KR100369764B1 - Ionic Molecular Conjugates of Biodegradable Polyesters and Bioactive Polpeptides - Google Patents

Abstract

The present invention relates to a sustained release pharmaceutical composition. The composition of the present invention comprises a polyester containing at least one free COOH group ionically bound to a bioactive polypeptide consisting of at least one effective ionic amine, wherein at least 50% by weight of the polypeptide present in the composition is ionically bound to the polyester.

Description

Ionic Molecular Conjugates of Biodegradable Polyesters and Bioactive Polpeptides

The present invention relates to sustained release of bioactive polypeptides.

Many drug delivery systems have been developed, tested, and used for controlled release of pharmaceutical compositions in vivo. For example, polyesters such as poly (DL-lactic acid), poly (glycolic acid), poly (ε-caprolactone) and various other copolymers have been used to release biologically active molecules such as progesterone; These were in the form of microcapsules, films or rods (Pitt CG, Marks, TA, and Schindler, A. 1980). As the polymer / therapeutic drug composition is implanted, for example subcutaneously or intramuscularly, the therapeutic drug is released over a certain period of time. The biocompatible, biodegradable polymer system is designed such that the captured therapeutic drug is released from the polymer matrix. Once the therapeutic drug is released, the polymer degrades in vivo to prevent surgical removal of the implant. The factors contributing to polymer degradation are not well understood, but the degradation of such polyesters can be controlled by causing ester bonds to degrade with non-enzymatic autocatalytic hydrolysis of the polymer component.

Several EPO publications and US patents describe polymer matrix designs and their role in controlling the rate and extent of release of therapeutic drugs in vivo.

For example, Deluca (EPO Publication No. 0 467 389 A2 / University of Kentucky) described the physical interaction between hydrophobic biodegradable polymers and proteins or polypeptides. The composition formed was a mixture of hydrophobic polymers that were introduced into the therapeutic drug and subject to sustained diffusion release of the drug from the matrix.

Hutchinson (US Pat. No. 4,767,628 / ICI) regulated its release by uniformly dispersing the therapeutic drug in a polymer device. Such formulations firstly exude the drug diffusion-dependently from the surface of the formulation; Second, it has been described that controlled release is provided by superposition of two phases of releasing the drug by an aqueous channel derived by decomposing the polymer.

Schematically, the present invention consists of a polyester containing at least one free COOH group ionically bound to a biologically active polypeptide consisting of at least one effective ionic amine, wherein at least 50% by weight of the polypeptide present in the composition is ion sustainedly bound to the polyester. It is characterized by a pharmaceutical formulation.

In a preferred embodiment, the polyester is modified such that the ratio of carboxyl to hydroxyl end groups rises to at least one infinity so that all hydroxyl groups can be substituted with carboxyl groups. Examples of suitable polyesters are L-lactic acid, D-lactic acid, DL-lactic acid, ε-caprolactone, p-dioxanone, ε-caproic acid, substituted and unsubstituted trimethylene carbonate, 1,5-diox Cepan-2-one, 1,4-dioxepan-2-one, glycolide, glycolic acid, L-lactide, D-lactide, DL-lactide, meso-lactide, alkylene oxalate, cycloalkyl From ethylene oxalate, alkylene succinate, (β-hydroxybutyrate), and any optically active isomers, racemates or copolymers thereof. Other heterochain polymers in connection with typical polyesters may also be used (eg polyorthoesters, polyorthocarbonates and polyacetals).

Preferably, the polyester is reacted with malic or citric acid to make it polycarboxylic.

In a preferred embodiment, the polyester is partially acid-tipped with glutaric anhydride. In another preferred embodiment, the polyester is fully acid-terminated with glutaric anhydride. Preferably, the polyester has an average degree of polymerization of 10 to 300, more preferably 20 to 50.

The ionic molecular conjugates of the present invention are preferably prepared from polycarboxylic acid-terminated polyesters in combination with monovalent bases and polyvalent basic bioactive polypeptides having at least one effective ionic amine group. Alternatively, any polyester can be used to form the ionic molecular conjugates of the present invention under conditions pretreated with a suitable base, such as NaOH. Moreover, any acid-stable peptide such as growth hormone secretion peptide (GHRP), progesterone-secreting hormone (LHRH), somatostatin, bombesin, gastrin isolation peptide (GRP), calcitonin, bradykinin, galanin, melano Site stimulating hormone (MSH), growth hormone secretion factor (GRF), amylin, tachykinin, secretin, parathyroid hormone (PTH), enkephalin, endoserine, calcitonin gene secreting peptide (CGRP), neuromedin (neuromedin), parathyroid gland Hormone-related proteins (PTHrP), glucagon, neurotensin, afferent cortical hormone (ACTH), peptide YY (PYY), glucagon secreting peptide (GLP), angiogenic visceral peptide (VIP), pituitary adenylate cyclase activating peptide ( PACAP), motiline, substrate P, neuropeptide Y (NPY), TSH and its homologues and fragments, and the like. Such ionic molecular conjugates can release their bioactive components in vivo at predetermined rates determined by the chemical structure, molecular weight, and pKa of the two components of the conjugate. The release mechanism of the drug requires that the insoluble binder form is partially transformed into a water soluble component through hydrolysis of the hydrophobic polyester. Thus, the release of bioactive polypeptides, respectively, independently represents (a) a decrease in the pKa difference between the bioactive polypeptide and the polyester, (b) the chemical reactivity of the polyester chain reflected by carbonyl nucleophilicity, and (c) the glass transition temperature. And decrease in polyester density in relation to minimum crystallinity, (d) increase in matrix hydrophilicity, and the like.

In a preferred embodiment, the polypeptide comprises 1-50% by weight of the total weight of the ionic molecular binder and at least 50%, preferably at least 85%, more preferably at least 95%, and even more preferred of the polypeptide present in the composition. Preferably 99% are ionically bonded to the polyester; The polyester component of the ionic molecular conjugate has a viscosity of about 0.05 to about 0.7 dl / gm in chloroform; Polyesters have an average molecular weight of about 1200-40,000.

Polymeric ionic molecular conjugates of the present invention can be readily prepared into injectable microspheres or particulates, and implanted films or rods, without the need for processing techniques requiring multiphase emulsions or non-aqueous dual phase systems. Preferably, the microparticles comprise (a) dissolving the composition in an organic solvent that is aprotic and miscible with water; (b) mixing the organic solvent in water; And (c) isolating the particulates from the water. In a preferred embodiment, the organic solvent is selected from the group of acetone, acetonitrile, tetrahydrofuran, dimethylformamide, and dimethoxy ethylene glycol.

In a preferred embodiment, the polyester / polypeptide ionic molecular conjugate can release a therapeutically effective amount of the bioactive polypeptide in vivo for a period of at least 20 days, more preferably at least 7 days and no more than 95 days. In another preferred embodiment, the release of the therapeutic ionic molecular conjugate is essentially a single step.

The sustained release composition of the present invention preferably comprises (a) providing a bioactive polypeptide having a polyester with a free COOH group and at least one effective ionic amine, and (b) ionically binding the polyester to the polypeptide in the composition. At least 50%, preferably at least 85%, by weight of the polypeptide present is prepared by the step of allowing the polyester to ionically bind. The polyester may have sufficient free COOH groups at the start, or when using such groups in quantities below the desired peptide loading level at the time of initiation, the polyester may be (1) for example an esterification or a functional group Reaction with malic acid or citric acid via exchange, (2) acid-terminated with, for example, glutaric anhydride, or (3) a polyester with a base such as NaOH to expose the acid groups. Can be. Finally, the polyester / polypeptide ionic molecular conjugate can be converted into implantable films or rods or injectable microspheres or particulates capable of releasing the polypeptide in vivo.

Preferably, the polyester condenses directly or catalytically or directly catalytically or autocatalytically one or more hydroxy acids, such as glycolic acid and lactic acid, in the presence of a predetermined concentration of polycarboxylic hydroxy acids such as malic or citric acid. By synthesis. The polyester thus formed preferably has acid-terminated hydroxyl end groups that are partially or fully acid-terminated.

The polyesters can also be catalyzed by ring-opening polymerization of lactones or by cyclic monomers such as ε-caprolactone, p-dioxanone, trimethylene carbonate, 1,5-dioxepan-2-one, or 1 , 4-dioxepan-2-one can be synthesized by polymerizing in the presence of a chain initiator, for example hydroxy polycarboxylic acid.

Another synthetic method is to react the hydroxy acid with a cyclic dimer and to condense the ring-opening system in the presence of a polycarboxylic acid.

Another method of synthesis is to react the organic polycarboxylic acid with the preformed polyester.

In the above-mentioned preferred embodiments, the acid-terminated polyesters have a ratio of carboxyl to hydroxyl end groups approaching at least one infinity (i.e., all hydroxyl groups are lost), with an average degree of polymerization of from 10 to 300, and 20-50 in a particularly preferred embodiment.

On the other hand, polyesters can form ionic molecular conjugates with bioactive polypeptides by treating a base such as NaOH.

Preferably, the polyester / polypeptide ionic molecular binder is synthesized by reacting, for example, the free polyester with the free polypeptide, for example, in a suitable liquid medium. In another preferred embodiment, a suitable solvent for forming the conjugate is a two-phase system of aprotic solvents such as acetone, tetrahydrofuran (THF) or ethylene glycol dimethyl ether and suitable solvents for peptides such as water. Will be a suitable proportion of mixture that can be miscible. Preferably, the polypeptide is a salt of monocarboxylic acid having a pKa of at least 3.5. Preferably, the polypeptide has one or more effective ionic amine groups.

In a preferred embodiment, the polypeptide is 1-50% by weight, preferably 10-20% by weight of the polyester / polypeptide ionic molecular conjugate. In a preferred embodiment, the reactive carboxyl groups of the polyester are partially neutralized with alkaline metal ions or organic bases. In another preferred embodiment, the alkali treatment provides for the chain breakdown of the polyester and the formation of low molecular weight binding sites.

As used herein, the term “peptide” refers to a protein, peptide, oligopeptide or synthetic oligopeptide.

As used herein, the term "polycarboxyl" refers to a compound having one or more carboxyl groups, for example malic acid and citric acid.

The term "average degree of polymerization" as used herein refers to the number of repeats of the monomer sequence.

The term "effective ionic amine" as used herein refers to a polypeptide containing one or more amine groups capable of forming ions under potent conditions.

The term "acid-tipped" as used herein refers to a compound having an acid terminus.

As used herein, the term “partially acid-tipped” refers to compounds wherein 1-99% of hydroxyl end groups are acid-terminated.

As used herein, the term “complete acid-tipped” refers to a compound wherein at least 99.9% of the hydroxyl end groups are acid-terminated.

As used herein, the term "hydroxy acid" refers to any compound containing hydroxyl and carboxyl groups.

The term "monocarboxylic hydroxy acid" as used herein refers to an organic acid having one carboxyl group and one or more hydroxyl groups.

The term "polycarboxylic hydroxy acid" as used herein, addresses a hydroxy acid having at least one carboxyl group.

As used herein, the term “organic azeotrope additive” refers to an organic liquid co-distilled with water.

As used herein, the term "bioactivity" refers to a molecule that causes or affects a biological phenomenon.

As used herein, the term “acyclize” refers to a chemical reaction that opens a ring.

As used herein, the term "polycondensation" refers to a reaction that forms a polyester by condensing two or more molecules.

The present invention provides novel pharmaceutical compositions in which biocompatible, biodegradable polyesters chemically bind polypeptides, peptides and / or proteins as homogeneous ionic groups. By chemically binding polyesters of different molecular weight to the therapeutic drug, the chemical properties of the composition can be precisely adjusted to meet the need for controlled single-step release of biologically active polypeptide molecules in vivo. Moreover, the compositions of the present invention are readily optimized to have organoleptic properties to confer more therapeutically active polypeptides.

Other features and advantages of the invention will be apparent in the following detailed description of preferred embodiments and in the claims.

synthesis

The biodegradable or absorbable polyesters of the present invention provide controlled chain hydrolytic properties by appropriately selecting constituent monomers, comonomers or comers to form chains with a predetermined composition and molecular weight, thereby providing a total positive charge at physiological pH. It is adjusted to have a desirable chemical reactivity that exhibits maximum binding to oligopeptides, polypeptides or proteins that exhibits (see, eg, FIG. 2).

To prepare the compositions of the present invention a three step synthesis scheme is used which is within the ability of one of ordinary skill in the art. This step comprises the steps of: (1) synthesizing the polycarboxylic acid-terminated polyester; (2) synthesizing the polyester / polypeptide ion conjugate by ion bonding the polycarboxylic acid-terminated polyester (or base treated polyester) with the biologically active polypeptide; (3) converting the ionic conjugate into an implant, rod, microsphere or particulate that can release the therapeutic drug in vivo for at least 7 days.

(1) Synthesis of Polycarboxylic Acid-terminated Polyester

The polycarboxylic acid-terminated polyester chains of the present invention may be prepared by direct condensation of 2-hydroxy acids with polycarboxylic organic acids, step-growth polymerization of ring-opened products, ring-opening-polymerization of lactones or lactone mixtures, or polycarboxyls It is synthesize | combined by the method of exchanging a functional group exchange reaction with the high molecular weight polyester which preformed organic acid (refer FIG. 1). Description of the synthesis of the polycarboxylic acid-terminated polyesters by the above-mentioned method is as follows.

Direct condensation of predetermined amounts of polycarboxylic organic acids with, for example, glycolic acid, DL-lactic acid, and DL-malic acid, in the presence or absence of an inorganic or organometallic catalyst, in optically active and / or inert forms Condensation of polycarboxylic acid in a glass reactor equipped with a mass stirring device and a device which provides a continuous flow of dry-nitrogen, generally monocarboxylic hydroxy acid or a mixture of two or more monocarboxylic hydroxy acids By heating in the presence of a fraction (named IA polyester, see Table I). Typically, the polycondensation is carried out at 150-170 ° C. for 4 to 72 hours. Nitrogen gas bubbles can be generated by a magnetic stirrer or through a polyester ingot to provide stirring of the reaction mixture. The polymerization continues until the desired average molecular weight (measured by solution viscosity) and / or acid value (measured by terminal titration) is obtained. Polyester analysis by end group titration is carried out as follows. Polyester samples (300 mg-500 mg) are accurately weighed and dissolved in a minimum amount of acetone (10-30 ml). After dissolution, the solution is diluted up to 100 ml with benzyl alcohol (Mallinckrodt, analytical reagent) and titrated to a pale red color end point (phenolphthalein) using potassium hydroxide in benzyl alcohol solution (standardized to HCI standard solution). The acid value for the polyester is determined by comparing the volume (ΔVs) of the base solution used for the sample with the volume (ΔVo) of the base used for the cosolvent.

At the end of the polymerization, the polyester is isolated and extracted with water or dilute aqueous sodium hydroxide solution to remove the water-soluble or soluble low molecular weight chain from a suitable organic solution.

Polyester analysis by GPC is performed as follows. The average molecular weight (MW) of the polyester is measured by GPC using a Waters Model 6000 solvent transport pump and a Dynamax (Rainin) model UV-D (UV-D) detector. It is operated in tetrahydrofuran (Burdick & Jackson UV grade) using a Jordin Gel DVB 1000 mm3, 50 cm x 10 mm column (Jordi Associates) at a flow rate of 1.2 ml / min at 25 ° C. Peak is detected as 1.0 AUFS at 220 nm. Columns are assayed using narrow band polystyrene standards (Polysciences Inc.) at molecular weights 4000, 9200 and 25000.

The modification of the direct condensation process requires the use of cation exchange resins (named IB polyesters, see Table 1) as organic azeotrope and condensation catalyst. This process requires filtration and distillation to remove catalyst and azeotrope, respectively. Typical examples and suitable analytical data for polyesters produced by the above process are shown in Table I.

Table I Polyesters Prepared by Direct Condensation

The hydroxy acid is reacted with a cyclic dimer, and the resulting open chain system is then reacted in the presence of a predetermined amount of polycarboxylic acid and with a suitable condensation catalyst such as glycolic acid, L-lactide and DL-malic acid. The step-growth polymerization of the ring-opening compound, condensed in the presence or absence, is carried out above except that it uses a mixture of monocarboxylic hydroxy acid, cyclic dimer of a second hydroxy acid, and hydroxy polycarboxylic acid. It is basically the same as the condensation method mentioned. Typical examples of the polyesters produced by the above process and appropriate analytical data are shown in Table II. If the cyclic dimer is pretreated with water, the system is treated with a single step-growth polymerization.

TABLE II Stage-Growth Polymerization of Ring Opening Products

As chain initiator, tin octoate in the presence of a predetermined concentration of hydroxy-polycarboxylic acid and a catalytic amount of an organometallic catalyst, for example a mixture of L-lactide, glycolide and DL-malic acid in the presence of tin octoate The ring-opening polymerization of the lactone or lactone mixture in the presence of uses dry cyclic monomers or mixtures of cyclic monomers, hydroxy-polycarboxylic acids and traces of tin octoate (concentration of 0.33 M in toluene), with dry oxygen In the absence atmosphere, transfer to a glass reactor equipped with a magnetic or mechanical stirrer. The polymerization reaction is continued under a nitrogen atmosphere until the desired molecular weight (measured solution viscosity on a scale) is obtained according to a suitable heating scheme. At the end of the polymerization scheme the temperature is lowered and the unreacted monomer is distilled off under reduced pressure. The polyester mass is cooled and the water soluble low molecular weight fraction is removed by cold extraction from a suitable organic solvent. The solution is then dried and the solvent is removed. Molecular weight is measured by intrinsic viscosity, and acid value is measured by terminal group titration. Table III shows examples and appropriate analytical data for the polyesters produced by the above process.

Table III Polyesters Prepared by Ring Open Polymerization

Functional group exchange, for example tin octo, in the presence of an organometallic catalyst, preferably with a high molecular weight polyester having a preformed COOH / OH ratio of polycarboxylic or hydroxy polyvalent base organic acid of 1 to ultimately 0 Reaction of 85/15 lactide / glycolide copolymers having a molecular weight of 5,000 or more and COOH / OH of 1 or less with DL-malic acid to produce a low molecular weight polyester having COOH / OH of 1 or less in the presence of an It is necessary to heat the high molecular weight polyester in the presence of trace amounts of organometallic catalysts such as tin octoate together with a predetermined amount of polycarboxylic acid or hydroxyl polycarboxylic acid. The reaction is heated at least 150 ° C. under vigorous stirring under a dry nitrogen atmosphere until the functional group exchange is complete (as measured by the depletion of residual unreacted polycarboxylic acid). Completion of the reaction is measured by monitoring the molecular weight of the low molecular weight polyester produced during the reaction (to measure solution viscosity using a capillary viscometer at 28 ° C.) and the presence of unreacted polycarboxylic acid. This is accomplished by aqueous extraction of polyester samples and analysis of the extract using high performance liquid chromatography (HPLC). Residual monomer, dimer and polycarboxylic acid content levels are determined by HPLC using a Waters Model 6000 solvent transport pump and a Dynamax (Rainin) model UV-D (UV-D) detector. 205 nm, 1.0 AUFS). It is operated using 0.025 N Na ₂ PO ₄ buffer with pH = 3.5 (equivalent flow rate = 1.0 ml / min) and using a Nucleosil C18, 5um, 25 cm × 4.6 mm column.

The desired polyester is isolated and purified as in the ring open polymerization. Examples of polyesters prepared by the above processing methods and appropriate analytical data are shown in Table IV.

Table IV: Polyesters Prepared by Functional Group Exchange

Other monomers suitable for the synthesis of polyesters used in the present invention include L-lactic acid, DL-lactic acid, ε-caprolactone, p-dioxanone, ε-caproic acid, trimethylene carbonate, 1,5-dioxepan 2-one, 1,4-dioxepan-2-one, glycolide, meso-lactide, and the like. Examples of useful polycarboxylic acid chain initiators and / or chain modifiers include malic acid, and citric acid.

(2) Synthesis of polyester / polypeptide ion conjugate by ionic reaction of polycarboxylic acid-terminated polyester and biologically active polypeptide

The above-mentioned polycarboxylic acid-terminated biodegradable polyesters are used to prepare ionic molecular conjugates in combination with mono- or polycarboxylic acid oligopeptides, polypeptides or proteins with effective ionic amine groups (see also FIG. 2). ). Moreover, certain polyesters tend to form ionic molecular conjugates with polypeptides when treated with a base such as 0.1 N NaOH or the like. This treatment exposes the acid groups of the polyester for multiple site ionic reactions with the cationic polypeptide.

Thus, the formation of the conjugate is achieved by direct intermolecular interactions of the components with or without inorganic base pretreated with polyester to maximize the binding rate performance for the basic drug in a suitable solvent. As noted above, the ionic interactions of their ionic binder components increase by the difference in their pKa levels.

The polyester is dissolved in a suitable aprotic solvent within a concentration range of 2% to 20% (w / v). Such solvents must dissolve the polyester and also be partially miscible with water. Suitable solvents used for this purpose include tetrahydrofuran, acetone, and ethylene glycol dimethylether. Bases such as aqueous solutions such as sodium hydroxide or sodium carbonate, potassium or ammonium are added to the solution to maximize the binding performance of the polyester. In general, the amount of base added corresponds to the amount of acid represented by the level of the counter-anion of the basic peptide used.

The polyester-base blend is mixed quickly, then an aqueous solution of the peptide or peptide salt is added to a peptide / polyester loading level of 2% to 50% (w / w) (peptide / polyester). The mixture is stirred for a period of time (up to 3 hours), then the solvent is removed and the product is dried in vacuo. The resulting material can be further processed into formulations. The resulting pharmaceutical composition is envisioned to be a chemically uniform composition made entirely of ionic molecular conjugates, basically lacking a fine or large dispersed region of the active drug in the biodegradable matrix. Examples of prepared ionic molecular conjugates and appropriate analytical data are shown in Table V.

TABLE V Ionic Molecular Binders-Peptide Binding ¹

(3) Conversion of ionic conjugates into implants, rods, microspheres, and microparticles capable of releasing therapeutic drugs for at least 20 days in a single step profile in vivo

The ionic conjugates of the present invention are (A) sterile microspheres for injection containing 1 to 50% by weight of polypeptides that can be released according to a single step profile basically to maintain pharmacological activity over 1 to 12 weeks (processing aids) (With or without 0.1 to 10% of a solid polyhydric alcohol): (B) prepared by casting, pressing or extruding in the presence or absence of a pharmaceutical inert processing aid, to provide a release profile similar to that in (A) Implantable Sterile Films: (C) Can be converted into a sterile rod for implantation, which is prepared by extrusion or compression and can provide a release profile similar to that in (A).

In vitro release analysis

Samples of dried and ground ionic binder material weighed 50 mg each were placed in a 25 mm diameter scintillation vial and modified PBS buffer (PBS buffer: 2.87 mg Na ₂ HPO ₄ , 0.654 mg NaH ₂ PO ₄ , 5.9 mg NaCl). 5 ml is added to each vial, and the vial is placed in a Lab-Line orbit Environ shaker and shaken at 37 ° C. at 120 RPM. Remove the vial periodically to pour out the supernatant and replenish with fresh PBS solution. The followed PBS solution is analyzed by HPLC to determine the amount of peptide released.

Peptide Extraction from Ionic Bonds

50 mg of ionic molecular binder samples are mixed in 20 ml of methylene chloride. The mixture is then extracted continuously in 50 mL, 20 mL and 20 mL portions of 2N acetic acid. Acetic acid extracts are collected and analyzed for peptide content by high performance liquid chromatography (HPLC). Peptide analysis by HPLC is as follows. HPLC analysis is performed using a Waters Model M-45 solvent transport pump and EM Science MACS 700 detector with wavelengths of 220 nm and 1.0 AUFS. Lichrospher (EM Separation) c18, 100 mm 3, 5 μm, 25 cm × 4.6 mm columns and peptides are analyzed using 30% acetonitrile / 0.1% TFA as equivalent elution buffer.

49: 49: 2 L-Lact / Glycol / Malic Acid / D-Trp ⁶ [LHRH] (Example # 8), 49: 49: 2 L-Lact / Glycol / Malic Acid / Somatostatin-Tumor Inhibiting Homolog (Example # 9 And 73.5: 24.5: 2 poly-L-lactide / glycol / malic acid: D-Trp ⁶ [LHRH] (Example # 10) in vitro showing the amount of peptide released over 28 days for the ionic molecular conjugate Details of the analysis (Table VI) are as follows. FIG. 3 is a graphical representation of the data.

Table VI In Vitro Analytical Data

Quantification of Peptides in Ionic Bonds

Ion binding peptides in the binder product are measured by dissolving 10 mg of sample in 5.7 mL of a 9: 1 mixture of acetone and 0.1 M aqueous trifluoroacetic acid. The solution is shaken at 25 ° C. for 15-24 hours and filtered through a 0.5 μm Teflon filter cartridge. The filtrate is then analyzed for peptide content by high performance liquid chromatography (HPLC). Peptide analysis by HPLC is performed at 220 nm using a Millipore model 717 Wisp Autosampler, a Model 510 pump and a Model 486 UV detector. Peptides were analyzed at a flow rate of 1.0 ml per minute using 35% acetonitrile in 0.14% sodium perchlorate buffer as an equal elution system on a Licrospher (EM isolated) c18, 100 mm 3, 5 μm, 25 cm × 4.6 mm column. do. The amount of peptide is quantified by comparing the area of the calibration peak of the working sample with that of the reference peptide injected.

Usage

Any acid-containing polyester / polypeptide ion conjugate described herein may be administered to the recipient alone or in combination with a pharmaceutically acceptable medium. Subcutaneous, intramuscular, parenteral, suppository or nasal administration may be easy, but therapeutic agents are administered depending on the condition to be treated. The composition concentration in the formulations of the present invention will vary depending on several factors, including the dosage and route of administration.

It is believed that one skilled in the art can readily utilize the present invention to the maximum extent possible using the methods described above. Accordingly, the following embodiments are merely exemplary and do not limit the remainder of the specification in any way.

Example 1 Direct Condensation Method

Synthesis of 50/50 Poly (D, L-Lactic Acid-Co-Glycolic Acid) Using Amerlyst 15 as Catalyst

In a round bottom flask equipped with a magnetic stirrer, Dean-Stark trap and water cooled concentrator, D, L-lactic acid (85% aqueous compound; 13.7 mg, 0.13 mole) was mixed with glycolic acid (10 mg, 0.13 mole) and glycol Mixed with acid (10 mg, 0.13 mole). Toluene (100 mL) and Amerist 15 beads (100 mg) were added and the mixture was refluxed under a nitrogen atmosphere for 72 hours to remove water from the mixture. The mixture was cooled, drained toluene from the solidified mass and the product was dissolved in methylene chloride (250 mL). Methylene chloride was treated with activated carbon (Darco, 500 mg), filtered and vacuum dried in a rotary evaporator. The polyester was further dried at 40 ° C. under high vacuum (1 mmHg) to give a white powder.

Eta _inh in CHCl ₃ = 0.3, acid value = 2499, Tg = 12 ° C

Example 2 Direct Condensation Method

Synthesis of 49/49 poly (L-lactic acid-co-glycolic acid / citric acid) using Amerlyst 15 as catalyst

Using a system similar to the above, L-lactic acid (88% aqueous mixture; 25.6 mg, 0.25 mole) was added to glycolic acid (19.2 mg, 0.25 mole), citric acid monohydrate (2.33 mg, 0.011 mole), toluene ( 150 mL) and Amerist 15 beads (500 mg). The mixture was heated to reflux for 51 hours and the water was removed by a Dean-Stark trap. Toluene was poured from the semi-solidified mass. The polyester was dissolved in acetone (300 mL) and dried on a rotary evaporator. The solid polyester was then dissolved in methylene chloride and washed twice with water (2 × 150 mL) to remove soluble oligomers. The organic solution was concentrated on a rotary evaporator and the product dried completely under vacuum to give a white solid (Table I, Polyester IB, Polymer # 4).

Eta _inh in CHCl ₃ = 0.11, acid value = 842, Tg = 15 ° C

Example 3 Step Growth Polymerization Method

Synthesis of 73.5 / 24.5 / 2 poly (L-lactide-co-glycolic acid / malic acid) using malic acid as catalyst

L-lactide (20 mg, 0.139 mole) was converted to glycolic acid (7.1 mg, 0.093 mole) and (d, l) -malic acid (1.0 mg, 0.0075 mole) using a 150 ml volumetric ampoule with air collection junction. Was taken. The mixture was stirred while generating nitrogen bubbles through a dust collector inlet (100 mL / min) and heated from 25 ° C. to 155 ° C. for 100 minutes. The reaction temperature was maintained at 155 ° C. for 70 hours and water was removed from the polymerization reaction in a cold trap on the reactor input line. After 70 hours the reaction was cooled to 100 ° C. and poured into a cold stainless steel receiver for curing. The solid polyester was then dissolved in methylene chloride and washed twice with water (2 × 150 mL) to remove soluble oligomers. The organic solution was concentrated on a rotary evaporator and the product dried completely in vacuo to give a white solid (Table II, Polyester Type II, Polymer # 2).

Eta _inh in CHCl ₃ = 0.13, acid value = 1800, Tg = 27 ° C

Example 4 Ring Open Polymerization Process

Synthesis of 75/25 Poly (L-Lactide-Co-Glycolide) Initiated by Malic Acid

L-lactide (12.0 mg, 0.0833 mole), glycolide (3.21 mg, 0.0277 mole), malic acid (0.3042 mg, 0.00227 mole) and tin octoate catalyst (0.33 M in toluene, 67 μl, 0.022 mmole) were dried It injected into the glass ampoule equipped with the magnetic kyo bar under nitrogen atmosphere. N ₂ was injected into the system and evacuated several times before the ampoule was sealed. The reaction was then melted at 140 ° C. and heated at 180 ° C., 190 ° C., 180 ° C. and 150 ° C. for 1, 4.5, 12 and 2 hours, respectively. After cooling to room temperature, the polyester was reheated to 110 ° C. for about 1 hour under reduced pressure of less than 1 mmHg, cooled to room temperature, cooled with liquid nitrogen, isolated and dried under reduced pressure.

Eta _inh in CHCl ₃ = 0.20, acid value = 2560, Tg = 39 ° C

Example 5 Ring Open Polymerization Process

Synthesis of 50/50 Poly (D, L-Lactide-Co-Glycolide) Initiated by Citric Acid

D, L-lactide (10.0 mg, 0.0694 mole), glycolide (8.06 mg, 0.0694 mole), citric acid (1.07 mg, 0.00555 mole) and tin octoate catalyst (0.33 M in toluene, 84 μl, 0.0278 mmole) Was put into a glass ampoule equipped with a magnetic stir bar under a dry nitrogen atmosphere and sealed. The reaction was melted and heated at 180 ° C., 185 ° C., 195 ° C. and 120 ° C. for 1, 2, 7 and 9 hours, respectively. After cooling to room temperature, the polyester was cooled down with liquid nitrogen, isolated and dried under reduced pressure.

Eta _inh in CHCl ₃ = 0.26, acid value = 970, Tg = 23 ° C

Example 6 Ring Open Polymerization Process

Synthesis of 50/50 Poly (D, L-Lactide-Co-Glycolide) Initiated by 1,6-hexanediol

D, L-lactide (10.0 mg, 0.0694 mole), glycolide (8.06 mg, 0.0694 mole), 1,6-hexanediol (0.656 mg, 0.00555 mole) and tin octoate catalyst (0.33 M in toluene, 84 [Mu] l, 0.0278 mmole) was put in a glass ampoule equipped with a magnetic stir bar under a dry nitrogen atmosphere and sealed under vacuum. The reaction was heated at 150 ° C., 185 ° C., 150 ° C. and 120 ° C. for 0.5, 4, 1, 5 and 3 hours, respectively. The resulting polyester was recovered and dried (Table III, Type III Polyester, Polymer # 5).

Eta _inh in CHCl ₃ = 0.39, acid value = 10.138, Tg = 30 ° C

Example 7 Functional Group Exchange Method

Synthesis of 50/50 poly (D, L-lactide-co-glycolide) containing carboxyl groups

50/50 poly (D, L-lactide-co-glycolide) (Borhringer A001, 8 g), citric acid (0.8 mg, 4.16 mole) and tin octoate (2 drops) were added to the glass ampoule under a dry nitrogen atmosphere. And sealed. The mixture was heated at 150 ° C. for 4 hours, cooled to room temperature, cooled with liquid nitrogen, isolated and dried under reduced pressure (Table IV, IV Polyester, Polymer # 1).

Ninh in CHCl ₃ = 0.26, acid value = 670, Tg = 23 ° C

Example 8

49: 49: 2 Synthesis of L-Lactic Acid / Glycolic Acid / Malic Acid (See Table I, Polymer # 4) and D-Trp ⁶ [LHRH] Ionic Molecular Bonds

49: 49: 2 500 mg of L-lactic acid / glycolic acid / malic acid (synthesized by direct condensation; Mw = 9,500; acid value = 1420) were dissolved in 10 ml of acetone (Mallinkrodt assay reagent). An aliquot of 0.1 N sodium hydroxide solution (1.14 ml) was added and the mixture was stirred at room temperature for 15 minutes. A 100 mg solution of D-Trp ⁶ [LHRH] (BIM-21003 peptide I: base content 87%, acetate content 7%) in 1.0 ml of water was added and the mixture was stirred at room temperature for 1 hour. Below 40 ° C. the solvent was first removed by Rotovap and then placed in a desiccator for 1 hour at room temperature under 1 mmHg vacuum. The dry solid was triturated and isolated in 100 ml of deionized water and filtered. The aqueous filtrate was tested by HPLC and found to contain less than 1 mg of soluble peptide. The solid material was dried in vacuo for several days to give 540 mg of white powder. This powder was used for in vitro testing (see Table VI, Example # 8).

Example 9

49: 49: 2 Synthesis of L-Lactic Acid / Glycolic Acid / Malic Acid (see Table I, Polymer # 4) and Somatostatin / Tumor Inhibitory Homolog Ionic Molecular Bonds

100 mg of 49: 49: 2 L-lactic acid / glycolic acid / malic acid (synthesized by direct condensation; Mw = 9,500; acid value = 1420) were dissolved in 2 ml of acetone (Mallinckrodt assay reagent). An aliquot of 0.1 N sodium hydroxide solution (0.32 ml) was added and the mixture was stirred at room temperature for 15 minutes. A 20 mg solution of somatostatin / tumor inhibiting homologue (BIM-23014 peptide II: 83% base, 9.8% acetate) in 1.2 ml of water was added and the mixture was stirred at room temperature for 1 hour. Below 40 ° C., the solvent was first removed by Rotovap and then placed in a desiccator for 1 hour at room temperature under 1 mmHg vacuum. The dry solid was triturated, isolated in 20 ml of deionized water and filtered. The aqueous filtrate was tested by HPLC and found to contain less than 0.05 mg of soluble peptide. The solid material was vacuum dried for several days to yield 106 mg of white powder. This powder was ground and used for in vitro testing (see Table VI, Example # 9).

Example 10

73.5: 24.5: 2 Synthesis of Poly L-Lactide / Glycolic Acid / Malic Acid (see Table II, Polymer # 2) and D-Trp ⁶ [LHRH] Ionic Molecular Bonds

73.5: 24.5: 2 800 mg of poly L-lactide / glycolic acid / malic acid (synthesis by step growth of ring-opening product: acid value = 1800) were dissolved in 16 ml of acetone. An aliquot of 0.1 N sodium hydroxide solution (2.8 ml) was added and the mixture was stirred at room temperature for 20 minutes. A 200 mg solution of D-Trp ⁶ [LHRH] (BIM-21003; 87% base, 7% acetate) in 2 ml of water was added and the mixture was stirred for 90 minutes. The solvent was removed and the resulting solid was triturated in deionized water as in Example 8 and found to contain less than 1% of soluble peptide. The isolated solid was vacuum dried for 4 days to give 839 mg of white powder. This powder was ground and used for in vitro release analysis (see Table VI, Example # 10).

Example 11

Formation of Peptide-Polymer Ion Binder Fine Particles 1.50 of L-Lactide / Glycolide / d, lmalic Acid Polyester (65; 33; 2)

The conjugate was synthesized by ring open polymerization as in Example 4 (molecular weight = 4700, bidispersity = 1.3, measured by GPC on a Jordi Gel gel 50 × 1 cm mixed linear column, THF eluate, Wyatt Mini Dawm light scatter detector dn / dc = 0.05, acid value 1475 by titration, Tg = 42 degreeC), and it dissolved in 40 ml of acetone. The acid groups were neutralized with 2.0 ml of a 0.5 g solution of BIM-23014 (peptide content 83.7%, acetate content 11.5) in 20 ml Milli-Q and slowly added while mixing into the polymer solution. An additional 40 ml of acetone was added in small portions while the peptide was added to prevent precipitation. The clear colorless solution was stirred for 1 hour and evaporated to dryness under reduced pressure. The resulting white solid was dissolved in a mixture of 20 ml of acetone and 2 ml of Milli-Q water to form a clear solution. The solution was injected into a 500 ml milli-Q water reservoir which was stirred at 4 ° C. through a 0.2 μ Teflon filter. The polymer / peptide binder phase separated into small particles upon contact with water. The slurry was mixed at 4 ° C. for 30 minutes, then residual acetone was removed under reduced pressure, the solid was isolated by centrifugation, resuspended in milli-Q water and centrifuged again. The isolated solid was lyophilized to give 1530 mg of white glass flow powder. The particle size was 2-100 μm. Ionic conjugates showed that the Tg occurred at 53 ° C. The total residual (unbound) peptide in all aqueous supernatants was 63 mg by HPLC analysis. The total initial peptide content was found to be 19.9% by weight of elemental nitrogen analysis. The percentage of peptide extractable from the conjugate was determined to be 16.9 wt% using acetone / 0.1 M TFA extraction technique. The resulting binder thus has an ionic (extractable) property of 84.8%.

As mentioned above, those skilled in the art can easily grasp the essential features of the present invention without departing from the spirit and scope of the present invention, and various changes and modifications of the present invention to apply to various uses and conditions. You can. Accordingly, other embodiments also belong to the present invention.

1 shows isomers of polycarboxylic acid-terminated lactide / glycolide (malic acid type) copolymers.

2 is a diagram of an ionic molecular conjugate showing the chemical interaction between lactide / glycolide (malic acid type) copolymer and somatoline (BIM-23014).

FIG. 3 is a graph depicting the percentage of peptide released from ionic molecular conjugates into PBS buffer over 28 days at 37 ° C. FIG.

Claims (39)

50 to 100 weights of the polypeptide present in the composition, comprising a polyester containing one or more free COOH groups and ionic bond with a bioactive polypeptide comprising one or more effective ionic amines, and containing malic acid, citric acid or a combination thereof % Is ion bonded to the polyester. The composition of claim 1 wherein the ratio of carboxyl to hydroxyl of the polyester is at least one. The method of claim 1, wherein the polyester is L-lactic acid; D-lactic acid; DL-lactic acid; ε-caprolactone; p-dioxanone; ε-caproic acid; Alkylene oxalate; Cycloalkylene oxalate; Alkylene succinates; β-hydroxybutyrate; Substituted or unsubstituted trimethylene carbonate; 1,5-dioxepan-2-one; 1,4-dioxepan-2-one; Glycolide; Glycolic acid; L-lactide; D-lactide; DL-lactide; Meso-lactide; And their optically active isomers; Composition consisting of members selected from the group consisting of racemates or copolymers. The composition of claim 1, wherein 1 to 99% of the hydroxyl end groups of the polyester are acid-terminated with glutaric anhydride. The composition of claim 1, wherein at least 99.9% of the hydroxyl end groups of the polyester are acid-terminated with glutaric anhydride. The composition of claim 1, wherein the polyester has an average degree of polymerization of 10 to 300. The composition of claim 1 wherein the polyester has a viscosity of 0.05 to 0.7 dl / g and an average molecular weight of 1,200-40,000 in chloroform. The composition of claim 1, wherein said bioactive polypeptide comprises 1 to 50% by weight of the total weight of said ionic molecular conjugate. The composition of claim 1, wherein 85% to 100% of the polypeptide present in the composition is ionically bonded to the polyester. The method of claim 1, wherein the polypeptide is LHRH, somatostatin, bombesin / GRP, calcitonin, bradykinin, galanin, MSH, GRF, amylin, tachykinin, secretin, PTH, CGRP, neuromedin, PTHrP, glucagon , Neurotensin, ACTH, GHRP, GLP, VIP, PACAP, enkephelin, PYY, motiline, substrate P, NPY, TSH, and homologues or fragments thereof. The composition of claim 1, wherein the ionic conjugate is capable of releasing a therapeutically effective amount of the polypeptide in vivo over seven days. (a) providing a bioactive polypeptide containing at least one free COOH and having a polyester containing malic acid, citric acid or a combination thereof and at least one effective ionic amine, and (b) bringing the polyester to the polypeptide Ionic bonding to form an ionic molecular conjugate in which 50-100% by weight of the polypeptide present in the composition is ionically bonded to the polyester, wherein step (b) of synthesizing the polyester / polypeptide ionic molecular conjugate (b1) dissolving the polyester in tetrahydrofuran, acetone, or ethylene glycol dimethyl ether, and then adding a base, and (b2) the load of the polypeptide is 2% to 50% (w / w) relative to the weight of the polyester. Preferably adding an aqueous solution containing the polypeptide or salt of the polypeptide. St. ways. 13. The method of claim 12, wherein the polyester contains acid-terminated hydroxyl end groups. The method of claim 13, wherein 1 to 99% of the hydroxyl end groups are acid-terminated with glutaric anhydride. The method of claim 13, wherein at least 99.9% of the hydroxyl end groups are acid-terminated with glutaric anhydride. The method of claim 12, wherein said polyester is synthesized using a hydroxy polycarboxylic acid chain initiator. 13. The method of claim 12, wherein the hydroxyl end groups of the polyester are acid-terminated. 18. The method of claim 17, wherein 1 to 99% of the hydroxyl end groups are acid-terminated with glutaric anhydride. 18. The method of claim 17, wherein at least 99.9% of the hydroxyl end groups are acid-terminated with glutaric anhydride. The method according to claim 12, wherein an average degree of polymerization of 10 to 300 is obtained as a result of the synthesis of the polyester. The method of claim 20 wherein the ratio of carboxyl to hydroxyl end groups of the polyester is at least one. The method of claim 12, wherein the polypeptide is a salt of an acid having a pKa of at least 3.5. The method of claim 12, wherein the polypeptide comprises 1 to 50% of the total weight of the ion binder. The method of claim 12, wherein 85% to 100% of the polypeptide present in the composition is ionically bound to the polyester. The method of claim 12, wherein as a result of the reaction, ionic bonds are formed between the reactants. A composition prepared by the method of claim 12. (a) dissolving the composition of claim 1 in an organic solvent which is aprotic and miscible with water; (b) mixing the organic solvent with water; And (c) isolating the microparticles from the water. The method of claim 27, wherein the organic solvent is selected from the group consisting of acetone, acetonitrile, tetrahydrofuran, dimethylformamide, and dimethoxy ethylene glycol. Containing at least one free COOH group, containing malic acid, citric acid or a combination thereof, having a ratio of carboxyl to hydroxyl groups of at least 1, L-lactic acid; D-lactic acid; DL-lactic acid; ε-caprolactone; p-dioxanone; ε-caproic acid; Alkylene oxalate; Cycloalkylene oxalate; Alkylene succinates; β-hydroxybutyrate; Substituted or unsubstituted trimethylene carbonate; 1,5-dioxepan-2-one; Glycolide; Glycolic acid; L-lactide; D-lactide; DL-lactide; Meso-lactide; And their optically active isomers; A polyester containing a member selected from the group consisting of racemates or copolymers. 30. The polyester of claim 29 having internal carboxyl groups in the chain of polyester. 30. The polyester of claim 29 wherein said malic acid or citric acid is inside of a polyester chain. 32. The polyester of claim 31 comprising L-lactic acid, D-lactic acid, or glycolic acid. 33. The polyester of claim 32 wherein the average molecular weight is between 1,200 and 40,000. 30. A composition comprising the polyester of claim 29 in ionized association with a bioactive polypeptide comprising at least one effective ionic amine, wherein 50 to 100% by weight of the polypeptide present in the composition is ionically bonded to the polyester. 35. The composition of claim 34, wherein the polyester has internal carboxyl groups in its chain. 35. The composition of claim 34, wherein said malic acid or citric acid is inside of a polyester chain. 37. The composition of claim 36, wherein the polyester contains L-lactic acid, D-lactic acid, or glycolic acid. 38. The composition of claim 37, wherein the polyester has an average molecular weight of 1,200-40,000. The composition of claim 38, wherein said polypeptide is somatostatin, LHRH or a homologue or fragment thereof.