JP4460665B2 - Bioabsorbable drug carrier - Google Patents

Description

【００３９】
表１より、調製例１で得られたヒアルロン酸ゲルは、４日後で３３％残存していたのに対し、ヒアルロン酸溶液は０％であり、本発明のヒアルロン酸ゲルは、生体内滞留時間が長いことが示唆される。
即ち、ヒアルロン酸溶液と比較して、生体内での分解吸収速度が遅く、薬物担体として有用な性質を有していることが示唆される。
【００４０】
実施例４：薬物保持試験
調製例１の難水溶性ゲルを各種薬物の２５ｍｇ／５ｍｌ水溶液に入れ、２５℃２４時間静置後のゲル周囲の水溶液中の各薬物濃度を吸光度により測定し、ゲルへの薬物の保持率を算出した。その結果を表２に示す。
【００４１】
【表２】

【００４２】
表２より、いずれの薬物もヒアルロン酸ゲルへの保持率は９５％以上と高かった。
【００４３】
実施例５：薬物放出試験
実施例２において薬物を保持したゲルを透析チューブに入れ、５００ｍｌのリン酸緩衝生理食塩水ｐＨ７．４に対して３７℃で透析した。経時的に透析外液の吸光度を測定し、ゲルからの薬物の放出率を算出した。その結果を表３に示す。
【００４４】
【表３】

【００４５】
表３より、各薬物ともいずれも、持続的に放出されていることがわかる。 即ち、持続放出システムにも利用できることが示唆される。
【００４６】
【発明の効果】
本発明により、ヒアルロン酸単独で形成された難水溶性のヒアルロン酸ゲルを含有する生体吸収性薬物担体を提供することができる。かかる本発明の生体吸収性薬物担体は、架橋剤等を使用していないため、安全性及び生体適合性に優れ、それ自体で、あるいは他の生体適合性物質と組み合わせて、注入、吸入、インプラント、及び経口投与等の薬物担体として利用でき、さらに、持続放出システムにも利用できる効果を奏する。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a bioabsorbable drug carrier containing a gel formed of hyaluronic acid alone, which is hardly soluble in a neutral aqueous solution.
[0002]
[Prior art]
In general, when a drug is administered into the body by a route such as oral, intravenous injection, or intramuscular injection, it is distributed throughout the body along the circulatory system, and is metabolized and excreted via the liver or kidney.
However, in order to make the most effective use of the pharmacological activity of the drug, it is desirable to allow the drug to act on the target site at an appropriate concentration for an appropriate time. Attempts have been made to devise dosage forms by using them and to exert more effective drug effects.
[0003]
The bioabsorbable material used for such applications is preferably biocompatible and can be molded into various forms depending on the application, and lactic acid / glycolic acid, collagen, hyaluronic acid, etc. are used. Yes. Among them, hyaluronic acid is a substance that is inherently present in the human body, and since it has a long track record of use as a pharmaceutical product such as an ophthalmic surgical aid and a joint injection, its biocompatibility has been sufficiently confirmed. However, since hyaluronic acid is rapidly absorbed in the body, its use as a drug carrier has been limited.
[0004]
Thus, attempts have been made to combine with other materials or to chemically modify hyaluronic acid to make it poorly water-soluble. For example, a preparation (Nikkei Biotech 1994.3.28) containing a mixture of erythropoietin and hyaluronic acid in a cylinder made of lactic acid / glycolic acid copolymer, hyaluronic acid and aliphatic, aromatic aliphatic, And the like (Patent No. 2569012) and the like for pharmacologically active substances consisting of complete or partial esters.
[0005]
However, these do not form a carrier with hyaluronic acid itself, the former is a substance in which other components substantially function as a carrier, and the latter is a substance whose chemical structure is different from that of the original hyaluronic acid. It has become.
[0006]
[Problems to be solved by the invention]
In order to take full advantage of the excellent biocompatibility inherent in hyaluronic acid itself, it forms a complex with a cationic polymer compound without using any chemical cross-linking agent or chemical modifier. No means has been developed so far to make the hyaluronic acid itself water-insoluble. We have found for the first time that a hyaluronic acid gel composed of hyaluronic acid alone can be produced by a simple method without using a cross-linking agent (PCT / JP98 / 03536), and this time, bioabsorption of this poorly water-soluble hyaluronic acid gel The present inventors have intensively studied the possibility of application to a sex drug carrier, found its usefulness, and completed the present invention.
[0007]
[Means for Solving the Problems]
That is, the present invention is (1) a bioabsorbable drug carrier containing a gel formed of hyaluronic acid alone that is hardly soluble in a neutral aqueous solution, and (2) satisfies the following requirements (a) and (b): A bioabsorbable drug carrier comprising a gel formed of hyaluronic acid alone, (a) a neutral 37 ° C. aqueous solution having a solubility in 12 hours of 50% or less, (b) hyaluron Hyaluronic acid solubilized by treating hyaluronic acid gel under acid-promoted acid hydrolysis conditions has a branched structure, and the solubilized hyaluronic acid has a molecular weight fraction with a degree of branching of 0.5 or more. (3) The gel formed of hyaluronic acid alone is one kind selected from the group consisting of sheet, film, crushed, sponge, lump, fiber, or tube (2) raw characterized by Absorbent drug carrier, (4) Neutral 37 ° C aqueous solution with a dissolution rate of 12% or less in 12 hours, solubilized by treating hyaluronic acid gel under accelerated acid hydrolysis conditions of hyaluronic acid A hyaluronic acid gel partially containing a molecular weight fraction having a degree of branching of 0.5 or more in the hyaluronic acid, and a bioabsorbable drug carrier containing non-gelled hyaluronic acid, (5) sheet, film, A bioabsorbable drug carrier comprising hyaluronic acid gel formed of crushed, sponge-like, lump-like, fiber-like, or tube-like hyaluronic acid alone and non-gelled hyaluronic acid.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The hyaluronic acid gel referred to in the present invention is a polymer having a three-dimensional network structure and a swollen body thereof. The three-dimensional network structure is formed by a crosslinked structure of hyaluronic acid.
[0009]
As an example, it is hardly soluble in a neutral aqueous solution in the form of a sheet, film, crushed, sponge, lump, fiber, or tube by freezing and then thawing an aqueous solution of hyaluronic acid having a pH of 3.5 or less. A hyaluronic acid gel can be obtained. More specifically, it will be described below.
[0010]
The hyaluronic acid used in the present invention may be extracted from animal tissue or manufactured by fermentation without regard to its origin.
The strain used in the fermentation method is a microorganism having the ability to produce hyaluronic acid such as Streptococcus genus isolated from the natural world, or Streptococcus equii FM-100 described in Japanese Patent Laid-Open No. Sho 63-123392 (90 pp. 90,000). No.), and a mutant that stably produces hyaluronic acid at a high yield, such as Streptococcus equi FM-300 described in JP-A-2-23489, is available. Those cultured and purified using the above mutant strains are used.
[0011]
The molecular weight of hyaluronic acid used in the present invention is preferably in the range of about 1 × 10 ⁵ to about 1 × 10 ⁷ daltons. Moreover, as long as it has a molecular weight within the above range, those obtained by subjecting it to a hydrolysis treatment or the like from a higher molecular weight can also be preferably used.
In addition, the hyaluronic acid said to this invention is used by the concept also including the alkali metal salt, for example, the salt of sodium, potassium, and lithium.
[0012]
Hyaluronic acid alone in the present invention means that no chemical cross-linking agent or chemical modifier other than hyaluronic acid is used, and that it is not complexed with a cationic polymer, which means self-crosslinking. It is.
[0013]
The hyaluronic acid gel as referred to in the present invention can be decomposed and solubilized by treating the hyaluronic acid gel under the conditions of accelerated acid hydrolysis reaction of hyaluronic acid. When the solubilized hyaluronic acid retains a crosslinked structure, it can be distinguished from linear hyaluronic acid in terms of polymer solution as hyaluronic acid having a branching point.
[0014]
As the conditions for the accelerated acid hydrolysis reaction of hyaluronic acid in the present invention, the pH of the aqueous solution and the temperature of 60 ° C. are suitable. The main chain cleavage reaction by hydrolysis of the glycosidic bond of hyaluronic acid is significantly accelerated in acidic and alkaline aqueous solutions as compared to neutral aqueous solutions. Further, the acid hydrolysis reaction is promoted at a higher reaction temperature.
[0015]
In the present invention, the GPC-MALLS method was used, and the molecular weight and the degree of branching of the molecular weight fraction separated by GPC were continuously measured online. In the present invention, the degree of branching is measured using an elution volume method that calculates the degree of branching by comparing the molecular weight of the solubilized hyaluronic acid in the same elution volume fraction with the molecular weight of the linear hyaluronic acid as a reference. It was. The degree of branching is the number of branch points present per polymer chain of solubilized hyaluronic acid and is plotted against the molecular weight of solubilized hyaluronic acid.
[0016]
The solubilized hyaluronic acid was diluted with a GPC solvent to adjust the concentration, filtered through a 0.2 μm membrane filter, and subjected to measurement.
When the hyaluronic acid gel referred to in the present invention has a crosslinked structure that exists stably even under the conditions of accelerated acid hydrolysis of hyaluronic acid, a branched structure is confirmed in the solubilized hyaluronic acid in a polymer solution theory. The degree of branching of the hyaluronic acid gel referred to in the present invention is 0.5 or more.
[0017]
As the acid used for adjusting the pH of the aqueous solution of hyaluronic acid, any acid can be used as long as it can be adjusted to pH 3.5 or lower. In order to reduce the amount of acid used, it is preferable to use a strong acid such as hydrochloric acid, nitric acid, sulfuric acid or the like.
[0018]
The pH of the aqueous solution of hyaluronic acid is adjusted to a pH at which the carboxyl group of hyaluronic acid is protonated at a sufficient rate. The pH to be adjusted is appropriately determined according to various properties such as the type of counter ion of hyaluronate, the molecular weight of hyaluronic acid, the concentration of the aqueous solution, the conditions for freezing and thawing, and the strength of the gel to be formed. .5 or less, preferably, pH 2.5 or less.
[0019]
Freezing and thawing are performed by placing an acidic aqueous solution of hyaluronic acid in an arbitrary container, freezing at a predetermined temperature, and thawing at a predetermined temperature after freezing. The temperature and time for freezing and thawing are appropriately determined within the range of temperature and time for freezing and thawing of an acidic aqueous solution of hyaluronic acid depending on the size of the container and the amount of the aqueous solution. Generally, the freezing temperature is below the freezing point and above the freezing point. The thawing temperature is preferred.
Since freezing and thawing time can be shortened, a freezing temperature of −5 ° C. or lower and a thawing temperature of 5 ° C. or higher are more preferable. The time is not particularly limited as long as it is longer than the time at which freezing and thawing are completed at that temperature.
[0020]
The number of repetitions of freezing and then thawing the adjusted acidic aqueous solution of hyaluronic acid depends on the molecular weight of the hyaluronic acid used, the concentration of the aqueous solution, the pH of the aqueous solution, the temperature and time of freezing and thawing, and the strength of the gel produced It is determined as appropriate according to various characteristics. Usually, it is preferably repeated one or more times.
Further, each time the operation of freezing and thawing is repeated, the temperature and time for freezing and thawing may be changed.
[0021]
In order to avoid acid hydrolysis of hyaluronic acid, the hyaluronic acid gel obtained by freezing and thawing the acidic solution with adjusted hyaluronic acid needs to remove components such as acid used for adjusting to acidic. In order to remove components such as acids, washing with an aqueous solvent or dialysis is usually performed.
The solvent to be used is not particularly limited as long as it does not impair the function of the hyaluronic acid gel. For example, water, physiological saline, phosphate buffer and the like are used, and preferably physiological saline, phosphoric acid A buffer solution or the like is used.
[0022]
The washing and dialysis methods are not particularly limited, but usually the batch method, filtration method, method of filling the column etc. and passing the liquid, etc. In the case of dialysis, dialysis membranes and ultrafiltration membranes are used. A method or the like is preferably used. These conditions include conditions such as the amount of liquid, the number of times, etc., as long as the components to be removed can be reduced to a target concentration or less, and can be appropriately selected depending on the form and use of the hyaluronic acid gel.
[0023]
This washed and dialyzed hyaluronic acid gel is bioabsorbed in a state of being immersed in a solvent, a wet state containing a solvent, an air-dried, a vacuum-dried or a freeze-dried state depending on the purpose of use. It serves as a sex drug carrier.
[0024]
Processes such as molding of hyaluronic acid gel can be made in the form of sheets, films, crushed, sponges, fibers, or tubes, depending on the choice of container and method when freezing the acidic solution with adjusted hyaluronic acid. It is possible to produce a hyaluronic acid gel of the desired form. For example, film-like and sheet-like forms can be obtained by casting on a plate and freezing, and a crushed form can be obtained by freezing and thawing with vigorous mixing and stirring with an organic solvent immiscible with water.
[0025]
By changing conditions such as the molecular weight and concentration of hyaluronic acid, various poorly water-soluble hyaluronic acid gels having different bioabsorbability can be obtained.
[0026]
The bioabsorbable drug carrier of the present invention can be formulated on its own or together with other biocompatible substances, generally known pharmaceutical additives, etc., and drugs such as injection, inhalation, implant, oral administration, etc. It can be used as a carrier.
[0027]
The bioabsorbable drug carrier containing the poorly water-soluble hyaluronic acid gel of the present invention can be held on the carrier without impairing the activity of the drug by noncovalent bonds such as ionic bonds. Quaternary ammonium salt (biochemical experiment course 4 carbohydrate chemistry (top) p136), chlorpromazine (N. Caram-Lelhametal. Biopoly, 41: 765), which is known to have an affinity for hyaluronic acid. -1997), various metal ions (Japanese Patent Application Laid-Open No. 5-124968, Japanese Patent Application Laid-Open No. 2-502547, Japanese Patent Application Publication No. 63-502670) and the like are also used in the poorly water-soluble hyaluronic acid gel of the present invention. Can be easily held. Furthermore, another drug can be held through these substances having affinity. For example, a metal protein can be retained via a metal ion.
[0028]
Specific examples of drugs having an affinity for hyaluronic acid include cetylpyridinium chloride, cetyltrimethylammonium chloride, benzethonium chloride, benzalkonium chloride, carnitine, butylscopolamine, barretamate, methylbenactidium, ipratropium, carbachol, ethylpipeta Examples include narate, thimepidium, butropium, prifinium, chlorpromazine, promazine, doxepin, and amitriptyline.
[0029]
Drugs that have no affinity for hyaluronic acid can be introduced into a carrier as an aqueous solution or dissolved in an aqueous solution of a biocompatible substance such as hyaluronic acid or serum albumin. It can be formulated by a method.
[0030]
The present invention can be used for treatment in various fields, and can be selected from the group consisting of sheet, film, crushed, sponge, lump, fiber, or tube depending on the application site. In the case of transmucosal administration, a sheet, film, or sponge is suitable for topical application, and in the case of pulmonary administration, crushing and dried size of 1 to 3 μm may cause coughing. It is suitable.
[0031]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
[0032]
Preparation Example 1: Preparation of poorly water-soluble hyaluronic acid gel Hyaluronic acid having a molecular weight of 2 × 10 ⁶ daltons was dissolved in distilled water to prepare a 1% solution. 1N nitric acid was added to this aqueous solution to adjust the pH to 1.5, and 25 mg of hyaluronic acid was placed in a vial and frozen in a freezer set at −20 ° C. The operation of taking out after 20 hours and thawing at 25 ° C. for 2 hours was repeated three times to obtain a slightly water-soluble gel.
Next, the gel was neutralized by immersion in 100 ml of 25 mM phosphate buffered saline at pH 7 for 24 hours, and then sufficiently filtered and washed with distilled water to obtain a lump-like slightly water-soluble gel.
[0033]
Example 1: Solubility test of poorly water-soluble hyaluronic acid gel A phosphate buffer component at a concentration of 50 mM was added to physiological saline to prepare a pH 7 phosphate buffered saline. The poorly water-soluble gel produced in Preparation Example 1 was immersed in 50 ml of phosphate buffered saline and gently stirred. The proportion of hyaluronic acid eluted in phosphate buffered saline at 37 ° C. was determined from the hyaluronic acid concentration in phosphate buffered saline.
[0034]
Measurement of Hyaluronic Acid Concentration Hyaluronic acid concentration in phosphate buffered saline was determined from the peak area of the differential refractive index detector using GPC.
[0035]
As a result, the dissolution rate of the hyaluronic acid gel obtained in Preparation Example 1 was 3% after 12 hours, 5% after 24 hours, and 85% hyaluron after 24 hours. An acid gel remained.
Therefore, it is suggested that the hyaluronic acid gel obtained in Preparation Example 1 is poorly soluble in a neutral aqueous solution.
[0036]
Example 2: Measurement of branching degree of sparingly water-soluble hyaluronic acid gel The sparingly water-soluble hyaluronic acid gel obtained in Preparation Example 1 was immersed in 15 ml of an aqueous hydrochloric acid solution having a pH of 1.5 and hydrolyzed at 60 ° C for 6 hours. It was. The gel is solubilized by hydrolysis, diluted twice with GPC solvent to a concentration of 0.05% by weight, filtered through a 0.2 μm membrane filter, and then injected with 0.1 ml of GPC- As a result of measuring MALLS, the degree of branching was 0.5 or more.
[0037]
Example 3 Bioabsorbability Test Mouse (ICR 8 weeks old) was placed in the abdominal cavity with the poorly water-soluble gel of Preparation Example 1 or 1 ml 1% hyaluronic acid solution as a comparison by laparotomy. 2, 4, and 8 days after the operation, the abdomen was opened, the remaining gel was collected, 2 ml of physiological saline was injected into the abdominal cavity, and the mixture was stirred well to collect the solution. The gel and liquid were subjected to alkali treatment (0.1 N NaOH, 2 h), neutralized, and the hyaluronic acid concentration was measured by gel filtration (ShodexOHpakKB806) to calculate the residual rate. The results are shown in Table 1.
[0038]
[Table 1]

[0039]
From Table 1, the hyaluronic acid gel obtained in Preparation Example 1 remained 33% after 4 days, whereas the hyaluronic acid solution was 0%. Is suggested to be long.
That is, compared with the hyaluronic acid solution, the decomposition and absorption rate in the living body is slow, suggesting that it has properties useful as a drug carrier.
[0040]
Example 4: Drug retention test The poorly water-soluble gel of Preparation Example 1 was placed in a 25 mg / 5 ml aqueous solution of various drugs, and each drug concentration in the aqueous solution around the gel after standing at 25 ° C for 24 hours was measured by absorbance. The retention rate of the drug was calculated. The results are shown in Table 2.
[0041]
[Table 2]

[0042]
From Table 2, the retention rate of all drugs in hyaluronic acid gel was as high as 95% or more.
[0043]
Example 5: Drug release test The gel retaining the drug in Example 2 was placed in a dialysis tube and dialyzed at 37 ° C against 500 ml of phosphate buffered saline pH 7.4. The absorbance of the external dialysis solution was measured over time, and the drug release rate from the gel was calculated. The results are shown in Table 3.
[0044]
[Table 3]

[0045]
From Table 3, it can be seen that all the drugs are released continuously. That is, it is suggested that it can be used for a sustained release system.
[0046]
【The invention's effect】
According to the present invention, a bioabsorbable drug carrier containing a poorly water-soluble hyaluronic acid gel formed with hyaluronic acid alone can be provided. Since the bioabsorbable drug carrier of the present invention does not use a crosslinking agent or the like, it is excellent in safety and biocompatibility, and it can be injected, inhaled, or implanted by itself or in combination with other biocompatible substances. It can be used as a drug carrier for oral administration and the like, and further has an effect that can be used for a sustained release system.

Claims (5)

The hyaluronic acid solubilized by treating the hyaluronic acid gel under accelerated acid hydrolysis conditions of hyaluronic acid was formed with hyaluronic acid alone, having a feature including a molecular weight fraction with a degree of branching of 0.5 or more A method for producing a sustained release carrier comprising a gel comprising :
a) a step of freezing and thawing a hyaluronic acid aqueous solution to produce a hyaluronic acid gel;
Including production methods. A method for producing the sustained release carrier according to claim 1 ,
b) a step of freezing and thawing twice for the hyaluronic acid gel or the aqueous solution containing the hyaluronic acid gel;
Including production methods. A method for producing a sustained release carrier according to claim 1 or 2 ,
The freezing time is 20 hours or more,
Production method. A method for producing a sustained release carrier according to any one of claims 1 to 3 ,
c) a step of immersing and neutralizing the hyaluronic acid gel obtained in step a) or b) with a buffer;
d) filtering and washing the hyaluronic acid gel neutralized by immersion in step c);
Including production methods. Obtained by the method of producing sustained-release agent carrier according to any one of claims 1 to 4, sustained release agent carrier.