MXPA99010284A - Sustained-release delayed gels - Google Patents

Abstract

The present invention relates to sustained-release formulations using alginate delayed gels and methods thereof.

Description

COMPOSITIONS OF DELAYED GELIFICATION, SUSTAINED RELEASE Field of the Invention The present invention relates to sustained release formulations using alginate delayed action gels and methods for manufacturing them.

Background of the Invention With advances in genetic and cellular engineering technologies, the availability of recombinant proteins has spawned advances in the use of proteins as medicines for therapeutic applications. Many diseases or conditions treated with pharmaceutical proteins require sustained protein levels to achieve the most effective therapeutic result. However, as with most pharmaceutical substances in proteins, the generally short biological half-life requires frequent administration. These repeated injections are given at various intervals which leads to fluctuating medication levels Ref.031946 and at a significant monetary and physical cost to patients. Since many conditions respond better to controlled levels of a pharmaceutical substance, there is a need for controlled release of a drug to provide longer periods of consistent release. Such sustained release medications could provide the patient not only with improved prophylactic, therapeutic or diagnostic effects, but also a reduction in the frequency of the injections as well as in the total costs. Current attempts or tests to sustain the levels of medication in humans or animals between doses have included the use of biodegradable polymers as matrices to control the release of the drug. For example, Patent No. 1,388,580 of Great Britain describes the use of hydrogels for the sustained release of insulin. The U.S. Patent No. 4,789,550 describes the use of alginate microcapsules coated with polylysine for the delivery of proteins by the encapsulation of living cells. Attempts or tests of sustained release have also used anionic or cationic polymeric compositions surrounded by ionic polymers of the opposite charge to encapsulate cells capable of producing biologically active compositions. U.S. Patent No. 4,744,933. Similarly, multiple coatings of anionic or cationic crosslinking polymers have also been described as means of obtaining controlled release. U.S. Patent Nos. 4,690,682 and 4,789,516. In addition, some additional attempts or tests describe the use of alginates alone, or alginates coated with other biodegradable polymers, for the controlled release of the polypeptide compositions or cationic precipitates thereof. PCT WO 96/00081, PCT WO 95/29664 and PCT WO 96/03116. These tests or attempts, however, have provided an insufficient means to obtain a sustained release supply of the pharmaceutical substances of the desired proteins. It is generally known that the use of certain biodegradable polymers, for example, polylactide co-glycolide, under in vivo conditions, exhibit high initial discharges of drug release. Johnson, 0. et al., Nature Med., 2/7: 795 (1996). Furthermore, it is generally known that proteins used with the common forms of sustained release preparations may undergo denaturation and lose their bioactivity during exposure to the encapsulating agent. Such preparations use organic solvents which can have deleterious effects on the protein of choice. Finally, as described below, the use of alginate alone has not provided the desired, controlled protein release necessary for effective therapeutic results. In general, alginates are anionic polysaccharides, which are naturally occurring, well known, of 1,4-linked β-D-mannuronic acid and L-guluronic acid. Smidsrod, 0. et al., Trends in Biotechnology, 8: 71-78 (1990); Aslani, P. et al., J. Microencapsulation, 13/5. 601-614 (1996). Alginates typically range from 70% anuronic acid and 30% guluronic acid, up to 30% mannuric acid and 70% guluronic acid. Smidsrod, supra. Alginic acid is insoluble in water while salts formed with monovalent ions similar to sodium, potassium and ammonium are soluble in water. McDowell, R.H., "Properties of Alginates" (London, Alginate Industries Ltd., 4th edition, 1977). Polyvalent cations are known to react with alginates to spontaneously form gels. Alginates have a wide variety of applications such as food additives, adhesives, pharmaceutical tablets, models for new cell growth, and wound dressings. Alginates have also been recommended for protein separation techniques. For example, Gray, C.J. et al., in Biotechnology and Bioengineering, 31 .: 607-612 (1988) trapped insulin in zinc alginate / calcium gels for the separation of insulin from other whey proteins. Alginate matrices have also been well documented for drug delivery systems, see for example U.S. Pat. No. 4,695,463 which discloses an alginate-based chewing gum supply system and pharmaceutical preparations. Alginate beads or globules have also been used for the controlled release of several proteins such as: the tumorigenic necrosis factor receptor in beads or alginate-cation beads coated with polycations, Wee, S.F., Proceed. Intern. Symp. Control. I laughed Bioact. Mater., 21: 730-31 (1994); the transformation of the growth factor encapsulated in beads or beads of alginate, Puolakkainen, P.A. et al., Gastroenterology, 107: 1319-1326 (1994); Angiogenic factors trapped in beads or calcium-alginate beads, Downs, E.C. et al., J. of Cellular Physiology, 152: 422-429 (1992); albumin entrapped in alginate-chitosan microcapsules, Polk, A. et al., J. Pharmaceutical Sciences, 83/2. 178-185 (1994), or beads or beads of calcium alginate-chitosan coated with polymers, Okhamafe, A. 0. et al., J. Microencapsulation, 13/5: 497-508 (1996); hemoglobulin encapsulated with beads or calcium alginate globules-chitosan, Huguet, M.L. et al., J. Applied Polymer Science, 51. 1427-1432 (1994), Huguet, M.L. et al., Process Biochemistry, 31: 745-751 (1996); and interleukin-2 encapsulated in microspheres of alginate-chitosan, Liu, L. S. et al., Proceed. Intern. Symp. Control. I laughed Bioact. Mater. 22: 542-543 (1995). Systems that use beads or beads of alginate gel, beads or alginate / calcium globules, to trap proteins, suffer from the lack of some sustained release effect due to a rapid release of the protein from beads or alginate globules . Liu, L. et al., J. Control. Laugh., 43: 65-74 (1997). To avoid such rapid release, several of the above systems use polycationic polymer coatings (eg, polylysine, chitosan) to retard the release of alginate beads or protein globules. See, for example, Wheatley, M.A. et al., J. Applied Polymer Science, 43: 2123-2135 (1991); Wee, S.F. et al. Supra; Liu, L.S. et al., supra; Wee, S. F. et al., Controlled Relay Society, 22: 566-567 (1995) and Lim, et al., Supra.

Polycations, such as polylysine, are positively charged polyelectrolytes which interact with negatively charged alginate molecules to form polyelectrolyte complexes that act as diffusion barriers on the surface of the bead or bead. Problems with the use of polycations can occur because: (1) such formulations can be cytotoxic due to polycations (Huguet, ML et al., Supra, Zimmermann, Ulrich, Electrophoresis, _13: 269 (1992), Bergmann, P. et al. al., Clinical Science, 67: 35 (1984)); (2) polycations are prone to oxidation; (3) beads or globules with polycationic coatings tend not to be erodible and accumulate in the body; (4) such formulations are made by means of laborious coating processes which include multiple coatings of the polycationic polylysine (Padol, et al., Proceed, Intern Symp. Control, Rei. Bioact. Mater, 2 ,: 216 (1986 ) and (5) the ionic interactions between the protein and the polycations can lead to the loss of the activity of the protein or cause the instability of the protein.In addition to the previous systems, there are also delivery systems which gel after The injection, or are based on organic substances and / or based on thixotropic substances.The gelled deposits which gel after injection in the body can sustain the release of trapped drugs.These could include the gelation induced by the temperature of poloxamers, (for example, Pluronics®, U.S. Patent No. 2,741,573). These are liquids at cold temperatures but gel at elevated temperatures such as body temperatures. These systems are difficult to control due to variations in ambient temperature conditions, and variations in anatomical temperatures especially if subcutaneous injections are used. As for organic-based gelling systems, such systems are not suitable for fragile protein drugs that are destabilized in the presence of organic solvents or non-physiological conditions. Thixotropic gels of aluminum stearate in oils have also been used to prolong the activity of penicillin. Buckwalter, et al, J. Am. Pharm. Assoc, 137: 472 (1948); Thompson, R.E., American Journal Clinical Nutrition, 1: 311 (1959); Thompson, Robert E., Sustained Relase of Parenteral Drugs, Bulletin of Parenteral Drug Assoc. , 1_4: 6-17, (1960); Chen, Y., Journal of Parenteral Science & Tech., 35: 106 (1981). Proteins tend to be destabilized in the presence of these types of systems that contain oils. Sustained-release drug delivery systems that involve a controlled time delay for gelation in the body are not generally known in the art. These types of delayed gel systems are known, however, in the context of plant tissue culture, immobilization of cells, microsphere formation, release of insecticides and the food industry. For example, alginates have been used with insoluble calcium complexes and d-gluconolactone as a proton donor for the release of calcium ions in the solution for gelation. See Draget, et al, Appl. Microbiol. Biotechnol., 3_l: 79-83 (1989). Similarly, similar systems have been used for the formation of beads or alginate globules by internal emulsification / gelation. The alginate microspheres were produced using alginate dispersed within the vegetable oil and gelation initiated by lowering the pH to release the calcium from the insoluble complex. See Poncelet, D. et al, Appl. Microbiol. Biotechnol, 43: 644-650 (1995). Alginate systems have also been used to immobilize cells. Burke, C. describes, in Methods of Enzymology, 135: 175-189 (1987), that dicalcium phosphate and d-gluconolactone can be used with alginates for the delayed gelation of cells. The U.S. Patent No. 4,053,627 discloses the use of alginate gel discs to control the release of an insecticide in an aqueous environment. These uses mentioned above are not designed for gelled deposits in the body for the sustained release of biologically active agents, especially proteins. Accordingly, there is a need to develop pharmaceutical formulations of delayed gelling which achieve a better sustained release medium for clinical applications. Numerous natural or recombinant proteins could benefit from constant long-term release through delayed-gel formulations and by means of which more effective clinical results are provided. The present invention provides such advances. The delayed gelling pharmaceutical compositions of the present invention are capable of providing protein protection, reduced degradation and slow dissolution with increased potency and stability of the protein. Also, the pharmaceutical compositions of the present invention provide a simple, rapid and economical means of a release of the controlled recombinant protein for effective prophylactic, therapeutic or diagnostic results.

Brief Description of the Invention The present invention relates to sustained release formulations using delayed alginate gels, and methods of producing them. In particular, the formation of delayed sustained release gels include thixotropic alginate gels with a biologically active agent. This approach provides an advantage of an efficient production and high loading of the biologically active agent within the delayed alginate gel for sustained release delivery while achieving protein protection, reduced degradation, and increased potency and stability of the agent. which is going to be supplied. In addition, timed or synchronized gelation provides more control over the gelling properties and the administration thereof.

Accordingly, an aspect of the present invention provides a delayed sustained release gel composition, comprising a hydrophilic polymer; a biologically active agent and at least one linked polyvalent metal ion. The speed of gelation is controlled by the level of free calcium, that is, the polyvalent metal ion released. The biologically active agent can be in a complex form. The formation of complex molecules and any related complexing agents is well known to those skilled in the art. In addition, the above composition may also contain the polyvalent metal ion which is a mixture of polyvalent metal ions bound and released. Due to the controlled time nature of these delayed gels, these mixtures can be placed in the body where they can gel after the injection. In addition, due to the thixotropic nature of the composition in the gelation state, these mixtures can be injected while in the gelled state, for example, by the pressure of the syringe, after which they can be regelified in the body . Another aspect of the present invention provides a delayed sustained release gelation composition, comprising a hydrophilic polymer; a biologically active agent; at least one linked polyvalent metal ion and further comprises at least one proton donor capable of releasing the bound polyvalent metal ion. The release of the protons from the proton donor liberates the cations of the bound polyvalent metal ion. Another aspect provides methods for producing the delayed release gelation compositions. One method comprises the steps of mixing a biologically active agent and a hydrophilic polymer with a solvent to form a first mixture and mixing the first mixture with at least one polyvalent metal ion linked to form a second mixture. An alternative method comprises the steps of mixing a biologically active agent and a hydrophilic polymer with a solvent to form a first mixture; mixing the first mixture with at least one polyvalent metallated ion to form a second mixture; and mixing with the second mixture at least one proton donor capable of releasing the bound polyvalent metal ion. The bound polyvalent metal ion and the proton donor can also be added to the first mixture together, or the proton donor can be added to the first premix to the bound polyvalent metal ion. In addition, a step to isolate the sustained release delayed gelation composition is also contemplated. When used herein, the term "linked polyvalent metal ion" refers to a polyvalent metal ion in a salt or chelate or ion complex form. The bound polyvalent metal ion can include a mixture of bound and unbound polyvalent metal ions. This could include, as noted above, the release of the polyvalent metal ion bound to unbound. When used here, the term "proton donor" capable of releasing the bound polyvalent metal ion refers to strong acids, weak acids, or materials capable of generating an acid, for example, lactones or esters (by aqueous hydrolysis), or a sparingly soluble acid or a slow dissolving acid such as adipic acid. When used herein, the term "solvent" refers to aqueous or non-aqueous based solvents capable of dispersing or dissolving biologically active agents, hydrophilic polymers, polyvalent metal ions, proton donors or complexing agents of choice. Such solvents are well known to one skilled in the art. Additions to form the first mixture and the second mixture can be made by methods well known to a person skilled in the art, including but not limited to pouring and stirring, droplet addition, dispersion, spraying or mixing using dew jets. , air jets, atomization, and electric fields. The term dispersion for purposes of this invention may mean liquid, solid or gaseous dispersions. When used herein, the term "isolation" refers to the process for the isolation of the delayed release gelation composition of the present invention. Such isolation and purification procedures are well known in the art. In yet another aspect, the present invention provides a delayed release delayed gelation composition produced by the above methods. Additional aspects include pharmaceutical formulations of delayed gelling of the above compositions in a pharmaceutically acceptable carrier, or adjuvant. In addition, the delayed gel composition can be contained in a syringe. In yet another aspect, the present invention provides methods of treating indications with sustained release delayed gelation compositions containing the biologically desired active agents.

DETAILED DESCRIPTION OF THE INVENTION Compositions Hydrophilic polymers including alginates and derivatives thereof can be obtained from various commercial, natural or synthetic sources well known in the art. When used herein, the term "hydrophilic polymer" refers to water-soluble polymers or polymers that have affinity for water absorption. Hydrophilic polymers are well known to a person skilled in the art. These include but are not limited to polyanions, including anionic polysaccharides such as alginate, gelan, carboxymethyl amylose, salts of polyacrylic acid, salts of polymethacrylic acid, maleyan anhydride ethylene copolymer (semi ester), carboxymethyl cellulose, dextran sulfate, heparin , carboxymethyl dextran, carboxy cellulose, 2,3-dicarboxylic cellulose, tricarboxylic cellulose, carboxy arabic gum, carrageenan carboxy, carboxy pectin, carboxy tragacanth gum, xanthan carboxy gum, pentosan polysulfate, carboxy starch, carboxymethyl chitin / chitosan, curdlan, inositol hexasulfate, ß-cyclodextrin sulfate, hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, heparin sulfate, carboxymethyl starch, carrageenan, polygalacturonate, carboxy guar gum, polyphosphate, carbonic acid-polyaldehyde, poly-1 -hydroxy-l-sulfonate-pro-eno-2, copolystyrene maieic acid, agarose, mesoglycan, polyvinyl sulfopropylated alcohols os, cellulose sulfate, protamine sulfate, guar gum, polyglutamic acid, polyaspartic acid, polyamino acids, derivatives or combinations thereof. One skilled in the art will appreciate various other hydrophilic polymers that are within the scope of the present invention. Similarly, polyvalent metal bound ions can be obtained from various commercial, natural or synthetic sources which are well known in the art. The polyvalent metal ions bound or sequestered within the scope of this invention include but are not limited to manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum or zinc. Metal ions can be obtained from the soluble salts of a complexing agent, acetates, phosphates, lactates, tartrates, citrates, sulfates, chlorides, carbonates, hydroxides, or fatty acid anions, such as oleates, thereof . One skilled in the art will appreciate various other complexes / linked polyvalent metal ions which are within the scope of the invention. The bound polyvalent metal ions may include a mixture of bound and unbound polyvalent metal ions. Proton donors, when used here, refer to the material that can generate acids. Proton donors are capable of releasing a sequestered or bound polyvalent metal ion. Proton donors are well known in the art, and include but are not limited to lactones such as gluconolactones, esters, buffer solutions and other acids that dissolve slowly. When acids which dissolve slowly are used herein, reference is made to acids such as solid acids which are of low solubility in the solvent. In addition, acids that dissolve slowly include coated devices or materials that slowly release acids. Acids well known within the scope of the art include acetic, adipic, citric, fumaric, gluconic, lactic, malic, phosphoric and tartaric acids. A person skilled in the art will appreciate other various proton donors that are within the scope of the invention.

When used herein, the term "buffer" or "buffer solution" refers to the use of organic and inorganic acids or bases or a combination thereof, to prepare a buffer solution as is known in the art. These also include amphoteric materials or amino acids. Organic acids within the scope of the present invention include hydrogen halide (e.g., hydrochloric acid), phosphoric, nitric or sulfuric acid. Other inorganic acids could be well known to a person skilled in the art and are contemplated herein. Organic acids within the scope of the invention include aliphatic carboxylic acids and aromatic acids such as formic, carbonic, acetic, propionic, butyric, valeric, capric, acrylic, malonic, succinic, glutaric, adipic, maieic, fumaric, glycine or phenol sulphonic. Other organic acids may be well known to a person skilled in the art. Organic bases include TRIS®, pyridine, PIPES®, and HEPES®. Amino acid buffers include glycine and mixtures of phosphoric acid / glycine. When used herein, biologically active agents refer to recombinant proteins or proteins that are naturally present, either from humans or from animals, useful for prophylactic, therapeutic or diagnostic applications, such as small molecules, oligonucleotides, and organic agents or inorganic The biologically active agent can be natural, synthetic, semi-synthetic or derivatives thereof. In addition, the biologically active agents of the present invention may be precipitable. A wide range of biologically active agents is contemplated. These include but are not limited to hormones, cytokines, hematopoietic factors, growth factors, anti-obesity factors, trophic factors, anti-inflammatory factors, and enzymes (see also U.S. Patent No. 4,695,463 for additional examples of biologically active, useful agents). A person skilled in the art will be able to easily adapt a biologically active agent to the compositions of the present invention. Such proteins could include but are not limited to interferons (see, US Pat. Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623 incorporated for reference herein, including the drawings), interleukins (see, US Patent No. 5,075,222, incorporated herein by reference). reference, including drawings), erythropoietins (see, US Pat. Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080 incorporated herein for reference, including drawings), colony-granulocyte stimulating factors (see, US Pat. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185, incorporated herein by reference, including the drawings), stem cell factor (PCT Publications Nos. 91/05795, 92/17505 and 95 / 17206, incorporated herein by reference, including the drawings), and the OB protein (see, PCT publications Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816, incorporated herein by reference). g, including the Figures). In addition, biologically active agents can also include but are not limited to products related to anti-obesity, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) ), follicle-stimulating hormone (FHS), human chorionic gonadotropin (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), tumor necrosis factor binding protein (TNF-bp), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor ( GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegerin (OPG), similar growth factors insulin (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte-derived growth factor (MGDF), growth factor of keratinocytes (KGF), thrombopoietin, platelet-derived growth factor (PGDF), growth stimulants of colonies (CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptokinase and kalikrein. The term "proteins", as used herein, includes peptides, polypeptides, unanimously agreed molecules, analogs, derivatives or combinations thereof. Derivatives of the biologically active agents can include the attachment of one or more chemical moieties to the protein portion. Chemical modification of biologically active agents has been found to provide additional advantages under certain circumstances, such as increased stability and circulation time of the therapeutic protein and reduction of immunogenicity. A person skilled in the art will be able to select the desired chemical modification based on the desired dosage, circulation time, resistance to proteolysis, therapeutic uses and other considerations. Derivatives such as polyethylene glycol and Fe fusion proteins are desirable.

Complexes The biologically active agent can be in complexed forms. This could include precipitated forms, structured forms, and association with other molecules. These forms bound with complexes of the agent can reduce the rate of diffusion of the agent out of the gel and therefore sustain the release of the agent. These complexed forms include but are not limited to the active agent biologically bound with complexes with antibodies, substrates, receptors, lipids, polymers, and precipitating agents. For example, biologically active agents, analogs or derivatives can be administered linked with complexes to a binder composition. Such a binder composition in addition to the above benefits can also have the effect of prolonging the circulation time of the agent, analog or derivative or improving the activity of the biologically active agent.

Such a composition can be a protein (or synonymously, a peptide), derivative, analog or combination or an agent other than a protein. By way of illustration, a binding protein for the OB protein is the OB protein receptor or a portion thereof, such as a soluble portion thereof. Other binding proteins can be ascertained by examining the OB protein, or the protein of choice, in the serum, or be selected empirically to verify the presence of agglutination. Such agglutination will typically not interfere with the ability of the OB protein or analog or derivative to bind to the endogenous OB protein receptor and / or effect signal transduction. In addition to the OB protein, the binding complexes will also be applicable to other therapeutic proteins of the present invention. Those skilled in the art will be able to ascertain the binding proteins appropriate for use with the present invention. Similarly, the precipitating agents used to precipitate the biologically active agent can be obtained from various commercial sources, natural or synthetic which are well known in the art. Precipitating agents include but are not limited to polyvalent metal ions or their salts such as acetates, citrates, chlorides, carbonates, hydroxides, oxalates, tartrates or hydroxides thereof, water-soluble acids or polymers. In particular, metal ions may include but are not limited to aluminum, barium, calcium, iron, manganese, magnesium, strontium and zinc. Preferably the metal ion is zinc or salts thereof, similar to chloride and acetate salts. Small molecules and water-soluble salts can also be used, such as ammonium sulfate, acetone, ethanol and glycerol. As for water-soluble polymers, these include but are not limited to polyethylene glycol, ethylene glycol / propylene glycol copolymers, hydroxyethyl cellulose, amylose, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3. , 6-trioxane, ethylene / maieic anhydride copolymers, polyamino acids, dextran, poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, ethylene oxide / polypropylene oxide copolymers, polyoxyethylated polyols, polyvinyl alcohol succinate, glycerin, oxides of ethylene, propylene oxides, poloxamers, alkoxylated copolymers, water soluble polyanions, derivatives or combinations thereof. The water soluble polymer may be of any molecular weight, and may be branched or unbranched. For example, the preferred molecular weight of polyethylene glycol is between about 700 Da and about 100 kDa because of the ease of handling and the efficiency of precipitation. Other sizes and types of precipitating agents can be used, depending on the desired therapeutic profile (eg, the duration of the desired sustained release, the effects, if any, on biological activity, ease of handling, degree or lack of antigenicity and other known effects of a desired precipitation agent for a therapeutic or analogous protein). One skilled in the art will appreciate other precipitating agents that are within the scope of the invention. In addition, the compositions of the present invention may also include extra excipients needed to stabilize the biologically active agent and / or the hydrophilic polymer. These may be contained in the buffer and may include, but are not limited to, condoms.

Pharmaceutical compositions The sustained release pharmaceutical compositions of the present invention can be administered by oral preparations (eg, liquid preparations that allow gelation in the stomach or intestines) and non-oral preparations (eg, intramuscular, subcutaneous, visceral, IV preparations). (intravenous), IP (intraperitoneal), intraarticular, placement in the ear, ICV (intracerebroventricular), IP (intraperitoneal), intraarterial, intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal, rectal, and uretine-transmucosal). In general, pharmaceutical compositions of sustained release delayed gel comprising effective amounts of the protein, or products thereof, with the sustained release compositions of the invention together with diluents, preservatives, solubilizers, emulsifiers, are encompassed by the present invention. pharmaceutically acceptable adjuvants and / or carriers necessary for administration. (See PCT patent 97/01331 incorporated herein for reference). The optimum pharmaceutical formulation for a biologically desired active agent will be determined by one skilled in the art depending on the route of administration and the desired dosage. Exemplary pharmaceutical compositions are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., 18 / a Ed., Easton, PA, pp. 1435-1712 (1990)). Due to the thixotropic nature of the delayed gelation formulation, the syringes can be used to be administered subcutaneously. The composition can be gelled in a syringe for subsequent injection. This gelling can be carried out in a delayed manner over time. The timing is controlled by the judicious adjustment of the amount of the gelling agent, and the proton donor if used, as well as the particle size of the polyvalent metal ion and the temperature of the mixture. Such a preparation could be used for subsequent re-gelling in the body after injection. The term thixotropic when used herein, refers to the viscosity of the gel mixture which is reduced under pressure, for example, from the syringe plunger, at such point the mixture can flow, for example, through the syringe needle, and then reform a gel at the site of the injection. The concept of delayed gelation can also be applied to the filling of a syringe wherein a sustained release gel composition is filled in a syringe and in gels of predetermined time in the syringe, for example, from a few minutes to many hours after the syringe. fill. This avoids the problem of filling a syringe with material that has already been gelled. These prefilled syringes can be stored for later injection in patients. Components that may be required for administration include diluents or buffers of various pH and strength or ionic strength (e.g., Tris-HCl, acetate); additives such as surfactants and solubilizing agents (e.g., Tween 80, HCO-60, Polysorbate 80), lipids, liposomes, antioxidants (e.g., ascorbic acid, glutathione, sodium metabisulfite), additional polysaccharides (e.g., carboxymethylcellulose, sodium alginate, sodium hyaluronate, protamine sulfate, polyethylene glycol), preservatives (for example, Thimersol, benzyl alcohol, methyl paraben, propyl paraben) and construction substances (for example, lactose, mannitol); the incorporation of the material with particulate preparations of the polymeric compounds such as the polylactic / polyglycolic acid polymers or copolymers, etc., or combined with liposomes. Hyaluronic acid can also be used as a component of administration and this can have the effect of further promoting the sustained duration in the circulation. Additionally, the sustained release compositions of the present invention can also be dispersed with oils (e.g., sesame oil, corn oil, vegetable oil), or a mixture thereof with a phospholipid (eg, lecithin), or triglycerides of intermediate chain fatty acids (eg, Migiyol 812) to provide an oily suspension. The compositions of the present invention may also be dispersed with dispersing agents such as water-soluble polysaccharides (e.g., mannitol, lactose, glucose, starches), hyaluronic acid, glycine, fibrin, collagen and inorganic salts (e.g., sodium chloride). ). In addition, mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, two-metered inhalers, and powder inhalers, are also contemplated for use in the administration of the sustained release compositions of the present invention. , all of which are familiar to those skilled in the art. The administration components can influence the physical state, stability, in vivo release rate, and the in vivo clearance rate of the present proteins and derivatives. A person of ordinary skill in the art will appreciate the appropriate administration components and / or mechanical devices suitable for use depending on the therapeutic use, the route of administration, the desired dosage, the circulation time, the resistance to proteolysis, the stability of the protein and other considerations.

Methods of Use Therapeutic. The therapeutic uses depend on the active agent biologically used. A person skilled in the art will readily be able to adapt an active agent biologically desired for the present invention, with its proposed therapeutic uses. Therapeutic uses for such agents are described in greater detail in the following publications incorporated herein for reference, which include the drawings. Therapeutic uses include but are not limited to uses for interferon-like proteins (see, US Patent Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623 incorporated herein for reference including drawings), interleukins (see, patent No. 5,075,222, incorporated herein by reference including the drawings), erythropoietins (see, US Pat. Nos. 4,703,008, 5,441,868, 5,618,689, 5,547,933, and 5,621,080 incorporated herein by reference including the drawings), the stimulatory factors of the colonies- granulocytes (see, US Pat. Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT Publication No. 94/17185, incorporated herein by reference including the drawings), stem cell factor (PCT Publication Nos. 91/05795 , 92/17505 and 95/17206, incorporated herein by reference including the drawings), and the OB protein (see, PCT publications Nos. 96/40912, 96/05309, 97/001 28, 97/01010 and 97/06816 incorporated herein by reference including the Figures). In addition, therapeutic uses of the present invention include the uses of biologically active agents including but not limited to anti-obesity related products, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (THS) , luteinizing hormone (LH), follicle-stimulating hormone (FHS), human chorionic gonadotropic (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF) , tumor necrosis factor binding protein (TNF-bp), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors ( FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage-stimulating factor (M-) CSF), the granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte-derived growth factor (MGDF), keratinocyte growth factor (KGF), thrombopoietin, platelet-derived growth factor (PGDF) , growth factors stimulate colonies (CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptokinase and kalikrein. The term "proteins", as used herein, includes peptides, polypeptides, unanimously agreed molecules, analogs, derivatives or combinations thereof. In addition, the present invention can also be used for the manufacture of one or more medicaments for the treatment or improvement of the conditions that the biologically active agent is proposed to treat.

Combination of therapies The present compositions and methods can be used in conjunction with other therapies, such as altered diet and exercises. Other medications, such as those useful for the treatment of diabetes (for example, insulin, and possibly amylin), medications to reduce blood pressure and cholesterol (such as those which reduce the levels of blood lipids) or other cardiovascular medications), medications to increase activity (eg, amphetamines), diuretics (for fluid elimination), and appetite suppressants. Such administration may be simultaneous or may be in seriatim. In addition, the present methods can be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of a body (e.g., laser or liposuction surgeries, designed to reduce body mass, or designed implant surgeries). to increase the appearance of body mass). The health benefits of cardiac surgeries, such as bypass surgeries or other surgeries designed to relieve or relieve a harmful condition caused by blockage of blood vessels by fat deposits, such as arterial plaque, can be increased with the concomitant use of the present compositions and methods. Methods for removing kidney stones, such as ultrasonic or laser methods, can also be used either prior to, during or after the course of the present therapeutic methods. In addition, the present methods can be used as an adjunct to surgeries or therapies for broken bones, damaged muscles, or other therapies which could be improved by an increase in lean tissue mass.

Dosages A person skilled in the art will be able to ascertain the effective dosages by administration and observation of the desired therapeutic effect. The dosage of the sustained release preparation is the amount necessary to achieve an effective concentration of the biologically active agent in vivo, for a given period of time. The dosage and frequency of the preferred administration of the sustained-release preparations varies with the type of the biologically active agent, the desired duration of the release, the target or target disease, the frequency of the desired administration, the animal species of subject. and other factors. Preferably, the formulation of the molecule will be such that between about 0.10 ug / kg / day and 100 mg / kg / day will produce the desired therapeutic effect. Effective dosages can be determined using diagnostic tools over time. By way of example, the present invention provides the dosages of the OB protein. For example, a diagnosis to measure the amount of OB protein in the blood (or in the plasma or in the serum) can be used first to determine the endogenous levels of the OB protein. Such a diagnostic tool may be in the form of an antibody assay, such as an antibody sandwich assay. The amount of the endogenous OB protein is quantified initially, and a baseline is determined. Therapeutic dosages are determined when the quantification of the endogenous and exogenous OB protein (i.e., the protein, analog or derivative found within the body, whether self-produced or administered) is continuous during the course of therapy. For example, a relatively high dosage may be necessary initially, until a therapeutic benefit is observed, and then lower dosages are used to maintain the therapeutic benefits.

Materials and methods Materials . Alginate in the form of the alginate salt can be found from various sources, or it can be prepared by methods well known in the art. The leptin, the GCSF and the unanimously approved interferon are from Amgen Inc. Other chemicals are from sources well known in the art.

Preparation of Delayed Gel - Introduction.

The delayed gel is prepared by combining a mixture of an anionic polymer and biologically active agent (eg, alginate) with a polyvalent cationic salt (eg, CaCO3) and a proton donor (eg, an acidified buffer or dissolving acid source or slow release such as d-gluconolactone), if used. For all cases, the anionic polymer, the protein and any precipitating agents / excipients can be prepared as a mixture. The gelation is initiated by the addition of the polyvalent cationic salt and the source of protons, if used, to this mixture. The addition of the proton, of the polyvalent metal ion to the biologically active agent mixture of the polymer can be simultaneous, or separately with the proton donor first or the polyvalent metal ion first. For the gelation induced by the buffer, the cationic polyvalent salt and the proton source can be mixed as an aqueous suspension before the time of gelation. After the gelation is initiated, the syringes are filled before gelation occurs (typically 5 to 10 minutes). The injections can be made before the gels of the material for gelation in situ, or after the gels of the material.

Alginate-protein mixture. A mixture of the solvent and precipitating agents / excipients (eg, zinc salts, buffers, etc.) is prepared. In rapid succession, a solution of the protein (e.g., leptin in 10 mM Tris HCl, pH 8) and sterile alginate (e.g., a 10% solution treated in an autoclave) are rapidly mixed. Where the biologically active agent is prepared as a fine suspension (for example, zinc-leptin is typically formed at 10-15 mg / ml), it may be desirable to concentrate the suspension. Calcium salt. The calcium salt can be prepared as an autoclaved suspension of fine powder in water (eg, 9.1% CaCO3, in water).

Proton source. For the gels induced by the buffer, the suspension of the calcium salt can be combined with the buffer, such as 1 M Tris HCl, pH 7.0 or 0.5 M PIPES, pH 6.7. For sources of slow release or dissolution acid (d-gluconolactone), a given weight of the powder is dissolved in water at a selected time briefly before use (eg, one minute before mixing). A pre-weighed mixture of sterile, dry powders, from the source of the acid and the calcium salt can also be used to simplify the process.

Solvent. The solvent may be aqueous, non-aqueous or mixtures thereof. Examples of non-aqueous solvents are dimethyl sulfoxide, dimethyl formamide, glycerol, polyethylene glycols, Pluronics® and so on.

Gelification The gelation of the polymer-drug mixture can be initiated by adding the calcium salt. The temperature of the mixture, and the amount and particle size of the salt can be used to control the rate of gelation. If a proton donor is additionally used, the gelation of the mixture can be initiated by adding either 1) calcium salt and acidified buffer suspension (jointly or separately), 2) calcium salt suspension followed by fresh solution / suspension of the slow release proton source or vice versa; or 3) calcium salt and proton source powder together or separately. After the addition of the calcium salt, other precipitating agents / excipients (eg, zinc salts, buffers, etc.) can be added to the mixture. After rapid and complete mixing, the gel mixture can be extracted into a syringe before it gels.

Loading of the gel. In general, the loading of the protein is already known. Unknown gel fillers can be determined as follows.

Method by Pop. Approximately 0.1 - 0.2 ml (taking the exact weight) of the gel is emptied into an Eppendorf tube, then dissolved in 1 ml of 0.01M sodium citrate. The mixture is incubated at room temperature with gentle agitation until the gel disintegrates (generally 2 hours until overnight). After the resulting suspension is centrifuged at 8K rpm for 2 minutes (Eppendorf, 5415 C) the absorbance at 280 nm of the supernatant is taken. Any residual solids are dissolved in 1 ml of 7M urea, the absorbance of this solution is recorded. From these absorbencies, the milligrams of the protein per gram of the gel can be calculated.

Cumulative method. This method is used in conjunction with in vitro release studies. The amount of protein released from the gel including the burned or fragmented at the end of the study, is totalized. For details, see the section on In Vitro Release Studies.

In Vitro Release Studies. The gel is formed either in an Eppendorf tube ("melted" samples) or in the syringe then emptied into the Eppendorf tube ("emptied" samples). In general, approximately 0.1-0.2 ml of gel are melted or extruded (exact exact amount). The release is started by adding a buffer solution (10 M Tris HCl, pH 8 for leptin, pH 7.4 for GCSF) to each tube and placing it in an incubator shaker (New Brunswick Scientific) at 37 ° C and 100-200 rpm. At selected time intervals, the sample is removed from the incubator. If the gel is intact, the supernatant is removed and centrifuged (Eppendorf, 8000 rpm, 2 minutes) and the supernatant is collected. Any solids are suspended in 1 ml of fresh Tris buffer and returned to the original release tube to resume the release. If the gel is extensively altered, the contents of the tube are centrifuged, the supernatant collected and the solids resuspended in 1 ml of the buffer to resume the release. The "time points" of the supernatant may require additional centrifugation (8000 - 13, 000 rpm, 8 minutes) to rinse them for UV scanning. The amount of protein released is determined from the absorbance of the supernatant. After the final release sample is taken, the amount left in the bead or bead is determined by the Burst Method (above). The percent released in a given time of the released cumulative protein, expressed as a fraction of either the original protein load (known from the weight of the gel and how it is formulated) or the final total protein released (including the burst) or final urea-citrate fractionation).

In Vivo Studies. Weight Loss in Mice. Six to eight week old female mice of type C57 / BLC are obtained from Charles River and Taconic Inc. They typically weigh 20 grams. Each group of a dose consists of five mice. The injections are subcutaneous.

Study of the PK of the Rat. Male rats are used in this study and they typically weigh 250-300 grams. The injections are done in a manner similar to that described in the mouse weight loss experiments. Blood is sampled by catheter collection at various time intervals after injection and the samples are analyzed to verify leptin by an ELISA assay.

EXAMPLES The following examples are offered to more fully illustrate the invention, but will not be proposed to limit the scope thereof. In addition, with respect to the above description or the subsequent examples, one skilled in the art will be able to make the necessary changes to the descriptions for large-scale production.

Example 1 This in vivo example shows that leptin is a delayed gel induced by the buffer solution, is active and exhibits a sustained release compared to a leptin solution. This example also illustrates a system that gels after injection into the animal. A sterile calcium carbonate suspension is prepared from the sifted solids of less than 75 microns in particle size. A premix of this with Tris pH 7 is added to the leptin in alginate (in 10 mM Tris pH 8) in such a way that the final concentrations of the ingredients were: 2% alginate (sterile filtrate), 10 mg / ml leptin, 7 mM Tris pH 8, 150 mM Tris pH 7 and 24 mM CaC03 (assuming a complete solution). Such a mixture gelled in the range of eight to nine minutes. When the leptin was omitted, the time of gelation was slightly longer (10-12 minutes). This allowed time to load the syringes. The delayed gel formulation was injected (while not yet gelled) in a group of mice on alternate days at 100 mg / kg / day (2 day value at 50 mg / kg). A second group was injected with leptin solution (in Tris buffer solution at 10 mg / ml) daily at 50 mg / kg and a third group received the leptin solution on alternative days at 100 mg / kg. The mice were weighed daily and the change in weight was expressed as a percentage of the initial weights of the mice. The dosing schedule of alternative days with the delayed gel provided almost the same weight loss as the leptin solution injected daily (8-9%). In contrast, the solution administered on alternate days showed a smaller weight loss (6%) of shorter duration. This last group returned to its baseline weight at 8 days, although the other groups regained their original weights at 10-11 days.

Example 2 This example shows that the production of a delayed gel of calcium alginate induced by the buffer solution can be prolonged in the sustained release in vitro compared to the ungelled formulations. The calcium carbonate suspension was prepared as a suspension of 100 mg / ml of a very fine powder. A premix of this with the buffer solution PIPES of pH 6.7 was added to a suspension of zinc leptin in alginate (Tris buffered to pH 8) that was generated from a concentrated leptin solution (83 mg / ml in Tris pH 8 in the time when the zinc was added). One mi of such mixture was melted or emptied into a 10 ml laboratory beaker. The final concentrations of all the ingredients were: 2% alginate (from the 10% solution treated in Keltone LVCR autoclave), 15 mM Tris pH 8, 50 mg / ml leptin, 1 mM ZnC12, 10 mM CaC03 (assuming complete dissolution) and 93 mM PIPES pH 6.7. In the absence of leptin, such a mixture gelled at 7 minutes. After gelling all night, pieces of 0.2 g of the gel were weighed and placed in twinkling vials for the release, in the agitator at 37 ° C. This release was compared to the release from the same formulation without calcium and PIPES (no 'calcium alginate gel). When the thickened formulation of zinc alginate and leptin (ungelled) was released, the time point of 5 hours was 75% and there was a 90% gradual release over the following ~ 3 days. However, zinc leptin in the delayed gel of calcium alginate exhibited a much more sustained release - - 35% at 5 hours, 60% in a day, then a more gradual release at 70% in 5 days.

Example 3 This example shows that a gel induced by d-gluconolactone containing leptin as a fine zinc precipitate, sustains the release of leptin. Leptin (~ 14 mg / ml in Tris pH 8) was added to a PIPES solution of pH 6.7 and a solution of ZnC12 was immediately mixed in, followed quickly by the alginate solution (autoclaved 10%) of So that the final concentrations of the ingredients were 1.1 M ZnC12, 2.2% alginate, 10 mM Tris (pH 8) and 22 mM PIPES (pH 6.7). This mixture was concentrated by centrifugation until the leptin level was 46 mg / ml. The gel mixture is then made by stirring in a suspension of CaC03 (fine powder) followed rapidly by a solution of d-gluconolactone. The final concentrations of the components were 2% alginate, 20 mM PIPES, 9 mM Tris, 1 mM ZnC12, 16 mM (assuming complete dissolution) CaC03 and 79 mM d-gluconolactone. The mixture is extracted in syringes before gelation, and gelation occurred in the syringes after 10 minutes. After storage overnight (3 hours at room temperature and then at 4 ° C), the gels were injected into the mice. The weight loss was verified in comparison with the control of the buffer solution. A five-day value of leptin was injected at 50 mg / kg / day, ie, a bolus of 250 mg / kg in the gel, and compared with the same bolus of free leptin in solution. The bolus of free leptin showed only a modest maximum weight loss of 4.4-4.8% at 2 to 3 days and started gaining weight on day 5. On the contrary, at 2 and 4 days, zinc leptin on the gel produced a weight loss of 9%. At five days, the weight loss for the gel was still 5%, and remained above the baseline (sustained weight loss) when the experiment ended at 7 days.

Example 4 This example shows that if the protein concentration of the alginate-leptin-zinc mixture is sufficiently high, the mixture forms a gel in the absence of calcium exhibiting a sustained release. Leptin (~ 14 mg / ml in Tris pH 8) is added to a buffer solution and a solution of ZnC12 was immediately mixed in, followed quickly by the alginate solution (10% autoclaved) in such a way that the concentrations Final ingredients were 1.1 mM ZnC12, either 1.1 or 2.2% alginate, 10 mM Tris (pH 8) and 22 mM PIPES pH 6.7 (if present). This mixture is concentrated by centrifugation until the desired concentration of the leptin solids is reached. The formulation of ~ 50 mg / ml had PIPES and 2.2% alginate. The formulation of ~ 100 mg / ml had no PIPES and 1.1% alginate. The final concentrations of the 50 mg / ml components of the calcium gel of Example 3 were 2% alginate, 20 mM PIPES, 9 mM Tris, 1 mM ZnC12, 16 mM (assuming complete dissolution) CaC03 and 79 mM d-gluconolactone. For the 100 mg / ml calcium gel, the formulation was similar except that the final concentrations of the components were 1% alginate, PIPES was omitted, 13 mM CaC03 and 67 mM d-gluconolactone. For the gels with calcium, the samples were extracted in syringes before gelation, and gelation occurred in the syringes after 10 minutes. Where calcium was not included in the gel, the Zn-leptin-alginate mixture was extracted into syringes and allowed to gel. It seems that the 50 mg / ml formulation without calcium did not gel. After storage overnight (3 hours at room temperature and then 4 ° C), the gels were injected into the mice. The weight loss was verified in comparison with the control of the buffer solution. The five-day value of leptin was injected at 50 mg / kg / day, that is, one bolus of 250 mg / kg in the gel. The calcium gels of Example 3 produced a weight loss of 9% within the range of 2-4 days; after five days, the weight loss for the gel was still at 5%, and remained above the baseline (sustained weight loss) when the experiment ended at 7 days. In comparison, the 50 mg / m formulation without calcium produced a 3% weight loss during days 2 to 4, however, the weight loss went to zero on day 5. In contrast, the leptin formulation of 100 mg / ml without calcium was at least as active as the leptin formulation of 100 mg / ml with calcium. The maximum weight loss for the 100 mg / ml gel, without calcium, was 9% on day 4, the maximum weight loss for the 100 mg / ml gel, with calcium, was 6%, also in on day 4. Both of the formulations start gaining weight on day 9.

Example 5 This example shows that sustained in vitro release of leptin from a delayed alginate gel can be prepared without first forming a fine leptin-zinc precipitate. Leptin (100 mg / ml, 10 mM Tris HCl, pH 8.8, pH adjusted from 8.0 to 8.8 with 1M NaOH) and 6% alginate (10 mM Tris HCl, pH 8.6) were cooled on an ice bath. Leptin (0.5 ml) was added to the 6% alginate (0.18 ml) and the mixture was stirred on an ice bath for 10-15 minutes; the final pH was 8.6-8-8. To this mixture is added a suspension of 1M CaCO3 (16 mcl) and the resulting suspension is stirred well. To this suspension is added dropwise, with stirring, a 0.1 M ZnC12 solution (100 mcl); the water was then added to bring the volume to 1 mi. The slurry mixture was mixed thoroughly and kept in an ice bath for 10-20 minutes. Then a 1.68M solution of d-gluconolactone (56 mcl) is completely stirred in this mixture. An amount of 0.1 ml of the final mixture (50 mg / ml leptin, 1% alginate) is emptied onto the inside of an Eppendorf tube and left overnight at 4C. After storage overnight an in vitro release in 10 mM histidine, pH 7.4, was carried out. The cast or melted gel exhibited a small amount of burst or rupture and a fairly constant release of leptin with 50% released in 6 days.

Example 6 This example shows that a leptin containing a gel induced by d-gluconolactone as a fine zinco precipitate, produces a more sustained release of leptin than the same suspension of zinc in the alginate that is not gelled. The gel with zinc leptin was prepared as in Example 3. A suspension of ungelled zinc leptin in alginate is prepared in the same manner, except that CaC03 and d-gluconolactone were omitted. The mice were dosed with bolus injections of 250 mg / kg and the weight loss experiment was done as described in Example 3. The ungelled suspension produced only a 3% by weight loss on days 2-4, while the gelled suspension led to approximately a 9% weight loss during the same period. The weight loss for the ungelled suspension returned to the baseline on day 5, while the weight loss for the gelled suspension remained above the baseline at seven days, when the experiment ended.

Example 7 This example shows zero order release characteristics in a pharmacokinetic / pharmacodynamic study in male rats, demonstrating both sustained release and simultaneous sustained effect of leptin (i.e., weight loss) provided by the gel composition described in FIG. Example 3. The concentration of leptin was 47 mg / ml, and pre-gelled in the syringe as described in Example 3. The rats were provided with a bolus dose of 0 mg / kg (control), 50 mg / kg and 250 mg / kg, then checked blood levels and weight loss for six days. The leptin-zinc precipitate without the gel was also injected into the rats at a dosage of 100 mg / kg. The high-dose leptin gel group exhibited a permanent blood level of ~2000 ng / ml throughout the period, while the lower-dose leptin gel group had a level of ~ l / 5 than the level for the four days. In contrast, leptin without the gel group exhibited a blood level of 2300 ng / ml that was maximized at 12 hours and then reduced by a factor of 100 during the same time period. The weight of the rat (against vehicle control) was gradually reduced during this period for the high-dose leptin gel group. The group weight of the leptin dosage gel followed the same course for almost 5 days; at this point, the leptin blood level of the lower dose group decreased. The bioavailability of the leptin drug was evaluated by comparing the normalized areas of the dose under the curve of the leptin gel formulation, the gel formulation without leptin and leptin administered intravenously. The bioavailability of the leptin gel formulation was 80% compared to a bioavailability of 63% of the gel formulation without leptin.

Example 8 This example shows the sustained in vitro release of G-CSF from delayed gel vehicles. Both "cast" and "extruded" materials are exemplified. The G-CSF in water of pH 3.4 was mixed with concentrated alginate (10%) to form a viscous, cloudy suspension. This mixture was gelled with CaC03 and d-gluconolactone, at 16 mM and 79 mM, respectively. Prior to gelation, the aliquots of the material were either melted or cast into tubes or extracted into syringes. After 1 hour at room temperature the gels were stored at 4 ° C (overnight). Before performing the release study, the gel in the syringes were extruded into tubes and allowed to harden for 20 minutes. Then a release buffer solution was added. The extruded gel exhibited a release of GCSF over a period of three days, i.e. 11% at one hour, 35% at one day, 75% at two days and ~ 90% at three days. The cast or cast gel released 22% at 1 hour, 80% at one day and 94% at two days.

Example 9 This example demonstrates the sustained in vitro release of leptin from a delayed alginate gel prepared from the calcium salt (CaS04) without the addition of a proton donor. Unbuffered leptin at pH 8 was used to form a zinc-leptin suspension in 2% alginate. The final concentrations of ZnCl2 and leptin were 1 M and 55 mg / ml, respectively. The fine CaS0 powder was mixed with the zinc-leptin-alginate suspension such that the final mixture was 10 mM in CaSO4. The aliquots of the material were emptied onto the walls of the eppendorf tubes. The mixture gelled at 4-5 minutes. After storage overnight at room temperature, a release study was carried out. The release of protein from the gel exhibited a low burst (< 5%) at 1 hour. In one day, 11% of the leptin was released. The release of leptin was gradual, 1.1% per day, for the following seven days.

Example 10 This example demonstrates sustained in vitro release of leptin from a delayed alginate gel prepared from a calcium salt in a non-aqueous solvent without the addition of a proton donor. A lyophilized powder of leptin is suspended in 2% alginate in dry dimethyl sulfoxide. A fine powder of calcium oleate is mixed with the alginate-leptin suspension in such a way that the final mixture is 10 mM in calcium. The aliquots of the material are emptied onto the walls of the test tubes. The mixture gels over the course of time. After storage overnight at room temperature, a release study is carried out. The release of the protein from the gel is gradual and sustained, during the following 7 days.

Example 11 This example demonstrates the sustained in vivo release of leptin from an alginate gel prepared from a calcium salt as described in Example 10. A weight loss study in mice is performed after a single injection of This gel contains leptin at 250 mg / kg. Weight loss is measured over several days.

Example 12 This example demonstrates sustained in vitro release of G-CSF from a delayed alginate gel prepared from a calcium salt in a non-aqueous solvent without the addition of a proton donor. A lyophilized powder of G-CSF is suspended in 2% alginate in dry dimethyl sulfoxide. A fine powder of the calcium oleate is mixed with the alginate-G-CSF suspension in such a way that the final mixture is 10 mM in calcium. The aliquots of the material are emptied onto the walls of the test tubes. The mixture gels over the course of time. After storage overnight at room temperature, a release study is carried out. The release of protein is gradual and sustained.

Example 13 This example shows the sustained in vitro release of the unanimously approved interferon, as described in U.S. Pat. No. 4,695,623, supra, from the delayed alginate gels. The water, the ZnCl2, the Tris buffer and the consensus interferon (in 10 mM Tris pH 7.5) are mixed with the alginate solution, then mixed with CaC03, and d-gluconolactone, in such a way that the final concentrations of the components were 1 mg / ml of the unanimously approved interferon, 10 M of ZnCl2, 1% of alginate, 20 mM of Tris, 10 mM of CaC03 and 40 mM of d-gluconolactone. The mixture is emptied onto an eppendorg tube (0.4 ml per tube), gelled at room temperature and stored overnight at 4 ° C. After storage overnight an in vitro release was performed in 10 mM histidine buffer, pH 7.4. The melted or cast gel exhibited a small initial burst or crack. The percent of the release was 3% in 1 hour, 14% in a day. For 4 days, 70% was released. At 5-6 days, the release rate was reduced to < 5% per day.

Example 14 This example shows that delayed alginate gels can be used for sustained release of consensus interferon. An alginate consensus interferon delayed gel was prepared according to example 13 except that the final concentrations were as follows: 0.2 mg / ml consensus interferon, 10 mM ZnCl2, 2% alginate, 20 mM Tris, 10 mM CaCO3 and 40 mM of d-gluconolactone. Another formulation was prepared with the same composition, except that the final concentration of interferon of unanimous approval was 1 mg / ml. The mixtures were extracted in syringes and gelled after 2 hours. The 0.2 mg / ml formulation was injected subcutusly at 1 mg / kg and the formulation at 1 mg / ml at the doses of 1 mg / kg and 5 mg / kg in male Syrian hamsters. The blood was collected by cardiac puncture and evaluated to verify the unanimously approved interferon to observe the sustained release of the drug.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (50)

1. A delayed sustained release gelation composition, characterized in that it comprises: a) a hphilic polymer, b) a biologically active agent, and c) at least one bound polyvalent metal ion.

2. The sustained release composition according to claim 1, characterized in that it further comprises (d) at least one proton donor capable of releasing the bound polyvalent metal ion.

3. The sustained release composition of claims 1 or 2, characterized in that the bound polyvalent metal ion is a mixture of the bound and unattached polyvalent metal ion.

4. The sustained release composition according to claim 1 or 2, characterized in that it further comprises excipients for stabilizing the biologically active agent or the hphilic polymer.

5. The composition according to claim 1 or 2, characterized in that the bound polyvalent metal ion is a salt selected from the group consisting of acetates, phosphates, lactates, tartrates, citrates, chlorides, sulfates, carbonates, hxide or fatty acid anions of the same.

6. The composition according to claim 5, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum or zinc.

7. The composition according to claim 6, characterized in that the metal ion is calcium.

8. The composition according to claim 1 or 2, characterized in that the hphilic polymer is a polyanion.

9. The composition according to claim 1 or 2, characterized in that the hphilic polymer is a polysaccharide.

10. The composition according to claim 9, characterized in that the polysaccharide is an acid polysaccharide.

11. The composition according to claim 10, characterized in that the polysaccharide is alginate.

12. The composition according to claim 11, characterized in that the alginate contains at least 30% guluronic acid.

13. The composition according to claim 11, characterized in that the alginate consists of at least 0.05% by weight.

14. The composition according to claim 1 or 2, characterized in that the biologically active agent comprises a protein.

15. The composition according to claim 14, characterized in that the protein consists of at least 0.001 mg / ml.

16. The composition according to claim 14, characterized in that the protein is selected from the group consisting of hematopoietic factors, colony stimulating factors, anti-obesity factors, growth factors, trophic factors, and anti-inflammatory factors.

17. The composition according to claim 14, characterized in that the protein is selected from the group consisting of leptin, G-CSF, SSCF, BDNF, GDNF, NT3, GM-CSF, IL-lra, IL2, TNF-bp, MGDF, OPG, interferons, erythropoietin, KGF, insulin and analogues and derivatives thereof.

18. The composition according to claim 1 or 2, characterized in that the biologically active agent is a biologically active agent in the form of a complex.

19. The composition according to claim 18, characterized in that the biologically active agent in the form of a complex is a precipitated protein.

20. The composition according to claim 19, characterized in that the precipitated protein is a zinc leptin precipitate.

21. The composition according to claim 2, characterized in that the proton donor is from an acid source.

22. The composition according to claim 21, characterized in that the acid source is selected from the group consisting of buffer solutions, esters, slowly dissolving acids or lactones.

23. A method of producing a delayed sustained release gelation composition, characterized in that it comprises the steps of: a) mixing a biologically active agent and a hphilic polymer in a solvent to form a first mixture; and b) mixing with the first mixture at least one polyvalent metal ion attached to form a second mixture.

24. The method according to claim 23, characterized in that it further comprises the step of c) mixing with the second mixture at least one proton donor capable of releasing the bound polyvalent metal ion.

25. The method according to claim 23 or 24, characterized in that the polyvalent metal ion is a salt selected from the group consisting of acetates, phosphates, lactates, citrates, sulfates, tartrates, chlorides, carbonates, hydroxides or fatty acid anions of the same.

26. The method according to claim 25, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum or zinc.

27. The method according to claim 26, characterized in that the metal ion is calcium.

28. The method according to claim 23 or 24, characterized in that the hydrophilic polymer is a polyanion.

29. The method according to claim 23 or 24, characterized in that the hydrophilic polymer is a polysaccharide.

30. The method according to claim 29, characterized in that the polysaccharide is an acid polysaccharide.

31. The method according to claim 30, characterized in that the polysaccharides is alginate.

32. The method according to claim 31, characterized in that the alginate contains at least 30% guluronic acid.

33. The method according to claim 31, characterized in that the alginate consists of at least 0.05% by weight.

34. The method according to claim 23 or 24, characterized in that the biologically active agent comprises a protein.

35. The method according to claim 34, characterized in that the protein consists of at least 0.001 mg / ml.

36. The method according to claim 34, characterized in that the protein is selected from the group consisting of hematopoietic factors, colony stimulating factors, anti-obesity factors, growth factors, trophic factors, and anti-inflammatory factors.

37. The method according to claim 34, characterized in that the protein is selected from the group consisting of leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, IL-lra, IL2, TNF-bp, MGDF, OPG, interferons, erythropoietin, KGF, insulin and analogues and derivatives thereof.

38. The method according to claim 23 or 24, characterized in that the biologically active agent is a biologically active agent in the form of a complex.

39. The method according to claim 38, characterized in that the biologically active agent in the form of a complex is a precipitated protein.

40. The method according to claim 39, characterized in that the precipitated protein is a zinc leptin precipitate.

41. The method according to claim 23 or 24, characterized in that it further comprises the step of isolating the sustained release composition.

42. The method according to claim 24, characterized in that the proton donor is from an acid source.

43. The method according to claim 42, characterized in that the acid source is selected from the group consisting of buffer solutions, esters, slowly dissolving acids or lactones.

44. A sustained release composition, characterized in that it is produced by the method of claims 23, 24 or 41.

45. A pharmaceutical formulation, characterized in that it comprises the sustained release composition according to claims 1 or 2 in a carrier, diluent or pharmaceutically acceptable adjuvant.

46. The pharmaceutical formulation according to claim 45, characterized in that the formulation is in a syringe.

47. A method of treating an indication with a sustained release composition according to claims 1 or 2 in a carrier, diluent or pharmaceutically acceptable adjuvant.

48. A method of treatment of a disorder selected from the group consisting of excess weight, diabetes, elevated blood lipid level, arterial sclerosis, arterial plaque, reduction or prevention of renal stone formation, insufficient lean tissue mass, Insufficient sensitivity to insulin, and attacks, with a sustained release composition according to claim 1 or 2 in a carrier, diluent or pharmaceutically acceptable adjuvant, wherein the biologically active agent is leptin, an analog or derivative thereof .

49. A method of treating a disorder selected from the group consisting of hematopoietic cell deficiencies, infection, and neutropenia with a sustained release composition according to claim 1 or 2 in a carrier, diluent, or pharmaceutically acceptable adjuvant, wherein the agent biologically active is G-CSF, an analog or derivative thereof.

50. A method of treating inflammation with a sustained release composition according to claim 1 or 2, in a carrier, diluent, or pharmaceutically acceptable adjuvant, characterized in that the biologically active agent is IL-lra, an analogue or derivative of the invention. same.