JPH1067687A - Biodegradable gel composition cross-linked by hyaluronic acid and biodegradable gel medicine cross-linked by hyaluronic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition capable of performing a controlled release of a medicine according to the degradation without performing a diffusion release of the medicine and manifesting an excellent responsibility against a stimulation inducing biodegradation and biocompatibility by dispersing a specific water-soluble compound into a hyaluronic acid-cross-linked gel. SOLUTION: The subject composition is obtained by dispersing (B) a water-soluble polyalkylene glycol (e.g. the one having 200-50,000 number average molecular weight and, concretely, polyethylene glycol, polypropylene glycol and these block copolymer) into (A) a hyaluronic acid-cross-linked gel. A medicine is selectively immobilized to the component B by adding the medicine such as insulin and dexamethasone phosphate to the composition and maintained stably until the component A is degraded. When the degradation of the component A progresses, the medicine elutes out with the component B and thereby the composition is preferably used as a medicine carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a crosslinked gel composition of hyaluronic acid which is degraded in vivo and is useful as a drug carrier, and a biodegradable crosslinked gel composition of hyaluronic acid using the same.

[0002]

2. Description of the Related Art Heretofore, a drug having a biodegradable polymer material carrying a drug, which has been administered to a living body by means of intravenous injection, oral administration, subcutaneous implantation, etc. Drugs for decomposing molecular materials and releasing drugs with the decomposition and supplying them to target cells, tissues, organs, and organs are widely known as so-called pharmaceutical sustained-release agents.

In recent years, in consideration of the homeostasis of the living body, an intelligent preparation which releases a drug only when the living body needs it, such as at the time of disease, can be manufactured using a stimuli-responsive polymer. I have. As one kind of such a stimuli-responsive polymer, a hyaluronic acid cross-linked gel that is specifically degraded by hydroxyl radicals generated during inflammation is known. Among them, a heterogeneous structural gel in which lipid microparticles are dispersed as a drug retention domain is known. Reported by the present inventors (N. Yui et al., J. Controlled Release,
25, 113 (1993)).

[0004]

However, the present inventor has found that the above-mentioned conventional heterogeneous structure gel in which lipid microparticles are dispersed is not sufficient in the following points. That is, in the preparation using the above-mentioned conventional heterogeneous structure gel containing lipid fine particles, the response to a stimulus that induces biodegradation, biocompatibility, and the range of applicable sites are still insufficient. Was not.

The present invention has been made in view of the above-mentioned problems of the prior art, and it is an object of the present invention to provide a sustained release of a drug without releasing the carried drug by diffusion and responding to the decomposition of the drug itself. A drug carrier that has a drug release behavior that is rate-limiting, that is, has excellent response and biocompatibility to stimuli that induce biodegradation, and has a sufficiently wide range of applicable sites. The purpose is to provide a carrier.

Further, the present invention has a drug release behavior in which the drug contained therein is not released by diffusion and is capable of sustained release of the drug in response to its own decomposition, that is, the drug release behavior is rate-controlled. It is a preparation, which also has excellent responsiveness and biocompatibility to stimuli that induce biodegradation,
It is another object of the present invention to provide a preparation having a sufficiently wide range of applicable sites.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, the above object was achieved by dispersing a water-soluble polyalkylene glycol in a hyaluronic acid crosslinked gel. And completed the present invention.

That is, the present invention provides a biodegradable crosslinked gel of hyaluronic acid, comprising a crosslinked gel of hyaluronic acid and a water-soluble polyalkylene glycol dispersed in the crosslinked gel of hyaluronic acid. is there.

Further, the present invention provides a crosslinked gel composition of hyaluronic acid comprising a crosslinked gel of hyaluronic acid and a water-soluble polyalkylene glycol dispersed in the crosslinked gel of hyaluronic acid, and a composition selected from the group consisting of the above water-soluble polyalkylene glycol. Characterized by containing a drug,
It is a biodegradable hyaluronic acid crosslinked gel preparation.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the biodegradable crosslinked gel composition and crosslinked gel preparation of the present invention will be described in more detail.

The biodegradable crosslinked gel composition of the present invention contains a crosslinked gel of hyaluronic acid and a water-soluble polyalkylene glycol dispersed in the crosslinked gel of hyaluronic acid.

The crosslinked gel of hyaluronic acid according to the present invention comprises:
It has a three-dimensional network structure in which hyaluronic acid molecules are linked to each other by crosslinking. Suitable crosslinking agents for forming such crosslinking include glycidyl methacrylate, polyfunctional glycidyl ether, epichlorohydrin and the like, with glycidyl methacrylate being particularly preferred. The molecular weight of the hyaluronic acid according to the present invention is not particularly limited, and hyaluronic acid having a normal molecular weight (5 × 10 ⁴ to 8 × 10 ⁶ ) is used. It should be noted that the hyaluronic acid mentioned here also includes its salts, hydrates and the like.

A water-soluble polyalkylene glycol is dispersed in the hyaluronic acid crosslinked gel. In the hyaluronic acid crosslinked gel composition of the present invention, even if the water-soluble polyalkylene glycol molecules are simply dispersed in the three-dimensional network structure of the hyaluronic acid crosslinked gel, or by covalent bonds such as crosslinks to the hyaluronic acid molecules. They may be combined.

As the water-soluble polyalkylene glycol according to the present invention, those having an alkylene group having 2 to 6 carbon atoms are preferable, and polyethylene glycol and polypropylene glycol having an alkylene group having 2 to 3 carbon atoms are particularly preferable. The water-soluble polyalkylene glycol may be a copolymer of a plurality of types, and a block copolymer of polyethylene glycol and polypropylene glycol is particularly preferable.

The number average molecular weight of the polyalkylene glycol according to the present invention is preferably from 200 to 50,000, particularly preferably from 1,000 to 10,000. When the number average molecular weight is less than 200, the drug that can be supported is limited to a low molecular weight drug, and the drug tends to leak easily.
If it exceeds 0000, the alkylene glycol tends to be hardly dispersed in the hyaluronic acid crosslinked gel.

The amount of the water-soluble polyalkylene glycol dispersed in the crosslinked gel of hyaluronic acid is not particularly limited, but it is preferable that the polyalkylene glycol is 10 to 10,000 parts by weight based on 100 parts by weight of hyaluronic acid. . When the content of the polyalkylene glycol is less than the lower limit, the amount of the drug that can be supported in the polyalkylene glycol tends to be insufficient, and when the content exceeds the upper limit, the drug that is supported in the polyalkylene glycol is easily released by diffusion. This is because it tends to be.

In the biodegradable hyaluronic acid crosslinked gel preparation of the present invention, a drug is carried (held) in the above hyaluronic acid crosslinked gel composition.

As the drug according to the present invention, any of so-called physiologically active substances can be used without particular limitation, and amino acids, physiologically active peptides,
A wide range of substances such as steroids, nucleic acids, yeasts and bacterial cells are targeted. Specific examples include insulin, interferon, anti-inflammatory steroids (eg, dexamethasone phosphate), superoxide dismutase as an active oxygen scavenger, leuprolide acetate as a luteinizing hormone-releasing hormone, various growth factors, and monoclonals. Antibodies and the like are mentioned, and insulin and dexamethasone phosphate are particularly preferred. The content of the drug in the preparation of the present invention is not particularly limited, and is appropriately selected depending on the type of the drug and the like.

As described above, the drug added to the crosslinked gel composition of the present invention comprising the crosslinked hyaluronic acid gel and the water-soluble polyalkylene glycol dispersed therein is a water-soluble polyalkylene glycol. Distribute preferentially inside. That is, in the hyaluronic acid crosslinked gel preparation of the present invention, the drug is selectively carried in the water-soluble polyalkylene glycol. And, although both the hyaluronic acid cross-linked gel and the water-soluble polyalkylene glycol are hydrophilic, in the preparation of the present invention, the hyaluronic acid cross-linked gel enclosing the water-soluble polyalkylene glycol diffuses the drug. Acts as a so-called barrier to emission.

Therefore, drug diffusion is remarkable inside a normal hydrophilic polymer having a high water content, and although the drug is originally released by the diffusion before the polymer is decomposed, the drug of the present invention has a water-soluble property. The drug selectively carried on the hydrophilic polyalkylene glycol is stably maintained until the hyaluronic acid crosslinked gel is decomposed. The decomposition of the crosslinked gel of hyaluronic acid proceeds from the surface in response to hydroxyl radicals and the like generated at the time of inflammation, and the drug is eluted together with the water-soluble polyalkylene glycol containing it. As described above, in the preparation of the present invention, the release of the drug is determined not by the drug diffusion but only by the biodegradation of the hyaluronic acid crosslinked gel. That is, although the hyaluronic acid crosslinked gel itself has an extremely high water content and the drug diffusivity in the gel is high, the release of the drug is limited by the decomposition of the gel. Thus, the amount of drug released from the formulation of the present invention will be (proportional) to the amount of the component that induces the degradation of the hyaluronic acid crosslinked gel.

In the preparation of the present invention, not only the crosslinked gel of hyaluronic acid but also the water-soluble polyalkylene glycol is hydrophilic, so that the action of the biodegradation-inducing component on the crosslinked gel of hyaluronic acid is inhibited by the water-soluble polyalkylene glycol. I will not do it. Furthermore, since the water-soluble polyalkylene glycol released together with the drug is hydrophilic, the preparation of the present invention is excellent in biocompatibility and has a sufficiently wide applicable site range.

The method for producing the crosslinked gel composition of the present invention and the crosslinked gel preparation of the present invention are not particularly limited, and may be prepared in the presence of a polyalkylene glycol and, if necessary, a drug.
A method of causing a cross-linking reaction of hyaluronic acid using a cross-linking agent or the like is appropriately adopted.

The dosage form of the biodegradable hyaluronic acid crosslinked gel preparation of the present invention can be widely used as an implant for intravenous injection, oral administration, subcutaneous implantation, intraarticular injection, and the like. Therefore, according to the present invention, it becomes possible to obtain various physiologically active substance-containing preparations by administration routes such as intravenous injection, transfusion, and subcutaneous implantation, which have been difficult so far. Therefore, the present invention can be applied to all preparations containing a physiologically active substance to be administered from all routes such as intravenous injection, oral administration, subcutaneous implantation, and implantation in tissue.

[0024]

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

Example 1 (Evaluation test of partitioning property of FITC-labeled insulin in PEG-HA two-phase system) Using a 0.1 M phosphate buffer (PBS, pH 7.4), the number average molecular weight Mn was 1000, 6000, respectively. A 50,000 polyethylene glycol (PEG) solution (40 wt.%), A sodium hyaluronate (HA) solution (3.0 wt.%), And a FITC-labeled insulin solution (1 mg / ml) were each prepared, and these were mixed at a volume ratio of 0.75: 0.75. : 0.5 (weight ratio 4: 4: 1), stirred for 1 minute, and then left for 30 minutes. Then, the solution was separated from the upper phase and the lower phase of the above mixed solution, and then measured with a fluorescence spectrophotometer (excitation wavelength).
490 nm) to measure the fluorescence intensity of FITC-labeled insulin at 520 nm to determine the insulin concentration in each of the upper and lower phases. In addition, as sodium hyaluronate, sodium hyaluronate (trade name: HA-
Q) was used (the same applies hereinafter).

FIG. 1 shows the obtained results.

As is clear from FIG. 1, it was confirmed that insulin was preferentially distributed to the upper phase, ie, the polyethylene glycol phase, for polyethylene glycol of any molecular weight.

Example 2 ( Preparation of methacrylated hyaluronic acid (MA-HA)) Sodium hyaluronate (HA) (0.5 g) was added to 30 ml of an aqueous solution of sodium chloride-sodium carbonate (NaCl-Na ₂ CO ₃ ) (0.15 M, p
H9.0) and then glycidyl methacrylate (2.0m
1, 14.2 mmol) was added dropwise. After the obtained heterogeneous system was degassed under reduced pressure, the mixture was stirred at room temperature in a nitrogen atmosphere for 5 days. The obtained reaction solution was added dropwise to 400 ml of ethanol, and the precipitate formed in the solution was filtered by suction and dried under reduced pressure to obtain methacrylated hyaluronic acid (MA-HA) as a white fibrous substance.

(Preparation of crosslinked gel preparation containing polyethylene glycol containing hyaluronic acid) 0.1 M phosphate buffer (PBS, pH 7.
Using 4), methacrylated hyaluronic acid (MA-HA) solution (7.4wt.%), Number average molecular weight Mn is 1000, 6000, respectively
50,000 polyethylene glycol (PEG) solution (40 wt.
%) And a FITC-labeled insulin solution (1 mg / ml) were prepared, and these were mixed at a weight ratio of 90: 5: 5 and stirred. Ammonium persulfate (APS) (2 mg) and N, N, N ′, N′-tetramethylethylenediamine (TMEDA) (50 μl) were added to the resulting solution as a redox reaction initiator.
After about 3 minutes, a gel formed in the solution (gelation). The resulting polyethylene glycol-containing hyaluronic acid cross-linked gel is
After being immersed in 0.1 M phosphate buffer for 24 hours, it was used for the following tests.

(Test of Drug Release Characteristics of PEG-Containing HA Cross-Linked Gel in the Presence of Hyaluronidase) The polyethylene glycol-containing hyaluronic acid cross-linked gel preparation obtained above was converted into a plate-shaped hydrogel (25 × 20 × 2 mm), and these were placed in a nylon bag. And a hyaluronidase (HAase) -0.1MPBS (pH 7.4) solution (10 units /
ml, 50 ml).

After immersion, the decomposition behavior of the gel and the amount of drug released were measured over time as follows. That is,
The gel degradation rate is measured continuously by measuring the change in weight at regular intervals, and the amount of released insulin is continuously measured using a flow cell by measuring the fluorescence intensity of FITC-labeled insulin at 520 nm using a fluorescence spectrophotometer (excitation wavelength: 490 nm). By doing so, each sought.

FIG. 2 (decomposition rate) and FIG.
(Insulin release).

Comparative Example 1 A crosslinked gel preparation of hyaluronic acid was prepared in the same manner as in Example 2 except that polyethylene glycol was not added, and the decomposition behavior and the amount of released drug were measured over time in the same manner as in Example 2. The obtained results are shown in FIG. 2 (degradation rate) and FIG. 3 (insulin release amount).

As apparent from FIG. 2, the crosslinked gel of hyaluronic acid showed a linear decomposition behavior almost proportional to time regardless of the presence or absence of polyethylene glycol. On the other hand, as apparent from FIG. 3, the drug release characteristics from the hyaluronic acid crosslinked gel showed different behaviors depending on the presence or absence of polyethylene glycol. That is, in the presence of polyethylene glycol, the drug was released almost in proportion to time, whereas in the absence of polyethylene glycol, the drug was released almost in proportion to the square root of time.

From the above results, it was confirmed that the drug release characteristics of the polyethylene glycol-containing hyaluronic acid cross-linked gel preparation of the present invention corresponded (proportionally) to the gel decomposition. On the other hand, in the case of the preparation of the hyaluronic acid cross-linked gel alone containing no polyethylene glycol, a relatively large amount of the drug was released in the initial stage, so that the drug was released not only by the decomposition of the gel but also by the diffusion. confirmed.

Example 3 (Preparation of biodegradable crosslinked gel preparation of hyaluronic acid) Aqueous phosphate buffer solution (40 wt.% PEG / PB) of polyethylene glycol (number average molecular weight Mn: 6000) prepared in Example 2
Dexamethasone phosphate (DmNaP) was added to and dissolved in the S solution) at a concentration of 2 mg / ml. The resulting solution;
The methacrylated hyaluronic acid phosphate buffer aqueous solution (7.4 wt.% MA-HA / PBS solution) prepared in Example 2 was mixed at a weight ratio of 1: 9 and stirred, and ammonium persulfate (APS) was used as a redox reaction initiator. ) (10 mg / g) and N, N,
N ', N'-tetramethylethylenediamine (TMEDA) (50μl
/ g) was added to allow a crosslinking reaction to obtain a gel. The resulting polyethylene glycol-containing hyaluronic acid crosslinked gel was separated, washed, crushed, filled into a syringe, and administered to the following living body.

(Efficacy Evaluation Test in Urate-Induced Acute Arthritis Model) The efficacy of the polyethylene glycol-containing hyaluronic acid crosslinked gel preparation obtained above on acute arthritis (effect on gait disorders) was evaluated using the urate-induced acute arthritis model. The evaluation was made as follows. In addition, the urate-induced acute arthritis model is an arthritis model in which acute arthritis is induced by injecting needle-like crystals of urate into a joint to cause physical stimulation.

That is, the above-mentioned test drug was injected into the right knee joint cavity of a rabbit (average body weight: 2.5 to 3.0 kg). Fifteen minutes later, 0.5 ml of a 5% urate crystal suspension was injected into the same site to induce arthritis. The gait state was observed over time, and the degree of the gait disorder was scored on a five-point scale based on the criteria shown in Table 1 below, and used as an index for drug efficacy evaluation.

FIG. 4 shows the results. It should be noted that, in place of the injection of the test drug, a rabbit prepared in the same manner as described above except that a physiological saline containing no test drug was injected was evaluated in the same manner, and the results are also shown in FIG. 4 (control).

[0040]

[Table 1] Comparative Example 2 A hyaluronic acid crosslinked gel preparation was prepared in the same manner as in Example 3 except that polyethylene glycol was not added, and the efficacy was evaluated in the same manner as in Example 3. FIG. 4 shows the obtained results.

As apparent from FIG. 4, the polyethylene glycol-containing hyaluronic acid cross-linked gel preparation of the present invention effectively acted on acute arthritis, and remission of walking disorder was observed.
Further, the medicinal effect of the polyethylene glycol-containing hyaluronic acid crosslinked gel preparation of the present invention was much more significant than that of the hyaluronic acid crosslinked gel preparation containing no polyethylene glycol.

[0042]

Industrial Applicability According to the present invention, a drug can be sustainedly released in response to its own decomposition without release of the carried drug by diffusion, that is, the drug release behavior is rate-limited. A biodegradable hyaluronic acid crosslinked gel composition which is excellent in response to stimuli inducing biodegradation and biocompatibility and has a sufficiently wide range of applicable sites, and is very useful as a drug carrier. Things are obtained.

Thus, according to the present invention, it is possible to obtain a biodegradable hyaluronic acid cross-linked gel preparation having extremely excellent properties as an intelligent preparation for inflammation and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the distribution of insulin between a polyethylene glycol solution having various molecular weights and a hyaluronic acid solution.

FIG. 2 is a graph showing the decomposition behavior of a hyaluronic acid cross-linked gel in the presence of hyaluronidase.

FIG. 3 is a graph showing the drug release characteristics of a hyaluronic acid cross-linked gel in the presence of hyaluronidase.

FIG. 4 is a graph showing the efficacy of a dexamethasone phosphate-containing hyaluronic acid crosslinked gel preparation for acute arthritis.

Claims (4)

[Claims] 1. A biodegradable crosslinked gel composition of hyaluronic acid comprising a crosslinked gel of hyaluronic acid and a water-soluble polyalkylene glycol dispersed in the crosslinked gel of hyaluronic acid. 2. The polyalkylene glycol is at least selected from the group consisting of polyethylene glycol having a number average molecular weight of 200 to 50,000, polypropylene glycol having a number average molecular weight of 200 to 50,000, and a block copolymer thereof. The biodegradable hyaluronic acid crosslinked gel composition according to claim 1, which is one kind. 3. A crosslinked gel composition comprising a crosslinked gel of hyaluronic acid and a water-soluble polyalkylene glycol dispersed in the crosslinked gel of hyaluronic acid, and selectively supported on the water-soluble polyalkylene glycol. , A biodegradable crosslinked gel formulation of hyaluronic acid. 4. The biodegradable hyaluronic acid crosslinked gel composition according to claim 3, wherein the drug is one selected from the group consisting of insulin and dexamethasone phosphate.