formulation prepared using 50 mM disodium pamoate resulted in a 15% burst (Formulation AL, Table 7) as compared to less than 4% burst for formulations prepared with 20 mM organic ion (Formulations AJ, AK, Table 7). This suggests that excess organic ion in the aqueous phase is deleterious to the in vitro release performance of the formulations. Table 7. The effect of organic ion concentration on the formation of octreotide-pamoate (Table Removed) Alternative organic ions in addition to pamoate were investigated to explore the general utility of the present invention. Microparticle formulations were prepared by an oil-in-water emulsion/solvent extraction method. PLGA polymer (MW 24,000,160 mg) was dissolved in EtOAc (1000 nL). Octreotide acetate (40 mg) was dissolved in BnOH (1000 fiL) and added to the polymer solution yielding a homogenous organic phase. The resulting organic phase was combined with a 1 % PVA aqueous phase containing 10-20 mM organic acid as its sodium salt to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This resulted in microparticle formulations with octreotide core loads between 6.8 and 15.3% as measured by RP-HPLC (Table 8). The effects of the tested organic ions on core load are revealing. Formulations AM - AP show no increase in the measured core load relative to control containing sodium pamoate (Formulations AT, AU, AY, Table 8). In contrast formulations AQ-AS, AV-AX and AZ-BB, which employed organic acids ranging from cholic acid to bicylic aromatics, provided peptide core loads comparable to pamoic acid (Table 8). These results imply that organic acids with appropriate physiochemical properties can be substituted for pamoic acid to produce comparable microparticle formulations. Table 8. The effect of various organic acids (sodium salts) in the aqueous phase on the formation of octreotide-complex microparticles. (Table Removed) Example 4 Encapsulation of Additional Peptides in PLGA Microspheres Using Organic Acid Salts in the Aqueous Emulsion Phase According to the Present Invention. Oxytocin acetate and leuprolide acetate were formulated in PLGA microparticles according to the present invention as described in the examples below. The results of these investigations demonstrate the utility of the present invention in relation to increased core load and encapsulation efficiency (Formulations Bl vs BJ-BK and BLvs BM) relative to conventional methodology (Table 9). Formulation Bl (Leuprolide) - Conventional encapsulation method PLGA polymer (MW 24,000, 160 mg) was dissolved in CH2CI2 (1000 µL). Leuprolide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation Bl (140 mg, 70.0% yield) with a median particle size 50.1 µm. The core load (1.99%), encapsulation efficiency (9.95%) and in vitro burst (1.63%) were determined by RP-HPLC assay. Formulation BJ (Leuprolide) - Organic ion assisted encapsulation method PLGA polymer (MW 24,000, 160 mg) was dissolved in CH2CI2 (1000 µL). Leuprolide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (100 mL) and stirred for 10 minutes. A secondary extraction solution consisting of 2% isopropanol (200 mL) was added and stirred for an additional four hours. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BJ (157 mg, 78.5% yield) with a median particle size 54.0 µm. The core load (9.4%), encapsulation efficiency (47.0%) and in vitro burst (5.31%) were determined by RP-HPLC assay. Formulation BK (Leuprolide) - Organic ion assisted method A microparticle formulation was prepared by an oil-in-water emulsion/solvent extraction method. PLGA polymer (MW 24,000,160 mg) was dissolved in CH2CI2 (1000 µL). Leuprolide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 50 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (100 ml) and stirred for 10 minutes. A secondary extraction solution consisting of 2% isopropanol (200 ml) was added and stirred for an additional four hours. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BK (120 mg, 60.0% yield) with a median particle size 43.1 µm. The core load (10.6%), encapsulation efficiency (53.0%) and in vitro burst (21.1%) were determined by RP-HPLC assay. Formulation BL (Oxytocin) - Conventional encapsulation method PLGA polymer (MW 13,000,180 mg) was dissolved in EtOAc (900 µL). Oxytocin acetate (20 mg) was dissolved in MeOH (100 µL) and added to the polymer solution yielding a milky suspension as the organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 5% EtOAc to provide an emulsion. The emulsion was collected directly into a 10 mM sodium phosphate (pH 8,0°C, 150 ml) solvent extraction solution and stirred for four hours while warming to room temperature to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BL (143 mg, 71.5% yield) with a median particle size 44.0 µm. The core load (1.67%), encapsulation efficiency (16.7%) and in vitro burst (46.3%) were determined by RP-HPLC assay. Formulation BM (Oxytocin) - Organic ion assisted encapsulation method PLGA polymer (MW 24,000,180 mg) was dissolved in EtOAc (1800 µL). Oxytocin acetate (40 mg) was dissolved in MeOH (200 µL) and added to the polymer solution yielding a milky suspension as the organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BM (158 mg, 79.0% yield) with a median particle size 144 urn. The core load (8.9%), encapsulation efficiency (44.5%) and in vitro burst (21.1%) were determined by RP-HPLC assay. Table 9. Peptide-pamoate complex microparticles by an in situ process. (Table Removed) Example 5 Insulin Encapsulation in PLGA Microparticles Using Organic Acid Salts in the Aqueous Emulsion Phase. Sodium Dodecylsulfate Microparticle formulations were prepared using an oil-in-water emulsion/solvent extraction method. The organic phase consisted of PLGA polymer (MW 11,800,150 mg) and PEGylated-insulin (50 mg) dissolved in CH2CI2(2 mL). The aqueous phase consisted of 1% PVA and 14 mM SDS. The homogeneous organic and aqueous phases were combined in a ratio of 1:5 to produce an organic in aqueous phase emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (100 mL) and stirred for 10 minutes before adding 100 mL 2% IPA. The solvent extraction solution was then stirred for an additional 3 hours to extract CH2CI2. Hardened microparticles were collected by filtration, washed with water, air dried and stored at -20°C. The resulting microparticle had a core load of 21% (encapsulation efficiency 84%). These microparticles were characterized by a large in vitro burst of 50% at 24 h in PBS at 37°C. Disodium Pamoate Microparticle formulations were prepared using an oil-in-water emulsion/solvent extraction method. The organic phase consisted of PLGA polymer (MW 11,800, 75 mg) and PEGylated-insulin (25 mg) dissolved in CH2CI2 (1 mL). The aqueous phase consisted of 1% PVA and 10 mM disodium pamoate. The homogeneous organic and aqueous phases were combined in a ratio of 1:5 to produce an organic in aqueous phase emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (50 mL) and stirred for 10 minutes before adding water (100 mL). The solvent extraction solution was then stirred for an additional 3 hours to extract CH2CI2. Hardened microparticles were collected by filtration, washed with water, air dried and stored at -20°C. The resulting microparticles had a core load of 18% (encapsulation efficiency 78%) and a final PEGylated-insulin/pamoate ratio of 1:2. In contrast to the microparticles made with SDS, these microparticles had a low in vitro burst of 5% in PBS at 37°C. Example 6 Evaluation of the Pharmacokinetics of Octreotide in PLGA Microparticles after Administration to Sprague Dawley Rats. Blood serum levels were measured for octreotide released from PLGA microparticle formulations injected subcutaneously in rats. Animals (n=6/group) were treated once by subcutaneous injection of a single dose level (~8-10 mg/kg) of six different octreotide PLGA microparticle formulations. At hours 1 and 6, and on days 1, 4, 7,11,14, 20, 28,42 and 54, serum samples were obtained from each animal to evaluate the octreotide pharmacokinetics. Serum concentrations were measured by a commercially available extraction-free radioimmunoassay kit (#8-2211) (Peninsula Labs). The Limit of Quantitation (LOQ) of the assay was 0.1 ng/mL. The mean octreotide serum concentrations for each time point are reported in Table 10. The preparation of the octreotide PLGA formulations tested is described below. Table 10. Mean octreotide serum levels (ng/mL) after a single subcutaneous treatment in rats. (Table Removed) Preparation and characterization of octreotide formulations used in the animal study. Formulation BC PLGA polymer (MW 24,000, 720 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (80 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BC (754 mg, 94% yield) with a median particle size 55.0 µm. The core load (8.5%), encapsulation efficiency (85.0%) and in vitro burst (7.4%) were determined by RP-HPLC assay. Formulation BD PLGA polymer (MW 24,000, 680 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (120 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BD (694 mg, 94% yield) with a median particle size 58.7 jam. The core load (11.8%), encapsulation efficiency (78.7%) and in vitro burst (4.1%) were determined by RP-HPLC assay. Formulation BE PLGA polymer (MW 24,000, 680 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (120 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BE (727 mg, 91% yield) with a median particle size 52.2 µm. The core load (11.6%), encapsulation efficiency (77.3%) and in vitro burst (2.75%) were determined by RP-HPLC assay. Formulation BF PLGA polymer (MW 24,000, 640 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (160 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BF (766 mg, 95.8% yield) with a median particle size 47.7 µm. The core load (14.7%), encapsulation efficiency (73.5%) and in vitro burst (5.5%) were determined by RP-HPLC assay. Formulation BG PLGA polymer (MW 28,000, 640 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (160 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BG (715 mg, 89.3% yield) with a median particle size 48.7µm. The core load (11.9%), encapsulation efficiency (59.5%) and in vitro burst (2.3%) were determined by RP-HPLC assay. Formulation BH PLGA polymer (MW 14,000, 560 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (240 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BH (680 mg, 85.0% yield) with a median particle size 40.6 ^im. The core load (17.4%), encapsulation efficiency (58.0%) and in vitro burst (6.8%) were determined by RP-HPLC assay. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. WE CLAIM: 1. A method of preparing a controlled release composition, said method comprises the steps of: a) combining a bioactive agent and a polymer with an organic solvent to obtain a homogeneous organic phase, wherein the bioactive agent is selected from the group comprising of proteins, peptides, LHRH agonists and synthetic analogs thereof, leuprolide, oxytocin, somatostatin and synthetic analogs thereof and hormones; b) combining an organic ion and water to obtain an aqueous phase, wherein the organic ion is selected from the group consisting of pamoate, trifluoromethyl-p-toluate, cholate, 2-naphthalene sulfonate, 2,3- naphthalene dicarboxylate, I-hydroxy-2-naphthoate, 3-hydroxy-2- naphthoate, 2-naphthoate, ana salicylsalicylate; c) contacti~g the homogeneous organic phase and the aqueous phase through the use of an emulsion process so that the molar stoichiometry of the bioactive agent relative to the organic ion ranges from 0.5 to 2.0; and d) recovering said controlled release composition in a manner known per se having diameter between 20.0 nm to 1.0 mm. 2. The method as claimed in claim 1, wherein the bioactive agent is octreotide, octreotide acetate and pharmaceutical equivalents thereof. 3. The method as claimed in claim 1, wherein in step (a) the said polymer used is selected from the group consisting of poly(lactide )s, poly(glycolide )s, poly(lactide-co-glycolide )s, poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-eo-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls, . polycyanoacrylates, polyetheresters, poly( dioxanone )s, poly( alkylene alkylate ), copolymers of polyethylene glycol and polyorthoester, qiodegradable polyurethanes, blends and copolymers thereof. 4. The method as claimed in claim 1, wherein the organic solvent is selected from th'e group consisting of methylene chloride, ethyl acetate, benzyl alcohol, acetone, acetic acid, propylene carbonate. 5. The method as claimed in claim 1, wherein the organic phase optionally comprises a cosolvent. 6. The method as claimed in claim 5, wherein the cosolvent is selected from the group consisting of methyl alcohol, ethyl alcohol, isopropyl alcohol, N-methyl pyrrolidinone, dimethyl sulfoxide, N ,N-dimethyl formamide, PEG200, PEG400, and benzyl alcohol. 7. The method as claimed in claim 1, wherein the stoichiometry of the bioactive agent relative to the organic ion ranges from 1.0 to 1.5. 8. The method as claimed in claim 1, wherein the aqueous phase optionally comprises an emulsifying agent. 9. The m.ethod as claimed in claim 8, wherein the emulsifying agent is selected from the group consisting of poly (vinyl alcohol), albumin, lecithin, vitamin (Equation Removed) E TPGS and polysorbates. 10. The method as claimed in claim 1, wherein. in step (d) the controlled release composition is recovered by removing organic solvent from the emulsion and ontain a droplet. 11. The method as claimed in claim 1, wherein the said controlled release composition is selected from the group consisting of microparticles and nanoparticles. 12. The method as claimed in claim 11, wherein the controlled release micro-particle composition have diameter between 1.0 to 200.0 microns. 13. The method as claimed in claim 11, wherein the controlled release nanoparticle composition have diameter between 20.0 nanometers to 2.0 microns. 14. The method as claim~d in claim 11, wherein the controlled release microparticle composition and the controlled release nano-particle composition are biodegradable. 15. A method of preparing a controlled release microparticle composition substantially as herein describe with reference to forgoing examples and drawings.

Full Text

CONTROLLED RELEASE COMPOSITIONS
Field of the Invention
The present invention relates to controlled release compositions including a water soluble bioactive agent, an organic ion and a polymer, wherein the content of said bioactive agent is about two to four fold higher than in the absence of organic ion.
BACKGROUND OF THE INVENTION
Currently there are numerous controlled release formulations on the market that contain various bioactive agents, such as GnRH analogs, human growth hormone, risperidone, and somatostatin analogs of which octreotide acetate is an example. Such formulations are preferred by physicians and veterinarians and by their patients because they reduce the need for multiple injections. Unfortunately, there are many problems with the formulations for controlled release compositions such as the fact that many popular bioactive agents are not good candidates for controlled release compositions due to their substantial water solubility. The use of highly soluble bioactive agents may result in tow drug content and an undesirable "burst" of bioactive agent upon contact with an aqueous solution, such as by administration to a patient or introduction to a physiological medium, which often limits the useable content of bioactive agent achievable in practice.
Various methods of solving the water solubility problem have been devised with some success. One such effort is disclosed in US Patent No. 5,776,885 by Orsolini et al. Orsolini converted water soluble peptides to their water insoluble addition salts with pamoic, tannic or stearic acid prior to their encapsulation in sustained release compositions. Encapsulation within a polymer matrix is then performed by dispersing the water insoluble peptide in a polymer solution and forming controlled release compositions via either extrusion or a cumbersome coacervation method. One disadvantage of this approach is the requirement to obtain the water insoluble addition salt prior to encapsulation within a polymer matrix. Furthermore, it was discovered in the present work that when pre-formed addition salts were employed in an emulsion process to form microparticles the result was release of substantial amounts of modified or degraded peptide from the composition
when placed in an aqueous physiological buffer. Modification was in the form of undesirable acylation of bioactive agent. Controlled release composition with a high drug load, low burst effect upon administration, and minimum degradation of the bioactive agent are greatly needed to realize the benefits of these types of composition as human or veterinary therapeutics.
STATEMENT OF THE INVENTION:
Accordingly, the present invention provides a method of preparing a controlled release composition, said method comprises the steps of:
a) combining a bioactive agent and a polymer with an organic solvent to obtain a homogeneous organic phase, wherein the bioactive agent is selected from the group comprising of proteins, peptides, LHRH agonists and synthetic analogs thereof, leuprolide, oxytocin, somatostatin and synthetic analogs thereof and hormones;
b) combining an organic ion and water to obtain an aqueous phase, wherein the organic ion is selected from the group consisting of pamoate, trifluoromethyl-p-toluate, cholate, 2-napthalene sulfonate, 2,3-napthalene dicarboxylate, I-hydroxy-2-naphthoate, 3-hydroxy-2-naphthoate, 2-.na phthoate, and salicylsalicylate;
c) contacting the homogenous organic phase and the aqueous phase through the use of an emulsion process so that the molar stoichiometry of the bioactive agent relative to the organic ion ranges' from 0.5 to 2.0; and
d) recovering said controlled release composition in a manner known per se having diameter between 20.0 nm to 1.0 mm.
SUMMARY OF THE INVENTION:
It has now been discovered that controlled release compositions capable of administration in a concentrated, low dose volume form which release active agent for a prolonged period of time can originate from water soluble bioactive agentswithout first converting them to a water insoluble form. This finding is in direct contrast to previous work in this field that suggested that water soluble bioactive agents would need to be manipulated in some way, for example 1:0 render them water insoluble, prior to forming a sustained release composition. Additionally, the conversion process required in previous compositions,
resulted in a high percentage of degradation of thtbioactive agent, whereas the compositions of the present invention are prepared with an understanding of how to reduce degradation. The presence of the bioactive
agent in a physiologically relevant form creates an increased core load with regard to previously prepared compositions, which are susceptible to significant degradation. Reduced degradation translates into increased release of unmodified bioactive agent, thus allowing for a smaller injection volume to deliver the same dose of bioactive agent. The compositions of the present invention offer a vast improvement over previous inventions in that they provide a way to deliver a relatively undegraded bioactive agent over a prolonged period of time using a reduced dose volume. In one embodiment, the controlled release compositions are micro particles and nanoparticles. In a particular embodiment, the microparticles and nanoparticles are biodegradable. In another particular embodiment, the polymer is selected from the group consisting of, but not limited to, poly (lactide)s, poly (glycolide)s, poly (Iactide-co-glycolide)s, poly (lactic acid)s, poly (glycolic acid)s, poly (lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly (amino acids), polyorthoesters, polyacetyls, polycyanoacrylates, polyetheresters,
poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of polyethylene glycol and poly(lactide)s or poly(lactide-co-glycolide)s, biodegradable polyurethanes, blends and copolymers thereof.
In a particular embodiment, the microparticle is made of poly(d,l-lactide-co-glycolide) (PLGA). PLGA degrades when exposed to physiological pH and hydrolyzes to form lactic acid and glycolic acid, which are normal byproducts of cellular metabolism. The disintegration rate of PLGA polymers will vary depending on the polymer molecular weight, ratio of lactide to glycolide monomers in the polymer chain, and stereoregularity of the monomer subunits. Mixtures of L and D stereoisomers that disrupt the polymer crystallinity will increase polymer disintegration rates. In addition, microspheres may contain blends of two or more biodegradable polymers, of different molecular weight and/or monomer ratio.
In other alternative embodiments, derivatized biodegradable polymers, including hydrophilic polymers attached to PLGA, can be used to form microspheres. In particular embodiments, the hydrophilic polymer is selected from the group consisting of, but not limited to, poly(ethylene glycol), poly(propylene glycol) and copolymers of poly(ethylene glycol) and polypropylene glycol).
Biodegradable Nanoparticles
In certain embodiments, the controlled release composition is a nanoparticle.
In certain embodiments, the bioactive agent, with or without a hydrophilic polymer attached, is associated with biodegradable submicron particles for controlled release of the bioactive agent. A nanoparticle has a diameter ranging from 20.0 nanometers to about 2.0 microns and is typically between 100.0 nanometers and 1.0 micron.
Nanoparticles can be created in the same manner as microparticles, except that high-speed mixing or homogenization is used to reduce the size of the polymer/bioactive agent emulsions to less than 2.0 microns and typically below 1.0 micron. Alternative methods for nanoparticle production are known in the art and may be employed for the present invention.
Production of Controlled Release Compositions
Controlled release compositions of the present invention may be made by any emulsion process known in the art.
In one embodiment an organic phase, containing one or more solvents, a bioactive agent and a polymer, is contacted with an aqueous phase, containing an
organic ion. In a particular embodiment, the organic phase additionally includes a cosolvent. In another particular embodiment, the aqueous phase additionally includes an emulsifying agent.
In a particular embodiment, the organic phase is contacted with the aqueous phase to form an emulsion wherein the emulsion comprises droplets of the organic phase dispersed in the aqueous phase. Solvent is subsequently removed from the emulsion droplets to form hardened microparticles. The hardened microparticles may then be recovered from the aqueous phase and dried.
In one embodiment, the organic phase may contain solvents including but not limited to, methylene chloride, ethyl acetate, benzyl alcohol, acetone, acetic acid, propylene carbonate and other solvents in which the biodegradable polymer is soluble. In a particular embodiment, the solvent of the organic phase may be selected from the group consisting of ethyl acetate and methylene chloride.
In one embodiment, the aqueous phase may contain solvents including but not limited to, methanol, benzyl alcohol, isopropyl alcohol, water, dimethylsulfoxide, N,N-dimethylformamide, methylene chloride and other solvents in which the bioactive agent is soluble.
In another embodiment, cosolvents may be added to the organic phase. They are optionally used to promote solubility of the bioactive agent in the organic phase. In a particular embodiment, they are selected from the group consisting of, but not limited to, methyl alcohol, ethyl alcohol, isopropyl alcohol, N-methyl pyrrolidinone, dimethyl sulfoxide, N,N-dimethyl formamide, PEG 200, PEG 400 and benzyl alcohol. In another particular embodiment, the cosolvent may be present between about 0 and 90% w/w of the solvent of the organic phase. In another particular embodiment, the cosolvent is present between about 0 and 50% w/w of the solvent of the organic phase. The bioactive agent may be dissolved first in an appropriate volume of the cosolvent which is then added to the solvent of the organic phase, preferably having the biodegradable polymer dissolved therein, so as to form a solution of all the components of the organic phase. A person of ordinary skill can adjust the volumes and order of addition to achieve the desired solution of bioactive agent and biodegradable polymer. In a certain embodiment, the bioactive agent will be present in the organic phase at a concentration of about 1 - 20% w/w. In a particular embodiment, the biodegradable polymer will be present in the organic
phase at a concentration of about 2-40% w/w. In another particular embodiment, the biodegradable polymer will be present in the organic phase at a concentration of about 5-20% w/w.
Organic ions are dissolved in the aqueous phase. In a certain embodiment, they are dissolved at a concentration of between about 0.1 and 1000 mM. In a particular embodiment, they are dissolved at a concentration of between about 1 to 100 mM. The concentration may be adjusted for each particular organic ion agent and bioactive agent to achieve the desired drug loading and encapsulation efficiency.
One or more emulsifying agents may be added to the aqueous phase to stabilize the emulsion. Emulsifying agents may be selected from the group consisting of, but not limited to, poly(vinyl alcohol), albumin, lecithin, vitamin E TPGS and polysorbates. The emulsifying agents are present at a concentration in the aqueous phase between 0 and 10% (w/w). In a particular embodiment, they are present at a concentration between 0.5 to 5% w/w.
Pharmaceutical Formulations
In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other alternative methods of administration of the present invention may also be used, including but not limited to intradermal administration, pulmonary administration, buccal administration, transdermal and transmucosal administration. Transmucosal administration may include, but is not limited to, ophthalmic, vaginal, rectal and intranasal. All such methods of administration are well known in the art.
In a particular embodiment, the controlled release composition of the present invention may be administered intranasally, such as with nasal solutions or sprays, aerosols or inhalants. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5.
Antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in any of the formulations. Preservatives and other additives may be selected from the group consisting of, but not limited to, antimicrobials, anti-oxidants, chelating agents, inert gases and the like.
Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.
In another embodiment, controlled release compositions of the present invention are applied topically. Such controlled release compositions include, but are not limited to, lotions, ointments, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Excipients, Carriers and Diluents
Controlled release compositions of the present invention can be formulated in any excipient the biological system or entity can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, polyethylene glycol and injectable organic esters such as ethyl oleate may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol or dextran.
Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Iris buffer, while examples of preservatives include thimerosol, cresols, formalin and benzyl alcohol.
Pharmaceutical carriers for controlled release compositions of the present invention are known to those skilled in the art. Those most typically utilized are likely to be standard carriers for administration to humans including solutions such as sterile water, saline and buffered solutions at physiological pH.
The controlled release compositions of the present invention may be suspended in any aqueous solution or other diluent for injection in a human or animal patient in need of treatment. Aqueous diluent solutions may further include a viscosity enhancer selected from the group consisting of sodium carboxymethylcellulose, sucrose, mannitol, dextrose, trehalose and other biocompatible viscosity enhancing agents. The viscosity may be adjusted to a value between 2 centipoise (cp) and 100 cp, preferably between 4 and 40 cp.
In a particular embodiment, a surfactant may be included in the diluent to enhance suspendability of the controlled release composition. Surfactants may be selected from the group consisting of, but not limited to, polysorbates and other biocompatible surfactants. Surfactants are used at a concentration of between 0 and 5% (w/w), preferably between 0.1 and 1% w/w.
A composition of the present invention comprising a bioactive agent, an organic ion and a polymer is found to show surprising properties. The composition therefore is found to be synergistic.
EXAMPLES
The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Conventional Preparation of Octreotide Acetate Encapsulated in Poly(lactide-co-glycolide) (PLGA) Microparticles Using Co-Solvents According to Previously Used Methods.
Octreotide acetate microparticle formulations were prepared to investigate the effect of different co-solvents in the organic phase. Formulations A-F, prepared using an oil-in-water emulsion/solvent extraction technique, are summarized in Table 1. PLGA polymer (50:50 lactide/glycolide, MW 24,000,180 mg) was dissolved in ethyl acetate (EtOAc, 900 µL), and octreotide acetate (20 mg) dissolved in a co-solvent (Table 1) was added to the polymer solution. The resulting homogeneous organic phase was added to an aqueous phase (2 ml) containing 1% poly(vinyl alcohol) (PVA) and the mixture was vortexed for 15-30 seconds. The emulsion was poured into a solvent extraction solution (10 mM sodium phosphate, pH 8.0,150 ml) and stirred for four hours to extract EtOAc. Particles were isolated by filtration, washed with water and air dried overnight. The formulations were characterized by particle size, scanning electron microscopy (SEM), morphology, octreotide core load and in vitro release profiles.
Formulation D was repeated using an emulsifying device, such as that disclosed in PCT Application Serial No. PCT/US04/11485, that combined a homogeneous organic phase (2 ml) consisting of octreotide acetate (20 mg), MeOH (100 µL), PLGA polymer (50:50 lactide/glycolide, MW 24,000,180 mg) and EtOAc (1.9 mL) with a 1 % PVA aqueous phase (4 mL). The emulsion was then added to a
solvent extraction solution and stirred for four hours to extract EtOAc. This process produced formulation D2 (Table 1).
The co-solvents investigated had a small influence on particle size and core load. Particle sizes were larger with the higher viscosity poly(ethylene glycol) (PEG) co-solvents. In contrast, core loads were similar for the methanol (MeOH) and PEG co-solvents (formulations A-C). The highest core loads were obtained for the MeOH cosolvent with a pH 8 buffered emulsion step (formulation D2) and for the dimethyl sulfoxide (DMSO) cosolvent (formulation F).
In vitro release kinetics were measured in either phosphate buffered saline (PBS, pH 7.2, 37°C) or 100 mM sodium acetate (NaOAc, pH 4.0, 37°C). An example is shown in Table 2 (Formulation D2). The PEG co-solvent systems showed the highest initial peptide burst (8-10%), while the remaining formulations had an initial burst in the range of 2-3%. All the formulations released peptide for at least 6 weeks. There was a decrease in the relative release rates for formulations prepared with polar aprotic solvents (formulations E - F) resulting in lower total release of peptide relative to the other formulations.
Octreotide acetate as the free peptide was measured to be 95% intact by high-pressure liquid chromatography (HPLC) following incubation in the release medium (PBS, pH 7.2, 37°C) after 49 days. In contrast, incubation of octreotide acetate PLGA microparticle formulations produced 55% of modified peptide species after 70 days in the release medium (PBS, pH 7.2, 37°C, Table 2). HPLC analysis showed that the new peptide entities were more hydrophobic than native octreotide acetate. HPLC/MS analysis revealed masses consistent with acylation of the parent peptide by PLGA polymer. The masses found were consistent with random acylation, for example, peptide plus one or two glycolic or lactic acid monomers in any combination. It may be that acylation products arise from attack on PLGA fragments or the polymer backbone by nucleophilic moieties in octreotide. At a lower pH these moieties likely would be protonated reducing their nucleophilicity and consequently the amount of acylated product. It was observed that formation of acylated byproducts for octreotide acetate PLGA microparticles incubated in 100 mM sodium acetate (NaOAc, pH 4.0) buffer was reduced to 1.25% at 49 days, in marked contrast to the results for PBS buffer (55%).
Table 1. Octreotide Acetate Encapsulation in PLGA Microparticles.
(Table Removed)
Table 2. In vitro release of formulations D2 and AG. NaOAc buffer contains 100 mM NaOAc (pH 4.0), 0.02% Tween-20 and 0.05% NaN3. PBS is phosphate buffered saline (pH 7.2) containing 0.02% Tween-20 and 0.05% NaN3. Samples were incubated in a shaking (150 Hz) water bath incubator at 37C. Peptide and acylated peptide release values are listed as cumulative percent released.
(Table Removed)
Example 2
Production of Water Insoluble Organic Acid Salts (Complexes) of Octreotide and Encapsulation in PL6A Microparticles According to Previously Used Methods.
Organic ion agents were investigated where the organic ion was initially complexed with octreotide acetate to form a water insoluble salt followed by encapsulation in PLGA microparticles.
Sodium Dodecvlsulfate (SDS). An octreotide-SDS complex was prepared by dissolving octreotide acetate (100 mg) in H2O (500 µL). SDS (1.5 equiv, 43.2 mg) dissolved in H2O (500 µL) was added drop wise to the octreotide acetate solution with vortexing at room temperature. A precipitate immediately formed. The sample was centrifuged at 10,000 rpm for 1 minute and the supernatant removed by pipette. The precipitate was washed with cold water and lyophilized providing an octreotide-SDS complex (95.3 mg). RP-HPLC analysis showed a pronounced broadening of the octreotide peak indicating formation of the octreotide/SDS complex. Formulations G-l were prepared using an oil-in-water emulsion/solvent extraction technique. PLGA

polymer (MW 24,000,180 mg) was dissolved in EtOAc (900 µL). Octreotide/SDS complex was dissolved in MeOH (100 µL) and added to the polymer solution. This resulted in a heterogeneous organic phase. In the case of formulation I (Table 3) an additional aliquot of MeOH (100 µL) was added to produce a homogeneous organic phase. The resulting organic phase was added to an aqueous phase (2 ml) containing 1% PVA and the mixture was vortexed for 15 - 30 seconds. The emulsion was poured into a solvent extraction solution (10 mM sodium phosphate, pH 8.0,150 mL) and stirred for four hours to extract EtOAc. Particles were isolated by filtration, washed with water and air dried overnight. The formulations were characterized by particle size, SEM morphology, octreotide core load and in vitro release profiles.
The measured core load for formulations G-l prepared from the octreotide-SDS complex were relatively low, between 0.6-2.6% (Table 3). Also the median particle size was reduced by approximately 40% relative to formulations (A-F) prepared with octreotide acetate.
The in vitro release profiles of formulations G-l in PBS were quite similar. Each had an initial burst of approximately 20% followed by three weeks of 1.5% release/week. After three weeks the rate of release increased to approximately 7.0 % release/week, culminating in approximately 80% total peptide release at 9 weeks.
The in vitro PBS release assay with these formulations resulted in the release of similar amounts of acylated (40-55%) and total peptide compared to octreotide acetate (formulations A-F).
Table 3. Octreotide-SDS Complex in the Organic Phase.
(Table Removed)
Benzoic Acid. Formulations (J-M) were prepared using one to ten
equivalents of benzoic acid co-dissolved in the organic phase with PLGA. PLGA polymer (MW 24,000,180 mg) and benzoic acid (2.4 - 24 mg) were dissolved in EtOAc (900 µL). Octreotide acetate was dissolved in MeOH (100 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was added to an aqueous phase (2 ml) containing 1% PVA and the mixture was vortexed for 15 - 30 seconds. The emulsion was poured into a solvent extraction solution (10 mM sodium phosphate, pH 8.0,150 ml) and stirred for four hours to extract EtOAc. Particles were isolated by filtration, washed with water and air dried overnight. The core loads measured between 0.88-1.67% over the range of 1-10 added equivalents of benzoic acid per equivalent of octreotide acetate (Table 4).
PamoicAcid. An octreotide-pamoate complex was prepared by
dissolving pamoic acid (19.4 mg, 0.05 mmol) in 0.2 N NaOH (500 µL) to provide the sodium pamoate salt. Octreotide acetate (100 mg, 0.10 mmol) was dissolved in deionized water (100 µL) and added drop wise with gentle vortexing to the sodium pamoate salt solution. This produced a flocculent light yellow precipitate. The precipitate was pelleted by centrifugation, and the supernatant removed by pipette. The pellet was washed with water (1.0 ml), re-suspended in water and lyophilized to a light yellow powder (113 mg). The octreotide/pamoate ratio of this preparation was 1.71 as measured by RP-HPLC.
A second octreotide-pamoate complex was prepared by dissolving pamoic acid (19.4 mg, 0.05 mmol) in 0.4 N NaOH (250 µL) and dioxane (250 µL) to provide a solution of sodium pamoate in dioxane/water (1:1). Octreotide acetate (50 mg, 0.05
mmol) was dissolved in dioxane/water (1:1, 200 µL). The octreotide acetate solution was added drop wise to the sodium pamoate with mixing to provide a light yellow, homogenous solution. This material was lyophilized to dryness providing a light yellow powder (65 mg). The octreotide/pamoate ratio of this preparation was 1.02 as measured by RP-HPLC. These two preparations were used to prepare new PLGA microparticle formulations.
Table 4. Benzoic Acid and Octreotide Acetate in Organic Phase.
(Table Removed)
Microparticle formulations (Table 5, Q-W) were prepared by an oil-in-water emulsion/ solvent extraction method. PLGA polymer (MW 24,000,180 mg) was dissolved in EtOAc (1000 µL). Octreotide pamoate (20 or 40 mg) was dissolved in benzyl alcohol (BnOH, 1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1 % PVA aqueous phase in a ratio of 1:2 to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C.
Formulation characterization (Table 5) revealed that the initial octreotide/pamoate ratio of 1.7 had little effect on the encapsulation efficiency and core load relative to the formulations prepared with the octreotide/pamoate ratio of 1.02. In contrast, changing the co-solvent to benzyl alcohol increased the encapsulation
efficiency by approximately 60% relative to methanol (e.g. Formulation S compared to T).
The in vitro release profiles of these formulations in PBS demonstrated total peptide released (79-92%, Table 5 Q-T) is comparable to PLGA octreotide acetate microparticles made by conventional methods (formulations D, F, Table 1) while the amount of acylated peptide released (28-40%, Table 5 Q-T) is decreased slightly relative to conventional formulations (44-55%, Table 1, A-D).
Formulations prepared using the octreotide/pamoate ratio of 1:1 did not show as strong a dependence of encapsulation efficiency and core load on the nature of the co-solvent as the 1.7 ratio formulations above. The differences in solubility for the complexes with different octreotide/pamoate ratios in the co-solvent is proposed as an explanation for this observation. The higher octreotide/pamoate ratio material had an increased solubility in benzyl alcohol relative to methanol resulting in higher encapsulation efficiency. In contrast, it was found that there was no significant difference in solubility in methanol versus benzyl alcohol for the 1:1 octreotide/pamoate complex. This resulted in similar encapsulation efficiencies and core loads independent of the co-solvents.
The in vitro release profiles of these 1:1 formulations (U-W) reveal similar trends as discussed above, namely, that the total percent of peptide released (85 -110%, Table 5 U-W) is again comparable to conventional formulations (Example 1) (ca. 85%, Table 2) while the amount of acylated product released (35 - 44% Table 5 U-W) is decreased somewhat relative to conventional formulations (44-55%, Table 1, A-D2).
Analysis of the final octreotide/pamoate molar ratio showed a wide variation among the formulations tested (Table 5) with a range from 2.1:1 (formulation W) to over 200:1 (formulation R). In all cases the ratio is more than twice the octreotide/pamoate ratio of the starting peptide salt complex. Thus the use of a preformed pamoate salt of the peptide octreotide yielded highly variable octreotide/pamoate molar ratios in the final sustained release formulation.
Table 5. Octreotide-Pamoate Microparticles Prepared Using Pre-Formed
(Table Removed)
Example 3
Octreotide Acetate Encapsulation in PLGA Microspheres Using Organic Acid Salts in the Aqueous Emulsion Phase According to the Present Invention.
Surprisingly it was discovered that the use of an organic acid salt in the aqueous phase of the emulsification process allowed use of a water soluble peptide and eliminated the need to prepare complexed species in an independent step prior to preparing the formulation. The present invention provided added benefits such as increased drug coreload, consistent octreotide/organic ion ratio, and decreased peptide degradation during in vitro release.
Microparticle formulations were prepared by an oil-in-water emulsion/solvent extraction method. PLGA polymer (MW 24,000,140 -180 mg) was dissolved in EtOAc (1000 µL). Octreotide acetate (20 -60 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogenous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10-50 mM
disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This resulted in a final octreotide/pamoate ratio of approximately 1-1.5 in the microparticle formulation measured by RP-HPLC (Table 6).
The effects of various experimental parameters on core load were investigated including organic to aqueous phase ratio, nature of co-solvent, and volume of co-solvent. BnOH was found to be a more suitable co-solvent than MeOH. It was possible to use BnOH in larger volumes than MeOH, as MeOH induced polymer precipitation in the organic phase. BnOH also led to a small increase in core load versus MeOH (Formulation Y, AB, Table 6). However, the use of BnOH without the organic ion in the aqueous phase did not provide high core loads or encapsulation efficiencies (Al, Table 6). It was also found that decreasing the organic to aqueous phase ratio increased the encapsulation efficiency slightly when BnOH was used as the co-solvent (Formulations AE, AF, Table 6). In all cases the molar ratio of octreotide to pamoate was tightly grouped between about 1.0 and 1.5, in contrast to the formulations of Example 2 (Table 5) where the use of a preformed octreotide/pamoate complex led to wide variations of the final octreotide/pamoate ratio from 2.1 to over 200.
Significantly, product with predictable and elevated drug core loads ranging from 5-17.5% could be formed with the method of the present invention, (formulations AD, AG, AH, Table 6), in contrast to the prior art methods of Examples 1 and 2 where the maximum drug core load achieved was about 8% (Table 5 - S) with averages ranging from 2-6% (Tables 1-5). In addition, the compositions of the present invention have consistent stoichiometry for the molar ratio of bioactive agent to organic ion (Table 6). This is in contrast to the compositions made using previous methods (Table 5). Furthermore, the relative production of acylated peptide is lower for microparticles made with the organic ion in the aqueous phase (Table 6) than for microparticles made with the use of preformed octreotide-pamoate (Table 5) or octreotide acetate (Table 2).
Table 6. Octreotide-Pamoate Complex Microparticles by an in situ Process.
(Table Removed)
The effect of organic acid concentration in the aqueous phase was explored to determine the optimal manufacturing parameters. PLGA polymer (MW 24,000,160 mg) was dissolved in EtOAc (1000 µL). Octreotide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogenous organic phase. The resulting organic phase was combined with a 1 % PVA aqueous phase containing
20 or 50 mM sodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. Formulations AJ - AL show that 20 or 50 mM disodium pamoate had no effect on core load relative to 10 mM disodium pamote (Table 7). However, the disodium pamoate concentration in the aqueous phase did have a measurable effect on the "day one" in vitro PBS release. The formulation prepared using 50 mM disodium pamoate resulted in a 15% burst (Formulation AL, Table 7) as compared to less than 4% burst for formulations prepared with 20 mM organic ion (Formulations AJ, AK, Table 7). This suggests that excess organic ion in the aqueous phase is deleterious to the in vitro release performance of the formulations.
Table 7. The effect of organic ion concentration on the formation of octreotide-pamoate
(Table Removed)
Alternative organic ions in addition to pamoate were investigated to explore the general utility of the present invention. Microparticle formulations were prepared by an oil-in-water emulsion/solvent extraction method. PLGA polymer (MW 24,000,160 mg) was dissolved in EtOAc (1000 nL). Octreotide acetate (40 mg) was dissolved in BnOH (1000 fiL) and added to the polymer solution yielding a homogenous organic phase. The resulting organic phase was combined with a 1 % PVA aqueous phase containing 10-20 mM organic acid as its sodium salt to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration,

washed with water, air dried and stored at 4°C. This resulted in microparticle formulations with octreotide core loads between 6.8 and 15.3% as measured by RP-HPLC (Table 8). The effects of the tested organic ions on core load are revealing. Formulations AM - AP show no increase in the measured core load relative to control containing sodium pamoate (Formulations AT, AU, AY, Table 8). In contrast formulations AQ-AS, AV-AX and AZ-BB, which employed organic acids ranging from cholic acid to bicylic aromatics, provided peptide core loads comparable to pamoic acid (Table 8). These results imply that organic acids with appropriate physiochemical properties can be substituted for pamoic acid to produce comparable microparticle formulations.
Table 8. The effect of various organic acids (sodium salts) in the aqueous phase on the formation of octreotide-complex microparticles.

(Table Removed)

Example 4
Encapsulation of Additional Peptides in PLGA Microspheres Using Organic Acid Salts in the Aqueous Emulsion Phase According to the Present Invention.
Oxytocin acetate and leuprolide acetate were formulated in PLGA microparticles according to the present invention as described in the examples below. The results of these investigations demonstrate the utility of the present invention in relation to increased core load and encapsulation efficiency (Formulations Bl vs BJ-BK and BLvs BM) relative to conventional methodology (Table 9).
Formulation Bl (Leuprolide) - Conventional encapsulation method PLGA polymer (MW 24,000, 160 mg) was dissolved in CH2CI2 (1000 µL). Leuprolide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation Bl (140 mg, 70.0% yield) with a median particle size 50.1 µm. The core load (1.99%), encapsulation efficiency (9.95%) and in vitro burst (1.63%) were determined by RP-HPLC assay.
Formulation BJ (Leuprolide) - Organic ion assisted encapsulation method PLGA polymer (MW 24,000, 160 mg) was dissolved in CH2CI2 (1000 µL). Leuprolide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (100 mL) and stirred for 10 minutes. A secondary extraction solution consisting of 2% isopropanol (200 mL) was added and stirred for an additional four hours. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BJ (157 mg, 78.5% yield) with a median particle size 54.0 µm. The core load (9.4%), encapsulation efficiency (47.0%) and in vitro burst (5.31%) were determined by RP-HPLC assay.
Formulation BK (Leuprolide) - Organic ion assisted method
A microparticle formulation was prepared by an oil-in-water emulsion/solvent extraction method. PLGA polymer (MW 24,000,160 mg) was dissolved in CH2CI2 (1000 µL). Leuprolide acetate (40 mg) was dissolved in BnOH (1000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 50 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (100 ml) and stirred for 10 minutes. A secondary extraction solution consisting of 2% isopropanol (200 ml) was added and stirred for an additional four hours. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BK (120 mg, 60.0% yield) with a median particle size 43.1 µm. The core load (10.6%), encapsulation efficiency (53.0%) and in vitro burst (21.1%) were determined by RP-HPLC assay.
Formulation BL (Oxytocin) - Conventional encapsulation method PLGA polymer (MW 13,000,180 mg) was dissolved in EtOAc (900 µL). Oxytocin acetate (20 mg) was dissolved in MeOH (100 µL) and added to the polymer solution yielding a milky suspension as the organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 5% EtOAc to provide an emulsion. The emulsion was collected directly into a 10 mM sodium phosphate (pH 8,0°C, 150 ml) solvent extraction solution and stirred for four hours while warming to room temperature to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BL (143 mg, 71.5% yield) with a median particle size 44.0 µm. The core load (1.67%), encapsulation efficiency (16.7%) and in vitro burst (46.3%) were determined by RP-HPLC assay.
Formulation BM (Oxytocin) - Organic ion assisted encapsulation method PLGA polymer (MW 24,000,180 mg) was dissolved in EtOAc (1800 µL). Oxytocin acetate (40 mg) was dissolved in MeOH (200 µL) and added to the polymer solution yielding a milky suspension as the organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (150 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BM (158 mg, 79.0% yield) with a median particle size
144 urn. The core load (8.9%), encapsulation efficiency (44.5%) and in vitro burst (21.1%) were determined by RP-HPLC assay.
Table 9. Peptide-pamoate complex microparticles by an in situ process.
(Table Removed)
Example 5
Insulin Encapsulation in PLGA Microparticles Using Organic Acid Salts in the Aqueous Emulsion Phase.
Sodium Dodecylsulfate Microparticle formulations were prepared using an oil-in-water emulsion/solvent extraction method. The organic phase consisted of PLGA polymer (MW 11,800,150 mg) and PEGylated-insulin (50 mg) dissolved in CH2CI2(2 mL). The aqueous phase consisted of 1% PVA and 14 mM SDS. The homogeneous organic and aqueous phases were combined in a ratio of 1:5 to produce an organic in aqueous phase emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (100 mL) and stirred for 10 minutes before adding 100 mL 2% IPA. The solvent extraction solution was then stirred for an additional 3 hours to extract CH2CI2. Hardened microparticles were collected by filtration, washed with water, air dried and stored at -20°C. The resulting microparticle had a core load of 21% (encapsulation efficiency 84%). These microparticles were characterized by a large in vitro burst of 50% at 24 h in PBS at 37°C.
Disodium Pamoate Microparticle formulations were prepared using an
oil-in-water emulsion/solvent extraction method. The organic phase consisted of PLGA polymer (MW 11,800, 75 mg) and PEGylated-insulin (25 mg) dissolved in CH2CI2 (1 mL). The aqueous phase consisted of 1% PVA and 10 mM disodium pamoate. The homogeneous organic and aqueous phases were combined in a ratio of 1:5 to produce an organic in aqueous phase emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (50 mL) and stirred for 10 minutes before adding water (100 mL). The solvent extraction solution was then stirred for an additional 3
hours to extract CH2CI2. Hardened microparticles were collected by filtration, washed with water, air dried and stored at -20°C. The resulting microparticles had a core load of 18% (encapsulation efficiency 78%) and a final PEGylated-insulin/pamoate ratio of 1:2. In contrast to the microparticles made with SDS, these microparticles had a low in vitro burst of 5% in PBS at 37°C.
Example 6
Evaluation of the Pharmacokinetics of Octreotide in PLGA Microparticles after Administration to Sprague Dawley Rats.
Blood serum levels were measured for octreotide released from PLGA microparticle formulations injected subcutaneously in rats. Animals (n=6/group) were treated once by subcutaneous injection of a single dose level (~8-10 mg/kg) of six different octreotide PLGA microparticle formulations. At hours 1 and 6, and on days 1, 4, 7,11,14, 20, 28,42 and 54, serum samples were obtained from each animal to evaluate the octreotide pharmacokinetics. Serum concentrations were measured by a commercially available extraction-free radioimmunoassay kit (#8-2211) (Peninsula Labs). The Limit of Quantitation (LOQ) of the assay was 0.1 ng/mL. The mean octreotide serum concentrations for each time point are reported in Table 10. The preparation of the octreotide PLGA formulations tested is described below.
Table 10. Mean octreotide serum levels (ng/mL) after a single subcutaneous treatment in rats.
(Table Removed)
Preparation and characterization of octreotide formulations used in the animal study.
Formulation BC
PLGA polymer (MW 24,000, 720 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (80 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BC (754 mg, 94% yield) with a median particle size 55.0 µm. The core load (8.5%), encapsulation efficiency (85.0%) and in vitro burst (7.4%) were determined by RP-HPLC assay.
Formulation BD
PLGA polymer (MW 24,000, 680 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (120 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BD (694 mg, 94% yield) with a median particle size 58.7 jam. The core load (11.8%), encapsulation efficiency (78.7%) and in vitro burst (4.1%) were determined by RP-HPLC assay.
Formulation BE
PLGA polymer (MW 24,000, 680 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (120 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BE (727 mg, 91% yield) with a median particle size 52.2 µm. The core load (11.6%), encapsulation efficiency (77.3%) and in vitro burst (2.75%) were determined by RP-HPLC assay.
Formulation BF
PLGA polymer (MW 24,000, 640 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (160 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 mL) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BF (766 mg, 95.8% yield) with a median particle size 47.7 µm. The core load (14.7%), encapsulation efficiency (73.5%) and in vitro burst (5.5%) were determined by RP-HPLC assay.
Formulation BG
PLGA polymer (MW 28,000, 640 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (160 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BG (715 mg, 89.3% yield) with a median particle size 48.7µm. The core load (11.9%), encapsulation efficiency (59.5%) and in vitro burst (2.3%) were determined by RP-HPLC assay.
Formulation BH
PLGA polymer (MW 14,000, 560 mg) was dissolved in EtOAc (4000 µL). Octreotide acetate (240 mg) was dissolved in BnOH (4000 µL) and added to the polymer solution yielding a homogeneous organic phase. The resulting organic phase was combined with a 1% PVA aqueous phase containing 10 mM disodium pamoate to provide an emulsion. The emulsion was collected directly into a 0.3% PVA solvent extraction solution (600 ml) and stirred for four hours to extract EtOAc. Hardened microparticles were collected by filtration, washed with water, air dried and stored at 4°C. This provided formulation BH (680 mg, 85.0% yield) with a median particle size 40.6 ^im. The core load (17.4%), encapsulation efficiency (58.0%) and in vitro burst (6.8%) were determined by RP-HPLC assay.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

WE CLAIM:
1. A method of preparing a controlled release composition, said method comprises
the steps of:
a) combining a bioactive agent and a polymer with an organic solvent to
obtain a homogeneous organic phase, wherein the bioactive agent is
selected from the group comprising of proteins, peptides, LHRH agonists
and synthetic analogs thereof, leuprolide, oxytocin, somatostatin and
synthetic analogs thereof and hormones;
b) combining an organic ion and water to obtain an aqueous phase, wherein
the organic ion is selected from the group consisting of pamoate,
trifluoromethyl-p-toluate, cholate, 2-naphthalene sulfonate, 2,3-
naphthalene dicarboxylate, I-hydroxy-2-naphthoate, 3-hydroxy-2-
naphthoate, 2-naphthoate, ana salicylsalicylate;
c) contacti~g the homogeneous organic phase and the aqueous phase through
the use of an emulsion process so that the molar stoichiometry of the
bioactive agent relative to the organic ion ranges from 0.5 to 2.0; and
d) recovering said controlled release composition in a manner known per se
having diameter between 20.0 nm to 1.0 mm.
2. The method as claimed in claim 1, wherein the bioactive agent is octreotide,
octreotide acetate and pharmaceutical equivalents thereof.
3. The method as claimed in claim 1, wherein in step (a) the said polymer used is
selected from the group consisting of poly(lactide )s, poly(glycolide )s,
poly(lactide-co-glycolide )s, poly(lactic acid)s, poly(glycolic acid)s, poly(lactic
acid-eo-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides,
polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls,
. polycyanoacrylates, polyetheresters, poly( dioxanone )s, poly( alkylene alkylate ),
copolymers of polyethylene glycol and polyorthoester, qiodegradable
polyurethanes, blends and copolymers thereof.
4. The method as claimed in claim 1, wherein the organic solvent is selected from
th'e group consisting of methylene chloride, ethyl acetate, benzyl alcohol, acetone,
acetic acid, propylene carbonate.
5. The method as claimed in claim 1, wherein the organic phase optionally
comprises a cosolvent.
6. The method as claimed in claim 5, wherein the cosolvent is selected from the
group consisting of methyl alcohol, ethyl alcohol, isopropyl alcohol, N-methyl
pyrrolidinone, dimethyl sulfoxide, N ,N-dimethyl formamide, PEG200, PEG400, and
benzyl alcohol.
7. The method as claimed in claim 1, wherein the stoichiometry of the bioactive
agent relative to the organic ion ranges from 1.0 to 1.5.
8. The method as claimed in claim 1, wherein the aqueous phase optionally
comprises an emulsifying agent.
9. The m.ethod as claimed in claim 8, wherein the emulsifying agent is selected from
the group consisting of poly (vinyl alcohol), albumin, lecithin, vitamin (Equation Removed) E TPGS and polysorbates.
10. The method as claimed in claim 1, wherein. in step (d) the controlled release
composition is recovered by removing organic solvent from the emulsion and
ontain a droplet.
11. The method as claimed in claim 1, wherein the said controlled release
composition is selected from the group consisting of microparticles and
nanoparticles.
12. The method as claimed in claim 11, wherein the controlled release micro-particle
composition have diameter between 1.0 to 200.0 microns.
13. The method as claimed in claim 11, wherein the controlled release nanoparticle
composition have diameter between 20.0 nanometers to 2.0 microns.
14. The method as claim~d in claim 11, wherein the controlled release microparticle
composition and the controlled release nano-particle composition are
biodegradable.
15. A method of preparing a controlled release microparticle composition
substantially as herein describe with reference to forgoing examples and
drawings.

Documents:

957-DELNP-2006-Abstract-(27-03-2009).pdf

957-DELNP-2006-Abstract.pdf

957-DELNP-2006-Claims-(01-11-2010).pdf

957-DELNP-2006-Claims-(27-03-2009).pdf

957-delnp-2006-claims.pdf

957-delnp-2006-Correspondence Others-(10-07-2014).pdf

957-DELNP-2006-Correspondence-301014.pdf

957-delnp-2006-correspondence-others 1.pdf

957-delnp-2006-Correspondence-Others-(02-07-2013).pdf

957-DELNP-2006-Correspondence-Others-(08-12-2010).pdf

957-DELNP-2006-Correspondence-Others-(09-12-2010).pdf

957-DELNP-2006-Correspondence-Others-(15-04-2009).pdf

957-DELNP-2006-Correspondence-Others-(27-03-2009).pdf

957-delnp-2006-correspondence-others.pdf

957-DELNP-2006-Description (Complete)-(01-11-2010).pdf

957-DELNP-2006-Description (Complete)-(27-03-2009).pdf

957-delnp-2006-description (complete).pdf

957-delnp-2006-Form-1-(02-07-2013).pdf

957-DELNP-2006-Form-1-(09-12-2010).pdf

957-DELNP-2006-Form-1-(27-03-2009).pdf

957-delnp-2006-form-1.pdf

957-delnp-2006-form-13-(27-03-2009).pdf

957-delnp-2006-form-18.pdf

957-DELNP-2006-Form-2-(27-03-2009).pdf

957-delnp-2006-form-2.pdf

957-DELNP-2006-Form-26-(27-03-2009).pdf

957-delnp-2006-form-26.pdf

957-DELNP-2006-Form-3-(27-03-2009).pdf

957-delnp-2006-form-3.pdf

957-delnp-2006-form-5.pdf

957-DELNP-2006-GPA-(09-12-2010).pdf

957-delnp-2006-pct-101.pdf

957-delnp-2006-pct-105.pdf

957-delnp-2006-pct-210.pdf

957-delnp-2006-pct-220.pdf

957-delnp-2006-pct-237.pdf

957-delnp-2006-pct-301.pdf

957-delnp-2006-pct-304.pdf

957-delnp-2006-pct-306.pdf

957-delnp-2006-pct-308.pdf

957-DELNP-2006-Petition-137-(27-03-2009).pdf

957-DELNP-2006-Petition-138-(27-03-2009).pdf

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Patent Number

264719

Indian Patent Application Number

957/DELNP/2006

PG Journal Number

04/2015

Publication Date

23-Jan-2015

Grant Date

17-Jan-2015

Date of Filing

23-Feb-2006

Name of Patentee

PR PHARMACEUTICALS, INC.

Applicant Address

1716 HEATH PARKWAY, FORT COLLINS, CO 80522, USA

Inventors:

#	Inventor's Name	Inventor's Address
1	COOK, GARY, P.	1426 S. BOWEN ST., LONGMONT, CO 80501, USA

PCT International Classification Number

A61K 9/14

PCT International Application Number

PCT/US2004/022817

PCT International Filing date

2004-07-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/489,402	2003-07-23	U.S.A.