MXPA99009388A - Sustained-release alginate gels - Google Patents

Abstract

The present invention relates to sustained-release formulations using alginate gel beads and methods thereof.

Description

ALGINATQ GELS OF SUSTAINED RELEASE CA PO ^ OF THE INVENTION The present invention relates to sustained release formulations using alginate gel beads and the methods thereof. ~ 7_ BACKGROUND With the advances in genetics and cellular engineering technologies, the availability of recombinant proteins has generated advances in the use of proteins as "Medicines for therapeutic applications." Many diseases or conditions treated with pharmaceutical proteins require sustained levels of protein. To achieve the most effective therapeutic result, however, as with most protein drugs, the generally short biological half-life requires frequent administration.These repeated injections are given at various intervals which result in fluctuating drug levels. at a significant physical and monetary cost to patients Since many conditions respond better to controlled levels of a drug, there is a need for controlled release of a drug to provide longer periods of time.

REF .: 31344 consistent release. Such sustained release drugs would provide the patient with not only better prophylactic, therapeutic or diagnostic effects, but also a decrease in the frequency of injections as well as in total costs. Current attempts to sustain drug levels in humans or animals between doses by using biodegradable polymers as matrices to control drug release. For example, Great Britain Patent No. 1,388,580 describes the use of hydrogels for sustained release of insulin. U.S. Patent No. 4,789,550 discloses the use of alginate microcapsules coated with polylysine for the release of proteins by encapsulation of living cells. Sustained release attempts have also utilized anionic or cationic polymeric compositions surrounded by ionic polymers of opposite charge to encapsulate cells capable of producing biologically active compositions. U.S. Patent No. 4,744,933. Likewise, multiple coatings of anionic or cationic crosslinked polymers have also been described as means to obtain controlled release. U.S. Patent Nos. 4,690.6 > 82 and 4,789,516. Further, "further attempts describe the use of alginates alone, or alginates coated with biodegradable polymeric compounds, for the controlled release of polypeptide compositions or cationic precipitates thereof PCT WO 96/00081, PCT WO 95/29664 and PCT WO 96/03116 Those attempts, however, have proven to be insufficient means to obtain the sustained release of the desired protein drug It is generally known that the use of certain biodegradable polymers, for example, co-glycolide poly-actide, In vivo conditions, exhibits high initial injections of drug release Johnson, O. et al., Nature Med /, 2/7: 795 (1996) In addition, it is generally known that proteins used with current formulas The sustained release preparations can undergo denaturing and lose their bioactivity after exposure to encapsulating agents. which can have harmful effects on the protein of choice. Finally, as discussed below, the use of alginate alone has not proven the controlled release of desired protein necessary for effective therapeutic results. In general, alginates are natural, well-known, anionic polysaccharides, comprised of 1,4-en-aza-D-mannuronic acid and a-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 vary from 70% mannuronic acid and 30% guluronic acid, 30% mannuronic acid and 70% guluronic acid. Smidsrod, supra. Alginic acid is insoluble in water whereas salts formed with monovalent ions such as sodium, potassium and ammonium are soluble in water. McDo Ell, R.H., "Properties of Alginates" (London, Alginate Industries Ltd, 4th edition 1977). It is known that polyvalent cations react with alginates to spontaneously form gels. Alginates have a wide variety of applications such as food additives, adhesives, pharmaceutical tablets 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 for drug delivery systems have also been documented, see for example, U.S. Patent No. 4,695,463 which discloses a chewing gum delivery system and pharmaceutical preparations based on alginate. Alginate beads have been used for the controlled release of various proteins such as: tumor necrosis factor receptor in cationic alginate beads coated with polycations, Wee, S. F. Proceed. Intern. Symp. Control. I laughed Bioact. Mater., 21: 730-31 (1994); transformation of the growth factor encapsulated in alginate beads, Puolakkainen, P. A. et al. , Gastroenterology, 107: 1319-1326 (1994); angiogenic factors trapped in calcium alginate beads, Downs, E.C. et al, J. of Cellular Physiology, 152: ~ 422-429 (1992); albumin "entrapped in chitosan alginate microcapsules, Polk, A. et al., J. Pharmaceutical Sciences, 83/2: 178-185 (1994), or chitosan calcium alginate beads coated with polymers, Okhamafe, AO et al. al., J. Microencapsultation, 13/5: 497-508 (1996); hemoglobin encapsulated with calcium chitosan-alginate beads, Huguet, ML et al., J Applied Polymer Science, 51: 1427-1432 (1994), Huguet, ML et al., Process Biochemistry, 31: 745-751 (1996), and interleukin 2 encapsulated in alginate-chitosan microspheres, Liu, LS et al., Proceed, Intern Symp. Control, Reí. Bioact. , 22_: 542-543 (1995) Systems using alginate gel beads, or alginate / calcium gel beads to trap proteins suffer from deficiencies of any sustained release effect due to the rapid release of the protein from the proteins. alginate beads, Liu, LS et al., • Control, Re., 43: 65-74 (1997) .To avoid such rapid release], a number of previous systems intended to use polycationic polymer coatings (eg, polylysine, chitosan) to retard the release of the alginate beads of protein. See, for example, Wheatley, M.A. et al. , J. Applied Polymer Science, 43: 2123-2135 (1991); Wee, S. F. et al. Supra; Lu, 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 pearl. The problems that can occur with the use of polycations are: (1) such formulations can be cytotoxic due to polycations (Huguet, ML et al., supra, Zimmermann, Ulrich, Electrophoresis, 13_: 269 (1992), Bergmann, P. et al., Clinical "Science, 61_: 35 (1984)); (2) polycations are prone to oxidation; (3) pearls with polycationic coatings tend not to be erodible and accommodated in the body; (4) such formulations are made via laborious coatings procedures , which include the multiple elements of polycationic polylysine (Padol, et al., Proceed, Intern Symp., Control, Rei, Bioact, Mater, 2: 216 (1986) and (5) ionic interactions. between protein and polycations can result in loss of protein activity or cause protein instability. Accordingly, there is a need to develop pharmaceutical formulations that achieve better sustained release media for clinical applications. Numerous recombinant or natural proteins could benefit from constant long-term release and, therefore, provide more effective clinical results. The present invention provides such advances. The pharmaceutical compositions of the present invention are capable of providing protection to the protein, decreasing degradation and a slow release with greater stability and potency of the protein. Also, the pharmaceutical compositions of the present invention provide simple, rapid and cheap means of controlled release of recombinant protein for effective prophylactic or diagnostic prophylactic results.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to sustained release formulations using beads or alginate gel particles, and methods for the same.

In particular, the formation of sustained release gels includes the coprecipitation of alginate gel beads with a biologically active agent. This method provides the advantage of producing a high and efficient load of biologically active agent within the gel of the alginate for sustained release, achieving at the same time the protection of the protein, less degradation, an increase in the stability and potency of the agent to be released. * Accordingly, an aspect of the present invention provides a sustained release agent comprising a hydrophilic polymer; a biologically active agent, and at least one precipitating agent During the formulation of the composition, the biologically active agent is co-precipitated with the hydrophilic polymer.In addition, precipitating agents may also be added to the composition. The term coprecipitation refers to the use of agents for the precipitation of the biologically active agent together with the hydrophilic polymer to form a matrix of the precipitated polymer and agent, for example, the formation of the alginate beads would be via precipitation. The precipitation of the molecules and any related precipitating agents is well known to those skilled in the art, Another aspect provides the methods for producing the sustained release compositions of the present invention. biologically active agent and a po hydrophilic polymer with a solvent to form a first mixture; dissolving at least one precipitating agent in a solvent to form a second mixture; adding the biologically active agent and the hydrophilic polymer solution of the first mixture with the precipitating agent and the solvent of the second mixture; and co-precipitating the biologically active agent within the hydrophilic polymer. The methods of the present may also include the use of additional precipitating agents. In addition, a step to isolate the sustained release composition was also contemplated. As used herein, the term "solvent" refers to water-based solvents capable of dispersing or dissolving the biologically active agents, hydrophilic polymers or precipitating agents of choice. Such solvents are well known to those skilled in the art. In addition to the first mixture with the second mixture to form the coprecipitation composition, it can be done by methods well known to those skilled in the art, including, but not limited to, droplet addition, dispersion, spray or mixing by the use of Spray jets, air jets, atomization and electric fields. The term dispersion, for the purposes of this invention, may mean liquid, solid or aqueous dispersions. As used herein, the term "isolation" refers to the process for the isolation of the sustained release composition of the present invention. Such isolation and purification procedures are well known in the art. In still another aspect, the present invention provides a sustained release composition produced by the above methods. Additional aspects include pharmaceutical formulations of the above compositions and a pharmaceutically acceptable carrier or adjuvant. In another aspect, the. present invention provides indications for methods of treatment with sustained release compositions containing the desired biologically active agents.

DETAILED DESCRIPTION OF THE INVENTION Compositions Hydrophilic polymers include alginates derived therefrom, can be obtained from various commercial sources, natural or synthetic well known in the art. As used herein, the term "hydrophilic polymer" refers to water-soluble polymers or polymers that have affinity for absorbing water. Hydrophilic polymers are well known to those skilled in the art. Those include but are not limited to polyanions, including anionic polysaccharides such as alginate, carboxymethyl amylose, polyacrylic acid salts, polymethacrylic acid salts, maleic anhydride ethylene copolymer (half ester), carboxymethyl cellulose, dextran sulfate, heparin, carboxymethyl dextran, carboxy cellulose, 2,3-dicarboxylic cellulose, tricarboxylic cellulose, carboxy gum arabic, carboxy carrageenan, carboxy pectin, carboxy gum tragacanth, carboxy xanthan gum, ponsonsan polysulfate, carboxy starch, carboxymethyl chitin / chitosan, curdla * h, hexasulfate of inositol, ß-cyclodextrin sulfate, hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, heparin sulfate, carboxylmethyl starch, carrageenan, polygalacturonate, carboxy guar gum, polyphosphate, polyaldehyde-carbonic acid, poly-l-hydroxy l-sulfonate-propen-2, malecopolystyrene-maleic acid, agarose, mesoglican, polyvinyl sulfopropylated alcohols, cellulose sulfate Bear, 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 which are within the scope of the present invention. Likewise, precipitating agents can be obtained from various commercial, natural or synthetic sources, which are well known in the art. Precipitating agents include but are not limited to polyvalent metal ions, salts, acetals, 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 and magnesium, strontium and zinc. Preferably the metal ions are calcium and zinc or salts thereof, such as zinc acetate, calcium acetate or chlorine salts. Small water-soluble molecules and salts such as ammonium sulfate, acetone, ethanol and glycerol can also be used. As for water-soluble polymers, these include but are not limited to polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6. -trioxane, ethylene / maleic anhydride copolymers, polyamino acids, dextran, poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene 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 to facilitate the handling and efficiency of precipitation. Other sizes and types of precipitating agents may be used, depending on the desired therapeutic profile (for example, 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 precipitating agent for a therapeutic protein or analogue). One skilled in the art will appreciate other precipitating agents that are within the scope of the invention. ~~ As used herein, the term "buffer" or "buffer" refers to the use of inorganic or organic acids or a combination thereof to prepare a buffer solution as is known in the art. Inorganic acids within the scope of the present invention include the hydrogen halide (e.g., hydrochloric acid), phosphoric, nitric or sulfuric acids. Other inorganic acids are well known to one skilled in the art and were contemplated here. Organic acids within the scope of the invention include the aliphatic carboxylic acids and aromatic acids such as formic, carbonic, acetic, propionic, butyric, valeric, caproic, acrylic, malonic, succinic, glutaric, adipic, maleic, fumaric, glycine or phenol sulphonic. Other organic acids are well known to those skilled in the art. The preferred buffer of the present invention includes glycine buffer systems and glycine phosphoric acid. As used herein, "biologically active agent" refers to recombinant or natural proteins, either human or animal, useful for prophylactic, therapeutic or diagnostic application. The biologically active agent can be natural, synthetic, semi-synthetic or derivatives thereof. The biologically active agents of the present invention must be precipitable. A wide variety of biologically active agents were contemplated. These include but are not limited to hormones, cytokines, hematopoietic factors, growth factors, anti-obesity factors, trophic factors, antiinflammatory factors, and enzymes (see also U.S. Patent No. 4,695,463 for additional examples of useful biologically active agents). One skilled in the art will be able to easily adapt a desired biologically active agent to the compositions of the present invention. Such proteins include but would not be limited to interferons (see, U.S. Pat. Nos. ,372,808, 5,541,293, 4,897,471, and 4,695,623 incorporated herein by reference, including the drawings), interleukins (see, U.S. Patent No. 5,075,222, incorporated herein by reference, including the drawings), erythropoietins (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,008, incorporated herein by reference, including drawings), granulocytic colony stimulating factors (see, US Patents Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823 and PCT Publication No. 94/17185 incorporated here for reference, including the drawings), undifferentiated cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206 incorporated herein by reference, including the drawings), and the OB protein (PCT Publications Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 incorporated herein by reference, including the figures). In addition, biologically active agents may also include but are not limited to related anti-obesity products, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone ( FSH), human chorionic gonadotropin hormone (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), necrosis factor binding protein tumor (TNF-bp), brain-derived neurotrophic factor (BDNF), glial tissue-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 (IGF), macrophage colony stimulating factor (M-CSF), colony stimulating factor of the s granulocytic macrophages (GM-CSF), megakeratinocyte-derived growth factor (MGDF), thrombopoietin, platelet-derived growth factor (PGDF), colony-stimulating growth factors (CSF), bone morphogenetic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptococcus and kallikrein. The term "proteins", as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof. Derivatives of biologically active agents may include the attachment of one or more chemical moieties to the protein moiety. It has been found that chemical modification of biologically active agents provides additional advantages in certain circumstances, such as increased stability and circulation time of the therapeutic protein and decreased immunogenicity. A person skilled in the art will be able to select the desired chemical modification based on the desired dose, circulation time, resistance to proteolysis, therapeutic uses and other considerations.

Complexes; The proteins, analogs or derivatives can be administered complexed to a binder composition. Such a binding agent can have the effect of prolonging the circulation time of the protein, analog or derivatives or increasing the activity of the biologically active agent, such a composition can be a protein (or its synonym, peptide), derivative, analog or combination. For example, a "binding protein" or binding agent for the OB protein is a receptor for the OB protein or portion thereof, - such as a soluble portion thereof. Other binding proteins can be obtained by examining the OB protein, or the protein of choice, in the serum, or being selected empirically for the presence of binding or agglutination. Such binding or 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 affect signal transduction. In addition to the OB protein, binding complexes to other therapeutic proteins of the present invention will also be applied. Those skilled in the art will be able to determine the appropriate binding proteins for use with the present invention.

Pharmaceutical Compositions The sustained release pharmaceutical compositions of the present invention can be administered orally (for example, capsules such as hard capsules and soft capsules, solid preparations such as granules, tablets, pills, troches or lozenges, seals, beads, powders). and lyophilized forms, liquid preparations such as suspensions) and non-oral preparations (e.g., intramuscular, subcutaneous, transdermal, visceral, IV (intravenous), IP (intraperitoneal), intraarterial, intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal , rectal, and transmucosal uterine preparations). In general, sustained release pharmaceutical compositions comprising effective amounts of protein, or products thereof, with the sustained release compositions of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers are encompassed by the invention. acceptable for administration. See PCT 97701331 incorporated herein by reference. The optimal pharmaceutical formulation for a desired biologically active agent will be determined by one skilled in the art depending on the route of administration and the desired dose. Exemplary pharmaceutical compositions are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th Edition, _ Easton, PA, pages 1435-1712 (1990)). Components that may be required for administration include diluents that contain several buffers (e.g., Tris-HCl, acetate), pH and ionic strength; additives such as surfactant and solubilizing agents (e.g., Tween 80, HCO-60, Polysorbate 80), antioxidants (e.g., ascorbic acid, glutathione, sodium metabisulfite), additional polysaccharides (e.g., carboxymethylcellulose, sodium alginate, hyaluronate) sodium, protamine sulfate, polyethylene glycol), preservatives (eg, Thimersol, benzyl alcohol, methyl paraben, propyl paraben) and diluents (eg, lactose, mannitol); the -incorporation of the material into particulate preparations of polymeric compounds such as polylactic or polyglycolic acid polymers or copolymers, etc., or combined with liposomes. Hyaluronic acid can also be used as a delivery component and this can have the effect of further promoting the sustained duration in the circulation. Additionally, the sustained release compositions of the present invention may also be dispersed with oil (eg, sesame oil, corn oil, vegetable oil), or a mixture thereof with a phospholipid (eg, lecithin), or triglycerides of medium chain fatty acid (e.g., Migliol-812) to provide the oil suspension. The compositions of the present invention can 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 the pulmonary delivery of therapeutics, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, were 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 components of the administration may have an influence on the physical state, stability, in vivo release rate, and speed of in vivo elimination of the proteins and derivatives herein. The person skilled in the art will appreciate the appropriate administration components and / or the appropriate mechanical devices to be used depending on the therapeutic use, the route of administration, the desired dose, the circulation time, the resistance to proteolysis, stability of the protein and other considerations.

Methods of Therapeutic Use. The therapeutic uses depend on the biologically active agent used. One skilled in the art will be able to easily adapt a desired biologically active agent to the present invention for its intended therapeutic uses. The therapeutic uses of such agents are set forth in more detail in the following publications incorporated herein by reference, including the drawings. Therapeutic uses include, but are not limited to, uses for interferon-like genes (see U.S. Patent Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623 incorporated herein by reference, including drawings), interleukins (see, U.S. Pat. No. 5,075,222, incorporated herein by reference, including the drawings), erythropoietins (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080, incorporated herein by reference, including the drawings), stimulating factors of the granulocytic colony _ (see, U.S. Patent Nos. 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), undifferentiated cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206 incorporated herein by reference, including the drawings), and the OB protein (PCT Publications Nos. 96/40912, 96 / 05309, 97/00128, 97/01010 and 97/06816 incorporated herein by reference, including the figures). In addition, therapeutic uses of the present invention include uses of biologically active agents including, but not limited to, related anti-obesity products, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) ), follicle stimulating hormone (FSH), human chorionic gonadotropin hormone (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), protein of binding of tumor necrosis factor (TNF-bp), brain-derived neurotrophic factor (BDNF), "glial tissue-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 (IGF), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakeratinocyte-derived growth factor (MGDF), thrombopoietin, "platelet derived growth factor" (PGDF), stimulating growth factors of colonies (CSF), bone morphogenetic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptokinase and kallikrein. The term "proteins", as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof. In addition, the compositions herein may also be used to manufacture one or more medicaments for the treatment or alleviation of the conditions that the biologically active agent intends to treat. By way of example, therapeutic uses of blood oxygenation and a decrease in bone resorption or osteoporosis in the absence of weight loss can also be achieved. ~ Combined therapies The compositions and methods herein can be used in conjunction with other therapies, such as altered diet and exercise. Other medications, such as those useful for the treatment of diabetes (eg, insulin, and possibly amylin), medications to lower cholesterol and blood pressure (such as those that reduce blood lipid levels or other cardiovascular drugs) , drugs that increase activity (for example), amphetamines), diuretics (for fluid elimination), and appetite suppressants. Such administration can be simultaneous or it can be in seriatum. In addition, the methods herein may be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of a body (e.g., liposuction or laser surgeries designed to reduce body mass, or designed implant surgeries) to improve the appearance of body mass). The health benefits of cardiac surgeries, such as diversion surgeries or other surgeries designed to alleviate a harmful condition caused by blockage of blood vessels by fatty deposits, such as arterial plaques, can be increased with the concomitant use of compositions and methods of the present. Methods for removing gallstones, such as ultrasonic or laser methods, can also be used either before, during or after a course of the therapeutic methods herein. In addition, the methods herein may be used as adjuncts to surgeries or therapies for broken bones, damaged muscles or other therapies that could be improved by an increase in lean tissue mass. Dose A person skilled in the art will be able to determine the effective doses by administration and observe the desired therapeutic effect. The dose of the sustained release preparation is the amount necessary to achieve the effective concentration of the biologically active agent in vivo, for a given period of time. The preferred dose and frequency of administration of the sustained release preparations varies with the type of biologically active agent, the desired duration of release, the target disease, the frequency of administration desired, the subject animal species 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 doses can be determined using diagnostic tools over time. By way of example, the present invention provides the dose of OB protein. For example, a diagnosis can first be used to measure the amount of OB protein in the blood (or plasma or serum) to determine the endogenous levels of OB protein. Such a diagnostic tool may be in the form of an antibody assay, such as an antibody sandwich assay. The amount of endogenous OB protein is quantified initially, and the basal level is determined. Therapeutic doses are determined as the quantification of endogenous and exogenous OB protein (i.e., the protein, analog or derivative found within the body, either produced or administered) continues during the course of therapy. For example, a relatively high dose may initially be necessary until a therapeutic benefit is observed, and then lower doses are used to maintain the therapeutic benefits.

Methods of Preparation Preparation of Protein / Alginate Beads. A typical procedure is illustrated by the following example which uses the OB protein or leptin as the protein of choice. One skilled in the art will understand and be able to apply those methods to other biologically active agents.

Preparation of the Drip Mixture. The term "drip mixture" as used herein refers to the mixture containing the hydrophilic polymer and the biologically active agent. One mL of 5% alginate mixture (10 mM TRIS, pH 8), with magnetic stirring, is added to 4 mL of leptiria (100 mg / mL, TRIS 10 tmM, pH 8) in a 10 mL beaker (in an ice bath). The mixture becomes cloudy. Then 40 mcL of 4 mM NaOH is added to the mixture and stirring is continued for 15 minutes (on ice). The mixture is rinsed and its final pH is between about 8.6 to 8.8. The concentration of alginate should be at least 0.05% by weight. In addition, the alginate should preferably be at least 30% guluronic acid. In addition to the above, polyethylene glycol can be added to the dropping mixture as discussed below. Similarly, buffers or excipients are useful for the stability of the protein of choice. One skilled in the art will be able to determine the appropriate ingredients that should be added for stability purposes depending on the protein chosen for the release.

Preparation of the Bath Mix. The term "bath mixture" as used herein refers to the mixture containing the precipitating agents used for the coprecipitation of the biologically active agent and the hydrophobic polymer. The bath typically contains 10 mL of mixture "in a 50 mL beaker consisting of 100 mM CaC12 plus other ingredients (see below) .The pH is preferably acidic to help decrease the effect of inrush. preferably be less than pH 4. The - absorber in the bath will also depend on the protein used.An expert in the art will be able to adjust the buffer capacity or strength on the basis of the protein used.Depending on this mode of stability of the protein, if the buffer concentration is too high, for example, with G-CSF, the protein may appear less stable and the sustained release will decrease.The bath may be comprised of CaC12, ZnC12, polyethylene glycols ("PEG") and acidic buffers, zinc interacts with the protein, precipitating it, thus helping to increase the charge of the pearl, decreasing the effect of the inrush and slowing the release of the pearl protein. Calcium helps form the alginate precipitate and the formation of the pearl. Calcium also helps the shape of the pearl, especially if the bath is viscous due to the addition of other additives such as PEG. Calcium can be increased when you have a higher viscosity to help maintain the shape of the pearl. The zinc concentration should be at least 0.1 mM and the calcium concentration should be at least 10mM. The addition of PEG helps increase the load. It is known that certain PEGs precipitate proteins. PEG can also be added to the protein / alginate mixture that is dripped in the bath to help maximize the load and sustained release. The molecular weight of the PEG can range from 700 kDa to 1000 kDa, but preferably 700 Da-100 kDa. One skilled in the art will be able to determine the amount of PEG that must be added to the bath mixture but this can be as high as 99%, preferably less than 75% by weight. One skilled in the art will be aware that the concentration of PEG can be limited by the viscosity of the bath.

Preparation of the Pearl. In general, the drops of the leptin / alginate drip mixture are sprayed, submerged or dispersed in a bath mixture (as described above). further, electrostatic means can be used for the formation of the bead. To produce small beads, that is, less than a few hundred microns in diameter, a flow chamber (nozzle) consisting of a needle with a coaxial air flow is used. one gas line and the other door to a syringe (3 mL) used to mold (at approximately 1 mL / min the protein / alginate mixture in the bath.) Typically, 2 mL of the mixture is injected into 10 mL of the bath mixture. The nozzle is placed "approximately 0.8 cm from the top of the bath beaker. The size of the bead is determined mainly with the gas flow rate, for example, at a flow rate of 8 L / min the size of the bead fluctuates from 50-150 microns in diameter. The flow velocity of leptin / alginate has a much smaller effect on the size of the bead. For large beads (ie, 1-3 mm in diameter), a 1-circuit tuberculin syringe equipped with a 24G needle is used to drip the leptin / alginate mixture into the bath mixture. The bath typically contains 1.5% CaC12 and ZnCl2 from 5 to 50 mM. The pearls are collected by pouring them through a 40 micron nylon cell scrubber. The beads are rinsed on the scrubber with 5 mL of sterile water and gently dried from the bottom side of the scrubber with a household cloth (gamma 67 sweep). The beads are stored in a microtube with a sterile screw cap.

Pearl Charge Irruption Method: The drug loading of a selected group of beads is determined by accurately weighing 100 mg of the beads, hydrated, in 1 mL of 0.5M sodium citrate, pH 8.5. The bead suspension is incubated at room temperature until the beads disintegrate usually forming a precipitate. The suspension is centrifuged at 14K rpm for 2 minutes, (eppendorf, 5415 C). The supernatant is cooled and the absorbance recorded at 280 nm. The precipitate is dissolved suspended in 1 mL of 7M urea. The absorbance of this mixture is recorded. The protein load of the charged, hydrated beads is expressed as mg of protein per mg of beads or mg of protein per mL of beads and is determined from the sum of the two absorbances.

~ - Cumulative Method: This method is used in conjunction with in vitro release studies. The amount of protein released from the beads includes the irruptions at the end of the study that are totaled. For details, see below.

In Vitro Release Studies Weigh charged, hydrated beads (100 mg) in a 1.5 mL microcentrifuge tube (eppendorf) and add 1 mL of buffer (10 mM histidine, pH 7.4). the sample was placed in an incubator shaker at 37 ° C and 100-200 rpm. At selected time intervals, samples were removed from the incubator, centrifuged (eppendorf, 1000 rpm, 2 minutes) and the supernatant was removed and replaced with 1 mL of fresh buffer. The amount of protein released was determined from the absorbance of the supernatant. After having taken the final liberated sample, the amount left in the pearls was determined by the Pearl Loading / Irruption Method. The percent released at a given time was determined from the sum of the total protein released and the remaining in the beads at the completion of the experiment. - In Vivo Studies Weight Loss in Mice: In general, the mice are injected once with a suspension of the loaded pearls or unfilled pearls. Female mice of six to eight weeks of age (type C57 / BLC) are used, with a typical weight of 20 grams. In the case of the bead samples, 350 mcL of buffer (50 mM MES pH6.7) is added to 100 mg of hydrated beads and vortexed. The suspension is extracted in an Ice syringe and all the beads and 300 mcL of the buffer are injected (23G needle) subcutaneously into the neck of the mouse. The mice are weighed daily.

Rat Pharmacokinetic Study: Six to eight week old female rats (Sprague Dawley type) are used, with a typical weight of 250 grams. The injections are performed in a manner similar to that described in the weight loss experiments in mice. Blood is sampled by catheter collection at various time intervals after injection and the samples are analyzed for leptin by an ELISA assay.

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

EXAMPLE 1 This example examines the effect of the leptin concentration in the beads on the release of leptin from alginate beads co-precipitated with zinc / leptin. The small beads were prepared as described above using 25 mM ZnC12 in the bath. The pearl of higher concentration, that is, 66 mg / mL of leptin, was prepared using 84 mg / mL of leptin in 1% alginate while the one of lower concentration, that is, of 21 mg / mL of leptin, was prepared from 21 mg / mL leptin in 1% alginate. When the concentration of leptin in the pearl increases, the fractional release of leptin from the pearl decreases. For the highest concentration, 25% of the leptin is released at 80h, while for the lowest concentration, 80% is released at 80h.

EXAMPLE 2 This example examines the effect of the level of ZnC12 in the bath on the release of leptin from the alginate beads co-precipitated with zinc / leptin.The small beads were prepared "as described above but the level of ZnCl2 in the bath is at 0.5 and 25 mM and the concentration of leptin in the beads is 37 mg / mL (by the cumulative method). This example shows that the level of ZnC12 in the bath increases the resulting beads that have a lower inrush and a lower release rate of leptin. At 0.5 mM ZnCl2, the beads have an irruption of 20% and a release of 50% at 40 h; while at 25mM ZnC12 the pearls have an irruption of less than 5% and a release of 25% at 40h.

EXAMPLE 3 This example compares a coprecipitated alginate bead with zinc / leptin with a control acetate buffer formulation (20 mg / mL) in a combined pharmacokinetic / bioactivity experiment. The small beads contain 64 mg / mL of leptin (ie, per mL of beads) and were manufactured as described above with 17 mM ZnC12 in the bath. Female rats (with a body weight of 220g) were given a single SC injection (subcutaneous) at a dose of 50 mg / kg. The plasma concentrations of the bead sample is sustained in relation to that of the control. Rats injected with samples of the bead maintain a plasma concentration of leptin of plus 50 mg / mL for 112 h compared with 12-18 h for control animals. The highest sustained blood leptin levels in the pearl group correlate with their more pronounced and sustained weight loss compared to the control group. Rats injected with pearl samples continuously lose weight for 120h; at 120h the total weight loss is 9% of the initial weight. In contrast, control rats lose 7% of their initial weight in 50h but gain again the weight at 120h.

EXAMPLE 4 This example shows the effect of several PEG in the bath, in addition to the 10 mM ZnCl2, on the efficiency of loading and the in vitro release of IL-lra. The small beads were prepared as described above with IL-lra in 10 mM PIPES pH 6.85. A bead bath (A) contains 100mM CaC12, 10mM znC12, 20% 1K PEG and 20% 2K PEG. A second bead bath (B) contains the same as A but without 20% 1K PEG. The concentration of IL-lra in beads A and B is 58 mg / mL7, that is to say, a loading efficiency of 74% according to what was determined from the emergence of sodium citrate. Formulation B has an invasion of 55% and a release of 75% after 18h. Formulation A has an irruption of 20% and a release of 50% after 18h. In this way the addition of PEG in the bath leads to highly charged beads that sustain the release on the protein, -. Also, the addition of 1K PEK leads to an even smaller irruption and a slower release of the protein.

EXAMPLE 5 This example shows the effect of having PEG, but not zinc, in the bath on the loading of and the initial irruption of IL-lra from alginate beads. The small beads were prepared as described above, except that the bath contains 20% 1K and 20% 2K PEG in addition to 100 mM CaC12. The load efficiency is 93% with 63 mg / mL of IL-lra in the bead. The initial irruption is 35%. In this way, the addition of PEG to the bath can lead to a high protein load without the presence of zinc ions.

EXAMPLE 6 In this example a comparison of the effectiveness of the release of the injection of a bolus of IL-lra in buffer (10 mM PIPES, pH 6.85) and the IL-lra in the alginate beads of Example 1 is made. SC (subcutaneously) mice were injected with female Balb / C (20 g body weight) at time zero with the different formulations, each containing 10 mg of IL-lra. At 18h the mice were injected IV (intravenously) with rhIL-lB (0.1 mcg per mouse) and then sacrificed 2h after blood sampling. The blood was analyzed to determine the glucose concentration and the number of lymphocytes. IL-lbeta normally causes a fall in glucose concentration and the number of lymphocytes, but the presence of a certain level of IL-lra protects against such loss. The result of the experiment shows that only the mice that received the IL-lra contained in the beads are protected against loss in the value of the blood parameters. These results demonstrate that the alginate beads sustain the release of the IL-lra at an effective level for at least 18h.

Example 7 This example is a control experiment illustrating the preparation and release of beads containing protein using GCSF, where the precipitation bath contains only CaC12 (100 mM). The large beads were prepared as described above. The syringe mixture contains 26 mg / mL GCSF (10 mmM TRIS pH7) in 1% alginate. The prepared beads contain 16 mg / mL of GCSF (from the emergence of citrate). In this way, with only CaC12 in the bath, the efficiency of the load is 35%. The fractional release of the protein shows an inrush of 60% and a release of 75% in a day. In this way, using a known procedure described in the literature, a low protein loading and a rapid release are obtained.

Example 8 This example shows the effect of ZnC12 in the bath on the loading and release of the GCSF in alginate beads. The large beads were prepared as described above except that 10 mM ZnC12 was added to the bath. The syringe mixture contains 46 mg / mL GCSF in 1% alginate. The prepared beads contain 28 mg / mL of GCSF (from the emergence of citrate). Thus with the addition of 10 mM ZnC12 to the bath (in addition to CaC12_100 mM) the efficiency of the charge is increased from 35% (Example 6) to 61%. The fractional release of the protein shows a reduced inrush of 40% and a release of 55% "in one day, thus the addition of ZnCl2 to the CaC12 bath leads to a greater efficiency of the load, a lower inrush and a lower release of the protein.

Example 9 This example shows the effect of having PEG in the bath with the pH of the bath being acid upon loading and releasing the GCSF with alginate beads. The large beads were prepared as described in Example 7 except that 20% PEG (Aldrich) was added to the bath and the pH of the bath is 1.7. The bath also contains 100 mM CaC12 and 10 mM ZnC12. The loading efficiency for the GCSF is 54% and the fractional release (25 mg / mL in the beads) shows a much lower irruption of less than 5% and 40% release after 100 hours. In this way an acid bath mixture which may contain PEG (in addition to CaC12 and ZnC12) leads to a lower inrush and a slower release of the protein.

EXAMPLE 10 This example shows the effect of having PEG and zinc in the bath and the pH of the bath decreased with acidifying agents on the loading and initial inrush of the GCSF of the alginate beads. The large beads were prepared as described above, except that the bath contains 25 mM ZnC12, 100 mM CaC12, and 5% PEG 1K and 5% PEG 10K. The pH of the bath was lowered with glycine buffer and phosphoric acid at pH 1.65. The resulting beads (20 mg / mL load) exhibited an irruption of less than 5% and a fractional release of 40% in 90h. In this way a combination of PEG and zinc '..- and a low pH in the bath leads to a low inrush and a slow release.

EXAMPLE 11 This example shows the preparation of GCSF in alginate beads with PEG in a low pH bath without the addition of zinc ions. The small beads were prepared as described above except that the bath contains 5% of 1K and 5% of 10K PEG. The pH of the bath was decreased to 1.43 using glycine buffer and phosphoric acid. The efficiency of the load is 42% per 14 mg / mL in the pearls (of the citrate irruption). The fractional release shows an irruption of 32% and a release of 35% after 70hT Example 12 The large GCSF / alginate beads of Example 12, and the subsequent Examples 13-15, were prepared in a manner similar to that described above but with stricter control of the timing of the different operations and an alternative method to determine load. More specifically, 1 mL of GCSF / alginate mixture was immersed in 10 mL of a bath with magnetic stirring for approximately 2 minutes. The beads were filtered "and washed with 5 mL of water.

The total procedure to produce the pearl takes approximately 5 minutes. The loading was determined (by A280) from the difference in the amount of protein in the alginate mixture immersed in the bath and the protein that was not incorporated in the beads formed, that is, the protein remaining in the bath mixture. and the washings. This amount of protein incorporated in the beads was divided by ~ the volume of 1 mL of the mixture added to the bath to obtain the load expressed in mg / mL of beads. Example 12 compares the presence of PEG in the bath on the GCSF loading in the beads. The large beads were prepared as described above, except that the bath contains 200 mM CaC12 and 15% PEG 8K (pH 5-6); The bath of the control beads have 200 mM CaC12. The addition of PEG to the bath increases the load from 21.8 mg / mL (70% efficiency) to 26.5 mg / mL (85% efficiency).

Example 13 This example shows the effect of lowering the pH of the bath on loading and releasing the GCSF with alginate beads. The formation of the bead charge was determined as in Example 12 with PEG except that one of the baths contains 0.5 M glycine buffer pH 2.1. The charge at pH 2.1, 24.9 mg / mL (efficiency of 80%), is similar to that of the control. "However, the initial release at one hour (29%) is lower and the release at 24 h is more sustained (32%) than the control (92% to 99% respectively).

Example 14 This example shows the effect of the addition of zinc to a PEG-containing bath on the GCSF loading in alginate beads. The formation and loading of the bead were determined as in Example 12 with PEG. Addition of 10 mM ZnC12 to the bath increased the load from 26.5 mg / mL (85% efficiency) to 30.3 mg / mL (97% efficiency).

Example 15 This example shows the initial irruption and sustained low release of the GCSF from the alginate beads. The formation, loading and release of the bead were carried out in a manner similar to that of EXAMPLE 13 except that the bath contains 100 mM CaC12, 5% PEG 1K and 5% PEG 2K, and 0.5 M glycine buffer (pH 2.1) . The loaded beads contain 24 mg / mL of GCSF. At 1/2 h the initial release is almost zero, at 19 h the release is 13.6% and at 44 h the release is 24%.

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

Claims (43)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A sustained release composition, characterized in that it comprises: a) a hydrophilic polymer; b) a biologically active agent; and c) at least one precipitating agent; characterized in that the biologically active agent co-precipitates within the hydrophilic polymer.

2. The composition according to claim 1, characterized in that the precipitating agent is selected from the group consisting of polyvalent metal ions or salts, acetates, citrates, chlorides, carbonate or hydroxides thereof.

3. The composition according to claim 2, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, aluminum or zinc.

4. The composition according to claim 3, characterized in that the precipitating agent is a polyvalent ion selected from a group consisting of zinc, calcium or a combination thereof.

5. The composition according to claim 1, characterized in that the hydrophilic polymer is a polyanion.

6. The composition according to claim 1, characterized in that the hydrophilic polymer is a polysaccharide.

The composition "according to claim 6, characterized in that the hydrophilic polymer is an acid polysaccharide

8. The composition according to claim 7, characterized in that the polysaccharide is alginate." "

9. The composition _ in accordance with claim 9, characterized in that the alginate contains at least 30% guluronic acid

10. The composition according to claim 8, characterized in that the alginate consists of at least 0.05% by weight

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

12. The composition according to claim 11, characterized in that the protein consists of at least 0.01 mg / mL.

The composition according to claim 11, characterized in that the protein is selected from the group consisting of haematopoietic factors, colony stimulating factors, anti-obesity factors, growth factors, trophic factors, and anti-inflammatory factors.

The composition according to claim 11, 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 and analogues or derivatives thereof.

15. The composition according to claim 1, characterized in that it also comprises at least two precipitating agents.

16. The composition according to claim 15, characterized in that at least one of the precipitating agents is selected from the group consisting of water-soluble polymers.

17. The composition according to claim 16, characterized in that the water-soluble polymer "is polyethylene glycol.

18. A method for producing a sustained release composition, characterized in that it comprises the steps of: a) dissolving a biologically active agent and a hydrophilic polymer with a solvent to form a first mixture; b) dissolving at least one precipitating agent in a solvent to form a second mixture; c) add the first mixture with the second mixture; and d) coprecipitating the biologically active agent with the hydrophilic polymer to form a coprecipitated particle.

19. The method according to claim 18, characterized in that the precipitating agent is selected from the group consisting of polyvalent metal ions or salts, acetates, citrates, chlorides, carbonate or hydroxides thereof.

Claim 19, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, aluminum or zinc.

The method according to claim 20, characterized in that the precipitating agent is a polyvalent ion selected from the group consisting of zinc, calcium or a combination thereof.

22. The method according to claim 21, characterized in that the precipitating agent in the second mixture consists of at least 1 mM of calcium and 0.1 M of zinc

23. The method according to claim 18, characterized in that the hydrophilic polymer It is a poly-anion.

24. The method according to claim 18, characterized in that the hydrophilic polymer is a polysaccharide.

25. The method of compliance with the claim 24, characterized in that the polysaccharide is an acid polysaccharide.

26. The method of compliance with the claim 25, characterized in that the polysaccharide is alginate.

27. The method of compliance with the claim 26, characterized in that the alginate contains at least 30% guluronic acid.

28. The method of compliance with the claim 27, characterized in that the first mixture consists of at least 0.05% by weight of alginate.

29. The method according to claim 18, characterized in that the biologically active agent comprises a protein.

30. The method according to claim 29, characterized in that the first mixture consists of at least 0.01 mg / mL of protein.

31. The method according to claim 29, 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.

32. The method according to claim 29, characterized in that the protein is selected from the group consisting of leptin, G-CSF, SCF, BDNF, OPG, GDNF, NT3, GM-CSF, IL-lra, IL2, TNF- bp, MGDF, interferons, erythropoietin, KGF and analogues or derivatives thereof.

33. The method according to claim 18, characterized in that it also comprises at least two precipitating agents in the second mixture. 3 .

The method in accordance with the claim 33, characterized in that at least one of the precipitating agents is selected from the group consisting of water-soluble polymers.

35. The method of compliance with the claim 34, characterized in that the water-soluble polymer is polyethylene glycol.

36. The method according to claim 18, characterized in that the addition of the first mixture to the second mixture occurs by spraying, electrostatic fields, drip addition, dispersion or mixing to form the coprecipitated particles.

37. The method according to claim 18, characterized in that it also comprises the step of isolating the coprecipitated particle.

38. The sustained release product, characterized in that it is produced by the method according to claims 18, 36 and 37.

39. A pharmaceutical formulation according to claims 1 or 15 in a pharmaceutically acceptable carrier, diluent or adjuvant.

40. A method for treating an indication with a sustained release composition according to claims 1 or 15 characterized in that it is in a pharmaceutically acceptable carrier, diluent or adjuvant.

41. A method of treating a condition, characterized in that it is selected from the group consisting of excess weight, diabetes, high level of blood lipids, arterial sclerosis, arterial plaque, reduction or prevention of gallstone formation, insufficient lean tissue mass, insufficient insulin sensitivity and stroke, with a sustained release composition according to claim 1 or 15 in a pharmaceutically acceptable carrier, diluent or adjuvant, wherein the biologically active agent is leptin, an analog or derivative of the same.

42. A method for treating a condition selected from the group characterized in that it consists of hematopoietic cell deficiencies, infection, or neutropenia with a sustained release composition according to claims 1 or 15 in a pharmaceutically acceptable carrier, diluent or adjuvant, wherein the biologically active agent is G-CSF, an analog or derivative thereof.

43. A method for treating inflammation with a sustained release composition according to claim 1 or 15 in a pharmaceutically acceptable carrier, diluent or adjuvant, characterized in that the biologically active agent is an IL-lra, an analog or derivative thereof. ,