CZ20004111A3 - Biologically degradable alginate gels exhibiting prolonged release - Google Patents

Abstract

The present invention relates to extended formulations releases that use biodegradable alginate delayed gels or particles and their methods production.

Description

Technical field

The present invention relates to sustained release formulations that use biodegradable alginate gel beads and / or delayed gels, and methods of making the same.

BACKGROUND OF THE INVENTION

The availability of recombinant proteins due to advances in genetic and cellular engineering technologies has developed the use of proteins as pharmaceuticals for therapeutic applications. Many diseases or diseases treated with pharmaceutical proteins require sustained protein levels to achieve the most effective therapeutic result. However, due to the generally short half-life, most protein drugs require frequent administration. These repeated injections are given at different intervals, resulting in fluctuations in drug levels under significant physical and financial burden on patients. Since many diseases better respond to controlled drug levels, controlled drug release is required to provide a longer sustained release period. Such sustained release drugs would provide the patient not only with an enhanced prophylactic, therapeutic or diagnostic effect, but also reduce frequent recurrence of injections as well as overall treatment costs.

Current attempts to maintain drug levels between doses in humans or animals involve the use of biodegradable polymers as a matrix for controlling drug release. For example, UK Patent No. 1,388,580 discloses the use of hydrogels for sustained release of insulin. United States Patent · ··· · • ·· · · · ·······

No. 4,789,550 discloses the use of polylysine-coated alginate microcapsules for administration of a protein by encapsulating living cells. Prolonged-release experiments also used anionic or cationic polymer formulations surrounded by counterion ionic polymers to encapsulate cells capable of producing biologically active formulations, see U.S. Patent No. 4,744,933. Similarly, multiple coatings of anionic or cationic crosslinked polymers have also been described as means to achieve controlled release, see U.S. Patent Nos. 4,690,682 and 4,789,516. In addition, further attempts describe the use of alginates or aiginates coated with other biodegradable polymers for the controlled release of polypeptide preparations or cationic precipitates thereof, see PCT WO 96/00081, PCT WO 95/29664 and PCT WO 96/03116.

However, these experiments have provided insufficient means to achieve sustained release administration of the desired protein therapeutics. It is generally known that the use of certain biodegradable polymers, eg, coglycolide polylactide, in vivo exhibits a high initial pulse in drug release (Johnson, O. et al., Nature Med., 2 (7): 795, 1996). In addition, it is well known that proteins used with current forms of sustained release formulations can undergo denaturation and lose their biological activity when exposed to encapsulating agents. These preparation processes use organic solvents which may have deleterious effects on the selected protein. Finally, as discussed below, the use of alginates alone did not provide the desired controlled release of protein necessary for effective therapeutic results.

In general, alginates are well known, naturally occurring anionic polysaccharides composed of 1,4-linked β-D-mannuronic acid and α-L-guluronic acid (Smidsrod, 0 et al.).

vary from 70 acid to 30% guluronic acid% guluronic acid is in water

Trends in Biotechnoi, 8, 71-78 (1990), Aslani, P. et al., J,

Microencapsulation, 13 (5), 601-614, 1996). Typically, alnurates of mannuronic acid and 30 mannuronic acid and 70 (Smidsrod, supra). Alginic acid insoluble, while salts formed with monovalent ions such as sodium, potassium, and ammonium are water soluble (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 variety of applications, such as food additives, adhesives, pharmaceutical tablets and wound dressings. Alginates have also been recommended for protein separation techniques. For example, Cray, C.J. et al, (Biotechnology and Bioengineering, 31, 607-612, 1988) captured insulin on zinc / calcium alginate gels to separate insulin from other serum proteins.

It has also been well shown to be alginate. matrices are good as drug delivery systems, see, for example, U.S. Patent No. 4,695,463, which discloses alginate-based chewing gum as a drug delivery system and for pharmaceuticals. Alginate beads have been used for the controlled release of various proteins such as: tumor necrosis factor receptor in polycation coated cationic alginate beads (Wee, S.T., Proceed, Intern, Symp. Control. Rel. Bioact. Mater., 21, 730-). 31, 1994), transforming growth factor encapsulated in alginate beads (Puolakkainen, PA et al, Gastroenterology, 107, 1319-1326, 1994), angiogenic factors trapped in calcium alginate beads (Downs, LC et al., J. of Cellular Physiology, 152, 422-429, 1992), albumin trapped in chitosan alginate microcapsules (Polk, A. et al.,

J. Pharmaceutical Sciences, 83 (2), 178-185, 1994), polymer-coated chitosan calcium alginate beads (Okhamafe, Ο.Ο. et al., J. Microencapsul., 13 (5), 497-508, 1996) , hemoglobin, encapsulated by chitosan calcium alginate beads (Huguet, ML et al., J. Applied Polymer Science, 51, 14271432, 1994; Huguet, ML et al., Process Biochemistry, 31, 746-751, 1996) and interleukin- 2 chitosan microspheres (Liu, encapsulated in alginate LS, et al., Proceed.

slowed

Wheatley,

Intern. Symp. Control. Rel. Bioact. Mater., 22, 542-543.

1995).

Systems using alginate gel beads or alginate / calcium gel protein capture beads suffer from a lack of any sustained or sustained release due to rapid release of protein from alginate beads (Liu, L. et al., J. Control. Rel., 43, 65-74) , 1997). To avoid such rapid release, a large number of the above systems have tried to use polycationic polymer coatings (e.g., polylysine, chitosan) to release protein from alginate beads (see e.g.

M.A. et al., J. Applied Polymer Science, 43, 2123-2135 (1991); Wee, S.F. et al., supra, Liu, L.S. et al., supra, Wee, S.F. et al., Controlled Release 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 polyeectrolyte complexes that act as diffusion barriers on the surface of the beads. When using polycations, problems may arise in that: 1) such preparations may be cytotoxic due to polycations (Huguet, ML et al., Supra, Zimmermann, Ulrich, Electrophoresis, 13, 269, 1992, Bergmann, P. et al., Clinical Science, 67, 35, 1984), 2) polycations are susceptible to oxidation, 3) polycation-coated pearls tend to be unbreakable and build up in the body, 4)

Ο -

the formulations are produced using laborious coating techniques that include multiple coatings with polycationic polylysine (Padol, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 2, 216, 1986) and 5) ionic interactions between the protein and polycations may result in loss of protein activity or cause protein instability.

Francesco et al. (U.S. Patent No. 5,336,668, and references cited herein) disclose complete and partial esters of alginic acid formed by various processes and having interesting pharmaceutical qualities. It is described how alginic acid esters can be used as biodegradable plastic materials for medical-surgical use, as additives for a variety of polymeric materials, or can be used in the preparation of various drugs. The possible use of alginic acid esters in sustained release formulations is not discussed, nor are ester alginate hydrogels described.

Nightlinger et al. (Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 22, 738-739, 1995) disclose esterified hyaluronic acid (HA) microspheres having controlled release properties. The references generally refer to different rates of degradation of HA derivatives and describe how the ester breaks to release alcohol and HA groups. There is no discussion of how or whether the HA backbone itself decomposes into lower molecular weight polymer units.

In order to be a polysaccharide-based sustained release drug delivery system, the polysaccharide must be biodegradable to non-toxic products. It has been found that certain alginate gel systems, although effective to provide sustained release of the drug, result in a "hump" (or knot) at the injection site due to very slow gel disintegration. In a therapeutic arrangement involving low doses of drug and sparse injections, this may not be a major problem. But in a treatment arrangement involving high doses of drug

• and more effective clinical application products. The sustained benefit of bringing numerous and thus ensuring and more frequent injections can be dissuasive. Means must be developed to increase the rate of disintegration of the alginate gel at the injection site.

Thus, there remains a need to develop pharmaceutical formulations that produce more versatile, sustained-release, sustained-release, recombinant or natural proteins, more effective clinical outcomes.

The present invention provides such processes. Pharmaceutical compositions using the biodegradable alginate gel particles or gels of the present invention are capable of providing increased bioavailability, protein protection, reduced degradation, and slow release with increased protein stability and efficacy. The pharmaceutical compositions of the present invention also provide a simple, rapid and inexpensive method of controlled release of recombinant protein for effective prophylactic, therapeutic or diagnostic results.

SUMMARY OF THE INVENTION

The present invention is based on studies using unmodified alginate (a group of anionic polysaccharides) hydrogels for sustained release of proteins. These unmodified protein-containing alginate hydrogels (see co-pending U.S. application Ser. No. 08 / 857,913 and 08 / 912,902) are formulated to prolong the release time, and the substances can be filled into a syringe and left to form a later gel in the same syringe as these gels have been found to be injectable. After a single subcutaneous injection, sustained protein is observed for many days in rodent models, but at

injection for a long period of time remains a palpable knot or knot with small size changes. This node consists of?. The alginate hydrogel filled with water and its size is a function of the volume of gel that is injected, and gel beads remain at the injection site.

Thus, the present invention relates to a new class of biocompatible polysaccharide hydrogels, e.g., ester alginate hydrogels, for sustained release of therapeutic proteins. Unexpectedly, esteralginate hydrogels, in addition to sharing the unmodified alginates' properties and sustained release, knot, which does not leave gelling, injectability at the injection site, means that the esteralginate hydrogels are biodegradable or degradable and are gradually resorbed into the surrounding tissues with a small reaction at the injection site.

The compositions of the present invention comprise alginic acid esters or derivatives thereof ionically crosslinked in a hydrogel (water retention) matrix containing a therapeutic agent, such as a protein.

The present invention further relates to a method of producing biodegradable sustained release formulations.

The present invention further relates to the use of ester alginate materials in liquid compositions for delayed gelling in the body.

The present invention further relates to compositions wherein the ester alginate hydrogels are in the form of beads or microspheres for sustained release of active agents, preferably therapeutic proteins.

In one embodiment of the present invention, the ester alginate hydrogels provide formulations for application to target sites in the patient. These preparations are useful; for preventing or inhibiting the formation of tissue adhesions after Φ φφ

Φ · φ

surgical procedures and traumas, in addition to tissue, in particular to supplement soft and hard tissues, to fill the confined space with resorbable material, as a skeleton for tissue growth and as a wound dressing.

In another embodiment, the ester alginate hydrogels provide a device for implantation into an organism containing an active agent, wherein the agent may either be bound or not bound to the alginate polymer.

In another embodiment, the ester alginate hydrogel formulations of the present invention provide a method of improving the bioavailability of the active agent in the formulation.

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

Preparations

Hydrophilic polymers including alginates and derivatives thereof can be obtained from various commercial, natural or synthetic sources well known in the art. The term "hydrophilic polymer" is used herein to refer to water-soluble or water-absorbent polymers. Hydrophilic polymers are well known to those skilled in the art. Hydrophilic polymers include, but are not limited to, polyanions, including anionic polysaccharides such as alginate, carboxymethylamylose, polyacrylic acid salts, polymethacrylic acid salts, ethylene maleate anhydride (half ester) copolymer, carboxymethylcellulose, dextran sulfate, heparin, carboxymethyl celluloseextran.

2,3-dicarboxycellulose, tricarboxycellulose, carboxylated gum arabic, carboxylated carrageenan, pectin, carboxypectin, carboxylated gum tragacanth, carboxylated xanthan gum

• 9

9 rubber, pentosan polysulfate

carboxylated starch carboxymethylchitin / chitosan, curdlan, inositol hexasulphate, β-cyclodextrin sulphate, hyaluronic acid, chondroitin-6 sulphate, dermatan sulphate, heparin sulphate, carboxymethyl starch, carrageenan, polygalacturonate, polycarboxylate sulphate, polycarboxylate sulphate, polycarboxylate sulphate, polycarboxylate sulphate 2, copoiystyrenmaleic acid, agarose, mesoglycan,. sulfopropylated polyvinylalcohol, cellulose sulfate, protamine sulfate, phosphogouar gum, polyglutamic acid, polyaspartic acid, polyamino acids, derivatives or combinations thereof. Those skilled in the art will recognize other various hydrophilic polymers that pertain to the field of the present invention.

Similarly, bound polyvalent metal ions can be obtained from various commercial, natural or synthetic sources well known in the art. 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 salts thereof, such as zinc acetate, calcium acetate or chlorides. Small water-soluble molecules and salts such as ammonium sulfate, acetone, ethanol and glycerol can also be used.

Aliphatic series alcohols for use as the esterification component of the alginic acid carboxyl groups of the present invention are, for example, those containing up to 34 carbon atoms, may be saturated or unsaturated, and may optionally also be substituted with other free or functionally modified groups such as amino, hydroxy groups , aldehyde groups, keto groups, mercapto groups, carboxyl groups, or groups derived therefrom, such as hydroxycarbyl groups or dihydrocarbylamino groups (the term "hydrocarbyl may further include not only monovalent radicals of hydrocarbons, e.g. type C _n H2 _n - _LD but also bivalent or trivalent radicals, such as "alkylene-C _n H ₂ or" alkylidene, = C _n H _2n), ether or ester groups, acetal or ketal groups, thioether or thioester groups and esterified with a carboxyl or carbamide groups or carbamide groups substituted with one or two hydroxy, nitrile or halogens.

In the aforementioned groups containing hydrocarbyl radicals, these radicals are preferably lower aliphatic radicals, such as heteroatoms such as oxygen, nitrogen and sulfur. Preferred are alcohols substituted with one or two of the above functional groups.

The alcohols of the above group preferably used under the conditions of the present invention are alcohols having a maximum of 12 and especially a maximum of 6 carbon atoms, in the radicals of the hydrocarbyl amino groups, the ether groups mentioned above, the thioether ester groups, thioester groups, acetal groups, ketal groups groups having at most alkyl groups of 4 carbon atoms, as well as esterified carboxy groups or substituted carbamide groups of hydroxycarbyl groups are alkyl groups with the same number of carbon atoms, and in which the amino or carbamide groups may be alkylene amino or alkylene carbonyl groups having at most 8 carbon atoms. Of these alcohols, saturated and unsubstituted alcohols such as methyl, ethyl, propyl, isopropyl alcohols, n-butyl alcohol, isobutyl, tert-butyl alcohols, amyl, pentyl, and the like are mentioned in the first place.

octyl, nonyl, and dodecyl alcohols, and in particular with a linear chain such as n-octyl or n-dodecyl alcohols. Among the substituted alcohols of this group, bivalent alcohols, such as ethylene glycol, hexyl, alcohols, acid, should be mentioned acid tt ttt tt tt ttt tt ttttttt tttttttt tttt tttt ttt ttt glycolic, citric, propylene glycol or butylene glycol, trivalent alcohols such as glycerin, aldehyde alcohols such as tartron alcohol, carboxyalcohol such as lactic acid, e.g. alpha-oxypropionic acid, tartaric acid, tartaric acid, tartaric acid, tartaric acid, tartaric acid amino alcohols such as aminoethanol, aminopropanol, n-aminobutanol and their dimethyl and diethyl amine derivatives, choline, pyrrolidinylethanol, piperidinylethanol, piperazinylethanol and the corresponding derivatives of n-propyl or n-butyl alcohols, monothioethylene glycol or alkyl derivatives thereof, for example the mercapto group derivative.

Among the higher saturated aliphatic alcohols deserving special mention are, for example, acetyl alcohol and myristyl alcohol, but for the purposes of the present invention higher unsaturated alcohols having one or two double bonds are particularly important, such as those contained in many essential oils and having affinity for terpenes such as citronelloi, geraniol, nerol, nerolidol, farnesoi, phytoi.

Of the lower unsaturated alcohols, attention should be paid to propargyl alcohol.

The aliphatic alcohols which should be mentioned in particular are alcohols having only one benzene moiety and an aliphatic chain having a maximum of 4 carbon atoms, in which the benzene moiety can also be substituted by 1 to 3 methyl or hydroxy groups or halogen atoms, in particular chlorine, bromine or iodine and wherein the aliphatic chain may be substituted with one or more functional groups selected from the group consisting of free amino groups or mono- or dimethyl groups or pyrrolidine or piperidine groups. Among these alcohols, benzyl alcohol and phenethyl alcohol are particularly preferred. The alcohols of the cycloaliphatic or aliphatic cycloaliphatic series may be derived from mono- or polycyclic hydrocarbons and may have a maximum of 34 carbon

• Φ • <

Atomic atoms. Of the alcohols derived from cyclic single ring hydrocarbons, mention should be made of those having a maximum of 12 carbon atoms, with rings containing preferably 5 to 7 carbon atoms, optionally substituted, for example, with one to three lower alkyl groups such as methyl, ethyl, propyl or isopropyl groups . Specific alcohols from this group are cyclohexanone, cyclonexanediol, 1,2,3-cyclohexanetriol and 1,3,5-cyclohexanetriol (fiorogiucitoi), inositoi, p-menthane derived alcohols such as carventolate, menthol, alpha and gamma-terpineol. - terpineol alcohols known as "terpineol, 1,4- and 1,8-terpine. Alcohols derived from condensed ring hydrocarbons are, for example, those of the thujane, pinane, and camphane groups, in particular thujano, sabinolpinolnyrate, D and L-borneol and D and L-isoborneol.

Also included are alcohols resulting from the esterification reaction of epoxy-containing compounds with alginates (see, e.g., U.S. Patent No. 2,463,824 and U.S. Patent No. 2,426,125).

The total and partial ester groups containing polyanions of the present invention are generally acidic polysaccharides wherein the glycoside oxygen is attached at the beta position to the carbonyl carbon of the ester. Without being bound by any specific mechanism, this arrangement of groups allows the polymer chain to break down through a beta-elimination mechanism that can occur under physiological conditions.

The alginic acid esters of the present invention consist of mannuronic acid residues (m-COOH or m-COO anion) and guluronic acid residues (g-COOH or g-COO anion) linked together by glycosidic ether oxygen bonds according to the following general formula 1:

- (M) n1- (M ') n2 - (G) n3- (G') n4- (Ά) n5 where:

M is a residue of mannuronic acid, m-COOH or m-COO anion,

M ^r is the residue of mannuronic acid ester, m-COOR 1,

G is a guiuronic acid residue, g-COOH or g-COO anion,

G 'is a guiuronic acid ester residue, g-COOR2,

A represents non-g or non-m chain units such as carbohydrates, carbohydrate oxidation products, or aliphatic, aromatic, araliphatic, alaromatic, cycloaliphatic radicals which may be substituted and interrupted by heteroatoms attached in the chain or at the chain ends, n1, n2 , n3, n4, and n5 are integers representing the average relative number of units incorporated,

R1 and R2 are independently aliphatic, aromatic, araiphatic, alaromatic, cycloaliphatic radicals which may be substituted and interrupted by heteroatoms, and derivatives (e.g., wherein hydroxy 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 = an aliphatic or alaromatic radical and further that 100 (n2 + n4) / (n1 + n2 + n3 + n4) be from 1 to 99 mol%, preferably 5 up to 50 mol%, more preferably 6 to 30 mol%, even more preferably 6 to 15 mol%, and most preferably 7 to 12 mol%, and 100n5 / (n1 + n2 + n3 + n4) + n5) is preferably less than 10 mol%.

In the partial esters of the invention, the non-esterified carboxy groups may be kept free or converted into salts. The bases for forming these salts are selected according to the end use of the product. The inorganic salts may be formed from alkali metals such as potassium and especially sodium and ammonium, or may be derived from alkaline earth metals such as calcium or magnesium or aluminum.

Of particular importance are salts with organic bases, in particular basic azo compounds and therefore aliphatic, araiphatic, cycloaliphatic or heterocyclic amines. These «and • ·

the ammonium salts may be derived from therapeutically acceptable amines or non-toxic but therapeutically inactive amines or amines with therapeutic effect. For the first type, aliphatic amines are preferred, for example mono-, di- and trialkylamines having alkyl groups of up to 8 carbon atoms or arylalkylamines having the same number of carbon atoms in the aliphatic moiety and wherein aryl is a benzene group optionally substituted with 1 to 3 methyl groups. Biologically inactive bases for salt formation may also be cyclic, such as monocyclic alkyienamines with cycles of 4 to 6 carbon atoms, optionally interrupted in the cycle by heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, such as piperazine or morpholine or may be substituted, for example, with an amino or hydroxy group such as aminoethano, ethylenediamino, ethylenediamine, ephedrine or choline.

The degree and type of esterification can be controlled by synthetic methods known in the art. Preferably, the alginate esters are prepared by treating quaternary ammonium salts of alginic acid with conventional alkylating agents in an aprotic organic solvent such as dimethylsulfoxide. The resulting esters are preferably esters of monovalent alcohols such as lower alkyl such as ethyl or aralkyl such as benzyl or mixtures thereof. They can also be formed by the reaction of aiginic acid with oxirane or epoxy containing compounds such as ethylene or propylene oxide.

It is also possible to form quaternary ammonium salts of partial esters, for example tetraalkylammonium salts having the above number of carbon atoms, and preferably salts of this type, in which the fourth alkyl group has 1 to 4 carbon atoms, for example methyl.

The degree of esterification (expressed in% (mole)) of aiginic acid refers to the desired rate of disintegration of the gel ib in the patient tissue. This gel disintegration rate is generally related to the desired rate of release of the active agent from the gel, which is for 5 years or less, usually 2 days to 270 days, more usually 2 days to 180 days, more usually 2 days to 90 days. The degree of esterification (D 1) is from 1 to 99 mol%, preferably from 5 to 50 mol%, more preferably from 6 to 30 mol%, even more preferably from 6 to 15 mol%, and most preferably from 7 to 12 mol%.

The term buffer or buffer solution as used herein refers to the use of inorganic or organic acids or a combination thereof for the preparation of a buffer solution, as is known in the art. Inorganic acids within the scope of the present invention include hydrogen halide (e.g., hydrochloric acid), phosphoric acid, nitric acid or sulfuric acid. Other inorganic acids are well known to those skilled in the art and are considered herein. Organic acids within the scope of the invention include aliphatic carboxylic acids and aromatic acids such as formic acid, carbonic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, acrylic acid, maionic acid, succinic acid, giutaric acid, adipic acid, maicinic acid, fumaric acid, gicycin or phenoisuifonic acid. Daishi organic acids are well known to those skilled in the art.

The term biologically active agent as used herein refers to recombinant or naturally occurring proteins, human or animal, useful for prophylactic, therapeutic or diagnostic applications, as well as non-protein based agents such as small molecules. The biologically active agents may be natural, synthetic, semisynthetic agents or derivatives thereof. The biologically active agent of the present invention must be able to precipitate. A variety of biologically active agents are contemplated. This includes, but is not limited to, hormones, cytokines, haemopoietic factors, growth factors. anti-obesity factors, trophic factors, anti-inflammatory factors and enzymes (see also U.S. Patent No. 4,695,463 for further examples of useful biologically active agents). One skilled in the art is readily able to adapt the desired biologically active agent to the composition 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, hereby incorporated by reference, including figures), interleukins (see U.S. Patent No. 5,075,222, hereby incorporated by reference). including erythropoietins (US Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080, hereby incorporated by reference), granulocyte colony stimulating factor (visas patents) No. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185, hereby incorporated by reference, including figures), stem cell factor (PCT Publication Nos. 91/05795, 92 / 17505 and 95/17206, hereby incorporated by reference, including figures, and OB protein (PCT Visas Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816, hereby incorporated by reference, including figures). In addition, biologically active agents may also include, without limitation, anti-obesity products, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human choriogonadotropin (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), tumor necrosis factor binding protein (TNF-bp), neurotrophic brain factor (BDNF) ), glial neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegerins (OPG), insulin-like growth factors (IGF), macrophage stimulating factor colony (M-CSF), macrophage and granulocyte colony stimulating factor (GM-CSF), megakeratinocyte growth factor (MGDFj, thrombopoietin, platelet growth factor (PGDF), rose colony stimulating factors (CSF), bone morphogenetic protein (BMP), superoxide-aismutase (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.

Derivatives of biologically active agents may include attachment of one or more chemical moieties to a protein moiety. Chemical modifications of biologically active agents have been found to provide additional benefits under certain conditions, such as increased stability and circulation time of the therapeutic protein and reduced immunogenicity. One skilled in the art will be able to select the desired chemical modification based on the desired dosage, circulation time, resistance to proteolysis, therapeutic use, and other considerations.

The term biodegradability in this specification refers to the disintegration of a higher molecular weight molecule of a particular polymer into a smaller number of units in the chain, i.e. disintegration into units of lower molecular weight. The term biodegradable gei refers to the dispersion of a gel in the environment in which it is used, the dispersion being conditional upon the disintegration of the higher molecular weight base polymer molecules resulting in fewer units in the polymer chain.

Complexes

Proteins, analogs or derivatives can be administered complexed with a binding agent. This binding agent may have the effect of prolonging the circulation time of the protein, analog or derivative or increasing the activity of the biologically active agent. Such a preparation may be a protein (or synonymously a peptide), a derivative, an analog, or a combination thereof. For example, the OB protein binding protein is the OB protein receptor or a portion thereof, such as a soluble portion thereof. Other binding proteins may be detected by examining the OB protein or protein of choice in serum or may be empirically tested for the presence of binding. Such binding typically does not interfere with the ability of the OB protein or analog or derivative to bind to the endogenous OB protein receptor and / or effect signal transduction. In addition to the OB protein, the binding complexes will also be useful as well for other therapeutic proteins of the present invention. Those skilled in the art are able to ascertain suitable binding proteins for use in the present invention.

Similarly, precipitating agents used to precipitate a biologically active agent 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, polyvaient metal ions or salts thereof, such as acetates, citrates, chlorides, carbonates, hydroxides, oxalates, tartrates, or hydroxides, acids, or water-soluble polymers thereof. Specifically, the metal ions may include, without limitation, aluminum, barium, calcium, iron, manganese, magnesium, strontium, and zinc. Preferably, the metal ion is zinc or salts thereof, such as acetate chloride. Water-soluble small molecules and salts such as ammonium sulfate, acetone, ethanol and glycerol can also be used.

• · · · ·

With respect to water-soluble polymers, these include but are not limited to polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly1,3,6-trioxane, ethylene maleate anhydride copolymers, polyamino acids. dextran, poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, polyvinyl alcohol-succinate, glycerin, ethylene oxides, propylene oxide, poloxamers, alkoxylated copolymers, or water-soluble copolymers thereof, in water. The water-soluble polymer can be of any molecular weight and can be branched or unbranched. For example, the preferred molecular weight of polyethylene glycol is between about 700 kD and about 100 kD for ease of handling and precipitation efficiency.

Depending on the desired therapeutic profile, other sizes and types of precipitating agents may be used (eg, desired sustained release duration, effect, biological activity, ease of handling, degree of deficiency or lack thereof, and other known effects of the desired precipitating agent on the therapeutic protein or analog). One skilled in the art is aware of other precipitating 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 hydrophilic polymer. These may be included in the buffer and may include, without limitation, preservatives.

• ·

Pharmaceutical preparations

The sustained release pharmaceutical compositions of the present invention may be administered orally (e.g., capsules such as hard capsules and soft capsules, solid preparations such as granules, tablets, pills, lozenges, powdered lozenges, pellets, powder liquid preparations such as parenteral administration (e.g. e.g.

intramuscular, subcutaneous, transdermal, visceral, i.v.

(intravenous), i.p. (intraperitoneal), intraarterial, intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal, rectal and uterine mucosal resorption).

or medical candies, and lyophilized forms, suspensions) and preparations

In general, the invention encompasses sustained release pharmaceutical compositions containing an effective amount of a protein or derived products, with the sustained release compositions of the invention together with pharmaceutically acceptable diluents, preservatives, solvents, emulsifiers, adjuvants and / or carriers required for administration. See PCT 97/01331 incorporated by reference. The optimal pharmaceutical formulation for the desired biologically active agent will be determined by one skilled in the art depending on the modes of administration and the desired dosage. Examples of pharmaceutical formulations are described in Remington's Pharmaceutical Sciences (Mack Publshing Co., 18th ed., Easton, PA, 1435-1712, 1990).

Because of the thixotropic nature of the delayed gel formulation, syringes for subcutaneous administration can be used. The formulation may be gelled in a syringe for later injection. This gelling can be performed in a delayed, i.e., delayed, manner. The timing is controlled by sensitively adjusting the amount of gelling agent and proton donor in the mixture, if necessary. Such a formulation will be used for later re-gelling in the body after injection. The term thixotropic, as used herein, refers to the viscosity of a gel composition that is reduced under pressure, eg, by a syringe plunger, whereby the mixture can flow through, eg, a syringe needle, and then recreate the gel at the injection site.

The concept of delayed gelling can also be applied to syringe filling, wherein the sustained release gel formulation is filled into the syringe and the preselected time in the syringe is gelatinized, eg, several minutes to many hours after filling. This avoids the problem of filling the syringe with the substance it has gelled. These pre-filled syringes can be stored for later injection to patients.

Components that may be required for administration include diluents with varying buffer contents (eg, Tris-HCl, acetate), pH and ionic strength, additives such as surfactants and solvents antioxidants metabisulfite

HCO-60, polysorbate 80), ascorbic, glutathione, (e.g., sodium, and carrier incorporation of the compound (e.g., Tween 80, (e.g., sodium acid)), other polysaccharides carboxymethyllulose, sodium alginate, hyaluronate protamine sulfate, polyethylene glycol), preservatives ( Thimersol, benzylalkonol, methylparaben, propylparaben) Substances or fillers (eg lactose, mannitol), polymeric particulate agents such as polylactic / polyglycolic acid polymers or copolymers, etc., or combined with liposomes. In addition, the sustained release formulations of the present invention may also be dispersed with oils (eg, sesame oil, corn oil, vegetable oil) or mixtures thereof with phospholipids (eg, lecithin). ) or triglycerides of fatty acids with moderate d with an alkali chain (eg Miglyoi 812) to form an oil suspension.

··

The compositions of the present invention may also be dispersed with dispersing agents such as water-soluble polysaccharides (eg, mannitol, lactose, glucose, starches), hyaluronic acid, glycine, fibrin, collagen, and inorganic salts (eg, sodium chloride).

In addition, it is also contemplated to use the mechanical sustained release devices of the present invention designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers and powder inhalers, all of which are known to the skilled person.

The components of the formulation required for administration may affect the physical and rate of state, stability, and in vivo release rate of the in vivo clearance of the present proteins and derivatives. The person skilled in the art is aware of the appropriate formulation components required for administration and / or the appropriate mechanical devices for use depending on the therapeutic use, the mode of administration, the desired dosage, the circulation time, proteolysis resistance, protein stability and other reasons.

Methods of use

Therapeutic use

The therapeutic use depends on the biologically active agent used. One of skill in the art will readily adapt the desired biologically active agent of the present invention for its intended therapeutic use. Therapeutic use for these agents is further detailed in the following publications, hereby incorporated by reference, including figures. Therapeutic uses include, but are not limited to, the use of proteins such as (see U.S. Patent Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623, hereby incorporated by reference, including figures), interleukins (see U.S. Patent No. 5,962,373, incorporated herein by reference). 5 075 222, hereby • · • ·

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), granulocyte colony stimulating factor (see patents) No. 4,999,291, 5,581,476, 5,582,823,

810 643 and PCT Publication Nos. 94/17185, hereby incorporated by reference. Including Figures), Stem Cell Factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including Figures) and OB protein (PCT Publication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816, hereby incorporated by reference, including figures).

In addition, the therapeutic uses of the present invention include the use of biologically active agents such as, without limitation, anti-obesity products, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTHj), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, interferon (alpha, beta, gamma), interleukins (IL-1 to 11-12), tumor necrosis factor (TNF), tumor necrosis factor binding protein (TNF-bp), neurotrophic brain factor (BDNF), glial neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegerins (OPG), insulin-like growth factors (IGF) , macrophage colony stimulating factor (M-CSF), macrophage and granulocyte colony stimulating factor (GM-CSF), megakeratinocyte growth factor tor (MGDF), thrombopoietin, platelet 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 includes peptides, polypeptides, consensus molecules, analogs, derivatives or derivatives thereof

ΦΦ Φ · φ · · φ

Φ ··· φφφφ combination. In addition, the compositions of the invention may also be used for the manufacture of one or more medicaments for treating or alleviating a disease state that the biologically active agent is intended to treat.

Examples of therapeutic use may also be to achieve blood oxygenation and decrease bone resorption or decrease osteoporosis without weight loss.

Combination therapies

The compositions of the invention may be used in conjunction and exercise with other treatments such as altered diet such as drugs useful for treatment. Other medicines, diabetes (eg insulin, and possibly amylin), cholesterol and blood pressure lowering medicines (such as medicines that lower blood lipid levels or other cardiovascular medicines), activity enhancing medicines (eg amphetamines), diuretics (for fluid secretion) ) and taste suppressors. Their administration may be simultaneous or may be administered one after the other. In addition, the present method can be used in conjunction with surgical procedures such as cosmetic surgeries aimed at altering the overall appearance of the body (eg liposuction or laser surgery aimed at reducing body mass or implant surgery aimed at increasing body mass). The health benefits of cardiological surgical procedures, such as bypasses or other surgeries aimed at alleviating the deleterious condition caused by blocking blood vessels by fat deposits, such as arterial plaques, can be enhanced by the simultaneous use of the present compositions and methods. Methods for the removal of gallstones, such as by ultrasonic or laser methods, may also be used, either before, during or after the course of the present therapeutic method. In addition, the present methods can be used as a supplement to surgical procedures. Nebo φ léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby léčby broken bones, damaged muscles, or other treatments that can be improved by enhancing poor tissue mass.

Dosage

One skilled in the art is able to determine the effective dosage by administering the drug and observing the desired therapeutic effect. The dose of the sustained release preparation is an amount necessary to achieve an effective concentration of the biologically active agent in vivo over a given period of time. The dosages and preferred frequency of administration of the sustained release formulation vary with the type of biologically active agent, the desired release time, the target disease, the desired frequency of drug administration, the animal species in question and other factors. Preferably, the formulation of the molecule will be such that a dose of between about 0.10 µg / kg / day to 100 mg / kg / day provides the desired therapeutic effect.

Effective dosages can be determined using diagnostic aids over time. By way of example, the present invention provides dosage of OB protein. For example, a diagnostic for measuring the amount of OB protein in blood (or plasma or serum) may first be used to determine endogenous levels of OB protein. Such a diagnostic tool may be in the form of an antibody assay, such as an antibody "sandwich assay." First, the amount of endogenous OB protein is quantified and the baseline value is determined. Therapeutic doses are determined, while quantification of the endogenous and exogenous OB protein (ie, a protein, analog, or derivative found in the body, either produced by the organism or administered) persists during therapy. For example, a relatively high dose may be needed initially until therapeutic benefit is observed, and then lower doses are used to maintain the therapeutic benefit.

DETAILED DESCRIPTION OF THE INVENTION

Material and methods

Material

The alginate in the form of sodium alginate can be found from sources well known in the art. Protein OB and GCSF are from Amgen Inc, other chemicals are known in the art.

Preparation of alginate hydrogel particles / beads

The preparation of particles and beads of an alginate hydrogel, with and without proteins, is described in detail in co-pending U.S. application Ser. No. 08 / 842,756, incorporated herein by reference.

Preparation of delayed gel

The preparation of delayed alginate hydrogels, with and without proteins, is described in detail in co-pending U.S. applications 08 / 857,913 and 08 / 912,902, incorporated herein by reference.

The following examples are provided to more fully explain and illustrate the invention, but are not intended to limit the scope thereof. In addition, in view of the above description or the examples below, one skilled in the art is able to make the necessary changes to the descriptions for large-scale production purposes.

Example 1

This example describes the preparation of the ester alginates used in the present invention.

* · ·

Tt tt tt

Preparation A: tetrabutylammonium (TBA) alginate

The sulfonic acid resin (Bio-rad, AG MP-50) is converted to the tetrabutylammonium form (TBA) by treatment with tetrabutylammonium hydroxide (Aldrich) using a periodic procedure at room temperature. To a solution of 10 g of sodium alginic acid in 800 ml of distilled water is added 60 ml of sulfone resin (Bio-rad, AG MP-50) as the tetrabutylammonium salt. The mixture is stirred for 0.5 hour at room temperature. The resin is removed by filtration and washed with distilled water. The TBA alginate in the filtrate is isolated by lyophilization (yield 16.8 g) and confirmed by ¹ H NMR.

Preparation B: Partial ethyl ester of alginic acid, degree of esterification (DE) = 30% (mole).

g (14.4 mmol of TBA units) The TBA alginate is dissolved in 500 mL of dimethylsulfoxif (DMSO) at room temperature. 673 mg of iodoethane (4.3 mmol, Aldrich) is then added. The mixture is stirred at 30 ° C for 15 hours and then cooled to room temperature.

To this solution is slowly added a solution of 2 g of NaCl in 20 ml of water to completely convert the TBA to the sodium salt. After stirring for 15 to 30 minutes, the solution is slowly poured into 1500 ml of ethyl acetate. The precipitate is collected by filtration and washed three times with acetone / water (8: 1 v / v) and three times with acetone, and then dried under vacuum. The compound is redissolved in -100 mL of distilled water and the pH is adjusted to ~ 6.5 with 0.2% NaHCO ₃ at 0 ° C. The solution is then dialyzed (MW 8000 limit) overnight against distilled water at 4 ° C, and then lyophilized. The yield of the partial ester is 2.8 g and the degree of esterification is 30 ± 1% ( ¹ H NMR, as maleimide internal standard).

ti 9 9 9

9 9 9 9 9 9 9 9 • 9 9 9 9 9 9 9 9

9 9 9 9 9

9999 9999 99 999 99

Preparation C: complete 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 B except that the amount of iodoethane added is adjusted to achieve the desired degree of esterification.

Preparation D: Partial propyl, hexyl, octyl and dodecyl esters of alginic acid.

The preparations are similar to those described in Preparations B and C above, but iodoethane is substituted with 1-iodopropane, 1-iodohexane, 1-iodooctane or 1-iodo-dodecane for each.

Preparation E: Partial benzyl ester of alginic acid, DE = 30%.

2.5 g of Ί'ΒΑ alginate (5.99 mmol of TBA units) are dissolved in -200 mL of DMlSO at room temperature. 307 mg of benzyl bromide (1.8 mmol, Aldrich) and 30 mg of TBA iodide (Aldrich) are added. The mixture is stirred at 30 ° C for 15 hours and then cooled to room temperature. To this solution is slowly added a solution of 0.6 g of NaCl in 10 ml of water to completely convert the TBA to the sodium salt. After stirring for 15 to 30 minutes, the solution is slowly poured into 500 ml of ethyl acetate. The precipitate is collected by filtration and washed three times with acetone / water (8: 1 v / v) and three times with acetone, and then dried under vacuum. The compound is redissolved in ~ 60 mL distilled water and the pH is adjusted to ~ 6.5 with 0.2% NaHCO ₃ at 0 ° C, and then the compound is dialyzed (MW 8000 limit) overnight against distilled water at 4 ° C . The yield of the partial ester is 1.3 g and the degree of esterification is 30 ± 1% 1 H NMR (maleimide internal standard).

9 · 9

Preparation F: complete 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 TBA iodide added is adjusted to achieve the desired degree of esterification.

Example 2

The following example illustrates the preparation of an ethyl ester alginate (DE-15% (mole) and 10% (mole)) gel containing a protein drug (leptin) and in vitro sustained release of the protein from the gel.

100 mg / ml leptin (10mM Tris HCl, pH 8.8, pH adjusted from 8.0 to 8.8 with 1M NaOH) and 6% ethyl ester alginate (15% (mole), 10mM Tris HCl, pH 8.6) are cooled in an ice bath. 0.5 ml of leptin is added to 0.18 ml of 6% ethyl ester alginate and the mixture is stirred in an ice bath for 10 to 15 minutes, the final pH is 8.6 to 8.8. To this mixture is added 16 μCO of 1M CaCO ₃ suspension and the resulting suspension is well mixed. To this suspension is added dropwise with stirring 100 μ 100 of 0.1 M ŽnCL ₂ solution, then water is added to make up the volume to 1 ml. The mixture is completely mixed and kept in an ice bath for 10 to 20 minutes. Then, 56 μΐ of 1.68M δ-gluconolactone (Aldrich) solution is thoroughly mixed into this mixture. The final mixture (50 mg / ml leptin, 1% ethyl ester alginate, 0.1 mL) is poured into the interior of the eppender tube and allowed to gel overnight at 4 ° C. After overnight storage, in vitro release was performed in 10 mM histidine buffer, pH 7.4. The cast gel with a 15% (mole) degree of esterification exhibits a minimum initial pulse release and a completely constant leptin release showing 60% release within six days.

0 · 0 · 4 4 · • 000 · · * 0 000 0 0 0 0 0 0 0 a a 000 · e

00000000 00 0 0 i 0 »0

The cast gel with a 10% (mole) degree of esterification shows a minimal initial pulse release and a completely constant leptin release showing 55% release within six days.

Example 3

This example = 15 mol% and drug (leptin) a

It implies the preparation of a non-ethyl ester alginate (DI 3 10% (mole)) gel containing protein in vitro prolonged release of protein from the gel.

This example is carried out by the podoiony method as exemplified except for the use of ethyl ester alginate.

Hexyl ester alginate gel with 15% (mole) and 10% degree of esterification shows a minimal initial described (mole) pulse release and exhibits a sustained release showing 50% release within six days.

Example 4

The following example shows the preparation of an ethyl ester alginate (DE = 15% (mole)) gel containing a protein drug (Znleptin) and an in vitro sustained release of the protein from the gel.

To 0.75 ml of a 4% w / v solution of ethyl esteraiginate (15% (mole)) is added 7.5 μΐ of 1M Tris HCl, pH 8.0, 33 μΐ of 0.5M PIPES, pH 6.8 and 8. , 5 μΐ 0.1 µM ZnCl2 · Mix well. To this solution is added 675 μΐ of the Zn-leptin suspension (100 mg / ml) and the mixture is thoroughly mixed. 24 μί of 1 M CaCO 3 suspension and 70 μί of 1.68 M δ-gluconolactone solution are thoroughly mixed into this mixture. 0.1 ml of the final mixture is poured into the interior of the eppendorf tube and allowed to gel overnight. After overnight storage, an in ° C release was performed in 10 mM histidine buffer, pn

7.4.

A 13 ethyl ester alginate gel exhibits low initial pulse release and sustained release of leptin showing 65% release over four days.

(mol) degree of esterification

Example 5

The following example shows the preparation of an ethyl drug ester alginate (DE = 30% (mol)) gel containing a protein drug (GCSF) and an in vitro sustained release of the protein from the gel.

To 0.5 ml of a solution of 2.39% ethyl ester alginate (30% (mole)) is added 100 μΐ 0.1M acetate buffer (at 4.5), 104 μΐ GCSF (48.2 mg / ml, HCl pH 3) and 24 6 ml of distilled water. The mixture is well mixed. 10 μΐ of a 1M Cah'PO ₄ suspension and 40 μΐ of a 1.68M δ-gluconolactone solution are thoroughly mixed into this mixture. 0.2 ml of the final mixture is poured into the interior of the eppendorf tube and allowed to gel overnight at 4 ° C. After overnight storage of the gel, in vitro release was performed in 10 µM Tris buffer, pH 7.5. The cast ethylesteralgate gel with a 30% (mole) degree of esterification exhibits less than 5% initial pulse release and sustained release of leptin showing 20% release within one day and 40% release within 2 days.

Example 6

The following example shows the preparation of an ethyl ester alginate (DE = 30% (mol)) gel containing a protein drug (GCSF) and an in vitro sustained release of the protein from the gel.

9

9 9 9 9999

Λ · ·

This example is carried out in a similar manner to that described in Example 5 except that the ethyl ester is replaced with benzyl ester alginate. Esteralginate gels during nighttime storage. Benzyl ester alginate gel with a 30% degree of esterification exhibits less than 5% initial pulsed release and sustained release of leptin showing 40% release within one day and 80% release within 2 days.

Example 7

This example shows the preparation of pearls from ethyl ester alginate.

Gel beads are prepared by dropwise adding a 2% esteralginate solution to a 100 mM calcium chloride solution (distilled water or 1M Trls HCl buffer, pH 7.0). The formed beads are washed with distilled water or buffer. Pearls are prepared using either a 30% or 50% degree of esterification.

Example 8

This example illustrates the preparation of leptin-containing esteraiginate beads.

The beads are prepared by dropwise adding a solution of 25 mg / ml leptin in 2% ethyl ester alginate (Tris HCl, pH 8.7) to a solution of 100 mM calcium chloride and 25 mM zinc chloride. Pearls are prepared using a 30% degree of esterification. The beads show a sustained release of leptin in vitro.

«« «*» • * «· 9 · 4 · 4 4 · · 4 9 ·

999 99

Example 9

This example illustrates the breakdown (or degradation) of high molecular weight esteralginate molecules in buffers with neutral physiological pH.

A 1% esteralginate solution is dissolved in either phosphate buffer (0.1 M sodium phosphate, pH 6.8) or 0.1 M Tris buffer (pH 7.0) and incubated at 37 ° C. Molecular weight decay is determined by measuring solution viscosity reduction (Brookfield, 25 ° C) at selected time intervals. It was found that unmodified sodium alginate is relatively stable, since its viscosity decreased only by 5% within 8 days (phosphate buffer), but for ethyl and benzyl esters of alginic acid (DE = 30%) the viscosity decreased by 35% within 8 days in the same buffer. The degree of degradation of alginic acid esters is also dependent on the degree of esterification, for example, in ethyl ester of lower degree of esterification (DE = 15%) the viscosity decreased by 25% within 8 days. Thus, the molecular weight breakdown is directly related to the degree of esterification.

Example 10

This example shows the in vivo degradation (or gradual loss) of protein-free esteralginate hydrogels and protein-containing hydrogels.

Esteralginate gels are prepared in a manner similar to that described in Example 3, but the final mixture is drawn into a syringe and allowed to gel in a syringe at 4 ° C overnight. Then, 100 μΐ of gel is injected subcutaneously into the back of the neck of mice (Charles River, female. 12 weeks old, BDF1, 20 g, 5 mice per group) and the site is periodically surgically examined on various Group members.

Using ethyl and benzyl ester alginate materials with DE = 30%, the results of a single injection study show that the ester alginate hydrogel disappears within 2 weeks. When using an ethyl ester alginate gel with EE = 15% gels still occur

On day 30 and 61 but have a smaller ethyl ester alginate gel with DE = 5 on day 30 and 61, with a small reduction in unsubstituted sodium alginates unchanged after day 61.

size. When% is used, gels still appear in size. In use, the gel persists relatively

The rate of loss of ester alginate gels with or without protein is the same.

Example li

This example provides loss and pharmacokinetic data for leptin-containing ester alginate in a rat.

weight hydrogels

Esteralginate gels are prepared in a manner similar to that described in Example 4, but the final mixture is taken up in a syringe and allowed to gel in the syringe at 4 ° C overnight. Rats are given a bolus dose of 0 mg / kg (controls) and 100 mg / kg, followed by blood levels and weight loss for seven days.

The results showed: esteralginate with DE = 5% (mole) showed a steady-state blood level of -2000 ng / ml for 3 days, then decreased to 2-3 ng / ml for another 3-4 days, esteralginate with DE = 15% ( mol.) showed even blood

surface -2000 ng / ml after 2 days, then the level dropped to 2 - 3 ng / ml v 5 days, esteralgi .Nat with DE = 30% (mol.) manifested even blood level -2000 ng / ml 1 day which has decreased na 2 -: 3 ng / ml at 4 days, blood level suspension Zn-leptin peaked and in 12 hours, and then decreased to 1-2 ng / ml at 6

«··· · · ·· days. All animals showed weight loss indicating that Zn-leptin was active. The results also showed that the incorporation of Zn-leptin into ethyl ester alginate gels (DE = 5% (mole) and 15% (mole)) almost doubled (factor 1.8-1.9) the area under the curve (AUC) of Zn-leptin suggesting a doubling of bioavailability, and the use of ethyl ester alginate gel (DE = 30% (mole)) showed the same bioavailability of Zn-leptin, based on AUC.

Claims (30)

PATENT CLAIMS 1. A delayed gel sustained release preparation comprising: a) hydrophilic polymer, (b) a biologically active agent; and c) at least one bound polyvalent metal ion, wherein the gel is biodegradable. 2. The sustained release preparation of claim 1, wherein the bound polyvalent metal ion is a mixture of bound and unbound polyvalent metal ion. The delayed gel sustained release preparation of claim 1, further comprising excipients for stabilizing the biologically active agent or hydrophilic polymer. The composition of claim 1 wherein the bound 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 of claim 4 wherein the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum, or zinc. 6. The composition of claim 5, wherein the metal ion is calcium. • · A composition according to claim 1, characterized in that: provided that the proton donor is from an acid source. A composition according to claim 7, characterized in that: wherein the acid source is selected from the group consisting of buffers esters, slow dissolving acids or lactones. 9. The composition of claim 1 wherein the hydrophilic polymer is a polyanion. 10. The composition of claim 1, wherein the hydrophilic polymer is a polysaccharide. 11. Preparation according to claim 10 confesses č u j - Lei s t i rn that the polysaccharide is sour polysaccharide. 12. Preparation according to claim 11 confesses č u 3 - Lei s t i rn that the polysaccharide is alginate • 13. Preparation according to claim 12 confesses č u j - Lei s wherein the alginate comprises at least 30% guluronic acid. 14. The composition of claim 12, wherein the alginate is at least 0.05% by weight. 15. The composition of claim 1, wherein the biologically active agent comprises a protein composition exhibiting improved bioavailability. The composition of claim 15 wherein the protein content is at least 0.001 mg / ml. Φ Φ · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ 17. The composition of claim 15 wherein the protein is selected from the group consisting of haemopoietic factors, colony stimulating factors, anti-obesity factors, growth factors, trophic factors, and anti-inflammatory factors. The composition of claim 15, wherein the protein is selected from the group consisting of leptin, GCSP, SCF, BDNF, GDNF, N'1'3, GM-CSF, IL-1ra, IL2, TNF-bp, MGDF, OPG , interferons, erythropoietin, KGF, insulin and analogs or derivatives thereof. The composition of claim 1, wherein the biologically active agent is a complexed biologically active agent. 20. The composition of claim 19, wherein the complexed biologically active agent is a precipitated protein. 21. The composition of claim 20 wherein the precipitated protein is a zinc-leptin precipitate. 22. A method of producing a delayed-release sustained-release gel composition wherein the gel is biodegradable, the method comprising the steps of: a) mixing the biologically active agent and the hydrophilic polymer in a solvent to form a first mixture, b) admixing at least one bound polyvalent metal ion to the first mixture to form a second mixture. · V v v v v v v v v v 23. The method of claim 22, further comprising the step of (c) admixing a second mixture of at least one proton chip capable of releasing bound polyvalent metal ion. 24. The method of claim 22 wherein the first mixture is concentrated prior to admixing a proton donor or bound polyvalent metal ion. 25. The process of claim 22 wherein the bound polyvalent metal ion is a salt selected from the group consisting of acetates, phosphates, lactates, tartrates, citrates, chlorides, carbonates, or hydroxides thereof. A method according to claim 22, characterized in that: The method of claim 1 wherein the method provides a substantially constant blood level of the biologically active agent for a period of time in a patient. 27. The composition of claim 24 wherein the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum, or zinc. 28. The composition of claim 26 wherein the metal ion is calcium. 29. The composition of claim 23, wherein the proton donor is from an acid source. 30. The formulation of claim 28, wherein the slowly dissolving acid is selected from 9 9 9 99. 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9999 9999 99 999 99 groups containing buffers, esters, slow-dissolving acids or lactones. 31. The composition of claim 29, wherein the acid source is δ-gluconolactone. 32. The composition of claim 22, wherein the hydrophilic polymer is a polyanion. 33. The composition of claim 22, wherein the hydrophilic polymer is a polysaccharide. 34. The composition of claim 32, wherein said composition is: The polysaccharide is an acidic polysaccharide. i c í se 35. The composition of claim 33, wherein said composition is: í o i se wherein the polysaccharide is alginate. 36. The composition of claim 34, wherein said composition is: í c í se wherein the alginate comprises at least 30% guiuronic acid. 37. The composition of claim 34, wherein said composition is: i c 1 se wherein the alginate is at least 0.05% by weight ) · 38. The composition of claim 22, wherein said composition is: i c 1 se The biologically active agent comprises a protein. 39. The composition of claim 37, wherein said composition is: i 0 i se wherein the protein content is at least 0.001 mg / ml. 40. The composition of claim 37 wherein the protein is selected from the group consisting of haemopoietic factors, colony stimulating factors, anti-obesity factors, growth factors, trophic factors, and anti-inflammatory factors. 41. The composition of claim 37 wherein the protein is selected from the group consisting of leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, IL-1ra, IL2, TNF-bp, MGDF, OPG, interferons, erythropoietin, KGF, insulin and analogues or derivatives thereof. 42 . The composition of claim 22 v y z n a č u j i c i s e t i rn, that a biologically active agent Yippee corplexed biological active agents. 4 3 . The composition of claim 41 v y z n a č u j i c i s e t i rn, that corplexed biologically active agents Yippee precipitated protein. 44. The composition of claim 42, wherein the precipitated protein is a zinc-leptin precipitate. 45. The method of claim 22, further comprising the step of isolating the sustained release formulation. 46. A sustained release preparation which has been produced by the process of claims 22 or 44. 47. A pharmaceutical composition comprising the sustained release composition of claim 1, 2, 3 or 45 in a pharmaceutically acceptable carrier, diluent or adjuvant. Pharmaceutical preparation according to claim 1, characterized in that the preparation is injectable The method of indicated treatment is characterized by treatment with the sustained release formulation according to 2, 3 or 45 in a pharmaceutically acceptable carrier. 48. confess the syringe 49. The method of claim 1, wherein the diluent or adjuvant is used. 50. A method of treating a disease state selected from the group consisting of excess weight, diabetes, high blood lipid levels, arterial sclerosis, arterial plaques, reducing or preventing gallstones, insufficient tissue tissue, insufficient insulin sensitivity, and stroke, characterized by by using the sustained release preparation of claims 1, 2, 3 or 45 in a pharmaceutically acceptable carrier, diluent or adjuvant, wherein the biologically active agent is ieptin, an analogue or derivative thereof. 51. A method of treating a disease state selected from the group consisting of haemopoietic cell deficiency, infection, and neutropenia comprising the use of the sustained release preparation of claims 1, 2, 3 or 45 in a pharmaceutically acceptable carrier, diluent or adjuvant, wherein the biologically active agent is G-CSF, analog or derivative thereof. 52. A method of treating inflammation comprising the sustained release formulation of claims 1, 2, 3 or 45 in a pharmaceutically acceptable carrier, diluent or adjuvant, wherein the biologically active agent is IL-1ra, an analogue or derivative thereof. ·· ·· * «« • t · f f 9 9 9 9 9 9 9 9 9 9 9 9 9 9 999 9999 99 999 99 999 53. An extended release formulation comprising: a) hydrophilic polymer, (b) a biologically active agent; and c) at least one precipitating agent, wherein the biologically active agent is co-precipitated with the hydrophilic polymer, wherein the composition is in the form of a gel particle and the particle is biodegradable. The composition of claim 52, wherein the precipitating agent is selected from the group consisting of polyvalent metal ions or salts, acetates, citrates, chlorides, carbonates, or hydroxides thereof. 55. The formulation of claim 53 wherein the metal ion is selected from the group consisting of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum, or zinc. 56. The composition of claim 52, wherein the precipitating agent is selected from the group consisting of zinc, calcium, or a combination thereof. 57. The composition of claim 52, wherein the hydrophilic polymer is a polysaccharide. 58. The composition of claim 56, wherein the polysaccharide is alginate. 59. A method of producing a sustained release preparation comprising the steps of: ·· ·· 4 4 4 a) dissolving the biologically active agent and the hydrophilic polymer in a solvent to form a first mixture, b) dissolving at least one precipitating agent in the solvent to form a second mixture, c) co-precipitating the biologically active agent with the hydrophilic polymer to form a co-precipulated gel particle, wherein the particle is biodegradable. 60. The method of claim 58 further comprising the step of isolating the co-preculture particles. 61. The composition of claim 59. sustained release by being produced by the process 62. A pharmaceutical composition according to claim 52 wherein the composition is in a pharmaceutically acceptable carrier, diluent or adjuvant. 63. A method of indicated treatment comprising treating the sustained release formulation of claim 52 in a pharmaceutically acceptable carrier, diluent or adjuvant.