MXPA00011231A - Biodegradable sustained-release alginate gels - Google Patents

Abstract

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

Description

BIODEGRADABLE ALGINATE GELS OF PROLONGED RELEASE Field of the Invention The present invention relates to sustained release formulations using biodegradable alginate gel beads and / or delayed-effect gels and methods for making these.

Background of the Invention With advances in the technologies of genetic and cellular manipulation, the availability of recombinant proteins has conceived advances in the use of proteins as drugs for therapeutic applications. Many diseases or conditions treated with pharmaceutical proteins require sustained levels of. proteins to achieve the most effective therapeutic result. However, as in most protein-based pharmaceutical products, a short-lived biological half-life often requires frequent administration. These repeated injections are given at various intervals that result in fluctuating medication levels at the expense of a significant physical and economic burden. Ref: 124595 in patients. Since many conditions respond better to controlled levels of a drug, there is a need to control the release of a drug to provide long periods of consistent release. These prolonged-release medications provide the patient not only with improved prophylactic, diagnostic or therapeutic effects, but also with a reduction in the frequency of injections as well as in overall costs. Current attempts to sustain the levels of medication in humans or animals between doses include the use of biodegradable polymers as matrices to control the release of the drug. For example, British Patent No. 1,388,580 teaches the use of hydrogels for the prolonged release of insulin. US Patent No. 4,789,550 describes the use of polylysine-coated alginate microcapsules for administration of the protein by encapsulation of living cells. The prolonged release attempts also use cationic or anionic compositions coated by ionic polymers of opposite charge to encapsulate cells that are capable of producing biologically active compositions; Patent of E. U. A. No. 4,744,933. Likewise, multiple coatings of cationic or anionic crosslinked polymers are described as a means to obtain a controlled release; US Patents A. Nos. 4,690,682 and 4,789,516. In addition, other attempts show the use of alginates alone, or alginates coated with other biodegradable polymers, for the controlled release of polypeptide compositions or cation precipitates thereof; PCT documents WO 96/00081; PC WO 95/29664 and PCT WO 96/03116. These attempts, however, provide insufficient means to obtain the prolonged release administration of the desired protein-based pharmaceutical products. Generally, it is known that the use of certain biodegradable polymers, for example, polylactide co-glycolide under in vi ve conditions, shows an initial outburst of drug release; Johnson, O. et al. , Nature Med., 2 (7): 795 (1996). In addition, it is generally known that proteins used with current forms of sustained-release preparations may undergo denaturation and lose their bioactivity upon exposure to the encapsulating agents. These preparations use organic solvents that can have harmful effects on the selected protein.

Finally, as discussed below, the use. of alginate alone, does not provide a desired controlled release of the protein, necessary to obtain effective therapeutic results. In general, alginates are well known, naturally found, are anionic, and are polysaccharides comprising 1-linked-β-D-mannuronic acid and α-L-guluronic acid; Smidsrod, O. et al. , Trends in Biotechnol., 8: 71-78 (1990); Aslani, P. et al. , J. Microencapsulation, 1_3 (5): 601-614 (1996). Typically, alginates vary from 70% mannuronic acid and 30% guluronic acid, and from 30% mannuronic acid and 70% guluronic acid; Smidsrod, supra. Alginic acid is insoluble in water whereas salts that are formed with monovalent ions such as sodium, potassium and ammonium are soluble in water; McDowell, R. H., "Properties of Alginates" (London, Alginate Industries Ltd, 4th edition 1977). It is known that polyvalent cations react with alginates and spontaneously form gels.

Alginates have a wide variety of applications such as for food additives, adhesives, pharmaceutical tablets and wound dressings. Alginates are also recommended for protein separation techniques. For example, Gray, C. J. et al. , in Biotechnology and Bioengineering, 1 607-612 (1988) entrapped insulin in calcium / zinc alginate gels for the separation of insulin from other serum proteins. Alginate matrices have also been well documented for drug delivery systems; see, for example, U.S. Patent No. 4,695,463 which discloses an alginate-based delivery system using chewing gum and its pharmaceutical preparations. Alginate beads are used for the controlled release of various proteins such as: the 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); that transform the growth factor encapsulated in alginate particles; Puolakkainen, P. A. et al. , Gastroenterology, 107: 1319-1326 (1994); to angiogenic factors trapped in alginate-calcium beads; Downs, E.C. et al. , J. of Cellular Physiology, 152: 422-429 (1992); the albumin trapped in microcapsules of alginate-chitosan; Polk, A. et al. , J. Pharmaceutical Sciences, 83 (2): 178-185 (1994); to alginate-chitosan particles coated with polymers; Okha afe, A. 0. et al. , J. Microencapsul. , 13 (5): 497-508 (1996); to hemoglobulin encapsulated with calcium alginate particles-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 to interleukin-2 encapsulated in alginate-chitosan microspheres; Liu, L. S. et al. , Proceed. Intern. Symp. Control. I laughed Bioact. Mater, 22r. 542-543 (1995).

Systems using alginate gel beads, or alginate / calcium gel particles, to entrap the proteins, suffer from a lack of any prolonged release effect due to the rapid release of the protein from the alginate beads; Liu, L. et al. , J. Control Reí., 4_3: 65-74 (1997). To avoid this rapid release, several of the systems mentioned above attempt to use polycationic polymer coatings (eg, polylysine, chitosan) to retard the release of the alginate beads with protein; see, for example, Wheatley, M.A. et al. , J. Applied Polymer Science, 4J3: 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 that interact with negatively charged alginate molecules to form polyelectrolyte complexes that act as diffusion barriers on the surface of the beads. Problems with the use of polycations can occur because (1) these formulations can be cytotoxic due to polycations; Huguet, M. L. et al. , supra; Zimmermann, Ulrich, Electrophoresis, 1 ^: 269 (1992); Bergmann, P. et al. , Clinical Science, 67: 35 (1984); (2) polycations are prone to oxidation; (3) beads with polycation shells tend not to wear out and accumulate in the body; (4) these formulations are made by laborious coating processes that include multiple coatings of the polylysine polycation; Padol, et al. , Proceed. Intern. Symp. Control. I laughed Bioact. Mater, 2: 216 (1986) and (5) the ionic interactions between the protein and the polycations can result in the loss of protein activity or cause instability in the protein. Francesco et al. , U.S. Patent No. 5,336,668 (and references cited herein) describe total and partial esters of alginic acid, which are made by various processes, and which possess interesting pharmaceutical qualities. It is described how alginic esters can be used as biodegradable plastic materials for medical-surgical uses, as additives for a wide range of polymeric materials; or to be used in the preparation of various medications. The potential use of esterified alginates in the extended-release formulations does not. discusses or describes alginate esterified hydrogels. Nightlinger et al. , Proceed. inter. Symp. Control. I laughed Bioact. Mater., 22: 738-739 (1995) describe esterified hyaluronic acid (HA) microspheres having controlled release capabilities. The references are generally directed to the different rates of degradation for their HA derivatives and describe how the ester breaks down to "release the alcohol and portions of HA." There is no discussion that relates to whether or not the main structure of the HA itself The polysaccharide itself must be biodegradable in non-toxic products, it has been found that certain systems of alginate gels are disintegrated into 'lower molecular weight polymers units, so that a prolonged administration system based on a polysaccharide is useful. , while effective to provide a prolonged release of the drug, result in a "protrusion" (or nodule) at the injection site due to very slow gel dissipation. In a therapeutic scenario that involves low doses of the drug and infrequent injections, but this can not be a major problem. However, in a therapeutic setting involving high doses of the drug and infrequent injections, this may not be a major problem. However, in a therapeutic scenario, which involves high doses of the drug and more frequent injections, this effect can be prohibitive. A means must be developed to increase the rate of dissipation of the alginate gel from the site of the injection.

There is still a need to develop pharmaceutical formulations that achieve a more versatile and effective means of prolonged release for clinical applications. Various recombinant or natural proteins can benefit from a constant and long-term release, and therefore, provide more effective clinical results. The present invention provides these advances. Pharmaceutical compositions using the biodegradable alginate gel particles or gels of the present invention are capable of providing increased bioavailability, protein protection, decreased degradation and slow release with increased protein stability and potency. Also, the pharmaceutical compositions of the present invention provide a simple, rapid and economical means of controlled release of recombinant proteins for effective prophylactic, therapeutic or diagnostic results.

Brief Description of the Invention The present invention is developed from studies using unmodified alginate hydrogels (a type of anionic polysaccharides) for the prolonged release of proteins. These unmodified alginate hydrogels, which contain proteins see co-pending US applications 08 / 857,913 and 08 / 912,902) are formed in a delayed manner while the materials can be filled into a syringe and then allowed to gel on the syringe. same syringe; It is discovered that these gels are injectable. After a single subcutaneous injection in rodent models, evidence of sustained protein levels over several days is observed; However, a perceptible protrusion or nodule remains at the site of injection for long periods of time with little change in its size. This protuberance consists of the alginate hydrogel filled with water and the size of the protrusion is a function of the volume of the gel that is injected. The gel beads also stay at the site of the injection. The present invention also relates to a new class of biocompatible biodegradable polysaccharide hydrogels, for example, alginate ester hydrogels for the prolonged release of therapeutic proteins. Unexpectedly, the alginate ester hydrogels, in addition to having the properties of gelation, injectability and prolonged release of the unmodified alginates, do not leave a bulge at the injection site that is, the alginate ester hydrogels are biodegradable or erodible and they are generally reabsorbed in the surrounding tissues with little reaction at the site of injection. The compositions of the present invention comprise alginate esters or their derivatives that are ionically crosslinked in a hydrogel matrix (containing water) and containing a therapeutic agent such as a protein. The present invention further relates to a method for producing biodegradable sustained release compositions. The present invention further relates to the use of alginate ester materials in liquid mixtures for delayed gelation in the body. The present invention furthermore relates to compositions where the hydrogels of alginate esters are in the form of microspheres or particles for the prolonged release of active agents preferably from therapeutic proteins. In one embodiment of the present invention, alginatb ester hydrogels provide compositions for application to specific target sites in a patient's body. These compositions are useful: to prevent or inhibit the formation of tissue adhesions after surgery and traumatic damage; for complementary tissues, especially for filling soft and hard tissues; to fill a confined space with a resorbable material; for a support of tissue growth; and as a bandage for wounds. In another embodiment, alginate ester hydrogels provide an active agent containing devices that contain the active agent for implantation in the body while the agent may be bound or unglued in the alginate polymer. In another embodiment, the alginate ester hydrogel compositions of the present invention provide a method for improving the bioavailability of the active agent in the composition.

Finally, the alginate ester hydrogel compositions of the present invention further provide a method for obtaining a substantially constant blood level over a period of time in the patient.

Detailed Description of the Invention Hydrophilic polymers including alginates and derivatives thereof, can be obtained from various commercial, natural or synthetic sources that are well known in the art. As used herein, the term "hydrophilic polymer" refers to water-soluble polymers or polymers that have an affinity for absorbing water. Hydrophilic polymers are well known to one skilled in the art. These include but are not limited to polyanions, including anionic polysaccharides, such as alginate, carboxymethylamyl, salts of polyacrylic acid, salts of polymethacrylic acid, ethylene maleic anhydride copolymer (half ester), carboxymethyl cellulose, dextran sulfate, heparin, carboxymethyl dextran, carboxycellulose, 2,3-dicarboxylic cellulose, tricarboxylic cellulose, carboxy gum arabic, carboxycargangen, pectin, carboxypectin, carboxy gum tragacanth, carboxy xanthan gum, pentosan polysulfate, carboxyalmidon, carboxymethyl chitin / chitosan, curdlan, inositol hexasulfate, -cyclodextrin, hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, heparin sulfate, carboxymethyl starch, carrageenan, polygalacturonate, carboxy guar gum, polyphosphate, polyaldehyde-carbonic acid, poly-l-hydroxy-1-sulfonate- propen-2, maleic copolystyrene acid, agarose, mesoglycan, polyvinyl sulfopropylated alcohols, sulphate of cellulose, protamine sulfate, phospho guar gum, polyglutamic acid, polyaspartic acid, polyamino acids, and derivatives or combinations thereof. One skilled in the art can appreciate another variety of hydrophilic polymers that are within the scope of the present invention. Similarly, agglutinated polyvalent metal ions can be obtained from commercial, natural or synthetic sources that are well known in the art. In particular, metal ions may include but are not limited to aluminum, barium, calcium, iron, manganese, magnesium, strontium and zinc. Preferably, the metal ions are calcium and zinc or the salts thereof, such as zinc acetate, calcium acetate, or chloride salts. Small water-soluble molecules and salts can also be used, such as ammonium sulfate, acetone, ethanol and glycerol. The alcohols of the aliphatic series for use as esterifying components of the carboxyl groups of alginic acid according to the present invention are, for example, those with a maximum of 34 carbon atoms, which may be saturated or unsaturated and which may possibly also be substituted by other free or functionally modified groups, such as the amino, hydroxyl, aldehyde, keto, mercapto, carboxyl groups, or by groups derived therefrom, such as hydrocarbyl or dihydrocarbyl (hereinafter the term 'hydrocarbyl' is taken to indicate not only monovalent hydrocarbon radicals such as of the CnH2n +? type, but also to indicate bivalent or trivalent radicals, such as the "alkylene" -CnH2 or * alkylidenes "= CnH2n), ether or ester groups, acetal or ketal groups, thio-ether or thio-ester groups and esterified carboxyl groups or carbamide or carbamide groups substituted by one or more groups hydroxyl, by nitrile groups or by halogens. In the above groups containing hydrocarbyl radicals, these are preferably lowered aliphatic radicals, such as heteroatoms, such as oxygen, nitrogen and sulfur. Preference is given to alcohols substituted by one or two of the functional groups mentioned above. The alcohols of the above group to be used preferentially within the terms of the present invention, are those with a maximum of 12 and especially with a maximum of 6 carbon atoms and wherein the hydrocarbyl radicals in the aforementioned amino, ether, ester, thioether, thioester, acetal and ketal groups representing alkyl groups with a maximum of 4 carbon atoms, and also in the esterified carboxyl groups or substituted carbamide groups, the hydrocarbyl groups are alkyls with the same number of carbon atoms, and wherein the amino or carbamide groups may be alkylene or alkylenecarbamide groups with a maximum of 8 carbon atoms. Of these alcohols those mentioned first and most frequently are saturated and unsubstituted alcohols such as methyl, ethyl, propyl, isopropyl, n-butyl alcohol, isobutyl alcohols, tert-butyl alcohols, amyl, pentyl, hexyl alcohols, octyl, nonyl and dodecyl and, above all, those mentioned above with a linear chain such as n-octyl and n-dodecyl alcohols. Of the substituted alcohols of this group, mention may be made of divalent alcohols, such as ethylene glycol, propylene glycol or butylene glycol, trivalent alcohols such as glycerin, aldehyde alcohols such as tartronic alcohol, carboxyl alcohols such as lactic acids, example, alpha-oxypropionic acid, glycolic acid, malic acid, tartaric acid, citric acid, aminoalcohols such as aminoethanol, aminopropanol, n-aminobutanol and its dimethyl and diethyl derivatives in the amino function, choline, pyrrolidinetanol, piperidinyl ethanol, piperazinyl ethanol and the corresponding derivatives of the n-propyl or n-butyl alcohols, monothioethylene glycol or their alkyl derivatives, for example, the ethyl derivative in the mercapto function. Of the mostly aliphatic saturated alcohols, those of special mention are, for example, cetyl alcohol and myristyl alcohol, but highly unsaturated alcohols with one or two double bonds, such as those contained therein, are especially important for the purposes of the present invention. especially in many essential oils and having an affinity with terpenes such as citronellol, geraniol, nerol, nerolidol, linalool, farnesol, phytol. Of the diminished unsaturated alcohols consideration is given to the propargyl alcohol. Of the aliphatic alcohols those mentioned above are all those with only one benzene residue and wherein the aliphatic chain has a maximum of 4 carbon atoms, and where also the benzene residue can be substituted between 1 and 3 methyl groups or hydroxyl or by halogen atoms, especially chlorine, bromine or iodine and wherein the aliphatic chain can be substituted by one or more functional groups selected from the group consisting of amino-free groups or mono- or dimethyl groups or by pyrrolidine groups or piperidine. Of these alcohols, benzyl alcohol and phenethyl alcohol are especially preferred. The alcohols of the cycloaliphatic or cycloaliphatic aliphatic series can be derived from mono- or polycyclic hydrocarbons and can have a maximum of 34 carbon atoms. Of the alcohols derived from the monoannular cyclic hydrocarbons, special mention is given to those with a maximum of 12 carbons, with rings preferably containing between 5 and 7 carbon atoms, possibly substituted for example between one and three lower alkyl groups, such as the methyl, ethyl, propyl or isopropyl groups. The specific alcohols of this group are cyclohexanol, cyclohexanediol, 1,2,3-cyclohexanothiol and 1, 3, 5-cyclohexanothiol. (floroglucitol), inositol, alcohols derived from p-menthane such as menthol carvomentol, alpha and terpineol range, 1-terpineol alcohols known as * terpineol ", 1, 4 and 1,8-terpine. Condensed rings are, for example, those from the tujano, pinano, cambano groups, particularly tujanol, sabinol pinol hydrate, D and L-borneol and D and L-isoborneol.

Also included are alcohols which are derived from the esterification reactions of epoxy-containing compounds with alginates (see for example, U.S. Patent No. 2,463,824 and U.A. Patent No. 2,426,125). The total and partial ester group containing the polyanions of the present invention are generally acidic polysaccharides in which the glycosidic oxygen is beta-linked to the carbonyl carbon of the ester. Although not limited by any specific mechanism, this portion configuration allows degradation of the polymer chain by a mechanism of beta-elimination that can occur under physiological conditions. The esters of the alginic acid of the present invention are comprised of residues of mannuronic acid (anion m-COOH or m-COO) joined together by oxygen glycosidic ether bonds of the following general formula I: - (M) nl - (M ') n2 - (G) n3 - (G') n4 - (A) n5- where: M is a residue of mannuronic acid, or an anion of m-COOH or -COO; M 'is a residue of mannuronic acid ester,. m-C00R1; G is a residue of guluronic acid, or an anion of g-COOH or g-COO; G 'is a residue of guluronic acid ester, g-C00R2; A represents non-g or non-m chain units, such as sugars, sugar oxidation products, or aliphatic, aromatic, araliphatic, aromatic, cycloaliphatic radicals that can be substituted and interrupted by linked heteroatoms within or at the ends of the chain. chain; ni, n2, n3, n4 and n5 are integers that represent the average relative number or the units incorporated; R1 and R2 are each aliphatic, aromatic, araliphatic, aromatic, cycloaliphatic radicals which can be substituted and interrupted by heteroatoms; and derivatives (for example wherein the hydroxyl groups are acetylated and reacted with isocyanates) and pharmaceutically acceptable salts thereof. In the esters of the present invention, it is desirable that R1 = R2 = aliphatic or alaromatic and in addition that 100 (n2 + n4) / (n1 + n2 + n3 + n4) is from 1-99 mol%, preferably 5-50 mol%, still more preferable 6-15 mol% and more preferably 7-12 mol% and 100n5 / (nl + n2 + n3 + n4 + n5) are preferably less than 10 mol%. In the partial esters of the invention the non-esterified carboxyl groups can be kept free or can be salified. The bases for the formation of these bases are chosen according to the final use of the product. The inorganic salts can be formed from alkali metals, such as potassium and in particular sodium and ammonia, or derived from alkaline earth metals such as calcium or magnesium or aluminum salts. Of particular interest are salts with organic bases, especially azoate bases (nitrogenated) and, therefore, aliphatic, araliphatic, cycloaliphatic or heterocyclic amines. These ammonium salts can be derived from therapeutically acceptable amines or from non-toxic but therapeutically inactive amines, or from amines with a therapeutic action. Of the first type, aliphatic amines are preferred, for example monoalkylamines, dialkylamines, and trialkylamines with alkyl groups with a maximum of 8 carbon atoms or arylalkylamines with the same number of carbon atoms in the aliphatic part and wherein aryl means a benzene group possibly substituted between one and three methyl groups or halogen atoms or hydroxyl groups. The biologically inactive bases for the formation of salts can also be cyclic, such as the monocyclic alkylene amines with cycles of between 4 and 6 carbon atoms, possibly interrupted in their cycle by heteroatoms which are chosen from the group consisting of nitrogen, oxygen and sulfur, such as piperazine or morpholine, or can be substituted, for example by amino or hydroxyl functions such as aminoethanol, ethylenediamol, ethylenediamine , ephedrine or hill. The degree and type of esterification can be controlled by synthetic methods that are known in the art. Preferably, the alginate esters are prepared by treating the quaternary ammonium salts of the alginic acid using conventional alkylating agents in an aprotic organic solvent such as dimethyl sulfoxide. The resulting esters of preference are the esters of monovalent alcohols such as decreased alkyl such as ethyl or aralkyl such as benzyl or their mixtures. Esters can also be formed by the reaction of alginic acid with oxirane or epoxy-containing compounds such as ethylene oxide or propylene oxide. It is also possible to form quaternary ammonium salts with partial esters for example the salts of * tetraalkylammonium with the aforementioned number of carbon atoms and preferably the salts of this type wherein the. fourth alkyl group has between 1 and 4 carbon atoms, for example a methyl group. The degree of esterification (which is expressed in mol%) of the alginate is related to the desired rate of fading of the gel in the patient's tissue. This rate of fading of the gel is generally related to the desired release rate of the active agent of the gel which is for a period of 5 years or less, normally from 2 days to 270 days, more usually from 2 days to 180 days, more usually from 2 days to 90 days. The degree of esterification (DE) is from 1 mol% to 99 mol%, preferably from 5 mol% to 50 mol%, more preferably from 6 mol% to 30 mol%, more preferably from 6 mol% to 15 mol%, more preferably from 7 mol% to 12 mol%. 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 hydrogen halide (eg, hydrochloric acid), phosphoric, nitric or sulfuric. Other inorganic acids are well known to someone skilled in the art and are contemplated herein. Organic acids within the scope of the invention include the aliphatic carboxylic acids and the 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 one skilled in the art. As used herein, biologically active agents refer to recombinant or naturally occurring proteins, either human or animal, useful for prophylactic, therapeutic or diagnostic applications, as well as non-protein based agents such as small molecules The biologically active agent can be natural, synthetic, semi-synthetic or derivatives thereof. The biologically active agents of the present invention must have the ability to precipitate. 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 useful biologically active agents). . One of skill in the art can readily adapt a desired biologically active agent to the compositions of the present invention. These proteins include but are not limited to interferons (see, U.S. Patent Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623 which are incorporated herein by reference and include the drawings), interleukins (see, U.S. Patent No. 5,075,222, which is 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 which are incorporated herein by reference and include the drawings), granulocyte colony-stimulating factors (see US Pat. of US Patents 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185, which are incorporated herein by reference and include the drawings), germ cell factor (Nos. of PCT publications 91 / 05795, 92/17505 and 95/17206, which are incorporated herein by reference and include the drawings), and the OB protein (see PCT publication nos 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 which are incorporated in the prese for your reference and include the drawings). In addition, biologically active agents may also include but are not limited to products related to antiobesity, insulin, gastrin, prolactin, adenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), stimulating hormone of follicles (FSH), human chorionic gonadotropin (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-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), factors of' fibroblast growth (FGF), neurotrophic growth factor ' (NGF), bone growth factor such as osteoprotegrin (OPG), insulin-like growth factors (IGF), macrophage colony stimulating factor (M_CSF), granulocyte macrophage colony stimulating factor (GM-CSF) , growth factor derived from megakeratinocytes (MGDF), thrombopoietin, platelet-derived growth factor (PGDF), colony stimulating growth factors (CSF), bone morphogenetic protein (BMP); superoxide dismutase (SOD), tissue plasminogen activator (TPA); urokinase, streptokinase and kallikrein. The term proteins, as used herein, include 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 modifications of the biologically active agents provide additional advantages under certain circumstances, such as an increase in the stability and time of circulation of the therapeutic protein and a decreased immunogenicity. One skilled in the art is able to select the desired chemical modifications based on the desired dose, circulation time, resistance to proteolysis, therapeutic uses and other considerations. As used herein, biodegradability refers to the disintegration of the molecular weight of a particular polymer into a smaller number of units in the chain, i.e., a disintegration into smaller units of molecular weight. The biodegradable gels refer to the dissipation of the gel in the environment of use, where the dissipation is contingent on the disintegration of the molecular weight of the constituent polymers, resulting in fewer units in the polymer chain.

Formation of Complexes Proteins, analogs or derivatives can be administered complexed in a binder composition. This binder composition can have the effect of prolonging the circulation time of the protein, analog or derivative or enhancing the activity of the biologically active agent. This composition may be a protein (or likewise, a peptide), derivative, analog or combination. For example, a binding protein for the OB protein is a receptor for the OB protein or a portion thereof, such as a soluble portion thereof. Other binding proteins can be determined by examining the OB protein, or the protein of choice, in serum, or detected empirically for the presence of agglutination. This agglutination does not normally interfere with the ability of the OB protein or the analog or derivative to bind to the endogenous receptor of the OB protein and / or to effect the transduction signal. In addition to the OB protein, the binding complexes are also applicable to other therapeutic proteins of the present invention. Those skilled in the art are able to determine the appropriate agglutination proteins for use with the present invention. Also, precipitation agents that are used to precipitate the biologically active agent can be obtained from various commercial, natural or synthetic sources that 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 its salts, such as the acetate chloride salts. The salts and small molecules soluble in water can also be used salts such as ammonium sulfate, acetone, ethanol and glycerol. With respect to water-soluble polymers these include but are not limited to polyethylene glycol, polyethylene 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, ethylene oxides, propylene oxides, poloxamers, alkoxylated copolymers, water soluble polyanions, and derivatives' or combinations of these. 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 for ease of handling and precipitation efficiency. Other sizes and types of precipitating agents can be used, depending on the desired therapeutic profile (e.g., the desired duration of prolonged 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 protein or its analogue). One of skill in the art appreciates other precipitation agents that are within the scope of the invention. In addition, the compositions of the present invention may also include additional excipients necessary to stabilize the biologically active agent and / or the hydrophilic polymer. These can be found in the buffer and can include but are not limited to condoms.

Pharmaceutical Compositions The sustained release pharmaceutical compositions of the present invention can be administered orally (e.g., capsules such as hard capsules and soft capsules, solid preparations such as granules, tablets, pills, lozenges, lozenges, pellets, powdered and lyophilized forms) , liquid preparations such as suspensions) and non-oral preparations (eg, intramuscular, subcutaneous, transdermal, visceral, IV (intravenous) preparations, IP (intraperitoneal), intraarthérial, intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal, rectal and that penetrate the uterine mucosa). In general, the invention encompasses sustained release pharmaceutical compositions comprising effective amounts of proteins, or products thereof, with the sustained release compositions of the invention together with pharmaceutically acceptable preservatives, solubilizers, emulsifiers, adjuvants and / or carriers that are They need for your administration. See PCT 97/01331 which is incorporated herein for reference. The optimal pharmaceutical formulation for a desired biologically active agent is determined by one skilled in the art depending on the route of administration of the desired dose. Exemplary pharmaceutical compositions are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton, PA, pages 1435-1712 (1990)).

Due to the thixotropic nature of the delayed-effect gel formulation, syringes can be used to administer it subcutaneously. The composition can be gelled in the syringe for later injection. This gelling can be carried out in a prolonged manner. The determination of the gelling time is controlled by judiciously adjusting the amount of the gelling agent and the proton donor in the mixture, if necessary. A preparation like this is used for a subsequent re-gelling in the body after the injection. The term "thixotropic" as used herein refers to the viscosity of the gel mixture which is lowered under pressure, for example, from the plunger of the syringe, at which time the mixture may flow, for example, through the syringe needle, and then re-form a gel at the injection site. The concept of delayed gelling can also be applied to the filling of a syringe wherein a prolonged-release gel composition is held in a syringe at a specified time it gels in the syringe, for example from a few minutes to several hours after being filled . This avoids the problem of filling a syringe with a material that has already gelled. These pre-filled syringes can be stored for a later injection in patients. Components that may be required for administration include diluents with various buffer contents (e.g., Tris-HCl, acetate), pH and ionic strength, additives such as surfactants and solubilizing agents (e.g., Tween 80, HCO- 60, Polysorbate 80), antioxidants (e.g., - ascorbic acid, glutathione, sodium metabisulfite), additional polysaccharides (e.g., carboxymethyl cellulose, sodium alginate, sodium hyaluronate, protamine sulfate, polyethylene glycol), preservatives (e.g. Thimersol, benzyl alcohol, methyl paraben, propyl paraben), and structuring substances (for example, lactose, mannitol); the incorporation of the material into preparations formed into particles of polymeric compounds such as polymers or copolymers of polylactic / polyglycolic acid, etc., or in combination with liposomes. Hyaluronic acid can also be used as a component of administration and this can have the effect of even more prolonged duration in the blood circulation. In addition, the sustained release compositions of the present invention can also be dispersed with oils (e.g., sesame oil, corn oil, and vegetable oil), or mixtures of these with a phospholipid (for example, lecithin), or triglycerides of medium chain fatty acids (e.g., Miglyol 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 intended for the pulmonary administration of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and metered dose inhalers, are also contemplated for use in the administration of the extended administration compositions of the present invention. pulverized content, of which all are known to those skilled in the art. The components of administration can influence the physical state such as stability, speed of in vivo release, and speed of in vivo clearance of current proteins and derivatives. One of skill in the art appreciates the appropriate administration components and / or appropriate mechanical devices to be used depending on the therapeutic use, route of administration, desired dose, circulation time, resistance to proteolysis, protein stability and other considerations.

• Methods of Therapeutic Use. The therapeutic uses depend on the biologically active agent that is used. One of skill in the art is easily able to adapt a desired biologically active agent of the present invention for therapeutic use. The therapeutic uses for these agents are set forth in greater detail in the following publications which are incorporated herein by reference and include the drawings. Therapeutic uses include but are not limited to uses for proteins such as interferons (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080 which are incorporated herein by reference and include drawings) , interleukins (see, U.S. Patent No. 5,075,222, which is incorporated herein by reference and includes drawings), erythropoietin (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080 which are incorporated herein). herein for reference and include drawings), granulocyte colony stimulating factors (see, U.S. Patent Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT Publication No. 94/17185, which are incorporated herein by reference and include drawings), germ cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, which are incorporated herein by reference and include drawings) and the OB protein (see Publication. is PCT Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 which are incorporated herein by reference and include drawings). In addition, therapeutic uses of the present invention include uses of biologically active agents that include but are not limited to products related to obesity, insulin, gastrin, prolactin, adenocorticotropic hormone.

(ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-) 1 through L-12), tumor necrosis factor (TNF), tumor necrosis factor binding protein (TNF-bp), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), factor 3 neurotrophic (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegrin (OPG), insulin-like growth factors (IGF), stimulating factor macrophage colony (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakeratinocyte-derived growth factor (MGDF), thrombopoietin, platelet-derived growth factor (PGDF), growth stimulating factors colo nias (CSF), bone morphogenetic protein (BMP); superoxide dismutase (SOD), tissue plasminogen activator (TPA); urokinase, streptokinase and kallikrein. The term proteins, as used herein, include peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof. In addition, the present compositions can also be used to make one or more medicaments for the treatment or reduction of conditions that the biologically active agent intends to treat. By way of example, therapeutic uses utilize oxygenation in the blood and a decrease in bone resorption or osteoporosis can also be achieved in the absence of weight loss.

Combination Therapy The present compositions and methods 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 (for example, insulin and possibly amylin), medications for lowering cholesterol and blood pressure (such as those that reduce blood lipid levels or other cardiovascular medications), medications for increased activity (for example, amphetamines), diuretics (fluid elimination), and appetite suppressants. This administration can be simultaneous or it can be in seriatim. In addition, current methods can be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of the body (eg, liposuction or laser surgery designed to reduce body mass, or implant surgery designed to increase the appearance of body mass). The health benefits of cardiac surgeries, such as diverting valve surgeries or other surgeries designed to alleviate a fatal condition caused by a blockage of blood vessels by fatty deposits, such as arterial plaque, can be increased by concomitant use of the present compositions and methods. Methods to eliminate stone formation in the bladder, such as ultrasonic or laser methods, can also be used either before, during or after a course of current therapeutic methods.

In addition, current methods can be used as an adjunct to surgeries or therapies for broken bones, damaged muscle, or. other therapies that can be improved by an increase in muscle mass.

Dosage Someone skilled in the art is 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 vi, during a given period of time. The preferred dose and frequency of administration of the sustained release preparations varies with the type of the biologically active agent, the desired duration of release, the target disease, the desired frequency of administration, the animal species of the subject and other factors. Preferably, the formulation of the molecule is between about 0.10 μg / kg / day and 100 mg / kg / day producing the desired therapeutic effect. The effective dose can be determined by using the diagnostic tools for a period of time. By way of example, the present invention provides the doses of the OB protein. For example, a diagnosis to quantify the amount of OB protein in the blood (or plasma or serum) can be used first to determine the endogenous levels of the OB protein. This diagnostic tool may be in the form of an antibody titre such as sandwich antibody titration. The amount of endogenous OB protein is quantified initially, and a baseline is determined. The therapeutic doses are determined as the quantification of the exogenous and endogenous OB protein (i.e., the protein, analog or derivative found within the body, whether self-produced or administered) and continues during the course of therapy. For example, a relatively high dose may be needed at the beginning, until the therapeutic benefit is observed, and then the lower doses are used to maintain the therapeutic benefits.

Methods and Materials Materials. Alginate in the form of sodium alginate can be found from sources that are well known in the art. The OB protein and GCSF are from Amgen Inc. other chemicals are from sources known in the art.

Preparation of Particles / Alginato Hydrogel Beads. The preparation of alginate hydrogel beads and particles, with or without proteins, as described in detail in the co-pending application of E. U. A. 08 / 842,756, which is incorporated herein by reference.

Preparation of the Delayed Effect Gel. The preparation of delayed-effect alginate hydrogels, with or without proteins, as described in detail in co-pending US applications Nos. 08 / 857,913 and 08 / 912,902, each of which is incorporated herein for reference .

The following examples are offered to further 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 must be able to make the necessary changes in the descriptions for large-scale production.

EXAMPLE 1 The following example describes the preparation of the alginate esters to be used in the present invention.

Preparation A: Tetrabutylammonium alginate (TBA) A sulfonic acid resin (Bio-Rad, AG MP-50) is converted to its tetrabutylammonium form (TBA) by treatment with tetrabutylammonium peroxide (Aldrich) when using the batch method at room temperature. To a solution of 10 g of sodium salt of alginic acid in 800 ml of distilled water are added 60 ml of sulphonic resin (Bio-Rad AG MP-50) in its tetrabutylammonium salt form. The mixture is stirred at room temperature for 0.5 hour. The resin is removed by filtration and washed with distilled water. The TBA alginate in the filtrate is isolated by lyophilization (production, 16.8 g) and confirmed by XH NMR.

Preparation B. Ethyl partial ester of alginic acid, degree of esterification (DE) = 30 mol%. The TBA alginate (6 g, 14.4 mmol TBA units) is dissolved in 500 ml of dimethyl sulfoxide (DMSO) at room temperature. Then iodoethane (Aldrich, 673 mg, 4.3 mmol) is added. The mixture is stirred at 30 ° C for 15 hours, then cooled to room temperature. To this solution is slowly added a solution of 2 g NaCl in 20 mL of water to completely convert it into TBA to the sodium salt. After being stirred for 15-30 minutes, the solution is poured slowly into 1500 mL of ethyl acetate. The precipitated product is collected by filtration and washed 3 times with acetone / water (8: 1 v / v) and 3 times with acetone, then dried in vacuo. The compound is redissolved in distilled water (-100 mL) and the pH is adjusted to approximately 6.5 with 0.2% NaHCO3 at 0 ° C. The solution is then subjected to dialysis (MW limit 8000) overnight against distilled water at 4 ° C and then lyophilized. The production of the partial ester is 2.8 g and the degree of esterification is 30 +/- 1% (XH NMR, maleimide as the internal standard).

Preparation C. Total and partial ethyl ester of alginic acid DE = 100%, 50%, 20%, 10% and 5%. The preparation of these compounds is similar to that described in preparation D, except that the amount of iodoethane that is added is adjusted to reach the desired degree of esterification.

Preparation D. Propyl, hexyl, octyl and dodecyl partial esters of alginic acid. The preparations are similar to those described in the above preparations B and C, but 1-iodopropane, 1-iodohexane 1-iodooctane or 1-iodododecane are replaced by iodoethane respectively.

Preparation E. Benzyl partial ester of alginic acid, DE = 30%. TBA alginate (2.5 g, 5.99 mmol TBA units) is dissolved in approximately 200 mL of DMSO at room temperature. The benzyl bromide (Aldrich, 307 mg, 1.8 mmol) and the TBA iodide (Aldrich, 30 mg) are added. The mixture is stirred at 30 ° C for 15 hours, and then cooled to room temperature. To this solution is added slowly a solution of 0.6 g of NaCl in 10 mL of water to completely convert the TBA to the sodium salt. After being stirred for 15-30 minutes, the solution is poured slowly into 500 mL of ethyl acetate. The precipitated product is collected by filtration and washed 3 times with acetone / water (8: 1 v / v) and 3 times with acetone, and ego dried under vacuum. The compound is redissolved in distilled water (~ 60 mL) and adjusted to pH ~ 6.5 with 0.2% NaHCO3 at 0 ° C and then subjected to dialysis (MW limit 8000 = overnight against distilled water at 4 ° C. The production of the partial ester is 1.3 g and the degree of esterification is 30 +/- 1% (1 H NMR, maleimide in the manner of the internal standard).

Preparation F. Total and partial benzyl ester of alginic acid with different DE. The preparation of these compounds is similar to that described in preparation E, except that the amount of benzyl bromide and the TBA iodide that are added are adjusted to reach the degree of esterification.

EXAMPLE 2 The following example shows the preparation of a protein drug (Leptin) containing an alginate gel ethyl ester (DE = 15 mol% and 10 mol%) and the prolonged release in vi tro of this gel. The leptin is cooled in an ice bath (lOOmg / mL, 10 M Tris HCl, pH 8.8, pH adjusted from 8.0 to 8.8 with 1M NaOH) and 6% ethyl ester alginate (15 mol%, lOmM Tris HCl, pH 8.6). Leptin (0.5 mL) is added to the 6% ethyl ester alginate (0.18 mL) and the mixture is stirred and an ice bath for 10-15 minutes; The final pH is 8.6-8.8. A suspension of 1 is added to this mixture; CaCO3 (16 μL) and the resulting suspension mixed very well. To this suspension is added by dripping, and with stirring, a 0.1M ZnCl 2 solution (100 μL); then water is added to make it have a volume of 1 mL. The mixture is mixed thoroughly and kept in an ice bath for 10-20 minutes. Then a solution of 1.68M d-gluconolactone (Aldrich, 56 μL) is stirred thoroughly in this mixture. The final mixture (50 mg / mL leptin, 1% ethyl ester alginate, 0.1 L) is emptied into the inside of an Eppendorf tube and kept overnight at 4 ° C until gel-forming. After storage overnight, the in vi tro release is carried out in lOmM of histidine buffer, pH 7.4. the gel cast with a degree of 15 mol% esterification exhibits a minimal exabrupt release and a reasonably constant leptin release that shows a 60% release for 6 days. The gel cast with a 10 mol% esterification degree exhibits a minimal outburst of release and reasonably constant release of leptin which shows a 55% release for 6 days.

EXAMPLE 3 The following example shows the preparation of a protein (leptin) drug containing the gel of hexyl ester of alginate (DE = 15 mol% and 10 mol%) and the prolonged release in vi tro of this gel. This example is carried out in a similar manner as that described in Example 2 except that the ethyl ester alginate is exchanged for the hexyl ester alginate. The gels of hexyl ester alginate with a degree of 15 mol% and 10 mol% of esterification exhibit minimal exabrupta release and exhibit a prolonged release which shows a 50% release for 6 days.

EXAMPLE 4 The following example shows the preparation of a protein drug (Zn-Leptin) containing the alginate ethyl ester gel (DE = 15 mol%) and the prolonged release in vi tro of this gel. To a solution of 4% (w / w) of ethyl ester alginate (15 mol%, 0.75 L) is added 1M Tris HCl pH 8.0 (7.5 μL), 0.5 M PIPES pH 6.8 (33 μL) and 0.1 M ZnCl2 ( 8.5 μL). The mixture is stirred well. To this solution is added a solution of Zn-Leptin (100 mg / mL, 675 μL) and the mixture is stirred thoroughly. A suspension of 1M CaC03 (24 μL) and a 1.68 M solution of d-gluconolactone (70 μL) are thoroughly stirred into this mixture. The final mixture (0.1 mL) is emptied into the inside of an Eppendorf tube and kept overnight at 4 ° C until a gel forms. After storage overnight the in vitro release is carried out in 10 mM of histidine buffer, pH 7.4. this alginate ethyl ester gel is flushed with a 15 mol% esterification grade and exhibits little exaggerated release and a prolonged release of leptin showing 65% release for 4 days.

EXAMPLE 5 The following example shows the preparation of a protein drug (GCSF) containing alginate ethyl ester gel (DE = 30 mol%) and prolonged release in vi tro from this gel. To a solution of 2.39% ethyl ester alginate (30 mol%, 0.50 mL) is added 0.1M acetate buffer (pH 4.5, 100 μL), GCSF (104 μL, 48.2 mg / mL, HCl pH3) and distilled water (246 mL). The mixture is stirred well. A suspension of 1M is thoroughly shaken CaHP0 (10 μL) and a solution of 1.68 M d-gluconolactone (40 dL) to this mixture. The final mixture (0.2 mL) is emptied into the inner part of a tube of Eppendorf and kept overnight at 4 ° C until a gel formed. After overnight storage of the gel, the in vitro release is carried out in 10 mM Tris buffer, pH 7.5. The gel drained of ethyl ester alginate with a degree of 30 mol% of esterification exhibits less than an exabrupta release of 5% and a prolonged release showing 20% release during one day and 40% release during the two days.

EXAMPLE 6 The following example shows the preparation of a protein drug (GCSF) containing a gel of benzyl ester of alginate (DE = 30 mol%) and the prolonged release in vi tro of this gel. This example is carried out in a manner similar to that described in Example 5 except that the ethyl ester is replaced by the benzyl ester alginate. The ester alginate gels during the storage period overnight. The benzyl ester alginate gel with a 30 mol% degree of esterification exhibits less than an exaggerated release of 5% and a prolonged release which shows 40% release during one day and 80% release in two days.

EXAMPLE 7 This example shows the preparation of the ethyl ester alginate beads. The gel beads are prepared by drip-loading the solutions of 2% ester alginate in 100 mM calcium chloride solutions (distilled water or 1M Tris HCl buffer at a> H of 7. 0). The beads that are formed are washed with distilled water or the buffer. Pearls are prepared by using either 30% or 50% degree of esterification.

EXAMPLE 8 This example shows the preparation of ester alginate beads containing leptin. The beads are prepared by dropwise adding a solution of 25 mg / mL leptin in 2% ethyl ester alginate (tris HCl, pH 8.7) in a solution of 100 mM calcium chloride and 25 mM zinc chloride. Pearls are prepared by using a 30% degree of esterification. The beads demonstrate a prolonged release of leptin in vi tro.

EXAMPLE 9 This example shows the disintegration of the molecular weight (or degradation) of the ester alginates in the buffers at a neutral physiological pH. The alginate esters (1% solution) are dissolved either in phosphate buffer (0.1M sodium phosphate, pH 6.8) or 0.1M Tris buffer (pH 7.0) and incubated at 37 ° C. The disintegration of the molecular weight is determined by quantifying the decrease in the viscosity of the solution (Brookfield, 25 ° C) at selected time intervals. Unmodified sodium alginate is found to be relatively stable since its viscosity decreases by only 5% for 8 days (phosphate buffer); However, with ethyl esters and benzyl esters of alginic acid (DE = 30%) the viscosity drops by 35% during 8 days in the same buffer. The amount of degradation of the alginic acid esters is also dependent on the degree of esterification, for example, with the ethyl ester of a lower degree of esterification (DE = 15%) the viscosity decreases by 25% for 8 days. Therefore, the disintegration of the molecular weight is directly related to the degree of esterification.

EXAMPLE 10 This example shows the degradation in vi vo (or gradual disappearance) of esterless alginate gels without protein and hydrogels containing protein.

The ester alginate gels are prepared in a similar manner described in Example 3 but the final mixture is taken in a syringe and allowed to gel in the syringe at 4 ° C overnight. Then 100 μL are injected subcutaneously into the back of the neck of a mouse (Charles River, 12-week-old female, BDF1, 20 g, 5 mice per group) and the site is examined surgically and periodically in different members of the group . When using the ethyl and benzyl ester alginate material with an SD = 30%, the results of the single injection site study show that the ester alginate hydrogels disappear within a period of 2 weeks. When using ethyl ester alginate gel with an SD = 15%, the gels are still present at 30 and 61 days but with a reduced size. When using the material of the ethyl ester alginate with an SD = 5%, the gels are still present at 30 and 61 days with a little reduction in size. By using the unsubstituted sodium alginate material, the gel persists relatively unchanged at day 61. The disappearance rate of the ester alginate gels is similar with or without the protein.

EXAMPLE 11 This example provides weight loss and pharmacokinetic data for hydrogels. of ester alginate containing leptin in rats. The ethyl ester alginate gels are prepared in a similar manner described in Example 4 but the final mixture is taken in a syringe and allowed to gel in the syringe at 4 ° C overnight. The rats are given a bolus dose of 0 mg / kg (control) and 100 mg / kg, then monitor blood levels and weight loss for 7 days. The results show: the ethyl ester alginate with a DE = 5 mol% exhibits a stable blood level of ~2000 ng / mL for 3 days, then declines to 2 ~ 3 ng / mL during the following 3-4 days; ethyl ester alginate with an ED = 15 mol% exhibits a stable blood level of ~2000 ng / mL for two days, then declines to 2-3 ng / mL after 5 days; ethyl ester alginate with a DE = 30 mol% exhibits a blood level of ~ 2000 ng / mL for 1 day, which decreases to 2-3 ng / mL after 4 days; the blood level of the Zn-leptin sequence has a peak at 12 hours, then decreases to 1-2 ng / mL after 6 days. All animals exhibit a weight loss that shows that Zn-leptin is active. The results also show that when Zn-leptin is incorporated in ethyl ester alginate gels (DE = 5 mol% and 15 mol%) it almost doubles (factor of 1.8 - 1.9) the area under the curve (AUC) of Zn-leptin, suggesting a duplication of bioavailability; and the use of ethyl ester alginate gel (DE = 30 mol%) shows a similar bioavailability for Zn-leptin, based on AUC. It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (63)

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

1. A prolonged-release delayed effect gel composition, characterized in that it comprises: a) a hydrophilic polymer; b) a biologically active agent; and c) at least one agglutinated polyvalent metal ion where this gel is biodegradable.

2. The prolonged release composition according to claim 1, characterized in that the bound polyvalent metal ion * is a mixture of a bonded and non-bonded polyvalent metal ion.

3. The sustained-release delayed gel according to claim 1, characterized in that it also comprises excipients to stabilize the biologically active agent or the hydrophilic polymer.

4. The composition according to claim 1, characterized in that the agglutinated polyvalent metal ion is a salt selected from the group consisting of acetates, phosphates, lactates, tartrates, citrates, chlorides, carbonates or hydroxides thereof.

5. The composition according to claim 4, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum or zinc.

6. The composition according to claim 5, characterized in that the metal ion is calcium.

7. The composition according to claim 1, characterized in that the proton donor is given from an acid source.

8. The composition according to claim 7, characterized in that the acid source is selected from the group consisting of buffers, esters, slowly dissolving acids or lactones.

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

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

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

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

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

14. The composition according to claim 12, characterized in that the alginate consists of at least 0.05% by weight.

?5. The composition according to claim 1, characterized in that the biologically active agent comprises a protein, and wherein the composition demonstrates an improved bioavailability.

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

17. The composition according to claim 15, 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.

18. The composition according to claim 15, characterized in that the protein is selected from the group consisting of leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, ILra, IL2, TNF-bp, MGDF, OPG, interferons, erythropoietin, KGF, insulin and analogues or derivatives thereof.

19. The composition according to claim 1, characterized in that the biologically active agent is a biologically active agent formed in complexes.

20. The composition according to claim 19, characterized in that the biologically active agent formed in complexes is a precipitated protein.

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

22. A method for producing a prolonged-release delayed-effect gel composition, characterized in that this gel is biodegradable and comprises the steps of: a) mixing a biologically active agent and a hydrophilic polymer in a solvent to form a first mixture; b) incorporating in the first mixture at least one polyvalent metal ion bonded to form a second mixture.

23. A method according to claim 22, characterized in that it further comprises the step of c) incorporating at least one proton donor capable of releasing the agglutinated polyvalent metal ion into the second mixture.

24, The method according to claim 22, characterized in that the first mixture is concentrated before incorporating the proton donor or the bonded polyvalent metal ion.

25. A method according to claim 22, characterized in that the agglutinated polyvalent metal ion is a salt selected from the group consisting of acetates, phosphates, lactates, citrates, tartrates, chlorides, carbonates or hydroxides thereof.

26. A method in accordance with the claim 22, characterized in that this method provides a substantially constant level of blood of this biologically active agent for a period of time in the patient.

27. The composition according to claim 24, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum or zinc.

28. The composition according to claim 26, characterized in that the metal ion is calcium.

29. The composition according to claim 23, characterized in that the proton donor is given from an acid source.

30. The composition according to claim 28, characterized in that the slowly dissolving acid is selected from the group consisting of buffers, esters, slowly dissolving acids or lactones.

31. The composition according to claim 29, characterized in that the acid source is d-gluconolactone.

32. The composition according to claim 22, characterized in that the hydrophilic polymer is a polyanion.

33. The composition according to claim 22, characterized in that the hydrophilic polymer is a polysaccharide.

34. The composition according to claim 32, characterized in that the polysaccharide is an acid polysaccharide.

35. The composition according to claim 33, characterized in that the polysaccharide is an alginate.

36. The composition according to claim 34, characterized in that the alginate contains at least 30% guluronic acid.

37. The composition according to claim 34, characterized in that the alginate consists of at least 0.05% by weight.

38. The composition according to claim 22, characterized in that the biologically active agent comprises a protein.

39. The composition according to claim 37, characterized in that the protein consists of at least 0.001 mg / ml.

40. The composition according to claim 37, 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.

41. The composition according to claim 37, characterized in that the protein is selected from the group consisting of leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, ILra, '? L2, TNF-bp, MGDF, OPG, interferons, erythropoietin, KGF, insulin and analogues or derivatives thereof.

42. The composition according to claim 22, characterized in that the biologically active agent of the biologically active agent formed in complexes.

43. The composition according to claim 41, characterized in that the biologically active agent formed in complexes is a precipitated protein.

44. The composition according to claim 42, characterized in that the precipitated protein is a zinc leptin precipitate.

45. The method according to claim 22, characterized in that it also comprises the step of isolating the prolonged release composition.

46. The prolonged release product produced by the method according to claim 22 or 44.

47. A pharmaceutical formulation comprising the sustained release composition according to claims 1, 2, 3 or 45 in a pharmaceutically acceptable carrier, diluent or adjuvant.

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

49. a method for treating an indication with a sustained release composition according to claims 1, 2, 3 or 45, in a pharmaceutically acceptable carrier, diluent or adjuvant.

50. A method of treating a disorder that is selected from the group consisting of excess weight, diabetes, high level of blood lipids, arterial sclerosis, plaque deposits in arteries, reduction or prevention of stone formation in the bladder, muscle tissue with insufficient mass, insufficient sensitivity to insulin, and strokes, with a prolonged release composition according to claims 1, 2, 3 or 45 in a pharmaceutically acceptable diluent or adjuvant carrier, wherein the agent Biologically active is a leptin, an analog or a derivative thereof.

51. A method for treating a disorder that is selected from the group consisting of hematopoietic cell deficiencies, infection and neutropenia, with a sustained release composition according to claims 1, 2, 3 or 45, in a carrier, diluent or pharmaceutically acceptable adjuvant, characterized in that the biologically active agent is G-CSF, an analog or derivative thereof.

52. A method for treating an inflammation with a prolonged release composition according to claims 1, 2, 3 or 45, in a pharmaceutically acceptable carrier, diluent or adjuvant, characterized in that the biologically active agent is IL-lra, an analog or a derivative of this.

53. A prolonged release composition comprising: a) a hydrophilic polymer; b) a biologically active agent; c) at least one precipitating agent, characterized in that the biologically active agent is co-precipitated within the hydrophilic polymer, where this composition is found in the form of a gel particle and where this particle is biodegradable.

54. The composition according to claim 52, characterized in that the precipitating agent is selected from the group consisting of polyvalent metal ions or metal salts, acetates, citrates, chlorides, carbonates or hydroxides thereof.

55. The composition according to claim 53, characterized in that the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, aluminum or zinc.

56. The composition according to claim 54, characterized in that the precipitating agent is a polyvalent ion that is selected from the group consisting of zinc, calcium, or a combination thereof.

57. The composition according to claim 52, characterized in that the hydrophilic polymer is a polysaccharide.

58. The composition according to claim 56, characterized in that the polysaccharide is an alginate.

59. A method for producing a sustained release composition, comprising 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) co-precipitating the biologically active agent with the hydrophilic polymer to form a gel particle precipitated together, characterized in that this particle is biodegradable.

60. The method according to claim 58, characterized in that it also comprises the step of isolating the precipitated particle together.

61. The prolonged release product prepared by the method according to claim 59.

62. A pharmaceutical formulation according to claim 52, in a pharmaceutically acceptable carrier, diluent or adjuvant.

63. A method for treating an indication using a sustained release composition according to claim 52, in a pharmaceutically acceptable carrier, diluent or adjuvant.