HK1108638B - Pharmaceutical compositions for controlled release delivery of biologically active compounds - Google Patents

Description

Pharmaceutical compositions for controlled release delivery of biologically active compounds

This application claims priority from U.S. provisional patent application serial No. 60/600,907 filed on 12.8.2004.

1. Field of the invention

The present invention relates to the field of controlled release delivery of biologically active compounds and to compositions and methods for controlled release delivery of biologically active compounds containing at least one basic group.

2. Background of the invention

The ability to deliver biologically active compounds in a controlled manner over a period of time has been a challenge. Controlled release delivery of bioactive compounds can improve bioavailability by protecting them from degradation in vivo, and at the same time replace multiple injections or continuous infusions, which are necessary because of the short half-life of these bioactive compounds. Reducing the frequency of administration can improve patient compliance. Biodegradable Polymers have been used as Drug carriers in implant devices for over thirty years [ Langer, R. and Chasin, M. (Eds.) Polymers as Drug Delivery Systems, Marcel Dekker, New York, NY, 1990 ]. The advantage of using biodegradable polymers as sustained delivery vehicles for bioactive compounds is that they do not need to be removed after delivery of their dose, since they are hydrolyzed to soluble, non-toxic oligomers or monomers. The rate of biodegradation depends on the physicochemical properties of the polymer, including crystallinity, hydrophobicity, chemical structure, molecular weight, and molecular weight distribution. In theory, these features can be designed or tailored to develop a drug delivery system in a controlled release manner over the desired duration of treatment.

The prior art has described a number of bioactive compounds in combination with biodegradable polymers to achieve extended release under physiological conditions by using appropriate polymers. The biologically active compound in the prior art compositions may be in the form of an uncharged molecule, molecular complex, salt, ether, ester or amide [ US 6,528,080, 5,739,176, 5,077,049 and US 4,938,763 ]. Specific examples of salts for injectable or implantable compositions include acetate, chloride, citrate, maleate, phosphate, succinate, sulfate, tartrate, and the like. However, the success of such formulations is limited to some biologically active compounds that are stable and have a wide therapeutic blood concentration range, e.g., leuprolide, gosorelin, and rhGH. Successful development of a controlled release delivery system for such biologically active compounds is challenging if the biologically active compound contains active functional groups and has a limited therapeutic blood concentration range. This is mainly due to the instability of the bioactive compound in the delivery system and the non-controllable release pattern of the bioactive compound from the delivery system, e.g. burst effects at the beginning, middle and later stages of release. Some bioactive compounds containing basic groups (including primary, secondary and tertiary amines) can pose serious obstacles to the successful development of controlled release delivery systems using biodegradable polymers. The compounds may alter (or catalyze) the hydrolysis process of the polymeric support in an uncontrolled manner and/or react with the polymers or their degradation products to form unwanted amide drug derivatives. The formation of these derivatives not only reduces the actual delivered dose, but can also cause unexpected side effects. The interaction/reaction between the biologically active compound and the polymeric carrier may occur in: 1) in the formulation process, the biologically active compound is incorporated into a polymeric carrier, such as by microencapsulation, injection molding, extrusion molding, mixing with a polymer solution in an organic solvent, and the like; 2) during storage, and 3) during biodegradation and during in vivo release of the biologically active compound.

In the formation of fine particles using a solvent evaporation/extraction method, an interaction/reaction between a bioactive compound containing a basic functional group (i.e., an amine group) and a polymer is reported, and during the formation of fine particles, the bioactive compound and the polymer are dissolved/dispersed in an organic solvent [ Krishnan m. and Flanagan dr., J Control release.2000 Nov 3; 69(2): 273-81]. A significant amount of amide moieties are formed. It is clearly shown that the solvents commonly used for the preparation of biodegradable polymeric drug delivery systems can allow for a fast reaction between the bioactive compound and the polymer. In a further study, accelerated degradation of polymers by organic amines was reported [ Lin WJ, Flanagan DR, lindardt rj. pharm res.1994 Jul; 11(7): 1030-4.]. It has also been reported that degradation of a polymer matrix containing simple drug salts such as epirubicin hydrochloride was found to promote degradation of the polymer and subsequently affect the release behavior from these microparticles [ Birnbaum DT, Brannon-Peppas l. molecular weight distribution changes during degradation and release of PLGA nanoparticles stabilizing excipient hcl. j Biomater Sci polymer ed.2003; 14(1): 87-102]. Domb et al report that drugs containing active amines and their salts also promote the degradation of biodegradable polymers in aqueous in vitro degradation media [ Domb AJ, Turovsky L, Nudelman r., Pharm res.1994 Jun; 11(6): 865-8]. For controlled release delivery of biologically active compounds for extended periods of time, both the reaction and the catalyzed degradation are undesirable.

When biodegradable polymers such as polylactic acid, polyglycolic acid, polyhydroxybutyric acid, polyorthoesters, polyacetals, etc. are used as drug delivery systems, biodegradation of the polymers (such as, for example, polylactide and polylactide-co-glycolide) causes water absorption and the creation of aqueous channels or pores from which biologically active compounds can leak (or diffuse) if they become water soluble. In addition, accumulation of polymer degradation products lowers the pH within the degrading polymer matrix, and local pH values between 1.5 and 4.7 have recently been reported (Na DH, Young YS, Lee SD, Son MO, Kim WA, Deluca PP, Lee KC. monitoring of peptide adsorption induced PLGA microspheres by capillary chromatography. J Control Release.2003 Oct 30; 92 (3): 291-9; and references cited therein). The acidic microenvironment inside the polymer matrix may induce some unwanted chemical degradation reactions, particularly for biologically active compounds containing reactive amine groups, such as peptides and proteins.

In the literature [ Schwendeman SP., Recent advances in the stabilization of protein encapsulated in injectable PLGA delivery systems. crit Rev the rDRUG Carrier System.2002; l9 (1): 73-98; sinha VR, Trehan a., biodegradableprospheers for protein delivery.j Control release.2003 Jul 31; 90(3): 261-80] further examples of prior art relating to instability or reaction/interaction of bioactive compounds and polymers during formulation, storage and in vivo release are reviewed, and are fully incorporated herein by reference.

Some organic acids, such as acetic acid, citric acid, benzoic acid, succinic acid, tartaric acid, heparin, ascorbic acid and non-toxic salts thereof, have been described in the prior art and are used as polymer degradation enhancers in a number of controlled release biodegradable systems. (PCT-patent application WO93/17668 (page 14, lines 4-13) and U.S. Pat. No. 4,675,189) (column 11, lines 5-19). Therefore, such acid additives are not expected to stabilize the polymer.

Various other approaches have been investigated, and successful controlled release delivery of biologically active compounds containing an active basic group has been obtained. However, despite the tremendous research efforts to try, only a small number of products for controlled release delivery of biologically active compounds are commercially available to date. See, e.g., US Pat. nos.4,728,721(Leuprolide, Lupron Depot); 4,938,763(Leuprolide, Eligard); 5,225,205(Triptorelin Pamoate, Trelstar); 4,767,628(Goserelin Acetate, Zoladex); 5,538,739(Octreotide, SANDOSTATIN LAR); 5,654,010(recombinant humangrowth hormone, Nutropin Depot); 4,675,189; 5,480,656; 4,728,721].

Clearly, there is a need for the development of new and suitable delivery systems that stabilize biologically active compounds, control degradation of polymers, limit burst effects, and maintain release of drug within the therapeutic limits over the duration of treatment. It is therefore an object of the present invention to address the above-listed deficiencies in the prior art and to provide a pharmaceutical composition for controlled release delivery of a biologically active compound to a subject comprising:

a) a complex of a biologically active compound having at least one basic functional group and a polyanion derived from hexahydroxycyclohexane, the polyanion having at least two negatively charged functional groups; and

b) a pharmaceutically acceptable carrier comprising a biodegradable, water-insoluble polymer.

The invention also provides methods for producing such controlled release pharmaceutical compositions and methods of using the same.

Summary of The Invention

The present invention provides compositions and methods for controlled release delivery of one or more bioactive compounds to a subject. Specifically, a pharmaceutical composition for controlled release delivery of a biologically active compound to a subject, comprising: a) a complex of a biologically active compound having at least one basic functional group and a polyanion derived from hexahydroxycyclohexane, the polyanion having at least two negatively charged functional groups; and b) a pharmaceutically acceptable carrier comprising a biodegradable, water-insoluble polymer. By complexing the biologically active compound with the polyanion, a robust, stable complex can be incorporated into a long acting drug delivery system that has a low initial burst release and a more desirable drug release-time profile than those found in many of the prior art.

It has surprisingly been found that the polyanions of the present invention can reduce or prevent the interaction/reaction between a biologically active compound containing a basic group and a polymer or degradation products thereof by forming a stable complex. The complexes may have low solubility in water or biological fluids. Preferably, the complex also has low solubility in the solvents used to prepare the dosage form. By reducing or preventing interactions/reactions between the bioactive compound and the polymer and/or its degradation products, these properties can stabilize the bioactive compound and slow down the degradation of the polymer not only during formulation, but also during release. More importantly, these properties can lead to the delivery of bioactive compounds from biodegradable polymeric carriers in highly desirable release patterns. It may allow for the sustained delivery of a biologically active compound to a subject over an extended period of time, e.g., from weeks to months, to benefit the subject.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for controlled release delivery of a biologically active compound to a subject comprising: a) a complex of a biologically active compound having at least one basic functional group and a polyanion derived from hexahydroxycyclohexane, the polyanion having at least two negatively charged functional groups; and b) a pharmaceutically acceptable carrier comprising a biodegradable, water-insoluble polymer.

It is another object of the present invention to provide a group of biologically active compounds containing at least one basic functional group which may benefit from a sustained controlled release delivery system.

It is another object of the present invention to provide a group of polyanions which can form stable complexes with biologically active compounds.

It is another object of the present invention to provide a process for preparing a complex between a biologically active compound of the present invention and a polyanion.

It is another object of the present invention to provide a complex that reduces or prevents unwanted degradation of the polymer by the bioactive compound, not only during formulation and storage, but also during polymer degradation and drug release in vivo.

It is another object of the present invention to provide a complex that stabilizes biologically active compounds not only during formulation and storage, but also during polymer degradation and drug release in vivo.

It is another object of the present invention to provide a pharmaceutically acceptable carrier comprising a biodegradable water-insoluble polymer having dispersed therein a bioactive compound/polyanion complex exhibiting sustained release of the bioactive compound.

It is another object of the present invention to provide a pharmaceutical composition having a biologically active compound/polyanion complex incorporated therein which can release the biologically active compound which retains its biological activity.

It is another object of the present invention to provide a pharmaceutical composition for medical applications such as drug delivery, vaccination, gene therapy, etc.

It is another object of the present invention to provide a pharmaceutical composition suitable for oral or parenteral administration; mucosal administration; ophthalmic administration; subcutaneous, intra-articular, or intramuscular injection; administration by inhalation; and topical application.

These and other objects of the present invention will become apparent upon reading the following detailed description of the disclosed embodiments.

Detailed Description

The present invention relates to a pharmaceutical composition for controlled release delivery of a biologically active compound to a subject, comprising: a) a complex of a biologically active compound having at least one basic functional group and a polyanion derived from hexahydroxycyclohexane, the polyanion having at least two negatively charged functional groups; and b) a pharmaceutically acceptable carrier comprising a biodegradable, water-insoluble polymer, and to methods of making and using such compositions. The compositions of the present invention may be prepared by methods known in the art in any conventional pharmaceutical administration form. Non-limiting examples of compositions of the present invention are solutions, suspensions, dispersions, emulsions, drops, aerosols, pastes, semisolids, pastes, capsules, tablets, solid implants, or microparticles. Advantages of the pharmaceutical compositions of the present invention include low burst and stable controlled release of the biologically active compound in vivo. It may allow for the delivery of bioactive compounds to a subject over an extended period of time, e.g., from days to months.

The terms "a", "an" and "an" when used herein are intended to be interpreted as "one or more" and "at least one".

The term "biologically active compound" is intended to include any substance having diagnostic and/or therapeutic properties including, but not limited to, small molecules, macromolecules, peptides, proteins, or enzymes. Non-limiting examples of therapeutic properties are antimetabolite, antifungal, anti-inflammatory, antineoplastic, anti-infective, antibiotic, nutritional, agonist and antagonist properties.

More specifically, the biologically active compound of the present invention may be any compound capable of forming a complex with a polyanion derived from hexahydroxycyclohexane, in particular a compound containing an electron donor basic group such as a basic nitrogen atom, for example, an amine, imine or ring nitrogen. The biologically active compound preferably comprises oneOne or more exposed protonatable amine functional groups, with a plurality of such groups being particularly preferred. Bioactive compounds useful in preparing the stabilized complexes of the invention include, but are not limited to, doxorubicin, doxycycline, diltiazemCyclobenzaprine, bacitracin, noscapine, erythromycin, polymyxin, vancomycin, nortriptyline, quinidine, ergotamine, benztropine, verapamil, flunarizine, imipramine, gentamicin, kanamycin, neomycin, amoxicillin, amikacin, arbekacin, bambenomycin, gatifloxacin, dibekacin, dihydrostreptomycin, fotiamycin, isepamicin, microscins (micronomicin), netilmicin, paromycin, ribomycin, rapamycin, sisomicin, streptomycin and tobramycin, amikacin, neomycin, streptomycin and tobramycin, pyrimethamine, naltrexone, lidocaine, prilocaine, mepivacaine, bupivacaine, ropivacaine, oxidized toxins, vasopressin, adrenocorticotropic hormone (ACTH), Epidermal Growth Factor (EGF), platelet-derived growth factor (PDGF), Prolactin, Luteinizing Hormone Releasing Hormone (LHRH), LHRH agonists, LHRH antagonists, auxins (including human, porcine and bovine), ghrelin, insulin, erythropoietin (including all proteins having erythropoietic activity), somatostatin, glucagon, interleukins, interferon-alpha, interferon-beta, interferon-gamma, gastrin, tetrapeptide gastrin, pentagastrin, urogastrin, secretin, calcitonin, enkephalin, endorphin, angiotensin, Thyrotropin Releasing Hormone (TRH), Tumor Necrosis Factor (TNF), parathyroid hormone (PTH), Nerve Growth Factor (NGF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-S), Heparinase, vascular endothelial growth factor (VEG-F), Bone Morphogenetic Protein (BMP), hANP, glucagon-like peptide (GLP-1), exenatide, peptide YY (PYY), renin, bradykinin, bacitracin, streptothricin, polymyxinE. Brevibacillin casein, Brevibacillin peptide, cyclosporine (including synthetic analogs and pharmaceutically active fragments thereof), enzymes, cytokines, antibodies, vaccines, antibiotics, antibodies, glycoproteins, follicle stimulating hormone, Goltoprofen, taftsin, thymopoietin, thymosin, thymidysin, thymic humoral factor, serum thymic factor, colony stimulating factor, motilin, bombesin, dinorphin, neurotensin, cerulein, urokinase, kallikrein, substance P analogs and antagonists, antagonist II, coagulation factors VII and IX, lysozyme, brevibacillin, melanotropin, thyroid hormone releasing hormone, thyroid hormone, thyrotropin, pancreatin, cholecystokinin, human placental lactogen, human chorionic gonadotropin, protein synthesis stimulating peptides, enterogastric inhibitory peptides, vasoactive intestinal peptides, platelet derived growth factors, and synthetic analogs and modified and pharmaceutically active fragments thereof.

As used herein, the term "polyanion" is intended to include any molecule containing at least 2 or more negatively charged functional groups. The polyanions of the present invention are derived from hexahydroxycyclohexane, which is formed by esterification with a phosphoric or sulfuric acid group capable of forming a stable complex with the biologically active compound. Myo-inositol is one of nine known cis isomers of hexahydroxycyclohexane, a 6-carbocyclic structure that is abundant in plants and animals. For example, phytic acid (InP6, phytic acid) is a natural dietary ingredient and makes up 0.4-6.4% (w/w) of most grains, legumes, nuts, oilseeds and soybeans. The expanded evidence population indicates that many, if not all, mammalian cells contain inositol polyphosphates with 5 or more phosphate groups. For example, InP6 is found in most mammalian cells where it can help to regulate a variety of important cellular functions. It is also shown that InP6 acts as an antioxidant, preventing the generation of reactive oxygen species responsible for cell damage and carcinogenesis by chelating with multivalent cations such as copper and iron. Some other examples of inositol polyanions include, but are not limited to, lower inositol phosphates, (i.e., inositol pentaphosphates, inositol tetraphosphates, inositol triphosphates, inositol diphosphates), and other polyphosphorated organic compounds, inositol hexasulfate (InS6) and lower inositol sulfate. The polyanion may be present as an acid or in the form of a salt.

Polyanions of at least two or more negatively charged groups are particularly preferred, in particular inositol hexaphosphate (InP6, phytic acid) and inositol hexasulfate (InS 6).

The term "stable complex" means physically and chemically stable complexes that are formed when the biologically active compound and the polyanion are properly combined under conditions such that a stable complex is formed, for example, by mixing an aqueous solution of the biologically active compound and the polyanion until the complex is formed. The complex may be in solid form (e.g., a paste, granules, powder, or freeze-dried agent), or the complex in powder form may be ground to a sufficient fineness so as to be uniformly dispersed in the biodegradable polymer carrier. Such complexes typically take the form of a precipitate that is produced when an aqueous formulation of the biologically active compound and the polyanion is combined. Optionally, one or more pharmaceutically acceptable excipients may be incorporated into the complex. Such excipients may act as stabilizers for the biologically active compound or complex thereof. Non-limiting examples include sodium bisulfite, p-aminobenzoic acid, thiourea, glycine, methionine, mannitol, sucrose, polyethylene glycol (PEG), and the like.

By way of example, a soluble antibiotic (e.g., doxorubicin) may be dissolved in water, and a solution of InP6 may be added thereto. Medicine preparation: the InP6 complex precipitates out. The precipitate may be washed and then separated by centrifugation or filtration. The isolated complex was dried in vacuo.

As another example, an aqueous solution of InP6 may be added to a solution of a local anesthetic (e.g., tetracaine hydrochloride). Medicine preparation: the InP6 complex precipitates out.

As another example, an aqueous solution of InP6 may be added to a peptide (e.g., glucagon-like peptide 1(GLP-1)) solution. Peptide: the InP6 complex precipitates out. The precipitate may be washed and then separated by centrifugation or filtration. The isolated complex was dried in vacuo.

As another example, an aqueous solution of InP6 may be added to an enzyme (e.g., lysozyme) solution. Enzyme: the InP6 complex precipitates out. The precipitate may be washed and then separated by centrifugation or filtration. The isolated complex was dried in vacuo.

The stabilized complexes of the biologically active compounds and polyanions of the present invention may optionally be incorporated with some excipients into a pharmaceutically acceptable carrier comprising a biodegradable water-insoluble polymer. The term "biodegradable water-insoluble polymer" is intended to include any biocompatible and/or biodegradable synthetic and natural polymer that can be used in vivo. "biodegradable water-insoluble polymer" is also intended to include polymers that are insoluble or become insoluble in water or biological fluids at 37 ℃. The polymer may be purified, optionally to remove monomers and oligomers, using techniques known in the art (e.g., U.S. patent 4,728,721). Some non-limiting examples of such polymers are polylactides, polyglycolides, poly (lactide-co-glycolide), polycaprolactones, polydioxanones, polycarbonates, polyhydroxybutyrates, polyalkylene oxalates, polyanhydrides, polyamides, polyesteramides, polyurethanes, polyacetals, polyorthocarbonates, polyphosphazenes, polyhydroxyvalerates, polyalkylene succinates, and polyorthoesters, and copolymers, block copolymers, branched copolymers, terpolymers, and combinations and mixtures thereof.

In addition, the biodegradable water-insoluble polymer may comprise end-capped, end-uncapped, or a mixture of end-capped and end-uncapped polymers. End-capped polymers are generally defined as having a capped carboxyl end group. The uncapped polymers have, in particular, free carboxyl end groups, as classically defined in the art.

The appropriate molecular weight of the polymer can be determined by one of ordinary skill in the art. Factors that may be considered when determining the molecular weight include the desired rate of polymer degradation, mechanical strength, and the rate of dissolution of the polymer in the solvent. Typically, a suitable range of polymers will have a molecular weight of from about 2,000 daltons to about 150,000 daltons, with a polydispersity of 1.1-2.8, depending on which polymer is selected, among other factors.

As used herein, the term "pharmaceutically acceptable carrier" is intended to include any carrier, injectable solution or suspension, particle, thin film, pellet, cylinder, disk, microcapsule, microsphere, nanosphere, microparticle, wafer, micelle, liposome, and other known polymeric configurations for drug delivery, having environmental response characteristics (e.g., thermosensitive, pH sensitive, electro-sensitive, etc.).

Methods for formulating various pharmaceutically acceptable polymeric carriers are well known in the art. For example, in U.S. patents: 6,410,044, respectively; 5,698,213, respectively; 6,312,679, respectively; 5,410,016; 5.529,914, respectively; 5,501,863, respectively; and PCT publication No. WO 93/16687; 4.938,763, respectively; 5,278,201; 5,278,202; a number of methods and materials are described in EP 0,058,481, which is incorporated herein by reference in its entirety.

In accordance with the present invention, a composition can be generated when the bioactive compound/polyanion complex is dispersed in a polymer matrix to form a solid graft that can be injected or transplanted into a subject. These implants may be prepared from the bioactive compound/polyanion complexes of the present invention, optionally with pharmaceutically acceptable excipients, using conventional polymer melt-processing techniques such as, but not limited to, extrusion, compression and injection molding, wherein high temperatures (preferably below 100 ℃) are used to melt the polymer matrix in the preparation of the implant. Preparation of such implants may be performed under sterile conditions, or alternatively may be terminally sterilized by irradiation using, but not limited to, gamma or electron beam sterilization.

According to one embodiment of the invention, a homogeneous mixture of the biologically active compound/polyanion complex and polymer may be prepared by dry mixing in any suitable apparatus, such as in a ball mill, and at room temperature or even at lower temperatures, such as < 10 ℃. The proportion of the powder constituents can vary within wide limits, for example from 0.1 to 30% by weight for the biologically active compound, depending on the desired therapeutic effect. A homogeneous mixture of the bioactive compound/polyanion complex and polymer may also be prepared by dispersing the complex in a solution of the polymer in an organic solvent, followed by removal of the organic solvent by evaporation or lyophilization. The resulting solid can be ground into a finely divided powder.

Once the given mixture is sufficiently homogenized, it can be molded according to the present invention using techniques known in the art. For example, it may be progressively compressed by progressively heating it prior to moulding. The compression ratio may vary depending on various factors, such as the geometry of the instrument or the particle size of the powder mixture. The control of the preheating and the control of the variation to which it is subjected are more important when the mixing is carried out: depending on the nature of the product to be treated (copolymer, biologically active compound), every effort is made to maintain a temperature gradient of not more than about 100 ℃. The initial temperature to which the powder mixture is subjected may be 25 ℃, and may be lower or higher depending on the circumstances.

The mould temperature should be kept as low as possible, preferably not exceeding 100 ℃, and the upper limit of the temperature is determined by the nature of the biologically active compound, which should not undergo deterioration. Sufficient pressure and sufficient temperature promote the best homogenization of the ingredients, and in particular, the uniform distribution of the composite throughout the copolymer entity can be readily determined by simple experimentation.

Alternatively, the homogenized powder may be compression molded at room temperature, similar to the preparation of FTIR beads.

In one embodiment of the invention, a copolymer of D, L-lactide and glycolide having a molar ratio of D, L-lactide to glycolide of 50/50 was dissolved in methylene chloride. To this solution tetracaine phytate was added and dispersed with a high shear mixer. The resulting mixture was placed in a rotary evaporator and most of the dichloromethane was removed under vacuum. The resulting thick dispersion was poured into a glass plate to form a film. The film thus obtained was melted and compression-molded into a film of about 0.5mm thickness.

Alternatively, the homogenous powder may be melted and extruded or injection molded into solid implants of different shapes known in the art, in accordance with the present invention. The actual extrusion can be performed by means of nozzles of standard shape and size. The cooling of the extrudate is achieved by any suitable means, such as cold sterile air or gas or simply by natural heat dissipation.

According to the present invention, these solid dosage forms, for example, fibers, rods, films or wafers, can be rendered into particulate form by crushing or grinding. The fully cooled extruded or moulded product as described above is then ground to a powder at low temperature, preferably at a temperature below 0 ℃, or even lower, e.g. -20 ℃. The product thus pulverized can then be screened to obtain the desired particle size. Preferred particle sizes may range from 1 □ m to 500 □ m, and these particulate delivery systems may be suspended in suitable conventional pharmaceutical injection vehicles.

According to another aspect of the invention, particularly effective and effective parenteral pharmaceutical formulations of biologically active compounds can also be prepared in pharmaceutically acceptable solvents in the form of a liquid or suspension of a polymer containing dispersed or dissolved drug/polyanion complexes. By complexing with polyanions, the reactive groups in the biologically active compound cannot interact with the polymer in solution. Thus, the stability of the biologically active compound in the composition of the invention is improved by complexing with the polyanion of the invention.

Thus, however, according to the present invention, there is provided a pharmaceutical composition comprising a biologically active compound complexed with a polyanion and a polymer for the long-term release of the biologically active compound, characterised in that the composition is in the form of an injectable solution/suspension comprising:

(a) a complex of a biologically active compound having at least one basic functional group and a hexahydroxycyclohexane derivative, the polyanion having at least two negatively charged functional groups; and

(b) a biodegradable, water-insoluble polymer;

(c) a pharmaceutically acceptable organic solvent which is a solvent for the polymer.

Suitable biologically active compounds and polyanions are those defined above, and particularly preferred polyanions are those containing at least 2 phosphate or sulphate groups as defined above, more preferably InP6 and InS 6.

The molar ratio of bioactive compound to polyanion in the complex varies from 0.1: 1 to 1: 0.1, depending on the nature of the bioactive compound and polyanion, and the desired time of peptide drug release.

Any suitable biodegradable polymer may be used, provided that the polymer is insoluble or becomes insoluble in aqueous media or body fluids at 37 ℃. Suitable biodegradable polymers are those defined above.

The type, molecular weight, and amount of biodegradable polymer present in the composition can affect the length of time the bioactive compound is released from the controlled release implant. The type, molecular weight, and amount of biodegradable polymer present in the composition can be selected by one of ordinary skill in the art in order to obtain the desired properties of the controlled release implant.

Suitable pharmaceutically acceptable organic solvents include, but are not limited to, N-methyl-2-pyrrolidone, N-dimethylformamide, dimethylsulfoxide, propylene carbonate, caprolactam, triacetin, benzyl benzoate, benzyl alcohol, ethyl lactate, triacetin, citrate esters, and polyethylene glycols, alkoxy polyethylene glycols, polyethylene glycol acetates, and the like, or any combination thereof.

The criteria for organic solvents for biodegradable polymers are that they are pharmaceutically acceptable and readily miscible for dispersion in aqueous media or body fluids. Suitable organic solvents can diffuse into the body fluid so that the liquid composition coagulates or coagulates in place to form the graft. Single and/or mixtures of such solvents may be used, and the suitability of such solvents can be readily determined by simple experimentation.

The pharmaceutical compositions of the present invention typically contain the biologically active compound in the range of 0.1-40% w/v. Generally, optimal drug loading depends on the desired release time and efficacy of the biologically active compound. Clearly, for biologically active compounds of low efficacy and longer release times, higher levels of binding may be required.

The viscosity of the solution composition of the present invention is determined by the molecular weight of the polymer and organic solvent used. For example, when poly (lactide-co-glycolide) is used, the polyester solution in NMP has a lower viscosity than in mPEG 350. Typically, the higher the molecular weight and concentration of the polymer, the higher the viscosity when the same solvent is used. Preferably, the concentration of polymer in the solution is less than 70% by weight. More preferably, the concentration of the polymer in the solution is between 20 and 50 wt.%.

Preferably, the complex should have low solubility in the organic solvent used. The active groups of the biologically active compound bind to the polyanion and are therefore not susceptible to interaction/reaction with the polymer or solvent. This greatly reduces the risk of adverse interactions/reactions with the polymer and its degradation products.

According to one embodiment of the invention, a simple salt, tetracaine chloride, was mixed with 50/50 poly (DL-lactide-co-glycolide) solution with carboxyl end groups in NMP. For in vitro studies, a mixture of droplets (about 100mg) was added to a phosphate buffered saline solution. The received fluid was replaced with fresh solution at selected time points and the removed PBS solution was analyzed for drug concentration using appropriate analytical methods.

According to another embodiment of the invention, tetracaine phytate is mixed with 50/50 poly (DL-lactide-co-glycolide) solution having carboxyl end groups in NMP. The drug complex is uniformly dispersed in the polymer solution. For in vitro studies, a mixture of droplets (about 100mg) was added to a phosphate buffered saline solution. The received fluid was replaced with fresh solution at selected time points and the removed PBS solution was analyzed for drug concentration using appropriate analytical methods.

According to another embodiment of the invention, ghrelin phytate and ghrelin acetate (octride acetate) are mixed with 50/50 poly (DL-lactide-co-glycolide) solution having carboxyl end groups in NMP and methoxypolyethylene glycol 350. The drug complex is uniformly dispersed in the polymer solution. The compositions were kept at room temperature and analyzed by HPLC to monitor the stability of the growth inhibitory peptide in the compositions over time. Complexation of the growth-inhibitory peptide with phytic acid significantly improves the stability of the growth-inhibitory peptide in the composition over time.

According to another embodiment of the invention, ghrelin phytate and ghrelin acetate (octride acetate) are mixed with 50/50 poly (DL-lactide-co-glycolide) solution having carboxyl end groups in NMP and methoxypolyethylene glycol 350. The drug complex is uniformly dispersed in the polymer solution. The composition was administered subcutaneously in Sprague-Dawley male rats to form grafts in place. The initial release of the aprotinin is determined by retrieving the graft at a predetermined time interval after administration and analyzing the aprotinin remaining in the graft. The stability of the growth-inhibitory peptide during formulation and release was also evaluated. Complexation of the aprotinin with phytic acid significantly reduces the initial release of the aprotinin and improves the stability of the aprotinin over time during release.

The release of bioactive compounds from these grafts formed in place will follow the same basic principles for drug release from a single polymeric device. The release of the biologically active compound may be affected by: the size and shape of the implant, the loading of the bioactive compound within the implant, the osmotic factors including the bioactive compound and the particular polymer, and the degradation of the polymer. Depending on the amount of biologically active compound selected for delivery, the above parameters can be adjusted by one skilled in the art of drug delivery to give the desired release rate and duration.

The amount of injectable solution composition administered will typically depend on the desired characteristics of the controlled release implant. For example, the amount of injectable solution composition used can affect the length of time that the biologically active compound is released from the controlled release implant.

According to another aspect of the invention, the composition in the form of microspheres is prepared by encapsulating the bioactive compound/polyanion complex in a polymeric carrier. Bioactive compound/polyanion complexes can be encapsulated using a variety of biocompatible and/or biodegradable polymers with unique properties suitable for delivery to different biological environments or for achieving specific functions. Thus, the rate of dissolution and delivery of the bioactive compound is determined by the particular encapsulation technique, polymer composition, polymer cross-linking, polymer consistency, polymer solubility, size and solubility of the bioactive compound/polyanion complex.

The bioactive compound/polyanion complex to be encapsulated is suspended in a polymer solution in an organic solvent. The polymer solutions must be sufficiently concentrated so that they are sufficient to completely coat the bioactive compound/polyanion complex when added to the solution. Such an amount is one that provides a weight ratio of bioactive compound/polyanion complex to polymer of between about 0.01 and about 50, preferably between about 0.1 and about 30. When they are coated by contact with the polymer, the bioactive compound/polyanion complex should remain suspended and not be allowed to aggregate.

Preferably, the complex should have very low solubility in the organic solvent used. The active groups of the biologically active compound should bind to the polyanion and therefore not readily interact with the polymer or solvent. This greatly reduces the risk of adverse interactions with the polymer.

Thus, the polymer solution of the bioactive compound/polyanion complex may be subjected to a variety of microencapsulation techniques including spray drying, spray congealing, emulsification, solvent evaporation emulsification.

According to one embodiment of the invention, the biologically active compound/polyanion complex is suspended in a polymer solution in an organic solvent. The suspended complex or microparticles are transferred with the polymer and organic solvent to a larger volume of aqueous solution containing an emulsifier. In the aqueous solution, the suspended complex is immersed in an aqueous phase where the organic solvent is volatilized or diffused from the polymer. The cured polymer encapsulates the bioactive compound/polyanion complex to form the composition. During the hardening phase of processing, emulsifiers help to reduce the interfacial surface tension between the phases of the material in the system. Alternatively, if the encapsulated polymer has some intrinsic surface activity, it may not be necessary to add a separate surfactant.

Emulsifiers useful in preparing the encapsulated bioactive compound/polyanion complexes according to the present invention include the poloxamers and polyvinyl alcohols exemplified herein, surfactants, and other surface active compounds that can reduce the surface tension between the polymer and the solution encapsulating the bioactive compound/polyanion complex.

Organic solvents useful in preparing microspheres of the present invention include acetic acid, acetone, methylene chloride, ethyl acetate, chloroform and other non-toxic solvents, which will depend on the characteristics of the polymer. Solvents should be selected that dissolve the polymer and are substantially (ultrastimate) non-toxic.

It is a preferred embodiment of the present invention that the integrity of the bioactive compound/polyanion complex is maintained during the encapsulation process. During the suspension process, the complexation is maintained by using an organic solvent in which the bioactive compound/polyanion complex has very low solubility. Subsequently, once the coated complex is transferred into an aqueous solvent, rapid hardening of the polymeric carrier and sufficient encapsulation of the bioactive compound/polyanion complex in the previous step prevents decomposition of the complex material.

The polymer used to encapsulate the bioactive compound/polyanion complex may be a homopolymer or a copolymer as described above.

In another embodiment, double-walled polymer coated microspheres may be advantageous. Double-walled polymer-coated microspheres can be prepared by preparing 2 separate polymer solutions in dichloromethane or other solvents that can dissolve the polymer. [ see Pekarek, k.j.; jacob, J.S. and Mathiewitz, E.double-walled polymer microspheres for controlled drug release, Nature, 1994, 367, 258-. The bioactive compound/polyanion complex is added to one of the above solutions and dispersed. Here, the bioactive compound/polyanion complex is coated with a first polymer. The solution containing the bioactive compound/polyanion complex coated with the first polymer is then combined with the solution of the second polymer. Now, the second polymer encapsulates the first polymer, which encapsulates the bioactive compound/polyanion complex. Ideally, this solution is then dropped into a larger volume of aqueous solution containing a surfactant or emulsifier. In the aqueous solution, the solvent is volatilized from the two polymer solutions, and the polymers precipitate to encapsulate the complex.

Although the formulations described above are primarily those intended for administration by the injectable or implantable route, the biologically active compound/polyanion complexes of the present invention may also be used in the preparation of oral, intranasal, or topical formulations.

Thus, in accordance with the present invention, compositions containing the bioactive compound/polyanion complexes can be administered to subjects in need of sustained controlled release delivery of the bioactive compound. As used herein, the term "subject" is intended to include a warm-blooded animal, preferably a mammal, more preferably a human.

As used herein, the term "administering to a subject" is intended to mean dispensing, delivering, or applying a composition (e.g., a pharmaceutical formulation) to a subject by any route suitable for delivering the composition to a location in need of the subject, including by oral administration, by nasal, by subcutaneous, intramuscular, intraperitoneal, intradermal, intravenous, intraarterial, or intrathecal injection and/or transplantation, by administration to the mucosa, or by in situ delivery, so as to provide the desired dose of the biologically active compound based on known parameters of treatment of various medical conditions with the biologically active compound.

As used herein, the term "controlled release delivery" is intended to mean continuous delivery of an agent in the body for a period of time after administration, preferably at least several days to weeks or months. Sustained controlled release delivery of an agent can be demonstrated, for example, by a continuous therapeutic effect of the agent over time (e.g., for GLP-1, sustained delivery of the peptide can be demonstrated by a continuous decrease in Alc over time). Alternatively, sustained delivery of the agent may be evidenced by detection of the presence of the agent in the body over time.

All books, documents, and patents referred to herein are incorporated by reference in their entirety.

Examples

The following examples illustrate the compositions and methods of the present invention. The following examples should not be considered as limiting, but merely teach how to prepare an effective drug delivery system.

EXAMPLE 1 preparation of Doxorubicin Phy-inositol hexaphosphate (DOX-PA)

A solution of doxorubicin hydrochloride (MW 578.98) at 2mg/mL in water (3.45mM) and a solution of dipotassium phytate (MW 736.22) at 20mg/mL in water (27.2mM) were prepared. While stirring the solution, 2.1mL of phytic acid solution was added to 100mL of doxorubicin hydrochloride solution. The desired ratio of phytic acid to doxorubicin is 1: 6. The mixture was centrifuged. The precipitate was washed 4 times with water and then lyophilized. The yield was 187mg (88.5%).

Solubility of doxorubicin phytic acid was determined in deionized water, phosphate buffered saline (PBS, pH 7.4), dimethyl sulfoxide (DMSO), Dimethylacetamide (DMAC), N-methyl-2-pyrrolidone (NMP), and methoxypolyethylene glycol 350 (mPEG). The results are shown in the following table:

solvent(s)	Solubility (□ g/mL)
		H2O	4.5
PBS(pH 7.4)	11.2
		DMSO	Soluble in water
DMAC	50
		NMP	50
mPEG	0

EXAMPLE 2 preparation of microspheres containing DOX-PA and DOX-HCl

121mg DOX-PA complex was dispersed in PLGA (DL 50503A, Alkermes) solution in Dichloromethane (DCM). The above organic phase was emulsified in 500mL of 1.0% (w/v) PVA solution pre-cooled in a refrigerator (. about.4 ℃). The emulsion was stirred at RT for a further 3h to volatilize DCM. The hardened microspheres were collected by decanting the supernatant, washed 3 times with deionized water, and then freeze-dried. Obtaining the reddish microspheres. The drug content in the microspheres was-5.1% as determined by HPLC.

Microspheres containing DOX-HCl were prepared by using DOX-HCl instead of DOX-PA, applying the same procedure described above.

Example 3 preparation of Encapsulated Doxorubicin Phy-hexaphosphate

The doxorubicin phytic acid prepared in example 1 was encapsulated in polylactide-co-glycolide (PLGA) using a double emulsification process. 1.4mg doxorubicin phytic acid was added to PLGA in methylene chloride (0.6g PLGA/ml solvent; 20 ml). The mixture was homogenized at 3,000rpm for 30sec using a homogenizer with a fine tip. The resulting suspension was transferred to a stirred tank (2000ml) containing 1% poly (vinyl alcohol) (PVA) and dichloromethane (4.5 ml). The solution was stirred at 1,000rpm for 1 min. The microspheres in the PVA solution were precipitated by immersion in distilled water, washed and filtered. The microspheres were then washed with distilled water containing 0.1% tween to reduce agglomeration and dried with nitrogen at 4 ℃ for 2 days.

Example 4 preparation of Tetracaine Phytic acid

1.0g tetracaine hydrochloride (3.33mmol) was dissolved in 40mL water and stirred vigorously. 20.5mL of the phytic acid solution of example 1 was added. After stirring for a further 30min, the precipitate was centrifuged and washed with water. The final product is in the form of a white powder. The solubility of the complexes in different solvents is shown below:

solvent(s)	Solubility (mg/mL)
		PBS(pH 7.4)	7.5
H2O(～pH 6.0)	4.5
		Acetic acid buffer (pH 4.5)	2.7

EXAMPLE 5 preparation of Polymer microspheres containing Tetracaine

Polymer (e.g., poly (lactide-co-glycolide) (PLGA)) microspheres are prepared by an oil-in-water (O/W) single emulsion technique. PLGA was dissolved in Dichloromethane (DCM). To encapsulate tetracaine, the drug was mixed with a PLGA solution in DCM. The mixed solution or suspension was emulsified in 500mL of 0.5-1% (w/v) PVA (PVA, 88% hydrolyzed, average molecular weight 31,000-50,000, Sigma-Aldrich) solution pre-cooled in a refrigerator at 4 ℃. The emulsion was stirred continuously at RT for 3h to evaporate DCM. The hardened microspheres were collected, washed 3 times with deionized water, and then freeze-dried.

In the case of microspheres containing tetracaine phytate (TCPA), 210mg TCPA was suspended in 5mL PLGA solution. The suspension was sonicated for 10 min. This suspension was slowly added to the continuous phase (1% PVA solution) pre-cooled at 4 ℃ while stirring. The emulsion was stirred continuously at room temperature for 3h to evaporate off the DCM. The hardened microspheres were collected, washed 3 times with deionized water, and then freeze-dried. Tetracaine loading was about 3.2%.

Polymeric microspheres containing tetracaine hydrochloride (TC-HCl) were prepared in a similar manner by replacing TCPA with TC-HCl.

EXAMPLE 6 preparation of pellets containing Tetracaine Phytic acid

Implantable pellets containing tetracaine phytate were prepared by compression molding. 249mg PLGA powder was thoroughly mixed with 25.7mg tetracaine phytate using a mortar and pestle. Then, 50mg of the mixture was molded using Delta Press to form pellets. Pellets containing tetracaine hydrochloride were also prepared for comparison.

Example 7 preparation of Tetracaine phytate containing grafts

2.56g of poly (lactide-co-glycolide) (PLGA) (RG504H, from Boehringer-Ingelheim) were dissolved in 7.73 g of dichloromethane. To this solution, 256mg tetracaine phytate was added and dispersed with a high shear mixer.

The resulting mixture was placed in a rotary evaporator and most of the dichloromethane was removed under vacuum. The resulting thick dispersion was poured into a glass plate and set to an adjustable blade spread of 0.7 mm.

The film thus obtained was melted at 80 ℃ and compression molded to give a film of about 0.5mm thickness. The films were incubated in phosphate buffer (containing 0.02% sodium azide) at pH 7.4 and 37 ℃, and the buffer solution was periodically checked with UV to determine the amount of tetracaine released.

Similar molded implants can be prepared using other biologically active compounds containing at least one basic functional group in place of tetracaine.

EXAMPLE 8 injectable formulations of Tetracaine Phytic acid and its in vitro Release

A40% (w/v) solution of poly (DL-lactide-co-glycolide) (PLGA) having carboxyl end groups in NMP was prepared by dissolving 160mg of PLGA (RG503H, from Boehringer-Ingelheim) in 0.4mL of NMP. By syringe flush, 39.9mg tetracaine phytate was mixed with the polymer solution. The droplet mixture (about 100mg) was added to phosphate buffered saline at pH 7.4. The received fluid was replaced with fresh solution at selected time points and the removed PBS solution was analyzed for drug concentration using UV detection at 280 nm.

Example 9 preparation of a Complex of Lidocaine and Phytic acid

1.0g lidocaine hydrochloride (3.69mmol) was dissolved in 400mL water and stirred vigorously. 28.8mL of the phytic acid solution of example 1 was added. After 30min, the pH was adjusted to 3.5 with 0.1N HCl solution. After stirring for a further 30min, the precipitate was filtered and washed 4 times with water. The final product was lyophilized.

Example 10 preparation of a Complex of Amoxicillin and Phytic acid

1.0g of amoxicillin hydrochloride (2.74mmol) was dissolved in 400mL of water and stirred vigorously. 21.3mL of the phytic acid solution of example 1 was added. After 30min, the pH was adjusted to 3.5 with 0.1N HCl solution. After stirring for a further 30min, the precipitate was filtered and washed 4 times with water. The final product was lyophilized.

Similar complexes can be prepared by replacing amoxicillin hydrochloride with other compounds containing at least one basic group.

Example 11 preparation of a Complex of growth inhibitory peptide and Phytic acid

A20 mg/mL solution of somatostatin was prepared by dissolving 215mg of the somatostatin in 10.75mL of water. 5mL of this solution was mixed with 1.45mL of a pH 3.12 solution of PA (1%, w/v). The mixture was shaken for 1min and then placed in a rotator to mix for 1 hour. The complex was separated by centrifugation and rinsed once with water. The precipitated product was freeze-dried for 48 h. The final product was obtained as a white powder.

Example 12 stability of growth-inhibiting peptides in injectable formulations

Injectable formulations of the growth-inhibiting peptides are prepared by dispersing the growth-inhibiting peptides in a polymer solution in a suitable solvent. For example, lactide with a ratio of 50/50: poly (DL-lactide-co-glycolide) (PLGA) of glycolide (PLG DL2.5A, from Alkermes) was dissolved in N-methyl-2-pyrrolidone (NMP) or methoxypolyethylene glycol (mPEG), or dimethyl ether of polyethylene glycol (PEGDM) to give a 40 wt% solution. Injectable formulations are prepared by dispersing the ghrelin phytic acid or acetic acid in the polymer solution. The mixture is thoroughly stirred until a homogeneous suspension or solution is obtained. 6 injectable formulations were prepared, shown below.

Note: mPEG: methoxypolyethylene glycol 350; NMP: n-methyl pyrrolidone;

PEGDM: polyethylene glycol dimethyl ether

The stability of the growth-inhibiting peptides in the above injectable formulations at room temperature was monitored by HPLC and the results are shown in the table below. Complexation of the aprotinin with phytic acid completely prevented degradation and/or acylation of the aprotinin in PLGA solutions in mPEG and PEGDM, whereas slight degradation of the aprotinin was observed in PLGA solutions in NMP over time at room temperature. When using the ghrelin acetate, a significant amount of ghrelin was degraded or reacted after 3 days at room temperature. In the case of PLGA solutions in NMP, there is almost 100% degradation or acylation of the ghrelin. Thus, the growth-inhibitory peptide phytic acid would be the preferred form for preparing stable formulations containing the peptide.

Note: mPEG: methoxypolyethylene glycol 350; NMP: n-methyl pyrrolidone; PEGDM: polyethylene glycol dimethyl ether; ac: an inhibitory growth peptide in the form of acetate; pa: an anti-growth peptide in the form of a phytate.

Example 13 stability of growth-inhibiting peptides in injectable formulations

Lactide with 50/50 ratio: poly (DL-lactide-co-glycolide) (PLGA) of glycolide (DL2.5A, from Alkermes) was dissolved in N-methyl-2-pyrrolidone (NMP) or methoxypolyethylene glycol (mPEG) to give a 40 wt% solution. Injectable polymer solutions are prepared by dispersing the ghrelin phytate or acetate or citrate. The mixture is thoroughly stirred until a homogeneous suspension or solution is obtained. Injectable formulations were prepared as shown below.

Note: mPEG: methoxypolyethylene glycol 350; NMP: n-methyl pyrrolidone.

The stability of the growth-inhibiting peptides in the above injectable formulations at room temperature was monitored by HPLC and the results are shown in the table below. It appears that both the salt form and the solvent of the growth-inhibitory peptide affect the stability of the growth-inhibitory peptide. With respect to stability of the growth-inhibitory peptide, mPEG is more preferred than NMP, and the phytate complex form of growth-inhibitory peptide is more preferred than acetate and citrate of growth-inhibitory peptide.

Note: mPEG: methoxypolyethylene glycol 350; NMP: n-methyl pyrrolidone; ac: acetic acid salt

A form of an aprotinin; ca: a citrate form of an inhibitory growth peptide; pa: phytic acid hexaphosphate

A form of an aprotinin.

Example 14 initial Release of growth inhibitory peptides in rats

Poly (DL-lactide-co-glycolide) (PLGA) was dissolved in N-methyl-2-pyrrolidone (NMP) or methoxypolyethylene glycol (mPEG) to give a 40 wt% solution. Injectable formulations are prepared by dispersing the ghrelin phytate or acetate. The mixture is thoroughly stirred until a homogeneous suspension or solution is obtained. The injectable formulations prepared are shown in the table below. These growth inhibitory peptide formulations (approximately 100. mu.L) were administered subcutaneously to the backs of Sprague-Dawley male rats. Release of the aprotinin was determined by recovering the graft at a predetermined time interval after administration (group G30 min, groups a-F24 h) and analyzing the aprotinin remaining in the graft. The stability of the somatostatin in the formulation and during release was also evaluated.

Note: mPEG: methoxypolyethylene glycol 350; NMP: n-methyl pyrrolidone; OCT: growth inhibitory peptide; OCT/Ac: an aprotinin acetate; OCT/Pa: ghrelin phytate.^*Including degradation peaks

Formulations a and G were similar, with G having a slightly higher drug content, but at different time points animals were collected and grafts were recovered. The results appear to indicate a gradual release of ghrelin over time. The growth inhibitory peptide released from the graft in group G at 0.5 hours post administration was about 3.29 ± 7.73%, and the growth inhibitory peptide released from the graft in group a at 24 hours post administration was about 10.82 ± 7.10%. The complexation of the growth-inhibiting peptide with phytic acid significantly improved the initial release and the stability of the peptide in the formulation and during release compared to formulation B. The results also show that mPEG is a better solvent than NMP in terms of stability of the growth inhibitory peptide. For the growth inhibitory peptide and PLGA, NMP appears to be a better solvent, which can facilitate the acylation reaction between the growth inhibitory peptide and PLGA or degradation products thereof.

The results regarding the stability of the somatostatin in the PLGA/NMP excipient relate to those obtained in vitro (reference examples 13 and 14). However, the degradation/reaction rate appears to be lower in vivo than in vitro (30% versus 85% after 24 hours). This difference can be explained by the fact that the implant is formed rapidly after administration by dispersing the solvent NMP in the surrounding tissue of the animal. Solvent dispersion will result in increased viscosity of the excipient or solidification of the PLGA, which causes a lower reaction rate between the growth-inhibiting peptide and the PLGA or degradation products thereof. However, since a significant amount of NMP (up to 35%) can still be detected in the graft 24 hours after administration, dispersion of the solvent is a slow process. This indicates that the remaining solvent can be trapped in the graft much longer than necessary. Therefore, the use of biologically active compounds in their more stable form is very important for the development of advantageous formulations.

Example 15 Release of growth-inhibiting peptides in rats

Injectable formulations are prepared by dispersing the somatostatin phytate in a poly (DL-lactide-co-glycolide) (PLGA) solution in mPEG 350. The mixture was stirred thoroughly until a homogeneous suspension was obtained. The injectable formulations prepared are shown in the table below. These growth inhibitory peptide formulations (approximately 100. mu.L) were administered subcutaneously to the backs of Sprague-Dawley male rats. Release of the aprotinin is determined by recovering the graft at a predetermined time interval after administration and analyzing the aprotinin remaining in the graft. The stability of the somatostatin in the formulation and during release was also evaluated.

Note: mPEG: methoxypolyethylene glycol 350; NMP: OCT: growth inhibitory peptide; OCT/Pa: ghrelin phytate. 5050 DL2.5A: poly (lactide-co-glycolide) with 50% lactide from Alkermes; RG 752S: poly (lactide-co-glycolide) with 75% lactide from Boehringer-ingelheim (bi).

Initial release of OCT from formulations a and B was 11.1 ± 1.7% and 14.0 ± 4.2%, respectively, while release from formulations C, D and E was 0.4 ± 2.0%, 1.5 ± 2.7% and 3.8 ± 4.5%, respectively. Although the difference was not statistically significant, there seems to be a trend that the initial release of OCT increases with decreasing polymer concentration. In addition, in these formulations, OCT is stable during formulation and in vivo release.

Example 16 preparation of a Complex of glucagon-like peptide 1(GLP-1) with Phytic acid

50mg of GLP-1 acetate (Mw 3297.7, 0.0152mmol) were dissolved in 5mL of water and vigorously stirred, and 1.01mL of a 1% solution of phytic acid pH 3.2 (molar ratio GLP-1: phytic acid 1: 1) was added. After stirring for a further 30min, the mixture was centrifuged. The supernatant was decanted off and the precipitate was rinsed 2 times with water and then freeze-dried. The final product was in the form of a white powder.

Example 17 preparation of a Complex of glucagon-like peptide 1(GLP-1) and inositol hexasulfate (InS6)

50mg of GLP-1 acetate (Mw 3297.7, 0.0152mmol) were dissolved in 5mL of water and vigorously stirred, and 1.35mL of a 1% potassium phytate (InS6) solution at pH 1.0 (molar ratio GLP-1: InS 6: 1) was added. After stirring for a further 30min, the mixture was centrifuged. The supernatant was decanted off and the precipitate was rinsed 2 times with water and then freeze-dried. The final product was in the form of a white powder.

Example 18 preparation of a Complex of PYY with Phytic acid

1.0g of PYY acetate (0.247mmol) was dissolved in 100mL of water and stirred vigorously and 11.5mL of the phytic acid solution prepared in example 1 was added (molar ratio PYY: phytic acid 1: 1). After stirring for a further 30min, the precipitate is filtered off and washed 4 times with water. The final product was lyophilized.

Example 19 preparation of lysozyme phytate

100mg of lysozyme (7.1 □ mol) was dissolved in 40mL of water and stirred vigorously, and 3.1 □ L of the phytic acid solution prepared in example 1 was added. After stirring for a further 30min, the precipitate is filtered off and washed 4 times with water and lyophilized. The final product was obtained as a white powder.

Similar complexes can be prepared by using naturally occurring peptides/proteins or their synthetic analogues in place of lysozyme.

Claims (27)

1. A pharmaceutical composition, comprising:

a) a complex of a biologically active compound having at least one basic functional group and a polyanion which is a hexahydroxycyclohexane esterified with at least two phosphate or sulfate groups; and

b) a pharmaceutically acceptable carrier comprising a biodegradable, water-insoluble polymer.

2. The pharmaceutical composition of claim 1, wherein the hexahydroxycyclohexane is selected from the group consisting of cis-inositol, epi-inositol, allo-inositol, neo-inositol, myo-inositol, muco-inositol, scylla-inositol, L- (-) -chiro-inositol, and D- (+) -chiro-inositol.

3. The pharmaceutical composition of claim 1, wherein the hexahydroxycyclohexane is myo-inositol.

4. The pharmaceutical composition of claim 1, wherein said polyanion is phytic acid.

5. The pharmaceutical composition of claim 1, wherein said polyanion is phytic acid.

6. The pharmaceutical composition of claim 1, wherein the biologically active compound has at least 1 basic nitrogen.

7. The pharmaceutical composition of claim 6, wherein the basic nitrogen is selected from the group consisting of amines, imines, and ring nitrogens.

8. The pharmaceutical composition of claim 1, wherein the biologically active compound is selected from the group consisting of small molecules and large molecules, wherein the large molecules are selected from the group consisting of proteins and enzymes, and the small molecules are peptides.

9. The pharmaceutical composition of claim 1, wherein the biologically active compound is selected from the group consisting of enzymes, cytokines, antibodies, vaccines, antibiotics, and glycoproteins.

10. The pharmaceutical composition of claim 1, wherein the biologically active compound is selected from the group consisting of: doxorubicin, doxycycline, diltiazemCyclobenzaprine, bacitracin, noscapine, erythromycin, polymyxin, vancomycin, nortriptyline, quinidine, ergotamine, benztropine, verapamil, flunarizine, imipramine, gentamicin, kanamycin, amoxicillin, amikacin, arbekacin, bambenomycin, butirosin, dibekacin, dihydrostreptomycin, fotemycin, isepamicin, microscine (micronomicin), netilmicin, paromomycin, ribostamycin, rapamycin, sisomicin, streptomycin and tobramycin, amikacin, streptomycin and tobramycin, pyrimethamine, naltrexone, lidocaine, prilocaine, mepivacaine, bupivacaine, tetracaine, ropivacaine, oxytocin, vasopressin, adrenocorticotropic hormone (ACTH), Epidermal Growth Factor (EGF), platelet-derived growth factor (PDGF), prolactin, growth factor (PDGF), Luteinizing hormone, Luteinizing Hormone Releasing Hormone (LHRH), LHRH agonists, LHRH antagonists, ghrelin, insulin, erythropoietin, somatostatin, glucagon, interleukin, interferon-alpha, interferon-beta, interferon-gamma, gastrin, tetrapeptide gastrin, pentapeptide gastrin, urogastrin, secretin, calcitonin, enkephalin, endorphin, angiotensin, Thyrotropin Releasing Hormone (TRH), parathyroid hormone (PTH), Nerve Growth Factor (NGF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEG-F), Bone Morphogenetic Protein (BMP), hANP, glucagon-like peptide (GLP-1), Exenatide, peptide YY (PYY), renin, bradykinin, bacitracin, polymyxin E, brevibacillin, gramicin, cyclosporine, follicle stimulating hormone, Gotuofen, taftsin, thymopoietin, thymosin, thymic stimulin, thymic fluid factor, serum thymic factor, colony stimulating factor, motilin, bombesin, neurotrophin, neurotensin, cyanin, urokinase, vasopressin, substance P and antagonists, angiotensin II, blood coagulation factors VII and IX, lysozyme, gramicidin, melanocyte, thyroid hormone releasing hormone, thyroid stimulating hormone, pancreatic hormoneZymoins, cholecystokinins, human placental lactogen, human chorionic gonadotropin, protein synthesis stimulating peptides, gastric inhibitory peptides, vasoactive intestinal polypeptides and platelet derived growth factors.

10. The pharmaceutical composition of claim 9, wherein the enzyme is heparinase.

11. The pharmaceutical composition of claim 9, wherein the cytokine is tumor necrosis factor.

12. The pharmaceutical composition of claim 9, wherein the antibiotic is neomycin.

13. The pharmaceutical composition of claim 9, wherein the auxin is a human, porcine, and bovine auxin.

14. The pharmaceutical composition of claim 9, wherein said erythropoietin comprises all proteins having erythropoietic activity.

15. The pharmaceutical composition of claim 8, wherein the biologically active compound is selected from the group consisting of: doxorubicin, rapamycin, naltrexone, Epidermal Growth Factor (EGF), LHRH agonists, LHRH antagonists, ghrelin-releasing factor, octreotide, interferon- α, interferon- β, interferon- γ, calcitonin, parathyroid hormone (PTH), glucagon-like peptide (GLP-1) and peptide yy (pyy).

16. The pharmaceutical composition of claim 1, wherein the biologically active compound is doxorubicin.

17. The pharmaceutical composition of claim 1, wherein the biologically active compound is glucagon-like peptide 1 (GLP-1).

18. The pharmaceutical composition of claim 1, wherein the biologically active compound is octreotide.

19. The pharmaceutical composition of claim 1, wherein the biologically active compound is peptide YY (PYY).

20. The pharmaceutical composition of claim 1, wherein the biodegradable, water-insoluble polymer is selected from the group consisting of: polylactides, polyglycolides, poly (lactide-co-glycolide), polycaprolactones, polydioxanones, polycarbonates, polyhydroxybutyrates, polyalkylene oxalates, polyanhydrides, polyamides, polyesteramides, polyurethanes, polyacetals, polyorthocarbonates, polyphosphazenes, polyhydroxyvalerates, polyalkylene succinates, polyorthoesters, and copolymers and mixtures thereof.

21. The pharmaceutical composition of claim 20, wherein the copolymer is a block copolymer, a branched copolymer, or a terpolymer.

22. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier comprises an environmentally responsive polymer or gel, wherein the environmentally responsive polymer or gel is thermosensitive, pH sensitive, or electrically sensitive.

23. The pharmaceutical composition of claim 1, in a form selected from the group consisting of: injectable solutions or suspensions, particles, thin films, cylinders, discs, microcapsules, nanospheres, wafers, and liposomes.

24. The pharmaceutical composition of claim 1 in the form of microspheres.

25. The pharmaceutical composition of claim 1, in particulate form.

26. The pharmaceutical composition of claim 1, in the form of a micelle.

27. The pharmaceutical composition of claim 1 in the form of a pellet.