Ionic conjugate with a long period of release of the peptide, the method of synthesizing an ionic conjugate, the method of synthesizing pm
(57) Abstract:The invention relates to medicine. Described ionic conjugate of a delayed release, which involves a complex polyester containing free COOH group conjugated with a bioactive polypeptide containing at least one effective ionogenic amine, and at least 50 wt.% polypeptide that is present in the conjugate ion difficulties with polyester. The proposed method of synthesizing an ionic conjugate by ion pairing, and the method for the synthesis of microparticles by dissolving ionic conjugate under item 1 in aprotic sadomasichism organic solvent. Conjugate provides regulated monophasic selection of biologically active polypeptide molecules in the body. 3 S. and 26 C.p. f-crystals, 6 tab., 3 Il. The invention relates to bioactive polypeptides, long lasting release of the active substance.Developed, tested and used in practice, a large number of so-called systems of drug delivery, regulatory allocation (release) in vivo pharmaceutical compositions. For example, polyesters, such as poly (DL-lactic acid), poly(glycolic to the main molecules such as progesterone (progesteron); they were made in the form of microcapsules, films or rods (Pitt CG, TA and A. Schindler 1980). After implantation of the composition of the polymer/therapeutic agent, for example, subcutaneously or venturimeter therapeutic agent is allocated within a certain period of time. Such biocompatible biodegradable polymer systems are intended to enable a prisoner in her therapeutic agent to diffuse through the polymer matrix. After the release of therapeutic agent, the polymer degrades in vivo without surgical removal of the implant. Although the factors that contribute to the decomposition of the polymer, is not well understood, it is believed that this decomposition for polyesters may be subject to availability of essential bridges to non-enzymatic autocatalytic hydrolysis of the polymer components.Several EPO publications and U.S. patents devoted to the development of polymer matrices and their role in regulating the rate and duration of release of therapeutic agents in vivo.For example, Deluca (EPO Publication 0467389 A2/Univ of Kentucky) describes the physical interaction between the hydrophobic biodegradable polymer and a white polymer, which extended its diffusive release from the matrix after the introduction of the subject.Hutchinson (U.S. patent N 4767628/ICI) regulate the release of therapeutic agent through a homogeneous dispersion in the polymeric device. Stated that this composition provides controlled continuous release of combining two methods: first - diffusion-dependent expired medicines from the surface composition and the second is the release of water through channels formed in the decomposition of the polymer.Brief description of the invention
In General, the invention is a pharmaceutical composition sustained release (discharge) formed a complex polyester containing one or more free groups-COOH, ion paired with a biologically active polypeptide, formed by at least one effective ionogenic an amine in which at least 50% of the weight of the polypeptide present in the composition, ionic difficulties with polyester.In preferred embodiments complex polyester modified with the aim of increasing the ratio of the number of carboxyl end groups to the number of terminal hydroxyl groups from more than a single the Rami of suitable esters are esters, obtained from such compounds as L-lactic acid, D-lactic acid, DL-lactic acid, caprolactone, p-dioxanone, -hexanoic acid, substituted and unsubstituted trimethylantimony, 1,5-dioxan-2-it, 1,4-dioxan-2-it, glycolide, glycolic acid, L-lactide, DL-lactide, meso-lactide, alkylaromatic, cycloalkylation, alkylresorcinol, (-hydroxybutyrate), and optically active isomers, racemates and copolymers of the above substances. Other heterochain polymers related to traditional complex polyesters, can also be used (for example, polyarteritis, polycholorinated and Polyacetals).Preferably complex polyester receive polycarboxylic reaction with malic or citric acid.In preferred embodiments complex polyester is a partially acid-closed polymer, a part of the hydroxyl groups which is closed by acid groups by reaction with glutaric anhydride. In other preferred embodiments complex polyester is a fully acid-gated polymer, in which all the hydroxyl groups are closed acid groups by reaction with glutaric anhydride. Preferably, the complex is-molecular conjugates of the invention are preferably made from a polycarboxylic acid-closed polyesters, conjugated with monobasic and poliennali bioactive polypeptides that have at least one effective ionogenic amine group. Alternatively, any complex polyester can be used for the formation of ion-molecular conjugate of the present invention provided that it is pre-treated with a suitable base, for example sodium hydroxide. In addition, you can use any acid-stable peptide, such as peptide secreting growth hormone (GHRP), luteinizing hormone - releasing hormone (LHRH), somatostatin, Balmain, gastronomically peptide (GRP), calcitonin, bradykinin, Galanin, melanocytestimulating hormone (MSH), factor secreting growth hormone (GRF), Amylin, tachykinin, secretin, parathyroid hormone (PTH), enkephalin, endothelin, calcitonin - gentilesse peptide (CGRP), neuromedin, a protein associated with thyroid hormone (PTHrP), glucagon, the neurotensin, adrenocorticotropic hormone (ACTH), peptide YY (PYY), glucuronidase peptide (GLP), vasoactive intestinal peptide (VIP), pituitary adenylate-cyclase activating peptide (PACAP), motilin, substance P, neuropeptide P, neuropeptide Y (NPY), TSH, and analogues and fragments. Such ion-molecule conjugates have spacetrucker, molecular weights and pKa of both components of the conjugate. The allocation mechanism of the drug causes the transformation of insoluble conjugate form in the water-soluble components, part by hydrolysis of the hydrophobic complex of the polyester. Thus, the selection of the bioactive polypeptide is increased independently
(a) with a decrease in the pKa difference between a bioactive polypeptide and a complex polyester
(b) with the chemical reactivity of the polyester chain, which is reflected carbonyl nucleophilicity,
(C) with decreasing density complex polyester, which is associated with the glass transition temperature and crystallization is minimized by the ability,
(d) with increasing hydrophilic ability of the matrix.In preferred embodiments the polypeptide is from 1 to 50 weight percent of the total weight of ion-molecular conjugate and preferably more than 82%, more preferably 95% and most preferably 99%, and is present in the composition of the polypeptide is conjugated with a complex ester; component of a complex of the polyester ion-molecule conjugate has a viscosity of from about 0.05 to 0.7 deciliter per gram in chloroform; and a complex polyester has Wed the gain can easily be made into microspheres or microparticles for injection or tapes or rods for implantation without the need to use the process leading to the multiphase emulsions or non-aqueous two-phase systems. Preferably, the microparticles are made (a) by dissolving the composition in an aprotic organic solvent with the ability to mix with water; (b) mixing the organic solvent with water, and (c) the selection of particles from the water. In preferred embodiments the organic solvent is chosen from the group comprising acetone, acetonitrile, tetrahydrofuran, dimethylformamide and dimethoxyacetophenone.In preferred embodiments the ion-molecular conjugate complex of the polyester with the polypeptide has the ability to distinguish in vivo a therapeutically effective dose of a bioactive polypeptide in a period equal to at least 20 days, more preferably for a period of up to 95 days, but not less than 7 days. In other preferred embodiments the selection of therapeutic ion-molecule conjugate is essentially monophasic.The compositions of this invention by sustained release of the active substance preferably made by (a) the availability of sophisticated polyester with free COOH groups and bioactive polypeptide having on the education of ion-molecular conjugate, in which at least 85% by weight of polypeptide present in the composition is an ionic conjugated with complex polyester. Complex polyester may be a polyester, which has a sufficient number of free COOH groups primarily or, if there is an insufficient number of such groups to obtain the necessary boot level, complex polyester may be (1) reaction, for example, malic or citric acid by esterification reaction or functional domestic currency or (2) the formation of acid end groups, for example, glutaric anhydride, or (3) a complex of the polyester can be treated with base, such as sodium hydroxide, to open acid groups. Finally, ion-molecular conjugate complex of the polyester with the polypeptide can be turned into a film or rods for implant or microspheres or microparticles for injection, is able to distinguish in vivo polypeptide.Preferably, the complex polyester synthesize catalytic or autocatalytic direct condensation of one sludge more hydroxy acid such as glycolic acid and lactic acid, in the presence of polycarboxylic hydroxy acid predefined concinna-closed hydroxyl terminal groups, which are preferably partially or completely acid-closed.Polyesters can also be synthesized by the catalytic polymerization of lactones with utilizarea (disclosure cycle) or by polymerization of cyclic monomers, such as caprolactam, p-dioxanone, trimethylantimony, 1,5-dioxan-2-he or 1,4-dioxan-2-he, in the presence of the initiator of the chain, for example, polycarboxylic hydroxy acid (Aha).Another method of synthesis involves the reaction of a hydroxy acid with a cyclic dimer with subsequent condensation system with open circuit in the presence of polycarboxylic acid.Another method of synthesis involves the reaction of organic polycarboxylic acid with a pre-obtained complex polyester.In the above preferred embodiments the acid-gated complex polyester has a ratio of the number of carboxyl to the number of hydroxyl end groups equal to more than the unit and approaching infinity (i.e., removing all hydroxyl groups), with an average degree of polymerization of from 10 to 300, in particularly preferred embodiments is from 20 to 50.Alternatively, a complex polyester in turn are capable of forming ion forefront of the/P> Preferably ion-molecule complex conjugate polyester with a polypeptide synthesized by the direct interaction of a complex of the polyester, for example, in a free form with the polypeptide, for example, in free form in a suitable liquid medium. In other preferred embodiments suitable solvents for the formation of the conjugate must be a mixture of aprotic solvent (e.g. acetone, tetrahydrofuran (THF) or etilenglikolevye) and a suitable solvent for the peptide (e.g., water) in such proportions that these two systems are mixed. Preferably the polypeptide is a salt of monocarboxylic acid having a pKa equal to or greater than a 3.5. Preferably the polypeptide has at least one effective ionogenic amine group.In preferred embodiments the polypeptide is from 1 to 50% by weight and preferably from 19 to 20 percent weight of ion-molecular conjugate of ester and of the polypeptide. In preferred embodiments of the available carboxyl groups of a complex of the polyester partially neutralized ion of an alkali metal or organic bases. In other preferred embodiments the alkaline processing provides the dissociation chain complex floor is m description means a protein, the peptide, Oligopeptide or synthetic Oligopeptide.The term "polycarboxylic used herein, refers to compounds having more than one carboxyl group, such as malic or citric acid.The term "average degree of polymerization" in this description is used to denote the number of repetitions Monomeric fragment.The term "effective ionogenic amine" used in this description, refers to a polypeptide that contains at least one amine group that can form under favorable conditions, ion.The term "acid-closed" refers in this description to compounds with acid end group.The term "partially acid-closed" in this description refers to compounds in which from 1 to 99 percent of their hydroxyl end groups of closed acid groups.The term "fully acid-closed" in this description refers to the indicated compounds in which more than 99.9% of their hydroxyl groups are closed acid groups.The term "hydroxy acid" herein refers to any compound containing hydroxyl and carboxylate with one carboxyl group and one or more hydroxyl groups.The term "organic azeotropically" refers to organic liquids that are distilled together with water."Bioactive" in this description is called a molecule that exerts or causes biological effects."Acilitate" in this description is the reaction leading to the disclosure of the loop.The term "polycondensation" refers to the formation of a complex of the polyester condensation of two or more molecules.This invention provides a new pharmaceutical composition, which chemically binds biocompatible biodegradable complex polyester with oligopeptides, polypeptides, peptides or proteins in a homogeneous structure of the ionic type. Chemical binding of polyesters of various molecular weights with therapeutic agents chemical characteristics of the composition can be accurately stored to ensure regulated monophasic selection of biologically active polypeptide molecules in vivo. In addition, the compositions of this invention are easily optimized with obtaining functional properties for better load therapeutically active polypeptide.Other characteristic features and advantages of the invention will be the Brief description of the drawings.In Fig. 1 shows isomers polycarboxylic acid-closed lactide/glycolide (Apple type) copolymer.In Fig. 2 depicts the molecular conjugate and shows the chemical interaction between the lactide/glycolide (Apple type) copolymer and somatuline (Somatuline BIM-23014).In Fig. 3 depicts a graph showing the release of the peptide, expressed in percent, of the ion-molecule conjugates in phosphate-saline buffer at a temperature of 37oC for 28 days.Description of the preferred embodiments
The synthesis.Biodegradable or absorbable polyesters of the present invention receive specially to achieve a desired chemical reactivity, providing controlled the ability of the circuit to hydrolysis, and the maximum binding capacity in respect of oligopeptides, polypeptides and proteins that have a positive charge at physiological pH, through precise selection of constituent monomers, comonomers or incoming fragments to form chains with defined structures and molecular weights (see, for example, Fig. 2).To obtain the compositions of the present invention use three-stage Sinto-closed polyesters; (2) synthesis of ionic complex conjugate polyester-polypeptide through ionic interactions polycarboxylic complex polyester or of ester-treated base, and biologically active polypeptides; and (3) the conversion of ionic conjugates in implants, rods, microspheres or microcapsules, are able to release therapeutic agent within at least 7 days.1) Synthesis of polycarboxylic acid-closed polyesters
Chain polycarboxylic acid-closed polyesters synthesized by several methods, such as direct condensation of 2-hydroxy acid and polycarboxylic organic acids, stepwise polymerization reaction products of utilizacii, polymerization of lactone or mixture of lactones, proceeding with disclosure of the cycle, or the functional currency organic polycarboxylic acid with a pre-obtained complex polyesters with large molecular weights (see Fig. 1). Below are descriptions of the synthesis of polycarboxylic acid-closed polyesters these listed methods.Direct condensation of 2-hydroxy acids in optically active and/or inactive form and a predefined number of the polycarboxylic organicism is ondensation glycolic acid, DL-lactic acid and DL-malic acid, usually carried out by heating monocarboxylic hydroxy acid or a mixture of two or more monocarboxylic hydroxy acids in the presence of a fraction of the polycarboxylic oxicity in a glass reactor, providing a continuous supply of dry nitrogen and the stirring of the reaction mixture (table I labeled as the Polyester type IA). Typically, the polycondensation is carried out at a temperature of 150 - 170oC over a period of time lasting from 4 to 72 hours. Stirring of the reaction mixture can be carried out using a magnetic stirrer or by ozonation nitrogen gas through the complex mass of the polyester. Polymerization continues until the desired average molecular weight, which is determined by solution viscosity and/or acid number, which is determined by the titration end groups. Analysis of polyesters by the method of titration end groups is carried out as follows. Samples polyesters (300 mg - 500 mg) was accurately weighed and dissolved in a minimal amount (10 - 30 ml) of acetone. After dissolving, the solution was diluted with benzyl alcohol, bringing the volume of each solution (100 ml benzyl alcohol - Malline - Krodt, Analytic sodium hydroxide in benzyl alcohol (standardized HCl sample). Solution volume of base used for sample Vaboutcompare with the volume of the base used for the control solution VFLgetting an acid number of complex polyester:
< / BR>When assessing the polymerization complex polyester is isolated and extracted with water or a diluted aqueous sodium hydroxide solution of a suitable organic solution to remove water-soluble or solubilizing chains with low molecular weights.Analysis of polyesters by the GPC method performs as follows. Average molecular weight (MB) of the polyesters is determined by the GPC method using a pump mark Waters Model 6000 and UV-D detector model Dynamax (rainin). Experiments performed in tetrahydrofuran (Burdick Jackson, UV grade) using divinylbenzene gel brand Jordin Gel DVB 1000 column size 50 cm x 10 cm (Jordi Associates) at a feed rate of 1.2 ml/min and a temperature of 25oC.Registration perform peaks at 220 nm or 1.0 AUFS. Column calibrated using polystyrene standards (Polysciences Inc.) with MB = 4000, 9200 and 25000.A variation of the process of direct condensation is the use of organic azeotropically and cation exchange resin as a catalyst andlaura stage filtration and evaporation to remove the catalyst and azeotropically respectively. Typical examples of the polyesters obtained using these methods and the corresponding data analysis are given in table I.Stepwise polymerization reaction products of utilizacii, in which the hydroxy acid reacts with a cyclic dimers and subsequent condensation of the resulting systems with an open chain in the presence of predetermined amounts of polycarboxylic acid and in the presence or in the absence of a suitable condensation catalyst, for example glycolic acid, L-lactide and DL-malic acid, essentially similar to the condensation process described above, except that it uses a mixture of monocarboxylic hydroxy acid, a cyclic dimer secondary hydroxy acid and polycarboxylic hydroxy acid. Examples of polyesters obtained by this method, and data analysis are presented in table II. In the case when the cyclic dimer is pre-treated water, the system is treated as a simple step polymerization.The polymerization of lactone or mixture of lactones, proceeding with disclosure cycle in the presence of polycarboxylic hydroxy acid predetermined concentration as the initiator rusticola and DL-malic acid, in
the presence of octoate divalent tin, using dry cyclic monomers or mixture of cyclic monomers and trace amounts of octoate divalent tin in the form of a 0.33 M aqueous solution in toluene, which in dry oxygen-free atmosphere is transferred to a glass reactor equipped with a magnetic or mechanical stirrer. The polymerization reaction is carried out in nitrogen atmosphere using an appropriate sequence of heating to achieve the desired molecular weight, which is determined by solution viscosity. At the end of the polymerization the temperature is reduced and the unreacted monomer is distilled off under vacuum. The weight of the polyesters was then cooled and water-soluble fractions with low molecular weights are removed from the reaction mixture by low-temperature extraction of a suitable organic solvent. The solution is then dried and the solvent is distilled off. After that, the characteristic viscosity determine molecular weight by titration end groups is calculated acid number. Examples of polyesters obtained by this method, and the corresponding results of the analysis are shown in table III.F is a complex polyesters large molecular weights with a ratio of COOH/OH from one to zero, preferably in the presence of an ORGANOMETALLIC catalyst, for example, the reaction in the melt 85/15 lactide/glycolide copolymer with a molecular weight of more than 5000 and the ratio of COOH/OH1 and DL-malic acid in the presence actinobacillus tin to obtain low molecular weight polyesters with a ratio of COOH/OH, less than or equal to the unit carried out by heating complex macromolecular polyester with a predetermined amount of polycarboxylic acid or polycarboxylic hydroxy acid in the presence of traces of an ORGANOMETALLIC catalyst, such as actinobacillus tin. The reagents are heated to temperatures above 150oC under an atmosphere of dry nitrogen with vigorous stirring until the completion of the functional currency, which is controlled by the dimension reduction of unreacted polycarboxylic acid. The result of the reaction are determined by control of the molecular weight (as determined by solution viscosity using capillary viscosimetry in the 28oC) complex obtained polyester with a lower molecular weight and the presence of unreacted polycarboxylic acid. This is carried out by aqueous extraction of the sample complex polyester and analysis of the extract by high performance liquid chromatography (VGH). Sod is mark Waters Model 6000 and UV-D detector model Dynamax (Rainin) (205 nm, 1,0 AUFS). Duct carried out using 0,025 N. buffer solution of Na2PO4, pH 3.5 (feed rate 1.0 ml/min) using a column (25 cm x 4.6 mm filled with Nucleosil C18, 5 mmThe desired complex polyester is isolated and purified from impurities similar to the method described for acyclic polymerization. An example of a complex of the polyester obtained in this way and the corresponding data analysis are presented in table IV.In this invention for the synthesis of polyesters used the following monomers: L-lactic acid, DL-lactic acid-caprolactone, p-dioxanone, -hexanoic acid, trimethylantimony, 1,5-dioxan-2-it, 1,4-dioxan-2-it, glycolic and mesolectal. Examples of the initiators of the growth of the macromolecule and/or modifiers circuits include malic acid and citric acid.2) Synthesis of ionic conjugate complex of polyester and polypeptide ion interaction polycarboxylic acid-closed polyesters and biologically active polypeptides.Polycarboxylic acid-closed biodegradable polyesters used for ion-molecular conjugates with mono - or polycarboxylic the oligopeptides, peptides, or bakeoven to form ion-molecule conjugate with the polypeptide provided he handled the basis of, for example, 0.1 N. sodium hydroxide. This treatment opens acid groups for numerous plots of ion interaction with a cationic polypeptide.Thus, these conjugates receive a direct molecular interaction between the components in a suitable solvent with or without pre-treatment of complex polyester inorganic base to obtain the maximum speed of the link with the main drug. As noted above, the ionic interaction of the components of the ionic conjugates increases within the difference in pKa values.Complex polyester is dissolved in a suitable aprotic solvent to obtain a concentration of from 2% to 20% weight/volume. Such a solvent should dissolve polyesters, and must be partially miscible with water. Suitable solvents for these purposes are tetrahydrofuran, acetone, and dimethyl ether of ethylene glycol. To obtain maximum binding capacity of the complex polyester to these solvents added an aqueous solution of base such as the hydroxide of sodium, potassium, ammonium or sodium carbonate, potassium. In General, the amount of added base is relevant to the data synthesis.After a short mixing in ester-based add an aqueous solution of the peptide or salt of the peptide at the level of dosing peptide complex polyester in the range from 2% to 50% wt./weight. (peptide/polyester). This mixture is stirred for a period of three hours and then the solvent is distilled off and the product dried under vacuum. The resulting product can be further used for the manufacture of metered dose of the composition. The resulting pharmaceutical composition is believed, must be made entirely of ion-molecular conjugates and essentially free from microscopic and macroscopic fields active drug dispersed in a biodegradable matrix. Examples of the obtained ion-molecule conjugates and the corresponding data analysis are presented in table V.3) Conversion of ionic conjugates in implants, rods, microspheres or microparticles, is capable of releasing therapeutic agent within at least 20 days in a monophasic profile.Ionic conjugate salts of this invention can be transformed into: (A) sterile microspheres for injection with or without 0.1 to 10% solid polyhydric alcohol as an auxiliary to the society, monophasic profile and to support pharmaceutical activity during the period of from one to 12 weeks; (B) sterile implantable films made by casting, pressing or extrusion, with or without pharmaceutically inactive adjuvants, capable of providing a release profile similar to those described in (A).Experience release in vitro.Samples of dried and ground material ionic conjugate weighing 50 mg each placed in scintillation containers with a diameter of 25 mm Aliquots of 5 ml of modified phosphate-saline buffer (phosphate-salt buffer: 2,78 g Na2HPO4, 0,654 g NaH2PO4, 5.9 g of NaCl, 0.5 g NaN3, 1 liter of deionized water; pH 7,27) is added to each vessel, the vessel is placed on the vibrator brand Lab-Line Orbit Environ-Shaker and rotate with a speed of 120 rpm at a temperature of 37oC. Capacity periodically get from a vibrator, decanted solution and fill the capacity of phosphate-saline buffer. The number of released peptide define exposing decanted solution analysis method VGH.Extraction of the peptide from the ion conjugates.The sample ion-molecular weight conjugate of 50 mg smety acetic acid are combined and examined for the content of the peptide by the method VGH. Analysis of the content of the peptide by the method WICH carried out as follows. Analysis VIH perform when using pump mark Waters Model M-45 and detector EM Sciens with a wavelength of 220 nm and 1.0 AUFS. The peptides isolated by using a Lichrospher (EM separation) C18, 100 5 nm, column 25 cm x 4.6 mm and as eluent buffer solution of 30% acetonitrile/0.1% TFUCK.Table VI presents data of the experiment in vitro, showing the number of peptide selected within the period of 28 days for the following ion-molecule conjugates: 49:49:2 L-lactic/glycolic/malic D-tryptophan6[LHPH] (example 8), 49:49:2 L-lactic/glycolic/malic somatostatin - appolinaire similar (example 9) and 73.5:24,5:2 poly-L-lactide/glycolic/malic D-tryptophan6(example 10). In Fig. 3 shows a graphical interpretation of these data.The determination of the number of peptides in ion conjugates.Ion-related peptide in the conjugate quantitatively determined by dissolving 10 mg of the samples was 5.7 ml of a mixture of acetone and 0.1 M aqueous triperoxonane acid in the ratio 9:1. The solution is subjected to vibration at a temperature of 25oC for 15-24 hours, and then filtered through a Teflon filter cartridges size of 0.5 μm. The filter is the so called Millipore Wisp Autosampler model 717, pump model 510 and UV detector 486 at a wavelength of 220 to them. Peptides pass through Lichrospher (EM separation) 25 cm x 4.6 mm C18 column, 5 μm, 100 angstroms, with a flow rate of 1.0 ml per minute when used as suantai system buffer 35% acetonitrile in 0.14% of sodium perchlorate. The number of peptides is determined by comparing the area of the corresponding peak in the analyzed sample area entered peptide standard.Application
Any acid-bearing ionic conjugates polyesters and polypeptides described in this invention can be administered to the recipient by themselves or in combination with pharmaceutically acceptable component. Despite the fact that they can be available for injection subcutaneous, intramuscular, parenteral, nasal, therapeutic drug is administered according to the disease to be treated. The concentration of the composition in the preparations of the invention will vary depending on a number of factors, including dose, which must be entered, and the method of administration.We believe that specialist in this area can, using the preceding description without additional clarification, most fully apply it. Thus, the above nightbreeze.Example 1. The direct condensation. Synthesis of 50/50 poly (D,L-lactic-co-glycolic) ether with a catalyst Amerlyst 15.D, L-lactic acid (85% solution in water; of 13.7 g of 0.13 mole) is mixed with glycolic acid (10 g, of 0.13 mol) in a round bottom flask, equipped with magnetic stirrer, water trap Dean-stark and a condenser cooled by water. To the mixture is added toluene (100 ml) and balls of catalyst Amberlyst 15, and the resulting mass is boiled for 72 hours under a nitrogen atmosphere, fending off the water from the mixture. The mixture is cooled, the toluene is decanted from the solidified mass, the product is dissolved in methylenechloride (250 ml). A solution of methylene chloride is treated with activated charcoal (Darco, 500 mg), filtered and dried under vacuum on a rotary evaporator. Complex polyester then dried under high vacuum (1 mm RT.CT.) at a temperature of 40oC, resulting in getting a white powder (harin chloroform = 0,3, acid number 2439, Tc= 12oC).Example 2. The direct condensation. Synthesis 49/49/2 poly(L-lactic-co-glycolic/citric) ether with a catalyst Amberlyst 15.Using a setting similar to those described above, L-lactic acid (88% aqueous solution of 25.6 g of 0.25 mole) is mixed with glycolic acid (19.2 g, and 0.25 mole), authorized the flask. The mixture is heated with stirring to boiling point and boiled for 51 hours, removing water by means of traps Dean-stark. The toluene is decanted from the semi-solid product. Complex polyester is dissolved in acetone (300 ml), filtered and dried on a rotary evaporator. Solid complex of the polyester is then dissolved again in methylene chloride and washed twice with water (2 x 150 ml) to remove soluble oligomers. The organic solution evaporated on a rotary evaporator, the residue is thoroughly dried under vacuum, the result is a product in the form of a white powder (see table I, complex polyester type IB, polymer 4) (xin chloroform = 0,11, acid number 842, Tc= 15oC).Example 3. The method of stepwise polymerization. Synthesis of 73.5/24,5/2 poly(L-lactide-co-glycolic/malic) ether when used as a catalyst malic acid.Using a cylindrical vial with a volume of 150 ml, provided with a device for input of nitrogen, L-lactide (20 g, 0,139 mole) is mixed with glycolic acid (7,1 g, 0,093 mol) and d,l-malic acid (1.0 g, 0,0075 mol). The mixture is stirred, barbotine nitrogen through the input device for injection of gas (100 ml/min), and heated from a temperature of 25oC to 155oC for 100 minutes. include process are removed through a cooled trap on the output line. After 70 hours the reaction mass is then cooled to 100oC and poured into a chilled steel receiver for solidification. Solid polyester is then dissolved in methylene chloride, washed twice with water to remove soluble oligomers (2 x 150 ml). The organic solution evaporated on a rotary evaporator and the product is carefully dried under vacuum, resulting in a gain substance in the form of a white powder (see table II, complex polyester type II polymer 2) (harin chloroform = 0,13, acid number 1800, Tc= 27oC).Example 4. The method of polymerization with the disclosure of the cycle. Synthesis of 75/25 poly(L-lactide-co-glycolide)broadcast initiated by malic acid.L-lactide (12.0 g, 0,0833 mol), glycolide (3,21 g, 0,0277 mol), malic acid (0,3042 g, 0,00227 mol) and the catalyst - actinobacillus tin (0.33 M in toluene, 67 μl, of 0.022 mole) is placed in a nitrogen atmosphere in a glass ampoule equipped with a magnetic stirrer. The system is rinsed with nitrogen and for some time create a vacuum in it, and then sealed. Then the reactants are subjected to melting at a temperature of 140oC, the resulting melt is heated and maintained at a temperature of 180oC, 190oC, 180oC and 150oC for 1, 4,5, 12 and 2 hours soonee 1 mm RT.article and maintained under these conditions for about one hour to remove monomer, again cooled to room temperature, quenched with liquid nitrogen, isolated and dried under vacuum (harin chloroform = 0,20, acid number 2560, Twith= 39oC).Example 5. The method of polymerization with the disclosure of the cycle. The synthesis of complex 50/50 poly(L,D-lactide-co-glycolide) broadcast initiated by citric acid.D, L-lactide (10.0 g, 0,0694 mole) is mixed with glycolide (8.06 g, 0,0694 mol), citric acid (1.07 g, 0,0555 mol) and actinobacillus tin (0.33 M th solution in toluene, 84 μl, 0,0278 mmole) under nitrogen atmosphere in a glass ampoule equipped with a magnetic stir bar and sealed under vacuum. The reactants are melted and maintained at a temperature of 180oC, 185oC, 195oC and 120oC for 1, 2, 7 and 9 hours, respectively. Complex polyester is cooled to room temperature, quenched with liquid nitrogen, the product is isolated and dried (harin chloroform = 0,26, acid number 970, Twith= 23oC).Example 6. The method of polymerization with the disclosure of the cycle. The synthesis of complex 50/50 poly(L-lactide-co-glycolide) broadcast initiated 1,6-hexandiol.Using the above system, D, L-lactide (10.0 g, wale, 85 μl, 0,0278 mmole) was placed under an atmosphere of dry nitrogen in a glass ampoule, which is then sealed under vacuum. The reagents are kept at a temperature of 150oC, 185oC, 150oC and 120oC for 0.5, 4, 1.5 and 3 hours, respectively. Complex polyester is isolated and dried (see table III, polyester type III polymer 5) (harin chloroform = 0,39, acid number 10138, Twith= 30oC).Example 7. The method of functional currency. The synthesis of complex 50/50 poly(D, L-lactide-co-glycolide) ether containing carboxyl group.50/50 poly(D,L-lactide-co-glycolide) ether (Boehringer A001, 8 g), citric acid (0.8 g, 4,16 mmole) and actinobacillus tin (2 drops) was placed in a glass ampoule under an atmosphere of dry nitrogen and sealed. The mixture is heated to 150oC and kept at this temperature for 4 hours, cooled to room temperature, quenched with liquid nitrogen, isolated and dried final product (see table IV, complex polyester type IV, polymer 1) (harin chloroform = 0,26, acid number 670, Tc= 23oC).Example 8. Synthesis of ion-molecular conjugate complex 49:49:2 (L-lactic/glycolic/malic) ether (see table I, polymer 4) and gonadotropinreleasing hormone D-Trp6[LHRH].can be the number 1420) dissolved in 10 ml of acetone (Mallinckrodt Anal. Reagent). To the resulting mixture add a solution of sodium hydroxide (0.1 N., 1,14 ml) and the mixture is stirred at room temperature for 15 minutes. A solution of 100 mg of gonadotropin-releasing hormone D-Trp6[LHRH] (BIM-21003 Peptide I, the basic content of 87%, a content of acetate 7%) in 1.0 ml of water added to the reaction mixture and the resulting mass is stirred at room temperature for 1 hour. Then the solvent is distilled off, first on a rotary evaporator under vacuum at a temperature of less than 40oC, and then desicator at room temperature for 1 hour under vacuum of 1 mm RT.article The dried solid is ground to powder and mixed with 100 ml of deionized water, and then allocate filtering. The aqueous filtrate is analyzed by the method VIH, detecting the content of soluble peptide of less than 1 mg of the Solid material is dried for several days under vacuum to obtain 540 mg of a white powder. The powder used in the experiment in vitro (see table VI, example 8).Example 9. Synthesis of ion-molecular conjugate complex 49:49:2 L-lactic/glycolic/malic polyester (see table I, polymer 4) and somatostatin/ puhelinnumero equivalent.100 mg complex 49: 49: 2 L-lactic/glycolic/malic polyester (synthetic is th mixture add a solution of sodium hydroxide (0.1 N., of 0.32 ml) and the mixture is stirred at room temperature for 15 minutes. A solution of 20 mg of somatostatin/puhelinnumero analogue (BIM-23014 Peptide II, the basic content of 83%, acetate content of 9.8%) in 1.2 ml of water is added to the mixture and the whole mass is stirred for 1 hour at room temperature. The solvent is then distilled off, first on a rotary evaporator at a temperature of less than 40oC, and then dessicator for 1 hour at room temperature under vacuum of 1 mm RT. tbsp. Dried solid is ground to powder and mixed with 20 ml of deionized water, and then allocate filtering. The aqueous filtrate is analyzed by the method VIH, detecting the content of soluble peptide of less than 0.05 mg of the Solid material is dried for several days under vacuum, the result is 106 mg of a white powder. The powder is ground and used in the experience of release in vitro (see table VI, example 9).Example 10. Synthesis of ionic conjugate complex 73,5:24,5:2 poly (L-lactide/glycolic/malic) ether (see table II, polymer 2) and gonadotropinreleasing hormone.80 mg complex 73,5: 24,5: 2 poly(L-lactide-glycolic/malic) ester (synthesized stepwise polymerization of acylated products; acid Ciaramella at room temperature for 20 minutes. The solution gonadotropinreleasing hormone 200 mg D-Trp6[LHRH] (BIM-21003; the main content of 87%, a content of acetate 7%) in 2 ml of water is added to the resulting mixture and the whole mass is stirred for 90 minutes. The solvents were removed and the resulting solid is triturated and mixed c deionized water as in example 8, which shows the content of water-soluble peptide less than 1%. The selected solid is dried for 4 days under vacuum, after which they receive 839 mg of white powder. The powder is crushed and used in the experience of release in vitro (see table VI, example 10).Example 11. Obtaining ionic conjugate the peptide-polymer in the form of microparticles 1,50 L-lactide/glycolide/d,l-malic complex polyester (65:33:2).The conjugates are synthesized by polymerization of disclosure cycle, as in example 4 (MB = 4700, the polydispersity of 1.3, as determined by gel permeation chromatography on a column of 50 x 1 cm with mixed filler Jordi Gel, eluent THF, detector light scattering dn/de = 0,05, acid number 1475 determined by titration, Tc= 42oC), dissolved in 40 ml of acetone. The acid groups are neutralized 2.0 g BIM-23014 (contents peptide 83,7%, acetate content of 11.5) in 20 ml of water is added slowly with Premia peptide to prevent the formation of sludge. Clear colorless solution is stirred for 1 hour and then evaporated to dryness under vacuum. The resulting white powder is again dissolved in a mixture of 20 ml of acetone and 2 ml of Milli - Q water c the formation of a transparent solution. This solution is introduced through a 0.2 μm Teflon filter into a container containing 500 ml of Milli - Q water with a temperature of 4oC with rapid stirring. Phase polymer/peptide complex stands immediately in small particles as a result of contact with water. After stirring for 30 minutes at a temperature of 4oC residual acetone is removed under vacuum and the solid particles are centrifuged again suspended in 100 ml of Milli - Q water and centrifuged again. The selected solid is dried leofiles drying with getting 1530 mg of a white free flowing powder. The particle size of the powder is from 2 to 100 μm, Tcionic conjugate equal 53oC. Total residue peptide that is not bound in all water supernatant, as found by the method VGH, is 63 mg Total initial content of the peptide, as determined by elemental analysis of nitrogen content, is 19.9% of the weight. The number extracted from the peptide conjugate is 16.9% of the weight that is determined by the extraction method acharacter.Qualified on the basis of the above description it will be easy to evaluate the characteristic features of this invention and, without distinguishing it from the scope of the present invention can perform various changes and modifications of the invention to adapt to various conditions. Therefore, other embodiments are also within the scope of this invention. 1. Ionic conjugate with a long period of release of the peptide containing complex polyester containing one or more free COOH groups, ionic conjugate with a biologically active polypeptide containing at least one effective ionogenic amine, and at least 50 wt.% polypeptide present in the composition, ion paired complex polyester.2. Ionic conjugate under item 1, where in the complex polyester respect to the number of carboxyl groups to the number of hydroxyl groups is greater than one.3. Ionic conjugate under item 1, where the polyester is formed by a component selected from the group comprising L-lactic acid, D-lactic acid, DL-lactic acid, E-caprolactone, p-dioxanone, E-Caproic acid, alkylaromatic, cycloalkylation, alkylresorcinol, -hydroxybutyrate, substituted and the D-lactide, DL-lactide, meso-lactide and their optically active isomers, racemates and copolymers.4. Ionic conjugate under item 1, where the complex polyester is partially acid-closed glutaric anhydride.5. Ionic conjugate under item 1, where the complex polyester is completely acid-closed glutaric anhydride.6. Ionic conjugate under item 1, where the complex polyester has a degree of polymerization ranging from 10 to 300.7. Ionic conjugate under item 1, where the complex polyester has a viscosity of from approximately 0.05 to approximately 0.7 gave 1 g in chloroform and the average molecular weight of about 1200 to 40,000.8. Ionic conjugate under item 1, where the bioactive polypeptide is from 1 to 50% by weight of the specified ion-molecule conjugate.9. Ionic conjugate under item 1, where more than 85% of the polypeptide present in the composition, ionic difficulties with polyester.10. Ionic conjugate under item 1, where the polypeptide is chosen from the group including luteinizing gammonvillage hormone (LHRH), somatostatin, Balmain/gastronomically peptide (GRP), calcitonin, bradykinin, Galanin, the drives of the working systems sistemului hormone (MSH), factor secreting growth hormone (GPF), Amylin, tachykinin, secretin, p is aveneu cancer (PTHrP), glucagon, neurotensin, adrenocorticotropic hormone (ACTH), growth hormone - releasing peptide (GHRP), glucuronidase peptide (GLP), vasoactive intestinal peptide (VIP), pituitary adenylate-cyclase-activating peptide (RASAR), enkephalin, peptide YY (RUU), motilin, substance P, neuropeptide N (N). S and their analogues and fragments.11. Ionic conjugate under item 1, which is capable of releasing in vivo a therapeutically effective dose of the polypeptide for a period of at least 7 days.12. The method of synthesizing an ionic conjugate, characterized in that (a) receive a complex polyester and bioactive polypeptide, having at least one effective ionogenic amine, and (b) are ion pair complex of the polyester to the polypeptide with the formation of ion-molecular conjugate, in which the polypeptide of the ion paired complex polyester.13. The method according to p. 12, wherein at least 50 wt.% polypeptide present in the composition, ionic difficulties with polyester.14. The method according to p. 13, characterized in that the polyesters have acid-closed hydroxyl end groups.15. The method according to p. 14, characterized in that hydroxyine fact, what terminal hydroxyl groups are completely acid-closed glutaric anhydride.17. The method according to p. 13, characterized in that the polyesters are synthesized using as initiators of education macromolecules polycarboxylic hydroxy acid (Aha).18. The method according to p. 13, characterized in that the hydroxyl end groups of polyesters acid-closed.19. The method according to p. 18, characterized in that the hydroxyl end groups of the partially acid-closed glutaric anhydride.20. The method according to p. 18, characterized in that the hydroxyl end groups are completely acid-closed glutaric anhydride.21. The method according to p. 13, characterized in that the synthesis of complex polyester leads to an average degree of polymerization in the range from 10 to 300.22. The method according to p. 21, characterized in that in a complex polyester respect to the number of carboxyl groups to the number of terminal hydroxyl groups is greater than one.23. The method according to p. 13, characterized in that the synthesis of ion-molecular complex conjugate polyester and polypeptide comprises: (a) dissolving a complex of the polyester in tetrahydrofuran, acetone or dimethyl ether of ethylene glycol with p is walsem level polypeptide/complex polyester from 2 to 50 wt%./weight. (polypeptide/complex polyester).24. The method according to p. 23, wherein the polypeptide is a salt of an acid having a pKa greater than or equal to 3.5.25. The method according to p. 23, wherein the polypeptide is from 1 to 50% of the total weight of the ionic conjugate.26. The method according to p. 23, characterized in that more than 85% of the polypeptide present in the composition, ionic difficulties with polyester.27. The method according to p. 23, characterized in that the reaction leads to the formation of ionic bonds between the reactants.28. Method for the synthesis of microparticles, characterized in that it comprises (a) dissolving an ionic conjugate under item 1 in aprotic sadomasichism organic solvent; (b) mixing the organic solvent with water, and (C) extraction of particles from the water.29. The method according to p. 28, wherein the organic solvent is chosen from the group comprising acetone, acetonitrile, tetrahydrofuran, dimethylformamide and dimethoxyacetophenone.
SUBSTANCE: invention proposes agent for suppression of snores noise designated for intramuscular administration. Agent comprises the highly purified clostridium toxin BONT/A or highly purified clostridium toxins BONT/B, BONT/C1, BONT/D, BONT/E, BONT/F and/or BONT/G. Also, agent can be used comprising a fused protein that involves light subunit of clostridium toxins among the group BONT/A, BONT/B, BONT/C1, BONT/D, BONT/E, BONT/F, BONT/G, TeNT, and heavy subunit of another clostridium toxin from the same group, or mixture of fused proteins, or complex comprising clostridium toxin or a fused protein, and one or more therapeutically good tolerable hemagglutinin, and/or one or more pharmaceutically good tolerable nontoxic protein. The proposed therapeutic agent expands assortment of medicinal agent used in the snore therapy. Invention can be used for suppression of snore noise.
EFFECT: enhanced effectiveness of agent.
10 cl, 3 ex