JP4990446B2 - Injection for treatment of arthropathy - Google Patents

Description

【００３７】
表１より、振り子摩擦試験による摩擦係数は実施例１のＨＡゲル及びリン脂質の混合製剤、及び実施例２のＨＡとＨＡゲル及びリン脂質の混合製剤が、比較例１、２より優位に低く、その値は正常な無処置群と同様な低い値を示した。
【００３８】
また、関節軟骨組織切片を切り出し、このＸ線像から骨の増殖性変化を評価した。
評価基準は、無処置の膝と比較して、軟骨下骨への効果と軟骨辺縁の骨棘の形成と軟骨面の滑らかさの３つの面から各々（２点：正常、１点：中程度の異常、０点：重度の異常）から評価し、０点から６点の７段階にグレーディングした。その結果を表２に示す。
【００３９】
【表２】

【００４０】
表２より、驚くべきことに実施例１、２は正常な無処置群に近い大きな治癒効果が認められた。それに対し、比較例１、２では軟骨の増殖変形が認められた。また、コントロールは著しい増殖性変形が認められた。
【００４１】
【発明の効果】
以上、本発明の生体適合性のＨＡゲル及びリン脂質、及び／又はＨＡを含有してなる構成の関節症治療用注入剤は、従来のＨＡ及びＨＡゲル、又はＨＡ及びリン脂質の構成の混合製剤では得られない製剤の投与回数の低減と高い治療効果を有するものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an injection for treating arthropathy using a biocompatible hyaluronic acid gel.
[0002]
[Prior art]
Joint fluid supplies nutrients to articular cartilage in living joints, and has an unparalleled lubrication function and shock absorber function, and its excellent viscoelastic function is one of the main components in joint fluid. Is largely controlled by hyaluronic acid (hereinafter abbreviated as HA). HA is a linear high-molecular polysaccharide in which β-D-N-acetylglucosamine and β-D-glucuronic acid are alternately bonded, and the molecular weight thereof may be as high as several million to 10 million. Are known.
[0003]
In general, according to the analysis results of HA concentration and molecular weight in joint fluids of patients with various arthropathy such as osteoarthritis and rheumatoid arthritis, the joint fluid of arthritic patients tends to decrease in concentration and molecular weight compared to normal joint fluid This is considered to be closely related to the occurrence of motor dysfunction and pain symptoms due to a decrease in the synovial fluid lubricating action and articular cartilage surface protecting action.
[0004]
Among these joint diseases, as an effective means for osteoarthritis of the knee, a method of injecting an HA solution into a diseased joint site has been adopted. As an example of the knee joint therapeutic agent, Alz (manufactured by Seikagaku Corporation, average The molecular weight is 900,000), Hyalgan (Fidia, average molecular weight <500,000), and high molecular weight can be expected to be more effective. It is also applied to rheumatoid arthritis developed by the present inventors. Svenir (Aventis Pharma Co., Ltd./Chugai Pharmaceutical Co., Ltd./Electrochemical Co., Ltd., average molecular weight 1.9 million) can be mentioned.
[0005]
Also, Synvisc (manufactured by Genzyme) containing a crosslinked HA gel which has been polymerized by chemically crosslinking HA to improve viscoelasticity has been developed. This cross-linked HA gel is an HA gel obtained by chemically cross-linking HA with a cross-linking agent divinyl sulfone and is called hylan. Methods for producing high runs and high run cross-linked gels are described in detail in US Pat. No. 4,713,448. Single and mixed gels based on HA are described in US Pat. Nos. 4,582,865 and 4,605,691.
[0006]
The treatment using these joint preparations requires 3 to 5 injections directly into the patient's joint every week, and the reduction is desired in view of the burden on the patient and the doctor.
[0007]
On the other hand, phospholipids exist on the surface of articular cartilage, keep the surface hydrophobic, and contribute to the formation of a fluid lubricating film by joint fluid between articular cartilage. Therefore, the lubricating composition for the treatment of arthritis that improves the lubrication of the joint surface can be provided by mixing this phospholipid with an aqueous HA solution. (Japanese Patent Application No. 63-507021)
Normally, it is said that the coefficient of motion friction of hyaluronic acid increases and the lubricating ability decreases at a steady load of about 3 kg / cm ² generated in a human knee joint, but the load on the knee joint is 20 kg by adding phospholipid. Even under severe conditions of / cm ^2, the coefficient of kinetic friction can be kept low at 0.003 to 0.05, and the lubricating ability can be improved. (“Multi-mode adaptive lubrication mechanism and friction characteristics in living joints” (Abstracts of the 73rd General Meeting of the Japan Society of Mechanical Engineers, 502-503 (1996)). Effect of intra-articular injection of phospholipid liposomes on sputum "(Journal of Japanese Society for Clinical Biomechanics, Vol. 19, 131-135 (1998), etc.).
[0008]
[Problems to be solved by the invention]
The conventional lubricating composition of HA and HA gel can further improve the viscoelasticity of joint fluid, which was difficult only by HA injection, and can be expected to improve the lubricating action and the articular cartilage surface protecting action.
However, the number of administrations required 3 to 5 injections directly into the patient's joint every week. In addition, it is difficult to reduce the coefficient of friction between cartilage under conditions where the joint is loaded, and in severe pathologies where the articular cartilage is worn, it is difficult to form a boundary lubrication film due to joint fluid. Could not be demonstrated.
[0009]
On the other hand, the lubricating composition composed of HA and phospholipid reduces friction between cartilage even under conditions where the joint is loaded, and phospholipid keeps the cartilage surface hydrophobic even in a pathological condition where articular cartilage is worn. Therefore, it can be expected to reduce the friction of the joint.
However, since the viscoelasticity of the joint fluid cannot be improved, it is difficult to obtain sufficient lubrication and articular cartilage surface protection.
[0010]
As a result of intensive studies on physical property tests and animal tests, the present inventors have found that a composition comprising HA gel and phospholipid and / or HA has a surprising synergistic effect in pharmacological tests using animals. The present inventors have found that the number of administrations of a prominent preparation can be reduced and that a high therapeutic effect is exhibited, and the present invention has been completed.
[0011]
[Means for Solving the Problems]
That is, the present invention provides (1) an injectable agent for treating arthropathy comprising (1) a biocompatible HA gel and phospholipid and / or HA, and (2) a concentration of the biocompatible HA gel is 5 to 5. The joint according to (1), wherein the joint is an infusion for treating arthritis according to (1), and (3) the biocompatible HA gel is pulverized. (4) The arthrosis treatment infusion according to (1), wherein the concentration of phospholipid is 1 to 200 mg / ml, (5) the phospholipid is phosphatidylcholine, phosphatidylethanol (1) The injectable agent for treating arthropathy according to (1), which is selected from the group consisting of amine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin and derivatives thereof, (6) , Α- Palmitoyl phosphatidylcholine (1), wherein the arthrosis therapeutic infusions, the concentration of (7) HA, is a 1-10 mg / ml (1), wherein the arthrosis therapeutic infusion.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The HA used in the present invention may be extracted from animal tissues or manufactured by fermentation, regardless of its origin.
The strain used in the fermentation method is a microorganism having the ability to produce HA such as Streptococcus isolated from the natural world, or Streptococcus equii FM-100 described in Japanese Patent Laid-Open No. 63-123392 (No. 9027 by Microtechnological Bacteria). ), A mutant that stably produces HA with 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.
The molecular weight of HA 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, a low molecular weight one obtained by hydrolysis treatment from a higher molecular weight can be preferably used as well.
In addition, HA as used in the field of this invention is used by the concept also including the alkali metal salt, for example, the salt of sodium, potassium, and lithium.
[0013]
The concentration of the HA of the present invention is preferably 1 to 10 mg / ml. When the concentration is lower than 1 mg / ml, the viscoelasticity is low and the effect as a pharmaceutical product for joints is low, and when the concentration is higher than 10 mg / ml, the viscosity is too high and the pressure becomes high and difficult to handle when injected into the joint.
[0014]
The molecular weight of HA can be measured by a GPC method using HPLC (liquid chromatography) generally used or a method derived from intrinsic viscosity measurement.
[0015]
The HA 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 the crosslinked structure of HA.
It is known that HA itself is a linear polymer and does not have a branched structure (Polysaccharide Biochemistry 1 Chemistry edited by Kyoritsu Shuppan 1969). The gel is defined as “a polymer having a three-dimensional network structure insoluble in any solvent and its swollen body” according to the new edition of the Dictionary of Polymers (Asakura Shoten 1988). According to the physics and chemistry dictionary (Iwanami Shoten 4th edition, 1987), it is defined as “a sol (colloidal solution) solidified into a jelly”.
[0016]
Any HA gel may be used as long as it has excellent biocompatibility when implanted or injected into a living body.
The term “biocompatibility” as used in the present invention means that there is no or minimal undesirable effects or side effects on the living tissue in contact with the HA gel of the present invention.
Possible side effects include toxicity, inflammation, immune reaction, foreign body reaction, encapsulation reaction, and the like.
[0017]
Examples of HA gels include HA gels (PCT / JP98 / 03536) consisting of HA alone that does not use chemical cross-linking agents or chemical modifiers, and methods that lead to the formation of ester bonds between carboxyl groups and hydroxyl groups of HA. Mention may be made of self-crosslinked HAs (see EP 0341745 B1). Further, there can be mentioned HA gel crosslinked using a bifunctional reagent such as divinyl sulfone, bisepoxide, formaldehyde or the like as a crosslinking agent (US Pat. No. 4,582,865, JP-B-6-37575). Gazette, JP-A-7-97401, JP-A-60-130601). Moreover, HA gel characterized by putting HA aqueous solution in presence of pH 2.0-3.8 and 20-80 mass% water-soluble organic solvent is mentioned (refer Unexamined-Japanese-Patent No. 5-58881).
[0018]
Among these, HA gel (PCT / JP98 / 03536) made of HA alone without using a chemical crosslinking agent or chemical modifier is particularly preferable from the viewpoint of biocompatibility.
[0019]
The concentration of the HA gel used in the present invention is preferably 5 to 60%. If it is less than 5%, the effect as a joint drug is low, which is not preferable. On the other hand, if it exceeds 60%, the aggregation of the HA gel becomes large and the handling at the time of injection becomes difficult.
[0020]
The HA gel used in the present invention is preferably contained in a pulverized state.
[0021]
The phospholipid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissue or artificially synthesized.
The phospholipid preferably comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin and their derivatives.
Particularly preferred is α-dipalmitoylphosphatidylcholine (α-DPPC). The α-DPPC may be an L-form, D-form, or racemic (DL) -form mixture.
[0022]
The concentration of the phospholipid used in the present invention is preferably 1 to 200 mg / ml.
When the concentration of phospholipid is less than 1 mg / ml, the effect as a joint drug is low, and when it exceeds 200 mg / ml, the aggregation of phospholipid increases, which is difficult in terms of storage and difficult to handle.
[0023]
Since phospholipids are amphipathic substances, they may be made into liposomes for the purpose of stability in an HA solution, but may be suspended in particular without being made into liposomes.
[0024]
To obtain a phospholipid suspension, a physiological saline suspension of phospholipid can be easily obtained by adding physiological saline to the phospholipid and sonicating.
[0025]
Phospholipids can be made into liposomes according to a conventional method as follows.
For example, α-DPPC is dissolved in chloroform so as to be 1% by mass, and this is taken into an eggplant type flask, and chloroform is removed by evaporation with a rotary evaporator, thereby forming an α-DPPC thin film on the surface of the eggplant type flask. Next, a physiological saline suspension is added to the liposome membrane of α-DPPC by adding physiological saline thereto and performing ultrasonic treatment.
[0026]
Furthermore, in the present invention, pharmaceutically acceptable physiological saline, phosphate buffer, or the like can be used for the HA gel and phospholipid, and / or HA.
[0027]
【Example】
Hereinafter, although detailed content is demonstrated using an Example, this invention is not limited to a following example.
[0028]
Preparation Example 1
Preparation of HA solution 6 g of sodium hyaluronate (manufactured by Denki Kagaku Kogyo) with a molecular weight of 2 × 10 ⁶ Dalton was weighed in a clean bench, and 450 ml of 5 mM pH 7.2 phosphate buffered physiological saline filtered aseptically with a Nalgen filter. In addition, it was dissolved in a roller bottle (high molecular weight HA sodium 1.3% by mass).
[0029]
Preparation Example 2
Preparation of HA gel HA sodium (manufactured by Denki Kagaku Kogyo) having a molecular weight of 2 × 10 ⁶ daltons was dissolved in water for injection to prepare a 1% by mass HA aqueous solution. The pH of this aqueous solution was adjusted to pH 1.5 with 1N hydrochloric acid to obtain an HA acidic aqueous solution. 100 ml of this HA acidic aqueous solution was placed in a 300 ml sample bottle and placed in a freezer set at −20 ° C. HA gel was obtained after freezing for 120 hours. Next, 10 ml of water for injection was added, and the washing which was allowed to stand for 10 minutes and then decanted was repeated three times, and then phosphate buffered physiological saline having a pH of 7 adjusted by adding a phosphate buffer component at a concentration of 50 mM to physiological saline. Neutralization by adding 200 ml and decanting was repeated twice, and then washed thoroughly with 5 mM pH 7.2 phosphate buffered physiological saline to obtain an HA gel.
[0030]
Preparation Example 3
Preparation of phospholipid solution To 1.0 g of L-α-DPPC (manufactured by Wako Pure Chemical Industries, Ltd.) was added 100 ml of 5 mM pH 7.2 phosphate buffered physiological saline, which was vigorously stirred for 3 minutes and further over 3 minutes. Suspended by sonication. This was sterilized by autoclaving at 121 ° C. for 15 minutes (becomes a milky white suspension).
[0031]
Example 1
Preparation of a mixed preparation of HA gel and phospholipid To 200 ml of 5 mM pH 7.2 phosphate buffered physiological saline filtered aseptically with a Nalgen filter, add 300 ml of the HA gel of Preparation Example 2 and 100 ml of the phospholipid solution of Preparation Example 3. It was. This mixed solution is subjected to HA gel crushing treatment and complete dispersion of phospholipid using a microhomogenizer (NISSEI EXCEL AUTO HOMOGENIZER), and the HA gel and phospholipid containing hyaluronic acid gel having an average particle size of 300 to 1,000 μm are dispersed. A mixed formulation was obtained.
These series of operations were performed in an aseptic and dust-free environment, and the chemicals used were sterilized in advance.
[0032]
Example 2
Preparation of HA, HA gel and phospholipid mixed preparation To 450 ml of the HA solution of Preparation Example 1 separately prepared, 100 ml of the HA gel of Preparation Example 2 and 50 ml of the phospholipid solution of Preparation Example 3 were added. Using this mixed solution, a HA homogenizer (NISSEI EXCEL AUTO HOMOGENIZER) is used to crush the HA gel and completely disperse the phospholipid. The HA and HA gel containing hyaluronic acid gel having an average particle size of 300 to 1,000 μm and A mixed phospholipid formulation was obtained.
These series of operations were performed in an aseptic and dust-free environment, and the chemicals used were sterilized in advance.
[0033]
Comparative Example 1
Preparation of Mixed Formulation of HA and HA Gel To 450 ml of the HA solution of Preparation Example 1 separately prepared, 100 ml of the HA gel of Preparation Example 2 and 50 ml of 5 mM pH 7.2 phosphate buffered physiological saline were added. This mixed solution was subjected to HA gel crushing treatment using a microhomogenizer (NISSEI EXCEL AUTO HOMOGENIZER) to obtain a mixed preparation of HA and HA gel containing hyaluronic acid gel having an average particle size of 300 to 1,000 μm.
These series of operations were performed in an aseptic and dust-free environment, and the chemicals used were sterilized in advance.
[0034]
Comparative Example 2
Preparation of Mixed Formulation of HA and Phospholipid To 450 ml of the HA solution of Preparation Example 1 prepared separately, 50 ml of the phospholipid solution of Preparation Example 3 and 100 ml of 5 mM pH 7.2 phosphate buffered physiological saline were added. This mixed solution was completely dispersed with phospholipid using a microhomogenizer (NISSEI EXCEL AUTO HOMOGENIZER) to obtain a mixed preparation of HA and phospholipid. These series of operations were performed in an aseptic and dust-free environment, and the chemicals used were sterilized in advance.
[0035]
Example 3
Pharmacological Effect of Mixed Formulation of HA, HA Gel and Phospholipid The pharmacological effect of the mixed formulation of HA gel and phospholipid of Example 1 and the mixed formulation of HA, HA gel and phospholipid of Example 2 is as follows. It was evaluated with.
60 Japanese white rabbits (weight approximately 3 kg) were anesthetized and the anterior cruciate ligament and medial collateral ligament of the right knee were separated. As a result, the right knee of the Japanese white rabbit is subjected to abnormal stress between the joints, so that the joint becomes inflamed within a week and becomes a model for osteoarthritis of the knee.
Seven weeks after the operation, the HA gel and phospholipid mixed preparation of Example 1 was applied to 10 chickens and the HA of Example 2 was applied to 10 chickens on the right knee of Japanese white rabbits in which inflammation of the joint was sufficiently induced. Mixed preparation of HA gel and phospholipid, mixed preparation of HA and HA gel of Comparative Example 1 in 10 birds, mixed preparation of HA and phospholipid of Comparative Example 2 in 10 birds, 0.3 ml twice at 10 day intervals Administered.
The remaining 10 birds were anesthetized only at the same interval without injection of an aqueous solution for control.
All patients were sacrificed 10 days after the second injection and radiographed. The ligament, the joint capsule and the soft tissue other than the half moon were excised from the knee joint, and immediately, the friction coefficient at a low amplitude region (0.03 to 0.01 rad) and a load of 3 kg was measured by a pendulum friction tester. The coefficient of friction was calculated based on the assumption that the potential energy loss due to the pendulum damping was all due to friction. The results are shown in Table 1.
[0036]
[Table 1]

[0037]
From Table 1, the friction coefficient by the pendulum friction test is significantly lower in the mixed preparation of HA gel and phospholipid in Example 1 and in the mixed preparation of HA, HA gel and phospholipid in Example 2 than in Comparative Examples 1 and 2. The value was as low as that in the normal untreated group.
[0038]
Further, articular cartilage tissue sections were cut out, and bone proliferative changes were evaluated from the X-ray images.
The evaluation criteria were compared to the untreated knee in each of three aspects: the effect on the subchondral bone, the formation of osteophytes at the cartilage margin, and the smoothness of the cartilage surface (2 points: normal, 1 point: medium) Grade from 0 to 6 points, and graded into 7 grades. The results are shown in Table 2.
[0039]
[Table 2]

[0040]
From Table 2, surprisingly, in Examples 1 and 2, a large healing effect close to that of the normal untreated group was recognized. In contrast, in Comparative Examples 1 and 2, cartilage proliferation and deformation were observed. In addition, remarkable proliferative deformation was observed in the control.
[0041]
【Effect of the invention】
As described above, the biocompatible HA gel and phospholipid of the present invention and / or the injectable agent for treating arthropathy comprising HA are mixed with the conventional HA and HA gel or HA and phospholipid composition. It has a high therapeutic effect and a reduced number of administrations of the preparation that cannot be obtained by the preparation.

Claims (8)

A biocompatible hyaluronic acid gel composed of hyaluronic acid alone without using a chemical cross-linking agent or a chemical modifier, and phospholipid,
And wherein the concentration from 5 to 60% of the biocompatibility of hyaluronic acid gel, the biocompatible hyaluronic acid gel is contained in crushed form, knee arthrosis therapeutic infusion. The knee arthrosis treatment according to claim 1, wherein the biocompatible hyaluronic acid gel is a biocompatible hyaluronic acid gel formed by adjusting an aqueous solution of a hyaluronic acid inorganic salt to an acid, freezing, and thawing. Injection agent. The injectable agent for treating knee joint disease according to claim 1 or 2, wherein the concentration of phospholipid is 1 to 200 mg / ml. The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin and their derivatives. An injection for treating knee joint disease according to any one of the above. The injection for treating knee joint disease according to any one of claims 1 to 4, wherein the phospholipid is α-dipalmitoylphosphatidylcholine. The injectable agent for treating knee joint disease according to any one of claims 1 to 5, further comprising a hyaluronic acid solution. The injectable agent for treating knee joint disease according to claim 6, wherein the concentration of hyaluronic acid is 1 to 10 mg / ml. Sonicating a solution containing phospholipid to prepare a phospholipid solution;
Preparing a mixed solution comprising the phospholipid solution and a biocompatible hyaluronic acid gel consisting of hyaluronic acid alone without using a chemical crosslinking agent or chemical modifier;
A step of crushing the hyaluronic acid gel and dispersing the phospholipid solution;
The concentration of the hyaluronic acid gel in the mixed solution is 5 to 60%,
A method for producing an injection for the treatment of knee osteoarthritis.