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    • 专利摘要:
    The present invention relates to a sustained release formulation form using a biodegradable sustained release alginic acid gel or particles and a method for preparing the same.
    公开号:KR20010025023A
    申请号:KR1020007012768
    申请日:1999-05-14
    公开日:2001-03-26
    发明作者:골든버그메릴시머；구지안화
    申请人:스티븐 엠. 오드레；암젠 인코포레이티드；
    IPC主号:
    专利说明:

    Biodegradable Sustained Release Alginate Gel {BIODEGRADABLE SUSTAINED-RELEASE ALGINATE GELS}
    [2] As genetic and cellular engineering techniques have advanced, the use of recombinant proteins in the use of proteins in therapeutic medicines has also increased. For many diseases or conditions treated with pharmaceutical proteins, it is essential to maintain protein levels continuously to obtain the best therapeutic effect. Most protein drugs, however, generally need to be administered frequently because their biological half-life is short. Repeated injections continue to change the level of the drug in the body, making a difference and putting a significant burden on the patient both physically and financially. Since many of the symptoms are alleviated sensitively to the level control of the drug, it is necessary to release the drug in a controlled manner so that it can be released constantly for a long time. These sustained release medications not only provide the patient with improved preventive, therapeutic or diagnostic effects, but also reduce the overall cost by reducing the number of injections.
    [3] There have recently been attempts to use biodegradable polymers as substrates to maintain drug levels upon administration to humans or animals. For example, British Patent No. 1,388,580 describes the use of hydrogels to slowly release insulin. US Pat. No. 4,789,550 describes the use of alginic acid microcapsules coated with polylysine for the transport of proteins by encapsulated live cells. U.S. Patent No. 4,744,933 has attempted to use anionic polymers or cationic polymer compositions to slowly release the drug, which is surrounded by ionic polymers having opposite charges to encapsulated cells capable of producing biologically active compositions. . Likewise, a method of coating the anionic or cationic crosslinked polymer multiple times is also described as a method of controlling release (see US Pat. Nos. 4,690,682 and 4,789,516). In addition, the use of alginic acid alone or by coating with other biodegradable polymers is described as another approach to controlled release of a polypeptide composition or its cationic precipitate [PCT WO 96/00081, PCT WO 95 /. 29664 and PCT WO 96/03116.
    [4] However, these methods have been insufficient to allow sustained release of the desired protein drug. Under in vivo conditions, the use of certain biodegradable polymers, such as, for example, polylactide co-glycerides, is known to release large amounts of drug initially [Nature Med by Johnson, O. et al. . 2/7: 795 (1996)]. Moreover, it is known that proteins used in the form of sustained release formulations are prone to denaturation and can lose bioactivity when exposed to encapsulating agents. Such formulations may use organic solvents which may have deleterious effects on the protein of choice. Consequently, the use of alginic acid alone does not provide the desired controlled release protein that is essential for effective therapeutic results, as described below.
    [5] Alginic acid is generally known as a naturally occurring anionic polysaccharide, consisting of 1, 4-linked-β-D-mannuronic acid and α-L-guluronic acid [Smidsrod, O. et al. Trends in Biotechnology, 8:71 -78 (1990); See Aslani, P. et al. J. Microencapsulation, 13 (5): 601-614 (1996). Alginic acid will generally consist of 70% mannuronic acid and 30% guluronic acid to 30% mannuronic acid and 70% guluronic acid (Smidsrod, supra). Alginic acid is insoluble in water, while salts formed with monovalent ions such as sodium, potassium and ammonium are water soluble [McDowell, RH, "Properties of Alginates" (London, Alginate Industries Ltd., 4th edition 1977). Is known to react with alginic acid to spontaneously form a gel.
    [6] Alginic acid is widely used in food additives, adhesives, pharmaceutical tablets and wound bandages. Alginic acid has also been used in protein isolation techniques. For example, Gray, C.J. In Biotechnology and Bioengineering, 31: 607-612 (1988), describe the entrapment of insulin in zinc alginate / calcium gels to separate insulin from other serum proteins.
    [7] Alginic acid substrates are also well known as drug transport systems, see, for example, US Pat. No. 4,695,463 which describes alginate based chewing gum transport systems and pharmaceutical formulations. Alginate beads have been used for the controlled release of several proteins such as: tumor necrosis factor receptors in cation-alginate beads coated with polycationic [Wee, S.F., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 21: 730-31 (1994); Transformation growth factor encapsulated with alginic acid beads [Puolakkainen, P.A. Gastroenterology, 107: 1319-1326 (1994) et al .; Angiogenesis factors captured by calcium alginate beads [Downs, E.C. J. of Cellular Physiology, 152: 422-429 (1992); Albumin captured in chitosan-alginic acid microcapsules [see J. Pharmaceutical Sciences, 83 (2): 178-185 (1994) to Pol, A. et al.) Or chitosan-alginate calcium beads coated with polymer [Okhamafe, A.O. J. Microencapsulation, 13 (5): 497-508 (1996); Hemoglobin encapsulated with chitosan-alginate calcium beads [Huguet, M.L. J. Applied Polymer Science, 51: 1427-1432 (1994), Huguet, M.L. Process Biochemistry, 31: 745-751 (1996) et al .; And interleukin-2 [Liu, L.S. encapsulated with alginic acid-chitosan centrosomes. Proceed as. Intern. Symp. Control. Rel. Bioact. Mater, 22: 542-543 (1995)].
    [8] Systems using alginic acid gel beads or alginic acid / calcium gel beads to capture proteins lack the sustained release effect due to the rapid release of proteins from the alginic acid beads [Liu, L. et al. J. Control. Rel. 43: 65-74 (1997). There have been several attempts to delay the release of protein alginic acid beads using polycationic polymer coatings (eg polylysine, chitosan) in the system to avoid this rapid release. For example, Wheatley, M.A. J. Applied Polymer Science, 43: 2123-2135 (1991); Wee, S.F. The above-mentioned documents; Liu, L. S. The above-mentioned documents; Wee, S.F. See Controlled Release Society, 22: 566-56 (1995), et al., Supra.
    [9] Polycationics such as polylysine are positively charged polyelectrolytes that work with negatively charged alginic acid molecules to form polyelectrolyte complexes, which act as diffusion barriers on the bead membrane. Problems arising from the use of polycationics include the following: (1) These formulations are cytotoxic due to polycationics [Huguet, M.L. The above-mentioned documents; Zimmermann, Ulrich, Electrophoresis, 13: 269 (1992); Bergmann, P. et al., Clinical Science, 67: 35 (1984); (2) polyvalent cations are susceptible to oxidation; (3) Beads coated with a polycationic coating tend to accumulate in the body without corrosion; (4) These formulations are prepared through a complex coating process, such as coating several times with polycationic polylysine [Proceed by Padol et al. Intern. Symp. Control. Rel. Bioact. Mater, 2: 216 (1986)]; (5) Ionic interactions between proteins and polycationic ions can cause loss of protein activity or protein instability.
    [10] US Pat. No. 5,336,668 (and citations) to Francesco et al. Describe fully esterified alginic acid and partially esterified alginic acid prepared by other methods and having pharmaceutical quality. It also describes a method in which alginic acid esters are used as biodegradable plastics for medical surgery, as additives to various polymeric materials, or in the manufacture of various pharmaceuticals. There is no literature describing the use of esterified alginic acid in sustained release formulations or the description of esterified alginic acid hydrogels.
    [11] Proceed by Nightlinger. Inter. Symp. Control. Rel. Bioact. Mater., 22: 738-739 (1995) describe esterified hyaluronic acid (HA) microparticles with controlled release capability. This document generally describes its HA derivatives with different degradation rates and describes how esters cleave alcohol and HA moieties. However, there is no document describing whether or how to cut the polymer units such that the HA backbone itself has a lower molecular weight.
    [12] In order for the polysaccharide-based continuous transport system to be useful, the polysaccharides must be biodegraded into non-toxic products. Certain alginic acid gel systems are effective in imparting sustained release to drugs while the delayed loss of these gels creates "bumps" (or nodules) at the injection site. Therefore, it is not a big problem in the case of the treatment method which has a low dose of drug and the number of injections, but this is a serious problem in the case of the treatment method which has a high dose of drug and a high injection frequency. Therefore, a method of increasing the rate of disappearance of the alginic acid gel at the injection site should be developed.
    [13] Thus, there is still a need to develop pharmaceutical formulations that are clinically applicable, more effective and have various sustained release properties. Many recombinant and natural proteins have the advantage of being released over a long period of time, resulting in more effective clinical results.
    [14] The present invention provides this advance. The pharmaceutical composition using the biodegradable long-lasting alginic acid gel particles or gels of the present invention not only enhances the stability and effectiveness of the protein, but also increases the bioavailability of the protein, protects the protein, reduces protein degradation, Can be dissolved. In addition, the pharmaceutical compositions of the present invention provide a simple, rapid and inexpensive method for the controlled release of recombinant proteins in effective prophylactic, therapeutic or diagnostic results.
    [15] Summary of the Invention
    [16] The present invention was developed as a study using an unaltered alginic acid (anionic polysaccharide group) hydrogel to continuously release a protein. Unmodified alginic acid hydrogels containing such proteins (see US Patent Application Nos. 08 / 857,913 and 08 / 912,902) are formed in a time-delayed manner, leaving the gel in the same syringe by filling the syringe with material. Become; Such gels have been found to be injectable. After a single subcutaneous injection into the rodent model, evidence was observed over several days of continued release of the protein. However, significant bumps or nodules remain at the injection site for a long time without change in size. This bump consists of an alginate hydrogel filled with water and the size of the bump is related to the gel volume injected. Gel beads also remain at the injection site.
    [17] The present invention thus relates to novel biodegradable and biocompatible novel polysaccharide hydrogels, such as alginic acid ester hydrogels, for sustained release of therapeutic proteins. Surprisingly, in addition to the gelling properties, injectability and sustained release of unaltered alginic acid, alginic acid ester hydrogels did not produce bumps at the injection site. That is, the alginic acid ester hydrogel is biodegradable or edible and is gradually reabsorbed into surrounding tissue without any reaction at the injection site.
    [18] The composition of the present invention comprises an alginic acid ester or derivative thereof ionic crosslinked with a hydrogel (water-containing) substrate containing a therapeutic agent such as a protein.
    [19] The present invention also relates to a method for preparing a biodegradable sustained release composition.
    [20] The present invention relates to the use of alginic acid ester materials in time delayed gelling liquid mixtures in the body.
    [21] The present invention also relates to compositions wherein the alginic acid ester hydrogel is in the form of beads or microparticles for the sustained release of the active agent, preferably the therapeutic protein.
    [22] In one embodiment of the invention, the alginic acid ester hydrogel provides a composition for application to a target site in a patient's body. Such compositions are useful for the following uses: to prevent or prevent the formation of tissue adhesions after surgery and trauma; Tissue supply, in particular soft and hard tissue supply; Space replenishment limited to resorbable materials; Scaffolding and wound care for tissue growth.
    [23] In another embodiment, the alginic acid ester hydrogel provides an active agent containing a device for implantation in the body, which active agent may or may not bind to the alginic acid polymer.
    [24] In another embodiment, the alginic acid ester hydrogel composition of the present invention provides a method for improving the bioavailability of the active agent in the composition.
    [25] Finally, the alginic acid ester hydrogel compositions of the present invention provide a method for obtaining substantially constant blood levels in a patient over a period of time.
    [1] The present invention relates to sustained release formulations using biodegradable alginic acid gel beads and / or persistent gels and methods for their preparation.
    [26] Composition
    [27] Hydrophilic polymers including alginic acid and derivatives thereof can be obtained from a variety of commonly available or natural or synthetic sources well known in the art. As used herein, the term hydrophilic polymer refers to a water soluble polymer or a polymer having affinity for water absorption. Hydrophilic polymers are well known to those skilled in the art. Such hydrophilic polymers include, but are not limited to, polyanions including anionic polysaccharides such as: alginic acid, carboxymethyl amylose, polyacrylates, polymethacrylates, ethylenemaleic anhydride copolymers (1 / 2 esters), carboxymethyl cellulose, dextran sulfate, heparin, carboxymethyl dextran, carboxy cellulose, 2, 3-dicarboxycellulose, tricarboxycellulose, carboxy arabic rubber, carboxy carrageenan, pectin, carboxy pectin, carboxy traga Kant gum, carboxy xanthan gum, pentosan polysulfate, carboxy starch, carboxymethyl chitin / chitosan, curdlan, inositol hexasulfate, β-cyclodextrin sulfate, hyaluronic acid, chondroitin-6-sulfate, dermatan (dermatan) sulfate, heparin sulfate, Carboxymethyl starch, carrageenan, polygalacturonate, carboxy guar gum, polyphosphate, polyaldehyde-carboxylic acid, poly-1-hydroxy-1-sulfonate-propene-2, copolystyrene maleic acid, agarose, meso Glycan, sulfopropylated polyvinyl alcohol, cellulose sulfate, protamine sulfate, phospho guar gum, polyglutamic acid, polyaspartic acid, polyamino acids or derivatives or combinations thereof. Those skilled in the art will recognize various other hydrophilic polymers within the scope of the present invention.
    [28] Likewise, bound multivalent metal ions can be obtained from a variety of commonly available sources, natural or synthetic sources, which are well known in the art. In particular, metal ions include, but are not limited to, aluminum, barium, calcium, iron, manganese, magnesium, strontium and zinc. Preferred metal ions are calcium and zinc or salts thereof, zinc acetate, calcium acetate or hydrochloride. Water soluble small molecules may also be used such as ammonium sulfate, acetone, ethanol and glycerol.
    [29] Fatty alcohols used as esterification components in the carboxyl groups of the alginic acids according to the invention have, for example, up to 34 carbon atoms, which are saturated or unsaturated, and also have one or two hydroxykeys, nitrile groups or halogens. Carbamide or carbamide groups or esterified carboxyl groups, hydrocarbyl or dihydrocarbylamino substituted by (hereinafter "hydrocarbyl" is a monovalent radical of a hydrocarbon such as C n H 2n + 1 type As well as "alkylene"-C n H 2- or "alkylidene" = C n H 2n ) or ether or ester groups, acetal or ketal groups, thio-ethers or Substituted by groups derived from the same as thioester groups, or functions such as amino, hydroxy, aldehyde, keto, mercapto, carboxyl groups Machine or variations may be substituted by other free radicals.
    [30] For those groups containing hydrocarbyl radicals, they are preferably lower aliphatic radicals which are heteroatoms such as oxygen, nitrogen and sulfur. Alcohols substituted with one or two of the foregoing functional groups are selected.
    [31] Alcohols having said groups preferably used within the term of the present invention are those having up to 12 carbon atoms, in particular up to 6 carbon atoms, such as the amino, ether, ester, thioether, thioester, acetal, up to 4 For ketal groups representing alkyl groups having 4 carbon atoms, also for esterified carboxyl groups or substituted carbamide groups, the hydrocarbyl radical is an alkyl having the same number of carbon atoms, and the amino or carbamide group has a maximum of 8 carbon atoms It may be an alkylene amino group or an alkylene carbamide group having a group. The first of these alcohols are saturated and unsubstituted alcohols such as methyl, ethyl, propyl, isopropyl alcohol, n-butyl alcohol, isobutyl, tert-butyl alcohol, amyl, pentyl, hexyl, octyl, nonyl , Dodecyl alcohol and all of the above alcohols having a straight chain such as n-octyl or n-dodecyl alcohol. Among the substituted alcohols of this group, dihydric alcohols are listed as follows: ethylene glycol, propylene glycol or butylene glycol, trihydric alcohols such as glycerin, aldehyde alcohols such as tartronic alcohol, carboxy alcohols such as lactic acid, For example alpha-oxypropionic acid, glycolic acid, malic acid, tartaric acid, citric acid, aminoalcohols, for example aminoethanol, aminopropanol, n-aminobutanol and its dimethyl, diethylamino group derivatives, choline, pyrrolidinylethanol, Alkyl derivatives such as piperidinylethanol, piperazinylethanol and n-propyl alcohol or n-butyl alcohol, the corresponding derivatives of monothioethylene glycol or ethyl derivatives of mercapto groups.
    [32] Among the highly saturated aliphatic alcohols, it is worth mentioning, for example, cetyl alcohol and myristyl alcohol, but of particular importance for the purposes of the present invention are citronellol, geraniol, nerol, nerolidol, linallo Highly unsaturated alcohols with one or two double bonds, which have affinity for terpenes such as ol, farnesol, phytol and are contained in essential oils in large quantities.
    [33] Among the lowly unsaturated alcohols contemplated are propargyl alcohols.
    [34] All of the above-mentioned aliphatic alcohols have only one benzene moiety, and the aliphatic chain has up to 4 carbon atoms and the benzene moiety is substituted with a halogen atom, in particular chlorine, bromine or iodine or with 1 to 3 methyl or hydroxy groups. The aliphatic chain may be substituted with one or more functional groups selected from free amino group or monomethyl group or dimethyl group, or may be substituted with pyrrolidine or piperidine group. Of these alcohols benzyl alcohol and phenethyl alcohol are particularly preferred. Cycloaliphatic alcohols or aliphatic cycloaliphatic families are derived from mono or polycyclo hydrocarbons and have up to 34 carbon atoms. Particular mention of alcohols derived from cyclo mononuclear hydrocarbons consists of up to 12 carbon atoms with rings containing 5 to 7 carbon atoms, for example 1 to 3 lower alkyl groups such as methyl, ethyl, propyl or isopropyl groups It may be substituted by. Particular alcohols of this group are carbomenthol menthol, alpha and gamma-terpineol 1-terpineol alcohols known as “terpineol”, and p-mentanes such as 1,4- and 1,8-terpin. Derived alcohols, cyclohexanol, cyclohexanediol, 1,2,3-cyclohexanetriol and 1,3,5-cyclohexanetriol (fluorogitol), inositol. Alcohols derived from hydrocarbons having condensed rings are, for example, tuzan, refuge, campane groups, in particular tuzanol, sabinol pinol hydrate, D-bornone, L-borneneol, D-isobornone and L-isobor Neol.
    [35] Also included are alcohols derived from ester reactions of alginic acid with epoxy-containing compounds (see, eg, US Pat. No. 2,463,824 and US Pat. No. 2,426,125).
    [36] Fully ester groups and partial ester groups containing the following ions of the present invention are generally acidic polysaccharides when glycoside oxygen is beta bonded to the carbonyl carbon of the ester. Although not associated with a particular mechanism, the arrangement of these components can cleave the polymer chain by beta-elimination mechanisms that can occur under physiological conditions.
    [37] Alginate esters of the invention are formulated with the following formula (I) and derivatives (if the hydroxy group is acetylated and reacted with isocyanic acid) and the mannuronic acid residues (m-COOH or m-COO anion) and guluronic acid residues (g-COOH or g-COO anion):
    [38] -(M) n1- (M ') n2- (G) n3- (G') n4- (A) n5-
    [39] From here
    [40] M is a mannuronic acid residue, m-COOH or m-COO anion;
    [41] M 'is a manuronic acid ester residue, m-COOR1;
    [42] G is a guluronic acid residue, g-COOH or g-COO anion;
    [43] G 'is a guluronic acid ester residue, g-COOR2;
    [44] A is an aliphatic, aromatic, araliphatic, alaromatic, cycloaliphatic radical or sugar that can be substituted and interrupted by a heteroatom in or at the end of the chain, or a sugar, such as a sugar oxidation byproduct. -g or non-m chain units;
    [45] n1, n2, n3, n4 and n5 are integers representing the average number of bonding units;
    [46] R 1 and R 2 are aliphatic, aromatic, araliparic, aralmatic, cycloaliphatic radicals which may be substituted and interrupted by heteroatoms, respectively.
    [47] In the esters of the invention, not only are R1 = R2 = aliphatic or aromamatic but also 100 (n2 + n4) / (n1 + n2 + n3 + n4) is 1-99 mol%, preferably 5-50 mol%, more preferably 6-30 mol%, even more preferably 6-15 mol% and most preferably 7-12 mol%, 100 n5 / (n1 + n2 + n3 + n4 + n5) is 10 It is preferably less than mol%.
    [48] For the partial esters of the invention, the unesterified carboxyl groups will be free or chlorinated. The base for this salt formation is chosen depending on the product finally used. Inorganic salts are formed from potassium, in particular alkali metals such as sodium and ammonium or derived from alkaline earth metals such as calcium or magnesium or aluminum salts.
    [49] Of particular interest are salts with organic bases, in particular nitrogenated bases and hence aliphatic, aralipatic, cycloaliphatic or heterocyclo amines. Such ammonium salts can be derived from therapeutically acceptable amines or non-toxic but therapeutically inert amines or from amines having therapeutic action. Preferred of the first form are aliphatic amines, for example mono-, di- and tri-alkylamines having alkyl groups of up to 8 carbon atoms or arylalkylamines having the same number of carbon atoms in the aliphatic moiety, wherein aryl is 1 To benzene group which may be substituted with from 3 methyl groups or halogen atoms or hydroxy groups. Biologically inert bases for salt formation are monocyclo alkyleneamines having rings of 4 to 6 carbon atoms, such that the ring may be interrupted by heteroatoms selected from groups formed by nitrogen, oxygen and sulfur. Ring, for example piperazine or morpholine, and may be substituted by amino or hydroxy functional groups such as aminoethanol, ethylenediamol, ethylenediamine, ephedrine or choline.
    [50] The degree and form of esterification can be controlled by synthetic methods known in the art. Preferably, the alginic acid ester is prepared by treating the quaternary ammonium salt of alginic acid with conventional alkylating agents in an aprotic organic solvent such as dimethyl sulfoxide. The resulting ester is preferably an ester of a monohydric alcohol such as lower alkyl such as ethyl or aralkyl such as benzyl or a mixture thereof. It is also possible to form an ester by an alginic acid reaction having an oxirane or an epoxy containing a compound such as ethylene oxide or propylene oxide.
    [51] It is also possible to form quaternary ammonium salts of partial esters, for example tetraalkylammonium salts having the above-mentioned number of carbon atoms and preferably salts of the form in which the fourth alkyl group has 1 to 4 carbon atoms, such as the methyl group.
    [52] The degree of esterification of alginic acid (expressed in mol%) is related to the desired rate of disappearance of the gel in the patient tissue. The rate of dissipation of such gels generally relates to the rate at which the active agent is released from the gel, the duration of which is 5 years or less, typically 2 to 270 days, more typically 2 to 180 days, and more usually 2 to 2 days. 90 days. The degree of esterification (DE) is 1 mol% to 99 mol%, preferably 5 mol% to 50 mol%, more preferably 6 mol% to 30 mol%, more preferably 6 mol% to 15 mol%, Even more preferably 7 mol% to 12 mol%.
    [53] As used herein, the term buffer or buffer refers to the use of inorganic acids, organic acids or combinations thereof to prepare buffers known in the art. Inorganic acids within the scope of the present invention include hydrogen halides (eg hydrochloric acid), phosphoric acid, nitric acid, sulfuric acid. Other inorganic acids are well known to those skilled in the art and are considered to be included herein. Organic acids within the scope of the present invention include aliphatic carboxylic acids and aromatic acids (eg, formic acid, carbonic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, acrylic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid). Acid, fumaric acid, glycine or phenol sulfonic acid). Other organic acids are well known to those skilled in the art.
    [54] As used herein, a biologically active agent refers to a protein that occurs recombinantly or naturally in the human body or animal, and the biologically active agent is useful in prophylactic, therapeutic or diagnostic applications as well as non-protein based drugs such as small molecules. Biologically active agents can be natural, synthetic, semisynthetic or derivatives thereof. The biologically active agent of the invention should be to precipitate. The range of biologically active agents is believed to be broad. These include hormones, cytokines, hematopoietic factors, growth factors, anti-obesity factors, nutritional factors, anti-inflammatory factors and enzymes (see US Pat. No. 4,695,463 for additional examples of useful biologically active agents). But it is not limited to this. Those skilled in the art will be able to modify the preferred biologically active agents to suit the compositions of the present invention.
    [55] Such proteins include, but are not limited to: interferon (see US Pat. Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623, herein incorporated by reference, including drawings), interleukin ( US Patent No. 5,075,222, incorporated herein by reference, including drawings, erythropoietin (US Pat. Nos. 4,703,008, 5,441,868, 5,618,698, incorporated herein by reference, including drawings, 5,547,933 and 5,621,080, granulocyte-colonal stimulating factors (US Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185), hepatocyte factor (herein with reference to the drawings, including references) PCT Publication Nos. 91/05795, 92/17505 and 95/17206) and OB proteins (PCT Publication Nos. 96/40912, 96 /, hereby incorporated by reference, including drawings). 05309, 97/00128, 97/01010 and 97/06816). Moreover, biologically active agents include, but are not limited to: anti-obesity related products, insulin, gastrin, prolactin, corticosteroids (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), Follicle stimulating hormone (FSH), placental gonadotropin (HCG), motilin, interferon (alpha, beta, gamma), interleukin (IL-1 to IL-12), tumor necrosis factor (TNF), tumor necrosis factor- Binding protein (TNF-bp), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factor (FGF), neurotrophic growth factor (NGF) , Bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte leukocyte macrophage colony stimulating factor (GM-CSF), macronucleus Cell derived growth factor (MDGF), Thrombopoietin, platelet-derived growth factor (PDGF), colon stimulatory growth factor (CSFs), bone morphogenic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase , Streptokinase and kallikrein. The term protein as used herein includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof.
    [56] Derivatives of biologically active agents have attached one or more chemical components to the protein component. Chemical modification of biologically active agents has been found to provide additional benefits under certain circumstances (eg, increased stability and therapeutic time of the therapeutic protein, reduced immunogenicity). Those skilled in the art will select the desired chemical modifications based on the desired dosage, cycle time, resistance to proteolysis, therapeutic use and other considerations.
    [57] As used herein, biodegradability refers to a particular polymer being cleaved to have fewer units in the chain, i.e., to less molecular weight units. Biodegradable gels refer to the loss of the gel in the environment of use, which involves loss of molecular weight cleavage of the constituent polymer and consequently fewer units in the polymer chain.
    [58] Complex
    [59] Proteins, analogs or derivatives can be administered in combination with the binding composition. Such binding compositions exhibit the effect of prolonging the circulation time of proteins, analogs or derivatives, or increasing the activity of biologically active agents. Such compositions may be proteins (or synonymously peptides), derivatives, analogs or combinations. For example, a binding protein suitable for an OB protein is an OB protein receptor or a portion thereof (eg a flexible portion thereof). Other binding proteins can be identified by examining OB proteins or other proteins in serum or screening for binding through experiments. Typically, such binding will not inhibit the ability of the OB protein, analog or derivative to bind to the endogenous OB protein receptor and / or will not affect signal transduction. In addition to the OB protein, the binding complex will also be applied to other therapeutic proteins of the invention. Those skilled in the art will be able to identify appropriate binding proteins used in the present invention.
    [60] In addition, precipitants used to precipitate biologically active agents can be obtained from a variety of commonly available natural or synthetic sources that are well known in the art. Precipitants include, but are not limited to: polyvalent metal ions or salts thereof (eg, acetates, citrates, hydrochlorides, carbonates, hydroxides, oxalates, tartarates or hydroxides thereof), acids or Water soluble polymer. In particular, metal ions include, but are not limited to aluminum, barium, calcium, iron, manganese, magnesium, strontium and zinc. Preferred metal ions include salts such as zinc or acetate chlorides. In addition, water-soluble small molecules and salts may be used ammonium sulfate, acetone, ethanol and glycerol.
    [61] Water soluble polymers include, but are not limited to: polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxo Dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid, dextran, poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide Ethylene oxide copolymers, polyoxyethylated polyols, polyvinyl succinate alcohols, glycerin, ethylene oxide, propylene oxide, poloxamers, alkoxylated copolymers, water soluble polyvalent anions or derivatives and combinations thereof. 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 Da and about 100 kDa, and polyethylene glycol having a molecular weight in this range is easy to settle and have good efficacy.
    [62] Preferred therapeutic profiles [e.g., desired sustained sustained duration, effects on (but only if) affected biological activity, ease of manipulation, degree or lack of antigenicity, and known precipitating agents regarding therapeutic proteins or analogs) Different sizes and types of precipitants can be used, depending on the different effects. Those skilled in the art will be able to recognize other precipitants that fall within the scope of the present invention.
    [63] Moreover, the compositions of the present invention comprise special excipients necessary to stabilize the biologically active agent and / or hydrophilic polymer. This excipient is contained in a buffer and may include, but is not limited to, preservatives.
    [64] Pharmaceutical composition
    [65] Sustained-release pharmaceutical compositions of the invention may be oral (e.g., capsules (hard capsules and soft capsules), solid preparations (granules, tablets, pills, intraoral tablets or tablets, cachets, crystallites, powders and frozen). Dried form), liquid preparations (suspensions)] and non-oral preparations [eg, intramuscular, subcutaneous, transdermal, visceral, IV (intravenous), IP (intraperitoneal), intraarterial, intradermal, intracapsular , Intraorbital, injectables, lung, nose, rectal and endometrial mucosa formulations.
    [66] In general, the present invention is understood as a sustained release pharmaceutical composition comprising an effective amount of a protein or derived product having the properties of a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier required for administration ( PCT No. 97/01331, incorporated herein by reference. The optimal pharmaceutical formulation for the preferred biologically active agent will be determined by one skilled in the art depending on the route of administration and the desired dosage. Typical pharmaceutical compositions include Remington's Pharmaceutical Sciences; Mark Publishing Company, 18th Ed., Easton, PA, pgs. 1435-1712 (1990).
    [67] Because of the thixotropic nature of the sustained gel formulation, syringes are used when administered subcutaneously. Later injections may gel the composition in a syringe. Such gelation can be carried out in time-lasting form. The time is controlled by appropriate adjustment of the gelling agent quantification and by the proton donor (if used) in the mixture. Such agents would have been used to gel again later in the body after injection. As used herein, the term thixotropy means that the viscosity of the gel mixture decreases under pressure (pressure from the syringe plunger), then the mixture is fluid (when passing through the syringe needle), and then back to the gel at the injected site. It means to be formed.
    [68] The concept of sustained gelation also applies to filled syringes wherein the sustained release gel composition is filled into the syringe and gelled in the syringe at a pre-controlled time (eg, from minutes to hours after filling). This avoids the problem of syringes already filled with gelled material. Such prefilled syringes can be stored for later injection into a patient.
    [69] The composition includes the following, which are necessary at the time of administration: various buffering ingredients (eg, tris-HCl, acetate), diluents of various pH and various ionic strengths; Additives such as surfactants and solubilizers (e.g. Tween 80, HCO-60, polysorbate 80), antioxidants (e.g. ascorbic acid, glutathione, metabisulfite sodium), special polysaccharides (e.g. Carboxymethylcellulose, sodium alginate, sodium hyaluronate, protamine sulfate, polyethylene glycol, preservatives (e.g. thimersol, benzyl alcohol, methyl paraben, propyl paraben) and constituents (e.g., lactose, Mannitol); Binding of a substance showing a particulate preparation of a high molecular compound such as a polylactic acid / polyglycolic acid polymer or copolymer or the like with a liposome. Hyaluronic acid can also be used as a dosage composition and may have the effect of further enhancing the duration of sustained in a circulating state. Moreover, the sustained release compositions of the present invention also provide a chain of fatty acid triglycerides for supplying oil (eg sesame oil, corn oil, vegetable oil) or mixtures of oils with phospholipids (eg lecithin) or oil suspensions. (Eg, migliol 812). The compositions of the present invention may also be dispersed in dispersants such as water soluble polysaccharides (eg mannitol, lactose, glucose, starch), hyaluronic acid, glycine, fibrin, collagen and inorganic salts (eg sodium chloride).
    [70] In addition, the administration of the sustained release composition of the present invention is not limited to medical nebulizers, measured dose inhalers, and mechanical devices designed for the pulmonary circulation of therapeutic products, including powder inhalers, everything apparent to those skilled in the art. Has been studied for use.
    [71] Dosage compositions determine the physical state, stability, rate of in vivo release and rate of in vivo clearance of proteins and derivatives of the invention. One skilled in the art selects suitable mechanical devices for use depending on the appropriate dosage composition and / or therapeutic use, route of administration, dosage regimen, cycle time, proteolytic resistance, protein stability and other considerations.
    [72] How to use
    [73] cure. Therapeutic use depends on the biologically active agent used. One skilled in the art will readily be able to tailor the desired biologically active agent to the therapeutic use intended for the present invention. Therapeutic uses for such medicaments are described in greater detail in the following publications, which are incorporated herein by reference, including the drawings. Therapeutic uses include, but are not limited to the use of the following proteins: interferon (US Pat. Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623, herein incorporated by reference, including the drawings. ), Interleukin (see US Pat. No. 5,075,222, incorporated herein by reference, including drawings), erythropoietin (US Pat. No. 4,703,008, 5,441,868, incorporated herein by reference, including drawings) Nos. 5,618,698, 5,547,933 and 5,621,080, granulocyte-collecting stimulating factors (US Pat. Nos. 4,999,291, 5,581,476, 5,582,823, incorporated herein by reference, including drawings) 4,810,643 and PCT Publication No. 94/17185), hepatocellular factor (herein PCT Publication Nos. 91/05795, 92/17505, and 95/17206, which are incorporated by reference in their entirety, and OB proteins (PCT Publication Nos., Incorporated herein by reference, including drawings). 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816).
    [74] In addition, therapeutic uses of the invention include the use of biologically active agents, including but not limited to: anti-obesity related products, insulin, gastrin, prolactin, corticosteroids (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), placental gonadotropin (HCG), motilin, interferon (alpha, beta, gamma), interleukin (IL-1 to IL-12), Tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF-bp), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factor (FGF), neurotrophic growth factor (NGF). Bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte leukocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte-derived growth Factor (MDGF), thrombopoietin, platelet-derived growth factor (PDGF), colon stimulatory growth factor (CSFs), bone morphogenic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator ( TPA), urokinase, streptokinase and kallikrein. The term protein as used herein includes peptides, polypeptides, cann's molecules and analogs, derivatives or combinations thereof. In addition, the compositions of the present invention can be used for the preparation of one or more medicaments for the treatment or amelioration of biologically active agents for therapeutic purposes.
    [75] By way of example, therapeutic use (blood oxygenation) and bone resorption or osteoporosis reduction may be achieved without weight loss.
    [76] Combination therapy. The compositions and methods of the present invention can be used in conjunction with other therapies such as dietary changes and exercise. Other medications include medications useful for treating diabetes (for example, insulin and potential amylin), cholesterol and blood pressure lowering agents (such as drugs that reduce blood lipid levels or cardiovascular medications), and drugs that increase activity ( For example, there are medications such as amphetamines, diuretics (for liquid feces) and appetite suppressants. Administration of these medications can be carried out simultaneously or sequentially. In addition, the methods of the present invention may be used for cosmetic surgery (eg, liposuction or laser surgery designed to reduce body mass, or transplant surgery designed to increase the appearance of body mass) designed to change the overall appearance of the body. Can be used in conjunction with surgical procedures. The health benefits of bypass surgery or other cardiac surgery planned to alleviate the toxic conditions caused by blockage of blood vessels by fatty deposits such as arterial plaques will increase the incidental use of the compositions and methods of the present invention. . Methods for removing gallstones, such as ultrasound or laser, may also be used before, during or after using the treatment methods of the present invention. Moreover, the method of the present invention can be used as an adjunct to fracture, muscle injury treatment or surgery or other therapies that are improved by increasing the fat free tissue mass.
    [77] Dosage
    [78] One skilled in the art can determine the effective dosage by observing the administration and the desired therapeutic effect. Dosages for sustained-release preparations are those necessary to obtain an effective concentration of the biologically active agent in vivo for a given time. Dosage and preferred frequency of administration of the sustained release preparations will vary depending on the type of biologically active agent, the desired duration of release, the subject disease, the desired frequency of administration, the species of the test animal and other factors. Formulation forms of the molecule would be desirable to achieve the desired therapeutic effect between about 0.10 μg / kg to 100 mg / kg per day.
    [79] Effective dosages can be determined using diagnostic methods for some time. By way of example, the present invention defines the dosage of OB protein. For example, a diagnosis that measures the amount of OB protein in the blood (or plasma or serum) may first be used to determine the endogenous level of the OB protein. Such diagnostic methods may appear in antibody assay forms such as antibody sandwich assays. The amount of endogenous OB protein is initially quantified and the baseline is determined. Therapeutic doses determined by quantifying endogenous and exogenous OB proteins (ie, proteins, analogs or derivatives found in the body that have been self-produced or administered) persist throughout the course of treatment. For example, relatively high doses are needed from the beginning until the therapeutic effect is seen, but after that low doses are used to sustain the therapeutic effect.
    [80] Materials and methods
    [81] material. Alginic acid in the form of sodium alginate can be found in a circle well known in the art. OB protein and GCSF are purchased from Amgen Incorporated. Other chemicals are obtained from sources well known in the art.
    [82] Alginic acid hydrogel particles / bead preparation. The preparation of protein containing and non-containing alginic acid hydrogel particles and beads is described in detail in co-pending US Application No. 08 / 842,756, which is incorporated herein by reference.
    [83] Permanent gel preparation. The preparation of protein-containing and non-sustainable alginic acid hydrogels is described in detail in co-pending US Application Nos. 08 / 857,913 and 08 / 912,902, each of which is incorporated herein by reference.
    [84] The following examples are intended to more fully illustrate the invention, but are not intended to limit the scope of the invention. Moreover, in connection with the above specification or the following examples, those skilled in the art will be able to make modifications to the specification as needed for large scale production.
    [85] Example 1
    [86] The following examples describe the preparation of alginic acid esters used in the present invention.
    [87] Preparation A: Tetrabutylammonium Alginate (TBA).
    [88] Sulfonic acid resins (obtained from Bio-Rad, AG MP-50) are converted to tetrabutylammonium (TBA) form by treatment with tetrabutylammonium hydroxide (obtained from Aldrich) using a batch method at room temperature. To a solution in which 10 g of sodium alginate salt is dissolved in 800 ml of distilled water is added 60 ml of sulfonic acid resin (available from Bio-Rad, AG MP-50) dissolved in the form of tetrabutylammonium salt. The mixture is stirred for 0.5 h at room temperature. Tetrabutylammonium alginate dissolved in the filtrate was freeze-dried to separate (output amount, 16.8 g) and confirmed by 1 H NMR.
    [89] Preparation B: partially esterified alginic acid ethyl ester, degree of esterification (DE) = 30 mol%.
    [90] Tetrabutylammonium alginate (6 g, 14.4 mmol TBA unit) is dissolved in 500 ml of dimethyl sulfoxide (DMSO) at room temperature. Then iodineethane (from Aldrich, 673 mg, 4.3 mmol) is added. The mixture is stirred at 30 ° C. for 15 hours and then cooled to room temperature. To this solution completely convert TBA to sodium salt, slowly add a solution of 2 g of NaCl in 20 ml of water. After stirring 15-30 minutes, the solution is slowly poured into 1500 ml of ethyl acetate. The precipitate is collected by filtration, washed three times with acetone / water (8: 1 v / v), three times with acetone and dried in vacuo. The compound is redissolved in distilled water (˜100 ml) and the pH is adjusted to ˜6.5 using 0.2% NaHCO 3 at 0 ° C. The solution is then dialyzed overnight at 4 ° C. against distilled water (MW cut-off 8000) and freeze-dried. The yield of partial ester is 2.8 g and the degree of esterification is 30 +/- 1% ( 1 H NMR, using maleimide as internal standard).
    [91] Preparation C: Fully esterified alginic acid ethyl ester and partially esterified alginic acid ethyl ester, DE = 100%, 50%, 20%, 10% and 5%.
    [92] The preparation of this compound is similar to that described in Preparation B, except that the amount of iodineethane added to reach the desired degree of esterification is adjusted.
    [93] Preparation D: partially esterified alginic acid propyl, hexyl, octyl and dodecyl esters.
    [94] The method of preparation is similar to that described in Preparations B and C above, but replaces iodineethane with 1-iodine propane, 1-iodinehexane, 1-iodineoctane or 1-iodinedecane, respectively.
    [95] Preparation E: partially esterified alginic acid benzyl ester, DE = 30%.
    [96] Tetrabutylammonium alginate (2.5 g, 5.99 mmol TBA units) is dissolved in ˜200 ml of DMSO at room temperature. Benzyl bromide (from Aldrich, 307 mg, 1.8 mmol) and TBA iodide (from Aldrich, 30 mg) are added. The mixture is stirred at 30 ° C. for 15 hours and then cooled to room temperature. To complete conversion of TBA to sodium salt, slowly add a solution of 0.6 g of NaCl in 10 ml of water to this solution. After stirring 15-30 minutes, the solution is slowly poured into 500 ml of ethyl acetate. The precipitate is collected by filtration, washed three times with acetone / water (8: 1 v / v), three times with acetone and dried in vacuo. The compound is redissolved in distilled water (˜60 ml) and the pH adjusted to ˜6.5 with 0.2% NaHCO 3 at 0 ° C. and then dialyzed overnight against distilled water at 4 ° C. (MW cut-off 8000). The yield of partial ester is 1.3 g and the degree of esterification is 30 +/- 1% ( 1 H NMR, using maleimide as internal standard).
    [97] Preparation F: Fully esterified alginic acid benzyl ester and partially esterified alginic acid benzyl ester with other DE.
    [98] The preparation of this compound is similar to that described in Preparation E except that the amounts of benzyl bromide and TBA iodide added to reach the desired degree of esterification are adjusted.
    [99] Example 2
    [100] The examples below show the preparation of protein drugs (leptin) containing alginic acid ethyl esters (DE = 15 mol% and 10 mol%) gels and in vitro sustained release experiments from these gels.
    [101] Leptin (100 mg / ml; 10 mM Tris HCl, pH 8.8; pH adjusted to 8.0-8.8 with 1M NaOH) and alginic acid ethyl ester (15 mol%, 10 mM Tris HCl, pH 8.6) are cooled in an ice bath. Leptin (0.5 ml) is added to 6% alginic acid ethyl ester (0.18 ml) and the mixture is stirred in an ice bath for 10-15 minutes; The final pH is 8.6-8.8. To this mixture is added a suspension of 1M CaCO 3 (16 μl) and the resulting suspension is mixed well. To this suspension 0.1 M ZnCl 2 (100 μl) solution is added dropwise with stirring; Then add water to make 1 ml volume. The mixture is thoroughly mixed and placed in an ice bath for 10-20 minutes. Then a solution of 1.68 M δ-glucononolactone (obtained from Aldrich, 56 μl) is added to this mixture and stirred thoroughly. The final mixture (50 mg / ml leptin, 1% alginic acid ethyl ester; 0.1 ml) is placed inside the Eppendorf tube and left overnight at 4 ° C. to gel. After overnight storage, in vitro release experiments are performed in 10 mM histidine buffer, pH 7.4. Cast gels with a degree of esterification of 15 mol% exhibited minimal burst and very constant leptin release with 60% release in 6 days. Cast gels with a degree of esterification of 10 mol% exhibited minimal release and very consistent leptin release with 55% release within 6 days.
    [102] Example 3
    [103] The following example shows the preparation of a protein drug (leptin) containing an alginic acid hexyl ester (DE = 15 mol% and 10 mol%) gel and an in vitro sustained release experiment from this gel.
    [104] This example is carried out in a similar manner to that described in Example 2 except for the alginic acid ethyl ester.
    [105] Alginic acid hexyl ester gels with 15 mol% and 10 mol% degree of esterification show minimal release and sustained release with 50% release within 6 days.
    [106] Example 4
    [107] The following example shows the preparation of protein drug (Zn-leptin) containing alginic acid ethyl ester (DE = 15 mol%) gel and in vitro sustained release experiment from this gel.
    [108] In a 4% (w / v) alginic acid ethyl ester (15 mol%, 0.75 ml) solution, 1 M Tris HCl (7.5 μl) at pH 8.0, 0.5 M PIPES (33 μl) and 0.1 M ZnCl 2 (8.5 μl) at pH 6.8 Add. The mixture is stirred well. To this solution is added Zn-leptin suspension (100 mg / ml, 675 μl) and the mixture is stirred thoroughly. Then a 1 M CaCO 3 (24 μl) suspension and 1.68 M δ-gluconolactone (70 μl) solution are added to this mixture and stirred thoroughly. The final mixture (0.1 ml) is placed inside the Eppendorf tube and left overnight at 4 ° C. to gel. After overnight storage, in vitro release experiments are performed in 10 mM histidine buffer, pH 7.4. Alginic acid ethyl ester cast gels with a degree of esterification of 15 mol% show little release, with sustained leptin release showing 65% release within 4 days.
    [109] Example 5
    [110] The following examples show the preparation of protein drugs (GCSF) containing alginic acid ethyl ester (DE = 30 mol%) gel and in vitro sustained release experiments from this gel.
    [111] To a solution of 2.39% alginic acid ethyl ester (30 mol%, 0.50 ml), 0.1 M acetate buffer (pH 4.5, 100 μl), GCSF (104 μl, 48.2 mg / ml, HCl at pH 3) and distilled water (246 ml) Add. The mixture is stirred well. A 1 M CaHPO 4 (10 μl) suspension and 1.68 M δ-gluconolactone (40 μl) solution are added to this mixture and stirred thoroughly. The final mixture (0.2 ml) is placed inside an Eppendorf tube and left overnight at 4 ° C. to gel. After the gels are stored overnight, in vitro release experiments are performed in 10 mM Tris buffer at pH 7.5. Alginic acid ethyl ester cast gels with a degree of esterification of 30 mol% exhibited sustained release with up to 5% release, 20% release in one day and 40% release in two days.
    [112] Example 6
    [113] The following examples show the preparation of protein drug (GCSF) containing alginic acid benzyl ester (DE = 30 mol%) gel and in vitro sustained release experiment from this gel.
    [114] This example is carried out in a similar manner to that described in Example 5, except that the alginic acid benzyl ester is used instead of the alginic acid ethyl ester. Alginic ester gels are stored overnight. Alginic acid benzyl ester gels with a degree of esterification of 30 mol% show up to 5% release, sustained release showing 40% release in one day and 80% release in two days.
    [115] Example 7
    [116] This example shows the preparation of alginic acid ethyl ester beads.
    [117] Gel beads are prepared by dropwise addition of a 2% alginic acid ester solution to 100 mM calcium chloride solution (distilled water or 1 M Tris HCl pH 7.0 buffer). The formed beads are washed with distilled water or buffer. Beads are prepared using those having an esterification degree of 30 mol% or 50 mol%.
    [118] Example 8
    [119] This example shows the preparation of leptin containing alginic acid ester beads.
    [120] Beads are prepared by dropwise addition of 25 mg / ml leptin solution dissolved in 2% alginic acid ethyl ester (Tris HCl, pH 8.7) to 100 mM calcium chloride and 25 mM zinc chloride solution. Beads are prepared using those having an esterification degree of 30 mol%. In vitro sustained leptin release experiments are performed with beads.
    [121] Example 9
    [122] This example shows the molecular weight breakdown (or degradation) of alginic acid esters in buffer at physiologically neutral pH.
    [123] Alginic acid ester (1% solution) is dissolved in 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 the decrease in viscosity in solution at selected time intervals. Unmodified sodium alginate decreases in viscosity by only 5% in 8 days and appears relatively stable; However, ethyl alginate and benzyl ester (DE = 30 mol%) drop to 35% viscosity in 8 days in the same buffer. The amount of decomposition of the alginic acid ester also depends on the degree of esterification. For example, ethyl esters with a low degree of esterification (DE = 15 mol%) have reduced viscosity to 25% within 8 days. Therefore molecular weight breakdown is directly related to the degree of esterification.
    [124] Example 10
    [125] This example shows in vivo degradation (or gradual loss) of alginic acid ester hydrogel containing protein and alginic acid ester hydrogel containing protein.
    [126] Alginic acid ester gels are prepared in a similar manner to that described in Example 3, but the final mixture is placed in a syringe overnight at 4 ° C. to become a gel in the syringe. Then 100 μl of gel was injected subcutaneously into the back of the neck of the mouse (obtained from Charles River, 12 week old female, BDF1, 20 g, 5 mice per group) and the site was periodically injected to the other members of the group. Examine surgically.
    [127] Using alginic acid ethyl ester and benzyl ester with DE = 30 mol%, the results of a single injection site study show that the alginic acid ester hydrogel disappears within two weeks. Using alginic acid ethyl ester gel with DE = 15 mol%, the gel is still present at 30 and 61 days but decreases in size. Using alginic acid ethyl ester with DE = 5 mol%, the gel is still present with little size reduction at 30 and 61 days. Using unsubstituted sodium alginate, the gel remains relatively unchanged throughout 61 days.
    [128] The loss rate of the alginic acid ester gel is similar to whether the protein is contained or not.
    [129] Example 11
    [130] This example provides weight loss and pharmacokinetic data in rats for leptin containing alginic acid ester hydrogel.
    [131] Alginic acid ethyl ester gel is prepared in a similar manner to that described in Example 4 but the final mixture is placed in a syringe overnight at 4 ° C. to become a gel in the syringe. The concentrates are administered to rats at doses of 0 mg / kg (control) and 100 mg / kg, and then blood levels and weight loss are measured for 7 days.
    [132] The results are as follows: Alginic acid ethyl ester with DE = 5 mol% shows stable blood levels of ~ 2000 ng / ml for 3 days, then drops to 2-3 ng / ml for the next 3-4 days; Alginic acid ethyl ester with DE = 15 mol% has a stable blood level of ˜2000 ng / ml for 2 days, then drops to 2-3 ng / ml on day 5; Alginate esters with DE = 30 mol% had blood levels of ˜2000 ng / ml for 1 day, then decreased to 2-3 ng / ml on Day 4; Blood levels of the Zn-leptin suspension peak at 12 hours and then decrease to 1-2 ng / ml on day 6. All animals show weight loss indicating Zn-leptin activity. In addition, the mixing of Zn-leptin with alginic acid ethyl ester gel (DE = 5 mol% and 15 mol%) almost doubled the area under the curve (AUC) of Zn-leptin, which was used in vivo. Suggests doubling of efficiency; The use of alginic acid ethyl ester gels shows similar bioavailability with Zn-leptin based on AUC.
    权利要求:
    Claims (63)
    [1" claim-type="Currently amended] Biodegradable and sustained release gel compositions comprising:
    a) hydrophilic polymer;
    b) biologically active agents; And
    c) at least one bound polyvalent metal ion.
    [2" claim-type="Currently amended] The sustained release composition of claim 1, wherein the bound polyvalent metal ion is a mixture of bound and unbound polyvalent metal ions.
    [3" claim-type="Currently amended] The sustained-release gel of claim 1, further comprising an excipient to stabilize the biologically active agent or hydrophilic polymer.
    [4" claim-type="Currently amended] The composition of claim 1, wherein the bound polyvalent metal ion is a salt selected from acetates, phosphates, lactates, tartarates, citrates, hydrochlorides, carbonates or hydroxides thereof.
    [5" claim-type="Currently amended] 5. The composition of claim 4 wherein the metal ion is selected from manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum or zinc.
    [6" claim-type="Currently amended] The composition of claim 5 wherein the metal ion is calcium.
    [7" claim-type="Currently amended] The composition of claim 1 wherein the proton donor is from an acid source.
    [8" claim-type="Currently amended] 8. The composition of claim 7, wherein the acid source is selected from buffers, esters, slowly soluble acids or lactones.
    [9" claim-type="Currently amended] The composition of claim 1 wherein the hydrophilic polymer is a polyvalent anion.
    [10" claim-type="Currently amended] The composition of claim 1 wherein the hydrophilic polymer is a polysaccharide.
    [11" claim-type="Currently amended] The composition of claim 10 wherein the polysaccharide is an acidic polysaccharide.
    [12" claim-type="Currently amended] 12. The composition of claim 11 wherein the polysaccharide is alginic acid.
    [13" claim-type="Currently amended] 13. The composition of claim 12, wherein the alginic acid contains at least 30% of guluronic acid.
    [14" claim-type="Currently amended] 13. The composition of claim 12, wherein the alginic acid is contained at least 0.05% by weight.
    [15" claim-type="Currently amended] The composition of claim 1, wherein the biologically active agent contains a protein and the composition exhibits improved bioavailability.
    [16" claim-type="Currently amended] The composition of claim 15, wherein the composition contains at least 0.001 mg / ml protein.
    [17" claim-type="Currently amended] The composition of claim 15, wherein the protein is selected from hematopoietic factor, colon stimulating factor, anti-obesity factor, growth factor, nutritional factor and anti-inflammatory factor.
    [18" claim-type="Currently amended] The composition of claim 15, wherein the protein is selected from: leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, IL-lra, IL2, TNF-bp, MDGF, OPG, Interferon, erythropoietin, KGF, insulin and analogs or derivatives thereof.
    [19" claim-type="Currently amended] The composition of claim 1 wherein the biologically active agent is a complex biologically active agent.
    [20" claim-type="Currently amended] 20. The composition of claim 19, wherein the complexed biologically active agent is a precipitated protein.
    [21" claim-type="Currently amended] The composition of claim 20, wherein the precipitated protein is zinc leptin precipitate.
    [22" claim-type="Currently amended] A process for preparing a biodegradable and sustained release gel composition comprising the following steps:
    a) mixing a biologically active agent with a hydrophilic polymer in a solvent to form a first mixture; And
    b) mixing at least one bound polyvalent metal ion into the first mixture to form a second mixture.
    [23" claim-type="Currently amended] 23. The method of claim 22, further comprising c) mixing at least one proton donor capable of releasing a bound polyvalent metal ion to a second mixture.
    [24" claim-type="Currently amended] 23. The process of claim 22, wherein the first mixture is concentrated before mixing the proton donor or the bound polyvalent metal ion.
    [25" claim-type="Currently amended] 23. The process according to claim 22, wherein the bound polyvalent metal ion is a salt selected from acetate, phosphate, lactate, citrate, tartarate, hydrochloride, carbonate or hydroxide thereof.
    [26" claim-type="Currently amended] The method of claim 22, wherein the method provides the patient with a substantially constant blood level of the biologically active agent for a period of time.
    [27" claim-type="Currently amended] 25. The composition of claim 24, wherein the metal ion is selected from manganese, strontium, iron, magnesium, calcium, barium, copper, aluminum, or zinc.
    [28" claim-type="Currently amended] 27. The composition of claim 26, wherein the metal ion is calcium.
    [29" claim-type="Currently amended] The composition of claim 23 wherein the proton donor is from an acid source.
    [30" claim-type="Currently amended] 29. The composition of claim 28, wherein the slowly soluble acid is selected from buffers, esters, slowly soluble acids or lactones.
    [31" claim-type="Currently amended] 30. The composition of claim 29, wherein the acid source is δ-glucononolactone.
    [32" claim-type="Currently amended] 23. The composition of claim 22, wherein the hydrophilic polymer is a polyvalent anion.
    [33" claim-type="Currently amended] 23. The composition of claim 22, wherein the hydrophilic polymer is a polysaccharide.
    [34" claim-type="Currently amended] 33. The composition of claim 32, wherein the polysaccharide is an acidic polysaccharide.
    [35" claim-type="Currently amended] 34. The composition of claim 33, wherein the polysaccharide is alginic acid.
    [36" claim-type="Currently amended] 35. The composition of claim 34, wherein the alginic acid contains at least 30% of guluronic acid.
    [37" claim-type="Currently amended] 35. The composition of claim 34, wherein the alginic acid is contained at least 0.05% by weight.
    [38" claim-type="Currently amended] 23. The composition of claim 22, wherein the biologically active agent contains a protein.
    [39" claim-type="Currently amended] 38. The composition of claim 37, wherein the composition contains at least 0.001 mg / ml protein.
    [40" claim-type="Currently amended] 38. The composition of claim 37, wherein the protein is selected from hematopoietic factor, colon stimulating factor, anti-obesity factor, growth factor, nutritional factor and anti-inflammatory factor.
    [41" claim-type="Currently amended] The composition of claim 37, wherein the protein is selected from: leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, IL-lra, IL2, TNF-bp, MDGF, OPG, Interferon, erythropoietin, KGF and analogs or derivatives thereof.
    [42" claim-type="Currently amended] 23. The composition of claim 22, wherein the biologically active agent is a complex biologically active agent.
    [43" claim-type="Currently amended] 42. The composition of claim 41, wherein the complexed biologically active agent is a precipitated protein.
    [44" claim-type="Currently amended] 43. The composition of claim 42, wherein the precipitated protein is zinc leptin precipitate.
    [45" claim-type="Currently amended] 23. The method of claim 22, further comprising the step of separating the sustained release composition.
    [46" claim-type="Currently amended] A sustained release product prepared by the process according to any one of claims 22 to 44.
    [47" claim-type="Currently amended] A pharmaceutical formulation comprising the sustained release composition of claim 1, 2, 3 or 45 comprising a pharmaceutically acceptable carrier, diluent or adjuvant.
    [48" claim-type="Currently amended] 47. The pharmaceutical formulation of claim 46, wherein the formulation is in a syringe.
    [49" claim-type="Currently amended] 45. A method of treating a symptom of a disease using the sustained release composition of claims 1, 2, 3 or 45 comprising a pharmaceutically acceptable carrier, diluent or adjuvant.
    [50" claim-type="Currently amended] A sustained-release composition of claim 1, 2, 3 or 45 comprising leptin or an analog or derivative thereof as a pharmaceutically acceptable carrier, diluent or adjuvant and biologically active agent is used to Methods of treatment: overweight, diabetes, high lipid levels in the blood, arteriosclerosis, arterial plaques, reduction or prevention of gallstone formation, inadequate fat tissue weight, inadequate insulin sensitivity and seizures.
    [51" claim-type="Currently amended] Hematopoietic cell deficiency using the sustained release composition of claim 1, 2, 3 or 45 comprising G-CSF or an analog or derivative thereof as a pharmaceutically acceptable carrier, diluent or adjuvant and biologically active agent. To treat a disease selected from among infections, neutropenia.
    [52" claim-type="Currently amended] Treatment of inflammation using the sustained release composition of claim 1, 2, 3 or 45 comprising IL-lra or an analog or derivative thereof as a pharmaceutically acceptable carrier, diluent or adjuvant and biologically active agent. How to.
    [53" claim-type="Currently amended] Sustained release compositions in the form of biodegradable gel particulates, characterized in that the biologically active agent is co-precipitated in the hydrophilic polymer:
    a) hydrophilic polymer;
    b) biologically active agents; And
    c) at least one precipitant.
    [54" claim-type="Currently amended] 53. The composition of claim 52, wherein the precipitant is selected from polyvalent metal ions or salts thereof, acetates, citrates, hydrochlorides, carbonates, or hydroxides thereof.
    [55" claim-type="Currently amended] 54. The composition of claim 53, wherein the metal ion is selected from manganese, strontium, iron, magnesium, calcium, barium, aluminum or zinc.
    [56" claim-type="Currently amended] 55. The composition of claim 54, wherein the precipitant is a polyvalent ion selected from zinc, calcium or a combination thereof.
    [57" claim-type="Currently amended] 53. The composition of claim 52, wherein the hydrophilic polymer is a polysaccharide.
    [58" claim-type="Currently amended] 59. The composition of claim 56, wherein the polysaccharide is alginic acid.
    [59" claim-type="Currently amended] Method for producing a sustained release composition, comprising the following steps:
    a) dissolving the biologically active agent and the hydrophilic polymer in a solvent to form a first mixture;
    b) dissolving at least one precipitant in the solvent to form a second mixture;
    c) addition of a second mixture to the first mixture; And
    d) co-precipitation of the biologically active agent and the hydrophilic polymer to form co-precipitated biodegradable gel particulates.
    [60" claim-type="Currently amended] 59. The method of claim 58, further comprising separating the co-precipitated particulates.
    [61" claim-type="Currently amended] A sustained release product prepared by the process according to claim 59.
    [62" claim-type="Currently amended] The pharmaceutical formulation of claim 52 comprising a pharmaceutically acceptable carrier, diluent or adjuvant.
    [63" claim-type="Currently amended] 52. A method of treating signs of disease using the sustained release composition of claim 52 comprising a pharmaceutically acceptable carrier, diluent or adjuvant.
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    同族专利:
    公开号 | 公开日
    BG104943A|2001-07-31|
    SK16752000A3|2001-09-11|
    BG65397B1|2008-06-30|
    BR9910553A|2001-01-30|
    EP1079811A1|2001-03-07|
    ZA200006299B|2001-05-30|
    CA2331446A1|1999-11-25|
    EA003670B1|2003-08-28|
    EP1079811B1|2006-10-25|
    AT343378T|2006-11-15|
    PT1079811E|2007-01-31|
    CN1309556A|2001-08-22|
    DE69933763D1|2006-12-07|
    AU3993999A|1999-12-06|
    US20020168406A1|2002-11-14|
    ES2275341T3|2007-06-01|
    HU0101895A2|2001-10-28|
    HU0101895A3|2004-01-28|
    NZ507943A|2003-08-29|
    WO1999059549A1|1999-11-25|
    YU69400A|2003-02-28|
    DE69933763T2|2007-10-04|
    NO20005564L|2001-01-18|
    RS49841B|2008-08-07|
    PL344337A1|2001-11-05|
    EA200001200A1|2001-04-23|
    IL139617D0|2002-02-10|
    US6432449B1|2002-08-13|
    DK1079811T3|2007-02-26|
    PL199499B1|2008-09-30|
    NO20005564D0|2000-11-03|
    JP2002515419A|2002-05-28|
    CA2331446C|2004-03-16|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-05-18|Priority to US09/080,832
    1998-05-18|Priority to US09/080,832
    1999-05-14|Application filed by 스티븐 엠. 오드레, 암젠 인코포레이티드
    1999-05-14|Priority to PCT/US1999/010737
    2001-03-26|Publication of KR20010025023A
    优先权:
    申请号 | 申请日 | 专利标题
    US09/080,832|US6432449B1|1998-05-18|1998-05-18|Biodegradable sustained-release alginate gels|
    US09/080,832|1998-05-18|
    PCT/US1999/010737|WO1999059549A1|1998-05-18|1999-05-14|Biodegradable sustained-release alginate gels|
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