KR100832552B1 - Injectable drug carrier using thermosensitive and biocompatable polyethyleneglycol/polyesters block copolymers - Google Patents

Abstract

An injection formulation of a drug is provided to release a desired amount of the drug gradually for a desired time to a portion desired by a drug person administered to the drug by using a bio-compatible and thermosensitive polyethyleneglycol/polyester block copolymer to make the drug semi-solid or gel. An injection formulation comprises a hydrophilic portion consisting of polyethyleneglycol having a molecular weight of 350-2,000g/mole and a biodegradable polyester hydrophobic portion containing a caprolactone(CL) segment as an essential ingredient and at least one of a paradioxanone(PDO) segment and a trimethylene carbonate(TMC) segment and is characterized in that it is prepared by mixing a protein or peptide-based drug with a bio-compatible and thermosensitive polyethyleneglycol/biodegradable polyester block copolymer having a molecular weight of 2,000-7,000g/mole, wherein the protein or peptide-based drug is albumin, calcitonin, octreotide, leuprolide, adenosine deaminase, L-asparaginase, urokinase, interferon alpha-2b, interferon beta-1b, human insulin, insulin-like growth factor-1(IGF-1), erythropoietin(EPO), granulocyte colony stimulating factor(G-CSF), human growth hormone(hGH), nerve growth factor(NGF), brain-derived neurotrophic factor(BDNF), neurotrophin-3(NT-3), fibroblast growth factor(FGF), epidermal growth factor(EGF), endothelial growth factor(VEGF), transforming growth factor-beta(TGF-beta) or platelet-derived growth factor(PDGF).

Description

Injectable drug carrier using thermosensitive and biocompatable polyethyleneglycol / polyesters block copolymers

Figure 1 shows the ¹ H-NMR of the methoxy polyethylene glycol- (polycaprolactone-co-polytrimethylene carbonate) block copolymer prepared in Example 1 according to the present invention.

Figure 2 shows the ¹ H-NMR of the methoxy polyethylene glycol- (polycaprolactone-co-polyparadioxanone) block copolymer prepared in Example 2 according to the present invention.

3 is a sol-gel phase transition behavior shown in the aqueous solution of the block copolymer prepared according to the present invention, (A) is a photo of the sol state taken at room temperature 25 ℃, (B) is a photo of the phase transition to gel at 37 ℃ (C) is a photograph showing the state that the aqueous solution of the copolymer in the sol state is ejected through the syringe.

4 is a sol-gel phase transition was measured by the change in viscosity according to the temperature change of the block copolymer carried out in Experimental Example 1 according to the present invention, (A) is the concentration of the MPEG-PCL / PTMC block copolymer Shows the size of the viscosity measured in the temperature range to be gelated, (B) is the size of the viscosity measured in the temperature range to gelate for each concentration of the MPEG-PCL / PPDO block copolymer, (C) at the same concentration Pluronic (F-127) and the present invention, the MPEG-PCL / PTMC, MPEG-PPDO block copolymer aqueous solution is measured for about 40 hours at a constant temperature of 37 ℃ for about 40 hours shows the change in the intensity of the viscosity.

5 is an animal experiment performed in Example 3 according to the present invention after mixing the bovine serum albumin to the aqueous solution of the block copolymer prepared in Example 1 of the sol state, it is injected into the subcutaneous of the mouse gelation in vivo As a confirmed photograph, (A) is a photograph of injection of a sol state polymer solution into the subcutaneous of the rat, (B) is a photograph of a gel formed from the injection of the rat subcutaneous and injected after 24 hours, (C ) Shows a picture of the gel formed in vivo from the subcutaneous tissue of the rat.

Figure 6 shows the amount of bovine serum albumin released in vitro performed in Example 4 according to the present invention, the same concentration of Pluronic (F-127), MPEG-PCL / PTMC, MPEG-PCL / It shows the tendency of release of bovine serum albumin in PPDO block copolymer hydrogel.

Figure 7 shows the release amount and concentration of bovine insulin in rats over time in vivo performed in Example 5 according to the present invention.

The present invention relates to the injection of a drug using a biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer, and more particularly, to preparing a biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer By using an intelligent drug delivery system, that is, a protein or peptide drug developed as an injectable hydrogel that can slowly release the drug to a desired area, to a desired time, and to a desired area by allowing the drug recipient to become semi-solid or gel on the spot during injection. It relates to the injection formulation of.

Since the 1980s, with the development of the biotechnology industry, a large number of peptides and protein drugs have been approved by the US Food and Drug Administration in large quantities and applied to clinical applications. Drugs are under development. Therefore, commercialization of protein and peptide drugs is urgently needed to develop a drug delivery system with stability and high efficiency. In particular, protein drugs cannot be absorbed by the body during oral administration, and their frequent injections cannot be avoided due to their ability to be easily lost by enzymes or acids. Therefore, it is important to study sustained-release injections using biodegradable polymers. It is considered to be. In recent years, the general drug delivery system has been attempted to maintain the minimum effective concentration of the drug in the blood and to minimize the side effects due to toxicity, but in most cases, constant blood concentration induces tolerance in pharmacological action, so that the specific receiving site There was a continuing need for high concentrations of drugs that would have better pharmacological effects than before. From this point of view, constant drug release, such as zero-order release, is not always efficient in all cases, and new drug delivery methods such as external control, self-controlled drug delivery systems, or intelligent drug delivery systems are needed. Injectable drug delivery system, which is a protein drug delivery method, is a phase-transfer polymer that can release a drug slowly as it is semi-solid and gel in place when injected, that is, hydrogel is sensitive to external stimuli such as temperature, pH, electric field and light. Refers to a polymer whose physical properties change.

Temperature-sensitive hydrogels are the most widely studied in cell transporters and drug delivery systems for tissue regeneration. This is because many polymers exhibit temperature transfer characteristics. When a hydrophilic group capable of dissolving or swelling in water is introduced into a general polymer, the polymer is dissolved or swelled by water. In general, polymers prepared in this way have low solubility in water as the temperature increases, but polymers composed of hydrophilicity and hydrophobic moieties such as methyl, ethyl, and propyl decrease in water solubility with increasing temperature. Will have When the polymer solution flows with the fluid at a normal temperature, the entrapment of the drug is possible by simple mixing. When heat is applied above the human body temperature, the polymer hydrogel becomes a gel, and in this case, the drug exhibits a sustained release behavior from the gel. When the polymer is crosslinked, the swelling and shrinkage behavior is shown, but when the polymer is not crosslinked, the phase transition behavior of the sol-gel is shown. Temperature sensitive polymers are represented by polymers with ether groups, alcohols with polymers and polymers with amide groups substituted with nitrogen.

Since the introduction of hydrogels using PHEMA (Polyhydroxyethyl Methacrylate) in 1960, various studies using hydrogels have been conducted continuously, and by 1980, the manufacture of hydrogels using calcium alginate became a big electricity in the field of biomaterials. Since then, the synthesis of hydrogels for biomaterials using natural or synthetic polymers has made great strides. A typical property of hydrogels is that they can contain large amounts of water as a network of hydrophilic polymers that can swell in water. The three-dimensional network structure of the hydrogel may be formed by various factors such as covalent bonds, hydrogen bonds, van der Waals bonds, or physical aggregation. Hydrogels can prevent degeneration of drugs from enzymes or pH in the intestine, and can also impart the characteristic properties of releasing drugs by stimulation in various human bodies. Hydrogels that contain sensory properties capable of irritating response can produce reversible volume changes or sol-gel changes within minutes of stimulation in the human body.

External stimuli can be classified into physical stimuli such as temperature, electricity, solvent change, light, pressure, sound, and magnetic force, and chemical stimuli such as ions and specific molecular recognition. The development is expected to be applicable to high-efficiency and sustained-release drug delivery systems that can minimize side effects of drugs.

Hydrogels made of polymers with LCST, such as the polyethyleneglycol / biodegradable polyester copolymers according to the invention, are elevated temperature hydrogels which condense and become gels when the temperature increases above LCST. The polymer composed of hydrophilic and hydrophobic moieties has a high hydrogen bonding force between the hydrophilic group and water molecules of the polymer at low temperature, so that it dissolves in water and becomes a sol state.However, if the temperature is increased, the hydrophobic portion of the polymer has a higher bond strength than the hydrogen bonding force. Therefore, the hydrophobic part of the polymer aggregates to cause phase transition to a gel state. Therefore, the increase of the hydrophobic moiety in the polymer lowers the LCST, and the LCST is changed by controlling the molecular chain of the hydrophilic and hydrophobic groups. When the polymer solution flows with the fluid in a sol state at a normal temperature, the entrapment of the drug is possible by simple mixing, and when the heat is applied above the human body temperature, the polymer hydrogel becomes a gel, in which case the drug exhibits a sustained release behavior from the gel. do. When the polymer is crosslinked, the swelling and shrinkage behavior is shown, but when the polymer is not crosslinked, the sol-gel phase transition behavior is shown. Poly (N-isopropylacrylamide) is most widely used because it has LCST near body temperature. Copolymers such as butyl methacrylate, polyethylene glycol, polypropylene glycol, and the like are changed in the human body through sol-gel changes at various temperature ranges. Is being used by. Copolymer of polyethylene oxide-polypropylene oxide (PEO-PPO) is also a polymer exhibiting a sol-gel change, and many copolymers are trade names such as Pluronic, Poloxamers, and Tetronic. US Patent No. 4188373.

On the other hand, the sol-gel polymer has a disadvantage that it must be released to the body by metabolism of the human body after being used in the human body, so that a polylactide-polyglycolide copolymer (PLGA) is introduced as a biodegradable polymer chain in the hydrophobic group. Many sol-gel polymers have also been reported (US Pat. Nos. 4,882,168, 4,716,203, 4,942,035, 5,476,909, 5,548,035).

Currently, many studies on intelligent hydrogels for application to drug delivery systems using physicochemical properties of polymers in response to external stimuli have been conducted. The application of such intelligent hydrogels for drug delivery in injection formulations requires low viscosity, fast gel formation and biodegradability, and low molecular weight for easy release to the body. In addition, in order to be used in biomaterials, it must have biocompatibility and there should be no damage to cells or metabolic organs during degradation or release.

Therefore, the present inventors have applied a thermosensitive hydrogel as a biomaterial as a drug carrier in an injectable form. As a hydrophilic polymer, the present inventors have high solubility in water and organic solvents, and are nontoxic and non-responsive to immune action. When applied to a living body by chemical bonding with ester-based polymers, polyethylene glycol is used to control the decomposition period by increasing the inflow of water into the copolymer, and also dissolves, chemical hydrolysis, and decomposition by enzymes in the human body. It is biocompatible because it is decomposed into biological metabolites through the back and is discharged to the outside of the body. It is an essential component of caprolactone (CL), a hydrophobic biodegradable ester family that can control the decomposition period by controlling molecular weight and chemical components. Paradioxanone (PDO) and trimethylene carbonate (TMC) Or they were simultaneously polymerized at a specific content ratio to study the sol-gel phase transition behavior of the aqueous solution in accordance with the temperature and concentration through the change of the type and chemical structure of the hydrophobic group. Gel formation was confirmed, and the hydrophilic portion composed of polyethylene glycol and caprolactone (CL) segment were included as essential components, and the paradioxanone (PDO) segment, trimethylene carbonate (TMC) segment, or these segments were simultaneously contained. As a block copolymer composed of a biodegradable polyester-based hydrophobic portion, a biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer having a molecular weight of 2,000 to 7,000 g / mole has been developed and patented. [Patent Application No. 2006-0023991].

Accordingly, the present inventors have applied the biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymers of Patent Application No. 2006-0023991 directly to protein or peptide drugs as injectable drug carriers in vitro and in vivo. As a result, the present invention was completed by confirming the possibility of controlling the release amount of the drug for a desired time at a desired site according to the concentration of the aqueous polymer solution.

Accordingly, an object of the present invention is to provide an injection formulation of a protein or peptide drug using a biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer as a drug carrier.

The present invention contains a hydrophilic part composed of polyethylene glycol and caprolactone (CL) segment as essential components, and a biodiode containing paradioxanone (PDO) segment, trimethylene carbonate (TMC) segment, or these segments simultaneously. Manufactured by mixing a protein or peptide-based drug with a drug carrier containing a biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer having a molecular weight of 2,000 to 7,000 g / mole. Characterized in that the injection form is used.

The present invention will be described in more detail as follows.

The present invention provides a biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer and uses this material to make an intelligent drug delivery system, ie, semi-solid or gel in situ upon injection, to the desired site, To an extent, the present invention relates to an injectable formulation of a protein or peptide drug developed into an injectable hydrogel which can release the drug slowly for a desired time.

First, the biocompatibility and temperature-sensitive polyethylene glycol / biodegradable polyester block copolymers used in the entire drug substance in the present invention will be described as follows.

Polyethylene glycol (PEG), which is used as a polymerization initiator of polyethylene glycol / biodegradable polyester block copolymer, has many advantages in the field of drug delivery, and can easily contain and release drug as a drug carrier and has high solubility in water and organic solvents. It is non-toxic and has no rejection of immune action. It has excellent biocompatibility and is approved by the US Food and Drug Administration for use in humans. In addition, since PEG has the largest protein adsorption inhibitory effect among hydrophilic polymers and improves the biocompatibility of blood contact substances, many applications have been made as biomaterials. However, there has been a problem that biodegradation does not occur while using biomaterials containing PEG. Because PEG is non-degradable and accumulates in the body, it has been reported to induce increased toxicity of plasma cholesterol and triglycerides after intraperitoneal injection. Therefore, in order to solve this problem, low molecular weight PEG having a molecular weight of 5,000 g / mole or less that can be easily removed from the body by filtration of the kidney and biodegradable esters that can be decomposed into biocompatible products by metabolism of the human body Copolymerization with the monomers resulted in the production of polyethylene glycol / biodegradable polyester block copolymers.

Biodegradable polymers of the ester series have the advantage of controlling the decomposition period by controlling the molecular weight and chemical components. Block copolymers of polyethylene glycol (PEG) and polycaprolactone (PCL), which have become basic models, have already been applied to biomaterials as temperature-sensitive copolymers having sol-gel phase transition properties. However, caprolactone is biodegradable and compatible with many polymers and has a feature of easily crystallizing. However, high crystallinity decreases compatibility with tissues and exhibits long-term degradation behavior when applied in vivo.

Thus, to caprolactone, paradeoxanone (PDO), trimethylene carbonate (TMC) of biodegradable ester series or these were added at a constant content ratio to lower crystallinity and control the biodegradation period. That is, it is represented by the following formula (1), each segment forms a random hydrophobic portion at random.

In Formula 1, x, y and z are each segments constituting the hydrophobic polyester portion, x is 50 to 95 mol%, y + z is 5 to 50 mol% (including when y or z is 0) .

Paradioxanone (PDO) and trimethylene carbonate (TMC), or these are simultaneously ring-opened to ester-based caprolactone (CL) using low molecular weight polyethylene glycol (Mn = 350 to 2000 g / mole) as a hydrophilic part in the present invention. Synthesis via copolymerization. Synthesis method is carried out by azeotropic distillation of polyethylene glycol, followed by addition of an ester monomer, methylene chloride (MC) or toluene as a reaction solvent, using an acid catalyst as the monomer activator, the reaction temperature is -40 ~ 130 The polymerization is carried out in the range of ° C. The acid catalyst is preferably one or more selected from HCl, HBr, CF ₃ COOH, CCl ₃ COOH, BrCH ₂ COOH, CH ₃ COOH, BCl ₃ , BBr ₃ and Camphorsulfonic acid.

The aqueous solution phase of the synthesized polyethylene glycol / biodegradable polyester block copolymer maintains the sol state having flow characteristics at room temperature and forms a gel over a certain temperature range (30 to 46 ° C.), and the critical temperature (44 to 47 ° C.) In the above, a thermosensitive material exhibiting a new sol-gel phase transition behavior showing flow characteristics was prepared, and thermal properties and crystallinity were analyzed by using a differential scanning calorimeter and an X-ray diffractometer. Injected into the rat to confirm the formation and integrity of the gel.

In addition, in order to apply such a temperature-sensitive block copolymer as an injectable drug carrier or a functional support for tissue regeneration, it is necessary to have a low viscosity, fast gel formation, and a low molecular weight to be easily discharged out of the body. Paradioxanone (PDO), trimethylene carbonate (TMC) or by introducing a constant content ratio at the same time was able to reduce the crystallinity by lowering the viscosity, and to obtain a molecular weight close to the expected value, biocompatibility and temperature sensitivity One of the requirements for hydrogels was the low viscosity and low molecular weight.

It is preferred to synthesize polyethyleneglycol / biodegradable polyester block copolymers having a total molecular weight in the range of 2,000 to 7,000 g / mole, but when the total molecular weight is less than 2,000 g / mole, the sol-gel in the human body temperature range for the purpose of the present invention is used. This is because there is a problem that the phase transition does not occur and is kept in a sol state, and when it exceeds 7,000 g / mole, there is a problem that it takes a long time for biodegradation to occur due to its large molecular weight.

As a representative example of the polyethylene glycol / biodegradable polyester block copolymer used in the present invention it can be represented by the following scheme 1.

In Scheme 1, n is an integer representing a repeating unit of polyethylene glycol constituting the hydrophilic portion, x, y and z are each segment constituting the hydrophobic polyester portion, x is 50 to 95 mol%, y + z is 5 ˜50 mol%, including when y or z is zero.

The polyethylene glycol / biodegradable polyester block copolymer prepared in this way has lower crystallinity than the polyethylene glycol-polycaprolactone block copolymer based on the basic model, so that the gel can be formed quickly and have a low viscosity that is easy to handle. It has a low molecular weight for easy discharge to the outside and can be applied as a drug carrier in injection form or a porous support for tissue engineering. In addition, it is possible to control the biodegradation period by adding a biodegradable ester-based paradioxanone (PDO), trimethylene carbonate (TMC), or these simultaneously at a constant content ratio to caprolactone showing long-term biodegradability. In addition, since the aqueous solution of polyethylene glycol / biodegradable polyester block copolymer shows sol-gel phase transition at various temperature ranges according to various kinds of biodegradable polymers such as paradioxanone (PDO) and / or trimethylene carbonate (TMC) As a biomaterial that is an object of the present invention, when applied to a human body, it may be applied to a gelling property at a temperature slightly higher or lower than the biological temperature as well as the gelling property at the biological temperature.

Therefore, the present invention uses the biocompatible and temperature-sensitive polyethylene glycol / biodegradable polyester block copolymer as a drug carrier to add a protein or peptide-based drug to the new injection formulation that can release the drug for 1 to 10 weeks. It is characterized by.

The mixing ratio of the copolymer and the protein or peptide-based drug is preferably 5 × 10 ^-5 to 50 parts by weight, more preferably 0.05 to 10 parts by weight, more preferably less than the range of the drug, relative to 100 parts by weight of the copolymer. In the case of the desired therapeutic and pharmacological effects can not be expected, if there is more than the above range there is a problem that can cause a serious immune response due to overuse of the drug.

In addition, the protein or peptide-based drug used in the present invention is albumin, calcitonin (calcitonin), octreotide (leutrelide), leuprolide, adenosine deaminase, L-asparaginase (L) Asparaginase, urokinase, interferon α-2b, interferon β-1b insulin, insulin-like growth factor-1; IGF- 1), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), nerve growth factor (NGF); brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3), fibroblast growth factor (FGF), epidermal growth factor (EGF), endothelial growth factor, VEGF), transforming growth factor-β, TGF-β, or Platelet-derived growth factor (PDGF) and the like, but not limited to all drugs requiring long-term administration and release is preferred for the subject of the present invention injection type hydrogel.

The preparation of such injection formulations is as follows.

The biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer was dissolved in PBS (pH 7.4) in a vial to form a sol, and then uniformly dispersed at 4 ° C. for one day. The content is added in an amount of 5 x 10 ^-5 to 50 parts by weight, more preferably 0.05 to 10 parts by weight, and vigorously stirred to prepare a physically mixed injection formulation with respect to 100 parts by weight of the copolymer.

Polyenisopropylacrylamide [Poly (NIPAAM)], which is a typical temperature-sensitive hydrogel polymer, exhibits an initial burst effect in drug release and shows a short release behavior due to diffusion for 8 hours. In addition, polymers including enisopropylacrylamide (NIPAAM) have limitations in terms of their application as drug carriers because they have the disadvantage of not being cytotoxic and biodegradable. In addition, copolymers of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO), which are used under the trade names Pluronic, Poloxamers, and Tetronic, are also representative of temperature-sensitive hydros. As a gel polymer, it has been widely studied in the form of an injection by mixing with a protein drug and various drugs, but the gel is dissolved in an excess aqueous solution, so that the gel cannot be dissolved within 2 to 3 days in the human body, so that the gel cannot be maintained. There is a limitation that should be applied only to sustained release preparations. On the other hand, the polyethylene glycol / polyester block copolymer used in the present invention has excellent biocompatibility compared to the conventional temperature sensitive hydrogel, so that there is no cytotoxicity and biodegradation occurs slowly in the body, and the gel formed through the control of biodegradability. Is long lasting and has several advantages for releasing the drug for the desired period.

Therefore, the injection preparation thus prepared is excellent in biocompatibility such as long-term release (1-10 weeks) of drug release, less immune rejection reaction to the living body, no inflammatory reaction, and decomposition gradually occurs in the body after injection. Since it is changed into harmless low-molecular substance and discharged out of the body, there is no separate surgical removal procedure after the release of the drug for a certain period of time, and frequent administration is not necessary, thus maintaining the efficacy of one-time administration, which facilitates the patient's convenience. It is expected to be useful as a carrier of protein or peptide drugs by enhancing the stability and effect of the drug.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1: Preparation of methoxypolyethylene glycol- (polycaprolactone-co-polytrimethylene carbonate) block copolymer [MPEG-PCL / PTMC]

To synthesize an MPEG-PCL / PTMC block copolymer with a molecular weight of 3150 g / mole, 1.7 g (2.26 mmol) of initiator methoxypolyethylene glycol (MPEG) and 80 mL of toluene were placed in a well-dried 100 mL round flask and Dean Stark Trap. Azeotropic distillation was carried out at 130 ° C. for 3 hours. After distillation, toluene was removed and methoxypolyethylene glycol (MPEG) was cooled to room temperature. Then, 5.15 g (2.26 mmol) of prepurified caprolactone (CL) and 0.27 g (2.25 mmol) of trimethylene carbonate (TMC) were added thereto. 25 mL of pre-purified methylene chloride (MC) was added as a solvent, followed by 4 mL of HCl as a polymerization catalyst, followed by stirring at room temperature for 24 hours. All procedures were carried out under high purity nitrogen. After the reaction, in order to remove unreacted monomer or initiator, the reactant was slowly precipitated in 600 mL of hexane and 150 mL of methanol. The precipitate was dissolved in methylene chloride (MC), filtered through a filter paper, and the solvent was removed through a rotary evaporator and dried under reduced pressure.

Molecular weight relative to the molar ratio of the components of the copolymer prepared above was measured using ¹ H-NMR [Fig. 1], molecular weight 3170 g / mole similar to the theoretical expected value was obtained, polydispersity The gel permeation chromatography (GPC) for the measurement of the result confirmed that it has a very narrow polydispersity of 1.19.

Example 2: Preparation of methoxy polyethylene glycol- (polycaprolactone-co-polyparadioxanone) block copolymer [MPEG-PCL / PPDO]

To synthesize an MPEG-PCL / PPDO block copolymer with a molecular weight of 3150 g / mole, 1.67 g (2.24 mmol) of methoxypolyethylene glycol (MPEG) and 80 mL of toluene were placed in a well-dried 100 mL round flask and Dean Stark trap. Azeotropic distillation was carried out at 130 ° C. for 3 hours. After distillation, all of toluene was removed, and methoxy polyethylene glycol (MPEG) was cooled to room temperature, and 5.11 g (2.24 mmol) of purified caprolactone (CL) and 0.38 ml (2.24 mmol) of paradioxanone (PDO) were injected. Then, 25 mL of methylene chloride (MC) purified beforehand as a reaction solvent was added thereto, followed by 4 mL of HCl as a polymerization catalyst, and the mixture was stirred at room temperature for 24 hours. All procedures were carried out under high purity nitrogen. In order to remove unreacted monomer or initiator after the reaction, the reactant was slowly dropped to 600 mL of hexane and 150 mL of heptane. The precipitate was dissolved in methylene chloride (MC), filtered through a filter paper, and the solvent was removed through a rotary evaporator and dried under reduced pressure.

Molecular weight relative to the molar ratio of the components of the copolymer prepared above was measured using ¹ H-NMR [Fig. 2], molecular weight 3200 g / mole similar to the theoretical expected value was obtained, polydispersity As a result of using GPC for the measurement of, it was confirmed that it has a very narrow polydispersity of 1.17.

Experimental Example 1 Determination of Sol-Gel Phase Transition Behavior with Temperature in Polyethyleneglycol / Biodegradable Polyester Block Copolymer

In order to observe the phase transition behavior of the polyethylene glycol / biodegradable polyester copolymer, the concentrations of 25 and 20 wt% of the MPEG-PCL / PTMC and MPEG-PCL / PPDO block copolymers prepared in Examples 1 and 2, respectively, were measured. After dissolving in distilled water, the mixture was refrigerated at 4 ° C. for one day to maintain equilibrium of uniformly dispersed polymer. The sol-gel phase transition behavior was measured at each temperature by increasing the spin rate at 0.2 rpm while increasing the prepared polymer solution by 1 ° C. per 3 minutes in the range of 10 ° C. to 60 ° C. using a viscosity meter. And in order to measure the change in the strength of the viscosity of the polymer solution dissolved in each concentration over time, the viscosity of the viscometer is fixed at 37 ℃, spin speed is 0.2 rpm and the change in viscosity for about 40 hours at intervals of 5 minutes It was. As a result, as shown in Fig. 4, when the concentration of the aqueous polymer solution is increased from 20 wt% to 25 wt%, the temperature range showing the gelling behavior is widened and the strength of the viscosity is also increased. MPEG-PCL / PTMC (A) And MPEG-PCL / PPDO (B), and the viscosity change at constant temperature of 37 ° C. was determined by Fluronic F-127, MPEG-PCL / PTMC, and MPEG-PCL at a concentration of 20 wt%. / PPDO was measured and it was observed that in the case of fluoric (F-127), the viscosity was gradually decreased after 15 hours, and then reached 45 hours, and the viscosity disappeared, whereas MPEG-PCL / PTMC In the case of and MPEG-PCL / PPDO, the strength of the viscosity was different, but both were observed to increase gradually with time (C).

Example 3 Polyethyleneglycol / Biodegradable Polyester Block Copolymer in vivo  Check gel formation

In order to confirm the sol-gel phase transition in the vicinity of the body temperature, the polyethylene glycol / biodegradable polyester block copolymer solution prepared in Example 1 was maintained in a sol state at room temperature, and then 1 mL of rats were removed using a disposable syringe. It was injected subcutaneously. After 24 hours, the injection site was excised to confirm gel formation. Therefore, it was confirmed that the polyethylene glycol / biodegradable polyester block copolymer solution rapidly formed a gel in vivo and the gel was maintained for a long time [FIG. 5].

Example 4 Elution Experiment of Albumin ( in vitro )

MPEG-PCL / PTMC or MPEG-PCL / PPDO block copolymers prepared by the methods described in Examples 1 and 2 contained bovine serum albumin in which the model drug of this experiment, the water-soluble protein, and the fluorescent substance FITC were bound. Hydrogels were prepared under the following conditions to confirm that albumin was continuously released in the hydrogels. As a comparative model, fluoric acid (F-127) and the present invention, MPEG-PCL / PTMC and MPEG-PCL / PPDO copolymers were dissolved in 5 ml vials of phosphate buffer solution (PBS, pH 7.4) at 20 wt% concentration. After forming a sol and uniformly dispersing at 4 ℃ for one day, albumin was measured at room temperature based on 100 parts by weight of fluoric (F-127), a comparative model of the present invention, MPEG-PCL / PTMC, MPEG-PCL / PPDO block copolymer. 1 part by weight (1 mg) was added and physically mixed to form a gel in a 37 ℃ thermostat. 4 ml of PBS was added to the hydrogel containing albumin prepared above, and 1 ml of analytical sample was taken at a predetermined time interval while stirring at a speed of 100 rpm in a thermostat maintained at 37 ° C. Replenished. Samples for confirming the release of the drug was blocked by light and quantitatively analyzed through a divergence intensity under conditions of excitation 495 nm and emission 525 nm using a fluorescence spectrometer (F-4500, Hitachi, Tokyo, Japan).

6 is a graph showing the amount of albumin released from the hydrogel containing albumin over time as a result of the release experiment according to the aqueous solution concentration of Pluronic (F-127) and each block copolymer. In the case of the comparative model fluoric (F-127) as shown in FIG. 6, the release was completed in two days, but the viscosity of the MPEG-PCL / PTMC and MPEG-PCL / PPDO block copolymer hydrogels prepared according to the present invention was measured. According to the strength of the drug can be controlled to control the release of the drug was confirmed.

Example 5 Elution Experiment of Insulin ( in vivo )

Containing bovine insulin MPEG-PCL / PTMC with MPEG-PCL / PPDO in the block, in order to ensure that in the hydrogel of Comparative model of the copolymer pluronic (F-127) insulin is continuously emitted from the next of the living body (in In vivo , the release characteristics of bovine insulin (Bovine Insulin) were measured.

MPEG-PCL / PTMC and MPEG-PCL / PPDO block copolymers prepared by the methods described in Examples 1 and 2 above were compared to fluoric (F-127), which is a comparative model, at 25, 20 wt%, 2 ml of PBS ( dissolved in 5 ml vials containing pH 7.4) to form a sol, and then uniformly dispersed at 4 ° C. for 1 day. Bovine insulin (F-127), a comparative model, and MPEG-PCL of the present invention 1 part by weight (1 mg) was added based on 100 parts by weight of / PTMC, MPEG-PCL / PPDO block copolymer, and vigorously stirred to prepare a completely physically mixed solution. The fluoric (F-127) containing the insulin thus prepared and the present invention MPEG-PCL / PTMC, MPEG-PCL / PPDO block copolymer hydrogel was extracted with a syringe (1cc, 26G needle) of each 0.6 ml 0.5 ml each was injected subcutaneously into three SD rats (male, 8 weeks old, about 350 g). Using a catheter, a blood sample (300 μl) was collected from the tail vein of the rat at a predetermined time in an EP tube containing 200 μl of saline (heparin: saline = 1: 499), and the sample was stirred. After that, plasma was separated by centrifugation at 10,000 rpm for 5 minutes and samples were stored frozen (-20 ° C.) until analysis. The concentration and release of bovine insulin in rat plasma were measured by Bovine Insulin ELISA kit (Merdia). In the enzyme-linked immunological method, the antigen was bound to the immobilized insulin receptor, the color plate was reacted on the microplate, and the absorbance was measured by measuring the wavelength of 450 nm to determine the amount of insulin released.

7 is a graph showing changes in the concentration of bovine insulin in the blood of rats over time. Pluronic (F-127), which had the highest viscosity intensity at the same concentration, had the lowest initial release of the drug and a very short release period of 3 days. In the case of MPEG-PCL / PTMC with the smallest relative strength, the initial release of drug was the highest, and both MPEG-PCL / PTMC and MPEG-PCL / PPDO block copolymers were continuously released for up to 70 days (A ). In addition, the release amount measured by increasing the aqueous solution concentration of the MPEG-PCL / PTMC and MPEG-PCL / PPDO block copolymers to 25 wt% was 25 wt%, and the initial release amount of the drug was 25 wt% having a relatively high viscosity strength than 20 wt%. It was confirmed that this was lowered (B). As a result, it was found that the drug was continuously released for about 70 days depending on the concentration in the hydrogel of the MPEG-PCL / PTMC and MPEG-PCL / PPDO block copolymer containing bovine insulin.

Therefore, it was confirmed that the present invention prepared according to the change of the conditions described above can be applied to the design of the sustained formulation of peptide-based and protein drugs similar to albumin or insulin until 5, 10, 20 or 70 days.

A copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO), which is used as a representative temperature-sensitive polymer, such as Pluronic and Poloxamers, is polyethylene glycol / Drug release experiments were performed in vivo by dissolving at 20 wt% of the same aqueous solution as the polyester block copolymer, and the results of drug release for 3 days were observed in Pluronic and Poloxamers. In the case of the polyethylene glycol / polyester block copolymer of the present invention, the drug was released for 1 to 10 weeks. In addition, Pluronic and Poloxamers were dissolved at a concentration of less than 20 wt%, and in vitro , the drug melted within 1 day, so the drug release could not be expected. In the present invention, the polyethylene glycol / polyester block copolymer is dissolved at a concentration of 15 wt% or 10 wt%, which is less than 20 wt%, and more continuous than the drug release result at 20 wt% when the drug release experiment is performed. One or more drug release results were shown and the gel formed was confirmed to be maintained. In addition, the hydrogel containing bovine insulin was found to continue to release the drug until about 70 days.

As described above, the drug injection formulation according to the present invention has excellent biocompatibility such as less immune rejection response to the living body than the conventional injection formulation of hydrogel, and thus does not cause an inflammatory reaction, and is gradually degraded in the body after injection. It is transformed into a low-molecular substance that is harmless to the human body and is discharged to the body.Therefore, there is no separate surgical removal procedure after the release of the drug for a certain period of time, and frequent administration is not necessary. The formed gel may be maintained for a long time to release the drug for a desired period of time, and have various effects, and have a drug release period of 1 to 10 weeks to allow the drug to be released continuously for a long time sustained release formulation. It is suitable to increase the stability and effect of the drug, and is very suitable as a carrier of protein or peptide drug. It is expected to be useful.

Claims (4)

A hydrophilic part composed of polyethylene glycol having a molecular weight of 350 to 2000 g / mole, Containing caprolactone (CL) segment as an essential ingredient and containing a paradioxanone (PDO) segment, trimethylene carbonate (TMC) segment, or a biodegradable polyester-based hydrophobic moiety represented by the following formula (1) Being, Injectable formulations prepared by mixing a protein or peptide-based drug with a biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer having a molecular weight of 2,000 to 7,000 g / mole; [Formula 1]

In Formula 1, x, y and z are each segments constituting the hydrophobic polyester portion, x is 50 to 95 mol%, y + z is 5 to 50 mol% (including when y or z is 0) . The injection formulation of claim 1, wherein the drug is mixed in an amount of 5 x 10 ^-5 to 50 parts by weight based on 100 parts by weight of the polyethylene glycol / biodegradable polyester block copolymer. According to claim 1, wherein the protein or peptide-based drug is albumin, calcitonin (calcitonin), octreotide (leutrelide), leuprolide, adenosine deaminase, L-asparaginase (L) Asparaginase, urokinase, interferon α-2b, interferon β-1b insulin, insulin-like growth factor-1; IGF- 1), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), nerve growth factor (NGF); brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3), fibroblast growth factor (FGF), epidermal growth factor (EGF), endothelial growth factor, VEGF), transforming growth factor-β, TGF-β, or blood plasma Injection formulation, characterized in that the platelet-derived growth factor (PDGF). The injection formulation of claim 1, wherein the injection formulation has a drug release period of 1 to 10 weeks.

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