KR100939983B1 - Hyaluronic Acid and Hydrophobic Poly Amino Acid Copolymer - Google Patents

Abstract

The present invention relates to a hyaluronic acid-hydrophobic polyamino acid copolymer comprising a hyaluronic acid unit and a hydrophobic polyamino acid unit.

Such hyaluronic acid-hydrophobic polyamino acid copolymer is a substance having biodegradable properties in the body, and can be used to transport active ingredients such as drugs, food additives and cosmetics, such as organic molecules including proteins, peptides and nucleotides. When used in the transport of drugs, they can lead to the maintenance and sustained release of the biological stability of the active ingredient, and show long-term persistence of more than a week.

Description

Hyaluronic Acid and Hydrophobic Poly Amino Acid Copolymer

The present invention relates to a biodegradable hyaluronic acid-hydrophobic polyamino acid copolymer comprising a hyaluronic acid unit and a hydrophobic polyamino acid unit, and includes an active ingredient such as drugs, food additives and cosmetic components such as organic molecules including proteins, peptides, nucleotides, and the like. It can be used to transport and particularly useful for the transport of drugs such as proteins and peptides.

Of the many polymers used to transport pharmaceutical, food and nutritional and cosmetically active ingredients, particularly biodegradable polymers used to transport the body with drugs as active ingredients, began with the study of polylactic acid (PLA) in the 1970s. It became.

Since studies on polylactic acid have been conducted, researches on polymers that exhibit biodegradation properties and are easily lost in the body, and in which the degraded unit substance is toxic and safe for the human body when degraded in the body, have been continued.

For example, polylactic acid-glycolic acid (PLGA), natural polysaccharides (Polysacc haride), polyamino acid, polyethylene glycol (PEG) -polylactic acid-glycolic acid (PLGA), polyethylene oxide (PEO) -polypropylene oxide (PPO) polymer Development has continued, and transport materials using them are commercially available.

However, synthetic polymers, such as PLGA, have the advantage of being biodegradable in the body because they are composed of ester linkages, but the active ingredient is caused by the degeneration of protein and peptide drugs by the release of hydrogen ions from the ester bonds. There is a problem that greatly impairs the stability of many attempts to solve this has been done.

In this regard, US Pat. No. 6,946,145 relates to a method for delivering a drug using a hydrophilic and lipophilic polyorthoester copolymer containing an amine group in the solubilization or encapsulation of a poorly soluble drug such as an anticancer agent. The technique is disclosed. The polyorthoester copolymer spontaneously associates in an aqueous solution to form micelles, and is a polymer used for sustained release of a drug.

U.S. Patent No. 5,904,936 uses a polymer based on a hydrophilic and lipophilic polyamino acid copolymer using two or more amino acids as a carrier of a drug, wherein the polymer spontaneously associates in an aqueous solution to form particles. Disclosed is a technique for transporting a drug such as a protein in a particle to be transported into the body so that the drug can be released slowly in the body.

PCT International Application WO 99/018142 is a copolymer of polyethylene glycol (PEG) -polylactic acid (PLA) -glycolic acid (PLGA), which exists in the liquid phase at room temperature, and forms a hydrogel when the temperature rises to body temperature. Techniques for drug delivery systems using sensitive polymers are disclosed.

U.S. Patent No. 6,800,663 discloses a copolymer of polyalphahydroxyacid-glycidyl methacrylate and polyethylene glycol to form a hydrogel through crosslinking to transport the drug and to control the release of the drug. Disclosed is a description of a delivery system.

In addition, Carbohydrate Polymers 69 (2007) p. 597-606 include Carboxymethyl Chitosan polymerized with Cholesterol, wherein the lipophilic cholesterol of the polymer spontaneously associates in an aqueous solution, and the hydrophilic portion of the polymer is negatively charged. Techniques for drug delivery systems using prominent nanoparticles have been disclosed.

Biomaterials 28 (2007) p.4132-4142 describes the use of copolymers of polyethylenimine and polycaprolactone to produce micelles with positive charges in aqueous solution, thereby delivering drugs such as nucleotides with negative charges into the body. A technique is disclosed.

As discussed above, although various polymers have been suggested for use in the delivery and delivery of drugs in the body, it is necessary to ensure that the degradation products are safe for the human body and that the sustained release of the drugs must be properly controlled without compromising the stability of the drugs. It is not enough to satisfy the essential conditions as a substance.

Accordingly, an object of the present invention is to solve the problems of the prior art and the technical problem that has been requested from the past.

That is, an object of the present invention is a polymer used for the delivery of pharmaceutical, food nutritional and cosmetic active ingredients, which can effectively transport the active ingredients in the body, can be easily degraded in the body, as well as decomposition products The present invention provides a hyaluronic acid-hydrophobic polyamino acid copolymer as a biocompatible, biodegradable novel substance which is safe for human body and enables sustained release of the active ingredient in the body.

The novel material according to the present invention for achieving this object relates to a hyaluronic acid-hydrophobic polyamino copolymer comprising a hyaluronic acid unit and a hydrophobic polyamino acid unit.

The copolymer of hyaluronic acid-hydrophobic polyamino acid forms micelles in an aqueous solution, and the hydrophobic polyamino acid and the active ingredient are physically combined in the micelle, thereby preventing the access of water (h ₂ o) in the body to Sustained release properties can be greatly increased. In addition, due to their biodegradation properties, they are easily degraded in the body and are used for the transport and sustained release of drugs such as proteins, organic molecules including peptides and nucleotides, active ingredients such as food additives and cosmetic ingredients, and especially drugs such as proteins and peptides. It can be useful.

The copolymer may form a copolymer of various structures, for example, a random copolymer, a block copolymer, a graft copolymer, and the like, but preferably, may be a structure of a block copolymer or a graft copolymer. More preferably, it may be a graft copolymer.

Hyaluronic acid, which is a component of the copolymer, is a biopolymer material in which repeating units consisting of N-acetyl-D-glucosamine and D-glucuronic acid are linearly connected, and various kinds of eyeglasses, synovial fluid of joints, chicken chives, etc. Extracted and purified from species and tissues by acid solubilization, alkali solubilization, neutral solubilization and enzyme solubilization. Such hyaluronic acid can be produced in various molecular weights according to extraction and purification methods, determination methods, etc., for the purposes of the present invention, its molecular weight is preferably 500,000 Da or more, more preferably 1,000,000 Da or more, particularly preferably And 1,000,000 to 3,000,000 Da.

The polyamino acid, which is another component of the copolymer, is composed of hydrophobic amino acids, which can increase the hydrophobicity of the formulation and consequently increase the sustained release properties of drugs such as physiologically active insulin. .

As used herein, the term "hydrophobic amino acid" refers to an amino acid having a relatively lower solubility than amino acid (hydrophilic amino acid) having high solubility in water. For example, leucine, isoleucine, and methionine. (Methionine), alanine (Alanine), phenylalanine (Phenylalanine), tryptophan (Tryptophan), valine (Valine) and the like, but are not limited to these. Especially, leucine, isoleucine, and phenylalanine are especially preferable. These hydrophobic amino acids may be used alone or in combination of two or more.

Such hydrophobic polyamino acids can be synthesized from N-carboxy anhydrides (NCAs) of hydrophobic amino acids and form the hydrophobic portion of the hyaluronic acid-hydrophobic poly amino acid copolymer.

In one preferred embodiment, the hydrophobic polyamino acid is a polyleucine synthesized from N-carboxy anhydride (NCA) of leucine, a polyisoleucine synthesized from N-carboxy anhydride (NCA) of isoleucine, or an N-carboxy anhydride of phenyl alanine. Polyalanine synthesized from (NCA).

The dry weight ratio of the hyaluronic acid unit and the hydrophobic polyamino acid unit in the copolymer is preferably 50: 1 to 1: 1, more preferably 30: 1 to 2: 1, particularly preferably 10: 1 to 3: 1. to be.

The synthesis of the copolymer can preferably be carried out on an organic solvent, in which case the hydrophilic group can be substituted with an organic salt such as a tetrabutyl ammonium (TBA) salt in order to dissolve the hydrophilic hyaluronic acid in the organic solvent. Then, N-carboxy anhydride (NCA) of the amino acid can be added to the organic solvent to carry out the polymerization. At this time, the polymerization reaction is initiated by the hydroxy (-OH) group of the hyaluronic acid. Since the polymerization of N-carboxy anhydride (NCA) of the amino acid is known in the art, a detailed description thereof is omitted below (see Kricheldorf, α-Aminoacid-N-carboxyanhydrides and Related Heterocycles, Chap. 2, 51-157, Springer-Verlag, Paris, 1987).

The present invention also provides a composition comprising a hyaluronic acid-hydrophobic poly amino acid copolymer, and one or more active ingredients, and a pharmaceutical or food nutritional or cosmetic composition comprising the same.

The active ingredient is not particularly limited as long as it is required to be transported in the body, and may be, for example, a protein, a peptide, a nucleotide, and an organic small compound having a hydrophobic or hydrophilic functional group.

In one preferred embodiment, the active ingredient may be a pharmacologically effective amount of insulin.

The term “therapeutically effective amount” refers to the reduction or reduction of one or more of the symptoms of the disorder requiring treatment to some extent or the initiation of a clinical marker or symptom of a disease requiring prevention. Means the amount of active ingredient effective to delay. Thus, a pharmacologically effective amount can be used to determine (1) the effect of reversing the rate of progression of the disease, (2) the effect of inhibiting further progression of the disease to some extent, and / or (3) one or more symptoms associated with the disease. It means the quantity which has the effect of reducing a grade (preferably, removing it). A pharmacologically effective amount can be determined empirically by experimenting with compounds in known in vivo and in vitro model systems for diseases in need of treatment.

The insulin is a polypeptide with a very short half life of about 30 minutes, and has a very fast rate of absorption and loss in the body. Therefore, the diabetic patient had the inconvenience of self-injecting insulin three times a day in order to maintain a constant blood level of insulin.

On the other hand, the copolymer of hyaluronic acid-hydrophobic polyamino acid according to the present invention adds hydrophobicity to the hydrophilic hyaluronic acid by polyamino acid to form microparticles spontaneously in hydrophobic parts in an aqueous solution. And insulin are physically bonded rather than chemically bound, thereby inhibiting the access of water (H ₂ O) in the body, thereby greatly increasing the stability of insulin drugs and sustained release properties of insulin. Therefore, there is an advantage that the discomfort of the patient due to insulin administration can be minimized.

The microparticles may be spherical, non-spherical or irregular in shape, and the size may preferably be 1 to 500 μm as an injectable size.

To improve the sustained release properties of the active ingredient, the copolymer of hyaluronic acid-polyamino acid is further enhanced synergistic sustained release effect when the active ingredient is included in an amount that can be continuously released and absorbed. Can be obtained.

As used herein, "amount sufficient to induce sustained release and absorption of the active ingredient" means an amount sufficient to inhibit the rate at which the active ingredient is released and absorbed into the blood. Thus, based on the total dry weight of the composition, the copolymer of hyaluronic acid and hydrophobic amino acid may be included at 50 to 99.9% by weight, preferably 70 to 99% by weight, more preferably 90 to 95% by weight.

In one preferred embodiment, the pharmaceutical composition of the invention comprises i) a hyaluronic acid-poly leucine copolymer 90-95 having a dry weight ratio of hyaluronic acid with a molecular weight of 1,000,000 to leucine N-carboxy anhydride (NCA) of from 10: 1 to 3: 1. Weight percent, and ii) a pharmacologically effective amount of insulin, and iii) a microparticle formulation in which insulin as an active ingredient is physically bound with a hyaluronic acid-poly leucine copolymer in aqueous solution.

When the composition according to the present invention is used as a food nutrition composition or a cosmetic composition, a food nutritionally acceptable carrier or cosmetically acceptable carrier may be further included.

A composition comprising a food nutritionally acceptable carrier can be used or added to, for example, a health food. Here, the term "health food" means a food product which has improved the function of the general food product by adding the composition according to the present invention to the general food product. The composition according to the invention can be added to general foods, or can be prepared by encapsulation, powdering, suspensions and the like. As such, when ingesting a health food containing the composition according to the present invention, unlike the general medicine because the food is a raw material has the advantage that there is no side effect that may occur when taking for a long time.

When using the composition according to the invention as a food additive, it may be added as it is or used with other foods or food ingredients, and may be suitably used according to conventional methods. The blending amount of the active ingredient may be appropriately determined depending on its purpose of use (prevention, health or therapeutic treatment).

There is no particular limitation on the kind of food. Examples of the food to which the metal-acid amino acid chelate can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, and the like. , Beverages, tea, drink, alcoholic beverages and vitamin complexes.

The composition according to the invention can be administered via several routes.

In the present invention, "administration" means introducing a predetermined substance into a patient by any suitable method and the route of administration of the composition of the present invention may be administered through any general route as long as the drug can reach the target tissue. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, nasal administration, ocular administration, topical administration, intranasal administration, intrapulmonary administration, for injection, to be administered rectally It may be formulated in a form such as oral or oral, but is not limited thereto. However, when oral administration, since the insulin is digested, oral compositions have disadvantages such as coating the active agent or formulating it to be protected from degradation in the stomach, and therefore, may be preferably administered in the form of an injection, more preferably Is administered subcutaneously. In addition, the compositions of the present invention may be administered by any device in which the active substance may migrate to target cells.

On the other hand, the composition of the present invention may optionally include a pharmaceutically acceptable carrier depending on the route of administration, may include a stabilizer for increasing the stability of the active ingredient. In particular, when the active ingredient is insulin, because it is not stable because it is easily aggregated or degraded as is known, it may include a stabilizer that can increase the stability of insulin. Such stabilizers may be covalently or non-covalently bound to the active ingredient, for example, sugars such as sucrose, lactose, glucose, mannitol, glycerol, and the like. Polyols, surfactants such as polysorbate, preservatives such as cresol, phosphates, inorganic salts, and the like, but are not limited thereto. In addition, the stabilizers may be used alone or in combination of two or more components, the type and amount of stabilizers suitable for insulin can be easily selected and used by those skilled in the art.

The present invention also relates to a sustained release injectable comprising dispersing the above-mentioned composition in a solution for injection.

As the solution for injection, an aqueous solution for injection such as distilled water for injection and buffer for injection can be preferably used. If necessary, the injection solution may further include a buffer, a pH adjuster, an isotonic agent, a dispersant, a preservative, a painless agent, a preservative, and the like.

The actual dosage of the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

The present invention also provides a method of increasing the in vivo persistence of an active ingredient exerting a pharmacological effect by including the composition (see FIG. 1).

For example, in the case of the injection of the microparticles (see Example 4) in which the hyaluronic acid-poly leucine copolymer and insulin are bound, the sustained release is significantly higher than that of the injection containing insulin alone (see Comparative Example 1).

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1 Preparation of Hyaluronic Acid Tetrabutyl Ammonium Salt

3 g of hyaluronic acid with a molecular weight of 1,000,000 were dissolved in 300 ml of 5% aqueous tetrabutyl ammonium hydroxide solution. Dialysis membrane of MWCO 12,000 was dialyzed in excess purified water for 18 hours to remove the unsubstituted tetrabutyl ammonium hydroxide. The dialysis solution was lyophilized for 3 days.

Example 1 Preparation of Hyaluronic Acid-Polyleucine Copolymer

1 g of hyaluronic acid tetrabutyl ammonium salt was placed in 200 ml of dimethylsulfoxide and dissolved at 60 ° C. with stirring. 0.2 g of leucine N-carboxy anhydride was dissolved in 20 ml of toluene, put into a solution of tetrabutyl ammonium hyaluronate and reacted at 60 ° C. for 18 hours. 100 ml of 4 M sodium chloride solution was added thereto, and 300 ml of ethanol was added to precipitate the reaction. 1 L of ethanol was washed and centrifuged at 3000 rpm for 10 minutes to recover the reaction. The ethanol was removed for 2 hours by vacuum drying. The dried reactant was dissolved in 100 ml of purified water and dialyzed in excess purified water with dialysis membrane of MWCO 12,000 for 18 hours to remove sodium chloride. The solution of the dialysis finished hyaluronic acid-poly leucine copolymer was lyophilized for 3 days to prepare a hyaluronic acid-poly leucine copolymer. The dried mass was 1 g with a yield of 83%.

Example 2: Preparation of Hyaluronic Acid-Poly Phenylalanine Copolymer

A hyaluronic acid-poly phenylalanine copolymer was prepared in the same manner as in Example 1, except that 0.1 g of phenylalanine N-carboxy anhydride was used instead of 0.2 g of leucine N-carboxy anhydride. The dried mass was 1 g with a yield of 91%.

Example  3: insulin and hyaluronic acid Poly  Preparation of Microparticles of Leucine Copolymer

20 mg of insulin was dissolved in 12 ml of 0.01 M hydrochloric acid solution. The pH of the insulin solution was adjusted to 7.4 using 1 N sodium hydroxide solution. 200 mg of the hyaluronic acid-poly leucine copolymer was dissolved in an insulin solution to prepare microparticles in which insulin was physically bound to the hyaluronic acid poly leucine copolymer.

Comparative Example 1: Preparation of Insulin Solution

20 mg of insulin was dissolved in 12 ml of 0.01 M hydrochloric acid solution. The pH of the insulin solution was adjusted to 7.4 using 1 N sodium hydroxide solution.

Experimental Example 1 Measurement of Microparticle Size of Hyaluronic Acid-Polyleucine Copolymer

The size of the microparticles of the insulin and hyaluronic acid-poly leucine copolymer prepared in Example 3 was measured by the light scattering method (Mastersizer 2000, Malvern Instruments). The results are shown in Table 1 below.

TABLE 1

Experimental Example 2: Proton NMR Measurement of Hyaluronic Acid-Polyleucine Copolymer

In order to confirm the grafted degree of the leucine peak and the poly leucine of the hyaluronic acid-poly leucine copolymer prepared in Example 1, it was measured using proton NMR spectroscopy. The results are shown in Tables 2 and 3.

TABLE 2

TABLE 3

As shown in the tables, it can be seen that poly leucine is bound at a rate of about 30% of the total units of hyaluronic acid.

Experimental Example  3: hyaluronic acid Poly  Association degree of leucine copolymer with interferon alpha

According to the mass ratio of the hyaluronic acid-poly leucine copolymer prepared in Example 1, the degree of the association of interferon alpha was measured using size SEC-HPLC. The results are shown in Table 4.

TABLE 4

Referring to Table 4, it can be seen that the hyaluronic acid-poly leucine copolymer and the active ingredient have an excellent association degree of 96.3% by having a weight ratio of 100: 1.

Experimental Example  4: insulin and hyaluronic acid Poly  Animals of Microparticles Prepared from Leucine Copolymer room Hum

In this experiment, it was confirmed that insulin was sustained by administering to the rat microparticles prepared from insulin and hyaluronic acid-polyleucine copolymer. An aqueous solution of insulin concentration 1.67 mg / ml was prepared in Example 3 and Comparative Example 1, respectively. The insulin solution of Comparative Example 1 was used as a control. 1, 2, 4, 8, 24 hour time points after administration of 0.5 ml of insulin solution and microparticle aqueous solution formed from insulin and hyaluronic acid-polyleucine copolymers subcutaneously in rats (Sprague Dawley Rat, male, 7-8 weeks old) Blood was collected at. Serum was separated and the blood serum concentration (μIU / ml) was measured by enzyme-linked immunosorbent assay (ELISA). The results are shown in FIG.

As shown in Figure 1, in the animal experiments of the microparticle formulation of Example 3 it can be seen that the maximum concentration of insulin in the blood is lowered, the half-life is also much increased compared to the insulin solution of Comparative Example 1. Therefore, it can be seen that the microparticle formulation formed by combining the hyaluronic acid-hydrophobic polyamino acid copolymer and insulin according to the present invention has a sustained-release effect on insulin.

As described above, the hyaluronic acid-hydrophobic polyamino acid copolymer according to the present invention spontaneously constitutes microparticles in an aqueous solution, induces sustained release in the body through physical bonding with the active ingredient, and secures the biological stability of the active ingredient in the body. can do.

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

1 is a graph showing the blood concentration of insulin when the microparticles of Example 3 according to the present invention and Comparative Example 1 were administered to mice.

Claims (18)

A hyaluronic acid-hydrophobic polyamino acid copolymer comprising a hyaluronic acid unit and a hydrophobic polyamino acid unit. The hyaluronic acid-hydrophobic polyamino acid copolymer according to claim 1, wherein the hyaluronic acid has a molecular weight of 1,000,000 or more. The hyaluronic acid-hydrophobic polyamino acid copolymer according to claim 1, wherein the polyamino acid is synthesized from N-carboxy anhydride (NCA) of hydrophobic amino acids to form a hydrophobic portion of the hyaluronic acid-hydrophobic polyamino acid copolymer. The method of claim 3, wherein the hydrophobic amino acid is selected from the group consisting of Leucine, Isoleucine, Methionine, Alanine, Phenylalanine, Tryptophan, and Valine. Hyaluronic acid-hydrophobic polyamino acid copolymer, characterized in that one or two or more. 4. The poly amino acid according to claim 3, wherein the poly amino acid is a poly leucine synthesized from N-carboxy anhydride (NCA) of leucine, a poly isoleucine synthesized from N-carboxy anhydride (NCA) of isoleucine, or an N-carboxy anhydride of phenyl alanine. It is a polyphenyl alanine synthesize | combined from (NCA), The hyaluronic acid-hydrophobic poly amino acid copolymer characterized by the above-mentioned. The hyaluronic acid-hydrophobic polyamino acid copolymer according to claim 1, wherein the weight ratio of hyaluronic acid to hydrophobic polyamino acid in the copolymer is 50: 1 to 1: 1. The hyaluronic acid-hydrophobic polyamino acid copolymer according to claim 1, wherein the copolymer is prepared by polymerizing N-carboxy anhydride (NCA) of hyaluronic acid and hydrophobic amino acid substituted with an organic salt in an organic solvent. A composition comprising a hyaluronic acid-hydrophobic poly amino acid copolymer according to any one of claims 1 to 7, and one or two or more active ingredients. The composition of claim 8, wherein the composition further comprises a pharmaceutically acceptable carrier, a food nutritionally acceptable carrier, or a cosmetically acceptable carrier. 9. The composition of claim 8, wherein the active ingredient is selected from the group consisting of proteins, peptides, nucleotides, and organic small compounds having hydrophobic or hydrophilic functional groups. The composition of claim 10, wherein said active ingredient is a pharmacologically effective amount of insulin. delete The composition of claim 8, wherein the content of the copolymer is 50 to 99.9% by weight based on the total dry weight of the composition. 9. The composition of claim 8, wherein the composition is in the form of microparticles. 15. The composition according to claim 14, wherein the microparticles are particles having an average particle size of 1 to 500 m and sustained release of the active ingredient in a predetermined solvent. The composition of claim 8, wherein the composition is formulated for injection, transdermal or oral so that it can be administered via the oral, nasal, ocular, subcutaneous, intravenous, intramuscular, intraperitoneal route. Sustained release injection in which the composition of claim 8 is dispersed in a solution for injection. delete

Images