KR20090049328A - Thermogelling pp-plx-pp block copolymers aqueous solution, and method for preparing the same - Google Patents

Abstract

The present invention provides poloxamer and poloxamer derivatives having a number average molecular weight of 600 to 15,000 Daltons (A- (propylene glycol) x- (ethylene glycol) y- (propylene glycol) xA or A- (ethylene glycol) x- (propylene glycol)). y- (ethylene glycol) xA) (hereinafter referred to as PLX) and a polypeptide having a number average molecular weight of 200 to 8,000 Daltons (PP) are combined, wherein A is a nucleophilic functional group such as a hydroxy group or an amine group -PLX-polypeptide (PP-PLX-PP) block copolymers and methods for their preparation.

The aqueous solution of the PP-PLX-PP block copolymer according to the present invention exists as an aqueous solution at a temperature below a certain concentration when above a certain concentration, but at a higher temperature, a transition to a hydrogel occurs and PP-PLX-PP is three times higher than that of PLX. The gel has a sustainability of the above, it is a material that significantly improved the persistence problem of the existing gel can be widely applied to pharmaceutical delivery media or tissue engineering.

PP-PLX-PP, Poloxamer, Polypeptide, Temperature Sensitive, Sol-Gel Transfer

Description

Temperature Sensitive Sol-Gel Transition PCP-PLK-PCP Block Copolymer and Method for Making the Same {THERMOGELLING PP-PLX-PP BLOCK COPOLYMERS AQUEOUS SOLUTION, AND METHOD FOR PREPARING THE SAME

The present invention relates to temperature sensitive PP-PLX-PP block copolymers and methods for their preparation.

Polymers reported so far to exhibit sol-gel transition reactions in aqueous solutions are poly (ethylene glycol) -poly (propylene glycol) -poly (ethylene glycol) (poloxamer), poly (N-isopropylacrylamide) and its airborne Coalesce, poly (ethylene glycol) / poly (lactide / glycolide), poly (ethylene glycol) / polypropylenephmaleate, chitosan / glycerol phosphate, polyphosphazene, poly (ethylene glycol) / polycaprolactone and the like.

Aqueous solutions of these polymers exist in solution or sol at room temperature or lower, but transition to hydrogels at temperatures near body temperature (37 ° C), so these polymers can be used as pharmaceutical delivery and tissue engineering materials. It is likely to be. In other words, by mixing with medicine or cells in a sol state, it is injected into the body through subcutaneous or intramuscular injection to instantaneously make depots at desired sites, thereby slowly releasing the drug or growing cells, Can be played back. This biodegradable material makes implants without the need for surgical operations and can be sterilized in a sol state through a simple Michael filter.

However, in the case of poloxamers, ie poly (ethylene glycol) -poly (propylene glycol) -poly (ethylene glycol), even hydrogels made from 20 to 30% by weight of aqueous solution are mostly dissolved within 1 to 3 days to maintain the shape of the gel. This has been a limitation as a material for drug delivery / tissue engineering.

Meanwhile, U.S. Pat.Nos. 6,117,949, 6,201,072, and 6,841,617 describe poly (ethylene glycol) / poly (lactide / glycolide) and their medical applications in which the transition from aqueous solution to hydrogel occurs when the temperature is raised. Doing. US Published Patent No. 20060018949 describes poly (ethylene glycol) / polypropylenephmalate and their medical applications in which a transition from aqueous solution to hydrogel occurs when the temperature is raised. However, poly (ethylene glycol) / poly (lactide / glycolide) and poly (ethylene glycol) / poly (propylene frmlate) generate acid upon decomposition, take a long time to dissolve, and also in aqueous solution When stored at room temperature, it is unstable and easily hydrolyzed. It also has the disadvantage that the minimum concentration at which the sol-gel transition occurs is relatively high, at least 10% by weight.

U.S. Patent Publication No. 20050020808 describes polyphosphazenes in which a transition from aqueous solution to hydrogel occurs when the temperature is raised, while U.S. Patent No. 6,344,488 describes chitosan / glycerol phosphate and their medical applications. However, poly (ethylene glycol) / poly (lactide / glycolide), poly (ethylene glycol) / poly (propylene fmarlate), polyphosphazene, etc. are malt-shaped and have a disadvantage in that it takes a long time to dissolve. have.

US Patent Publication No. 20040077780 describes a wide range of poly (ethylene glycol) / polypeptide copolymers consisting of two or more amino acids and their medical applications in which the transition from aqueous solution to hydrogel is increased at elevated temperatures.

Korean Patent Laid-Open Publication No. 2002-0023441 describes an isopropylacrylamide copolymer causing sol-gel phase transition and vascular occlusion using the same. However, since they are not degraded in vivo, there is a limitation that they should be removed from the body when applied as a material for drug delivery or tissue engineering. In addition, acrylamide is a carcinogen, and the toxicity of residual monomers may be a problem.

U.S. Patent Publication 20050175573 A1 is characterized in that poloxamers are prepared using a chemical bond of urethane or urea to produce multiblock poloxamers. That is, poloxamer was connected to diisocyanate or the like to increase the molecular weight by urea or urethane bonds. However, this is very different from the present invention characterized by introducing a polypeptide block into a poloxamer as a PP-PLX-PP triple block polymer.

The inventor has filed a patent application (application number: 2006-78581) on polyethylene glycol / polypeptide block copolymer and its medical application. It is prepared from polyethyleneglycol having an amine group at the end and N-carboxy amino acid anhydride. Currently, polyethyleneglycol having an amine group at the end is very expensive. Accordingly, the present inventors have conducted research on the basis of the above-described matters, and as a result, a temperature-sensitive sol-gel transition PP-PLX-PP copolymer is prepared using poloxamer, and the material significantly solves the problem of poloxamer. The present invention has been completed by discovering the advantages of improving. In addition, the PP-PLX-PP according to the present invention can be easily obtained commercially available poloxamers of various compositions as a raw material and the cost of the PPP-PLX-PP copolymer is finally produced at a significantly lower value It has an excellent advantage that can significantly reduce.

An object of the present invention is to solve the problems of the conventional sol-gel transition material described above, but exists in an aqueous solution (sol) state at room temperature or below, but biodegradable PP-PLX- that changes to a hydrogel at body temperature By providing a PP block copolymer and a method for preparing the same, it is to solve the problem of high polymer concentration required to cause 1) short gel persistence and 2) sol-gel transition of existing poloxamers.

As one embodiment for achieving the above object, the present invention is a polypeptide-PLX-polypeptide (PP-PLX-PP) to which poloxamer or poloxamer derivative (hereinafter referred to as PLX) and polypeptide (PP) are bound ) Block copolymer.

The poloxamer or poloxamer derivative used as block A in the block copolymer according to the present invention is specifically (A- (propylene glycol) x- (ethylene glycol) y- (propylene glycol) xA or A- (ethylene glycol) x It is a polymer represented by the formula-(propylene glycol) y- (ethylene glycol) xA). In this case, A is a nucleophilic functional group, preferably a hydroxy group or an amine group, but is not limited thereto. The poloxamer or poloxamer derivative is bound to the polypeptide (PP) by A. In addition, in said formula, x is 1-100, y is 10-150.

The number average molecular weight of the poloxamer or poloxamer derivative is preferably 600 to 15,000 daltons. In addition, the poloxamer or poloxamer derivative is composed of ethylene glycol and propylene glycol as can be seen in the above formula, the weight ratio of ethylene glycol / propylene glycol of the poloxamer or poloxamer derivative used in the present invention is 30/1 To 2/1 is preferred.

In addition, the polypeptide (PP) block used as the block B in the block copolymer according to the present invention is composed of one or two or more kinds of amino acids, and the amino acids are alanine, arginine, asparagine, aspalic acid, asparagine, cystine, glutamine And glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Also included are both copolymers in which the amino acids are in DL- and L-forms. One polypeptide (PP) is attached to each of both ends of the poloxamer or poloxamer derivative (PLX). The number average molecular weight of the polypeptide is preferably 1,000 to 30,000.

As mentioned above, the PP-PLX-PP copolymer of the present invention is a polypeptide (PP) linked to both ends of a poloxamer or poloxamer derivative (PLX), wherein the weight ratio of PLX / PP is 5/1 to 1 / 5 is preferred. Moreover, the molecular weight of the preferable PP-PLX-PP copolymer of this invention is 1,000-30,000.

When the molecular weights of the blocks A and B are in the above ranges, hydrophilicity and hydrophobicity can be easily adjusted. In addition, the molecular weight range of a PP-PLX-PP block copolymer is the value which considered the viscosity of the solution which can be used as an injection. In other words, if the molecular weight of the polypeptide is too large, the solubility in water is low, and the dissolution process takes a lot of time. In addition, if the poloxamer is too long compared to the polypeptide, extracorporeal excretion after injection is not only a problem, but also requires a large concentration causing sol-gel transitions and sometimes at physiologically useful temperatures (10-37). ^o C) The problem arises that no sol-gel transition is observed.

The aqueous solution of PP-PLX-PP block copolymer according to the present invention exists in an aqueous solution (sol) at room temperature or lower temperature, but at higher temperatures such as body temperature, transition occurs to hydrate gel and biodegradation. In addition to having the properties, the conventional poloxamer has a very excellent effect of having a gel persistence of three times or more that the gel persistence is maintained for 10 days or more, compared to having a short gel persistence of less than 3 days.

Furthermore, while conventional poloxamers induce sol-gel transitions at relatively high concentrations of 15 to 40% by weight, the PP-PLX-PP block copolymers according to the present invention have sol-gels at low concentrations of less than 10% by weight. It can be used for the delivery of medicine even if a small dose is used. Therefore, the polymer which causes the sol-gel transition which does not have physiological activity and performs only the drug delivery function should be used in a small amount as much as possible, and the polymer of the present invention can use a much smaller amount than the conventional poloxamer. In addition, it is economically very advantageous compared to the conventional poloxamer, and thus can be effectively applied as a medicine delivery system.

Thus, in one preferred embodiment, the PP-PLX-PP block copolymer of the present invention exhibits a sol-gel transition concentration within 10% by weight.

In another aspect, the invention provides a PP according to the invention, comprising reacting a poloxamer or poloxamer derivative (PLX) having at least one terminal amine group or hydroxy group with an N-hydroxy anhydride amino acid. A method for producing a -PLX-PP block copolymer.

The poloxamer or poloxamer derivative (PLX) used in the production process according to the invention is (A- (propylene glycol) x- (ethylene glycol) y- (propylene glycol) xA or A- (ethylene glycol) x- (propylene Glycol) y- (ethylene glycol) xA), wherein A is a nucleophilic functional group at the terminal, specifically a hydroxy group or an amine group. The poloxamer or poloxamer derivative (PLX) has one or more hydroxy or amine groups.

In addition, in the preparation method of the present invention, N-hydroxy amino acid anhydride is reacted with PLX in order to link the polypeptide (PP) to the terminal of the poloxamer or poloxamer derivative. At this time, the N-hydroxy amino acid anhydride is alanine, arginine, asparagine, aspalic acid, asparagine, cystine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan , N-hydroxy anhydride of one or more amino acids selected from the group consisting of tyrosine and valine.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the examples are only illustrative of the present invention, and the scope of the present invention is not limited to these examples.

Example

Hydrogen nuclear magnetic resonance spectra were measured using a Bruker DPX-250 NMR spectrophotometer and infrared absorption spectra respectively using a Nicolet Impact 400 FT-IR spectrometer.

Sol-gel transition temperatures were analyzed using Thermo Haake, Rheometer RS1. At this time, after filling the polymer solution between a diameter of 25 mm and a gap of 0.5 mm, the sample was strained at a stress of 4.0 dyne / cm ² and a cycle of 1.0 rad./sec, and the heating rate was 0.2 ° C / min.

Example  One: PP - PLX - PP  Preparation of Block Copolymers (1)

Poloxamine (molecular weight: 2,000 Daltons) and 50 ml of dried toluene are placed in a flask, followed by distillation under a stream of nitrogen to remove water. Into this, chloroform (7 ml) without water was added to dissolve polyethylene glycol, and N-carboxy alanine anhydride was dissolved in N, N-dimethylformamide (4.0 ml) without water, followed by chloroform (with water). 10.0 ml) is added and reacted at 30 ° C. for 24 hours. After the reaction was completed, 50 ml of chloroform was poured to form a solution, poured into diethyl ether to reprecipitate, and the residual solvent was removed by a vacuum pump. The yield is 50-80%.

¹ H-NMR (acetic acid-d ₄ ): 4.8 (-CO CH (CH ₃ ) NH-), 3.6 ( -CH ₂ CH ₂ O-), 0.7 to 1.9 (-COCH ( CH ₃ ) NH-) and ( -CH ₂ CH ( CH ₃ ) O-).

Example  2: PP - PLX - PP  Preparation of Block Copolymers (2)

Poloxamine (molecular weight: 2,000 Daltons) and 50 ml of dried toluene are placed in a flask, followed by distillation under a stream of nitrogen to remove water. Add water-free chloroform (7 ml) to dissolve polyethylene glycol, and dissolve N-carboxy DL-alanine anhydride and L-alanine anhydride in N, N-dimethylformamide (4.0 ml) without water. Add chloroform (10.0 ml) from which water was removed, and react at 30 ° C for 24 hours. After the reaction was completed, 50 ml of chloroform was poured to form a solution, poured into diethyl ether to reprecipitate, and the residual solvent was removed by a vacuum pump. The yield is 50-80%.

¹ H-NMR (acetic acid-d ₄ ): 4.8 (-CO CH (CH ₃ ) NH-), 3.6 ( -CH ₂ CH ₂ O-), 0.7 to 1.9 (-COCH ( CH ₃ ) NH-) and ( -CH ₂ CH ( CH ₃ ) O-).

Example  3: PP - PLX - PP  Preparation of Block Copolymers (3)

Poloxamine (molecular weight: 900 Daltons) and 50 ml of dried toluene are placed in a flask, followed by distillation under a stream of nitrogen to remove water. Add chloroform (7 ml) without water to dissolve polyethylene glycol, and dissolve N-carboxy L-phenylalanine anhydride and L-alanine anhydride in N, N-dimethylformamide (4.0 ml) from which water was removed. Add chloroform (10.0 ml) from which water was removed, and react at 30 ° C for 24 hours. After the reaction was completed, 50 ml of chloroform was poured to form a solution, poured into diethyl ether to reprecipitate, and the residual solvent was removed by a vacuum pump. Yield is 60-80%.

¹ H-NMR (ethyl _{-d 4): 7.1 ~ 7.3 (} - C 6 H 5), 4.8 (-CO CH (CH 3) NH-),, 3.6 (- CH 2 CH 2 O-), 2.8 ~ 3.4 ( -CH ₂ C ₆ H ₅ ) 0.7 to 1.9 (-COCH ( CH ₃ ) NH-) and (-CH ₂ CH ( CH ₃ ) O-).

Example  4: Sol-Gel Transition Characterization Experiment

0.5 ml of a certain aqueous solution of a predetermined concentration (1.0 ~ 12% by weight) in a 4 ml test tube, and check the state while raising the temperature from 0 ℃ to 80 ℃ at 1 ℃ intervals. In each step, the temperature at which the test tube is not turned over and defined as the sol-gel transition temperature is defined. 1 is a phase transition diagram created from the results of this experiment.

In addition, sol-gel transition properties were analyzed using rheological properties. Using a Ther Haake Rheometer RS 1 instrument, an aqueous polymer solution (8.0% by weight) was placed between flat plates with a diameter of 25 mm and a gap of 0.5 mm, with a cycle of 1.0 rad / sec at a stress of 4.0 dyne / cm ² . The strength was measured while raising the temperature of the aqueous solution at a rate of 0.5 ° C./min while giving a vibration. The transparent aqueous solution of 15 ° C. or lower transitions to the gel at 20 ° C. or higher, resulting in a sharp increase in modulus (G ′). Figure 2 is a graph showing the strength and viscosity changes with temperature of the 5.0 wt% polymer aqueous solution.

According to the present invention, a temperature sensitive PP-PLX-PP block copolymer having promising applications for drug delivery or tissue engineering has been prepared. The aqueous solution of the PP-PLX-PP copolymer according to the present invention exists as an aqueous solution below a certain temperature when the concentration is higher than a certain concentration, but at a higher temperature, the transition to the hydrogel occurs. While the gel persistence of conventional poloxamers is within 2 to 3 days, PP-PLX-PP is a material that significantly improves the gel persistence problem and can be applied as a drug delivery medium.

1 is a phase transition graph showing the sol-gel transition behavior according to the temperature and concentration of the aqueous polymer solution of the present invention.

Figure 2 is a graph showing the strength and viscosity changes with temperature of the aqueous polymer solution of 5.0% by weight concentration.

FIG. 3 shows the results of investigating the sustainability of poly (L-alanine) -PLX-Poly (L-alanine) hydrogel in 37 ^° C. excess phosphate buffer. Hydrogels made from an aqueous solution of F127 (MW = 12,000 Daltons) (30 wt.%), A representative sol-gel transition poloxamer, were compared under the same conditions.

Claims (8)

PP-PLX-PP block copolymer consisting of a poloxamer or poloxamer derivative (PLX) block, and a polypeptide (PP) block, wherein a transition from sol to gel occurs when the temperature is increased. According to claim 1, wherein the poloxamer or poloxamer derivative (PLX) is (A- (propylene glycol) x- (ethylene glycol) y- (propylene glycol) x A or A- (ethylene glycol) x- (propylene glycol) y- (ethylene glycol) xA) Wherein A is an amine group or a hydroxy group; x is from 1 to 100, y is from 10 to 150 PP-PLX-PP block copolymer. The method of claim 1, wherein the number average molecular weight of the poloxamer or poloxamer derivative (PLX) block is 600 ~ 15,000 Daltons, the number average molecular weight of the polypeptide (PP) block is 200 ~ 8,000 Daltons, the molecular weight of the copolymer PP-PLX-PP block copolymer that is 1,000 to 30,000. The PP-PLX-PP block copolymer according to claim 1, wherein the content of poloxamer or poloxamer derivative (PLX) is 16.6% to 83.3% by weight, and the content of polypeptide is 16.7 to 83.4% by weight. The PP-PLX-PP block copolymer according to claim 1, wherein the polyethylene glycol contained in the poloxamer or poloxamer derivative (PLX) is 33.3-96.7 wt% and the polypropylene glycol is 3.3-66.7 wt%. . The method of claim 1, wherein Polypeptides are composed of glycine, alanine, arginine, asparagine, aspalic acid, asparagine, cystine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine PP-PLX-PP block copolymer, which is one or two amino acids of choice. The PP-PLX-PP block copolymer according to claim 6, wherein the polypeptide comprises one or two selected from DL-type amino acids and L-type amino acids. PP-PLX-PP block according to claims 1 to 7, comprising the step of reacting a poloxamer or poloxamer derivative (PLX) having at least one amine or hydroxy group at its end with an N-hydroxy anhydride amino acid. Method of Making Copolymers.

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