JPS61210034A - Remedy for arthropathy - Google Patents

Abstract

PURPOSE:To provide a remedy for arthropathy, consisting of crosslinked hyaluronic acid, giving a solution similar to the normal human synovia when dissolved in physiological saline water, etc., having high safety, and resistant to the tissue enzyme such as hyaluronidase, etc. CONSTITUTION:The objective remedy for arthropathy is composed of a crosslinked hyaluronic acid (abbreviated as crosslinked HA) produced by crosslinking HA or its salt with a crosslinking agent, preferably a polyfunctional epoxy compound [e.g. epichlorohydrin, 1,2-bis(2,3-epoxypropoxy)ethane, etc.]. Crosslinked HA can be produced efficiently by adding a water-soluble organic solvent to an alkaline aqueous solution of HA, collecting the precipitate, and reacting the precipitate with a polyfunctional epoxy compound at >=50 deg.C. The agent is useful as a remedy for various arthropathy such as osteoarthritis, rheumatoid arthritis, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a therapeutic agent for arthropathy, and more particularly, to a therapeutic agent for arthropathy that can be applied to the woody treatment of the pathological condition of arthropathy itself.

[Prior art and its problems] Among joint diseases, in osteoarthritis and rheumatoid arthritis, hyaluronic acid (hereinafter referred to as rHAJ) in the joint fluid of patients decreases in both concentration and molecular weight. is the lubricating function of synovial fluid.

It is thought to be one of the causes of deterioration of various functions such as protection of the surface layer of articular cartilage. Conventional drug treatments for these joint diseases are symptomatic treatments and are not appropriate from the point of view of repairing or improving damaged joint tissues.

On the other hand, if degeneration of articular cartilage can be suppressed or articular cartilage that has already degenerated can be partially repaired, this could be an essential treatment for the disease state itself.

From this point of view, by injecting HA, which is the main component of synovial fluid, into the joint cavity, it is possible to treat traumatic arthritis in racehorses [Acta Vet Scandinavia].

5cand, ), 11, 139 (1970)], human osteoarthritis [Pathol Paiol,
Biol, ) 22.731 (1974, some degree of effectiveness has been recognized.

However, in vivo, HA forms a complex with a specific protein, has stable and strong stringiness, and has high viscoelasticity, and has a special function. HA exhibits almost no stringiness, and it is extremely difficult to extract and purify highly viscous HA using conventional techniques. In addition, after administration in vivo, especially to pathological sites, enzymatic decomposition and non-enzymatic redox decomposition [Arthritis rheumatism (A.
rthritis Rheumatism), 4,
240 (1981)] because it is difficult to maintain its viscosity.

Satisfactory results have not been obtained.

Therefore, the inventors of the present invention have conducted extensive research to resolve the deficiencies and drawbacks of the prior art as described above, and as a result, the i! The present invention was completed by discovering that the object of the present invention can be achieved by using bridged hyaluronic acid (hereinafter referred to as cross-linked HAJ).

[Structure of the Invention] The therapeutic agent for open-face disease of the present invention is characterized by comprising crosslinked HA.

The cross-linked HA used in the present invention may be any water-soluble material obtained by cross-linking HA or its salt with a suitable cross-linking agent.

Among these, those with high viscosity, for example, the viscosity of a 1% physiological saline solution (20°C, sliding speed 1 sec') is 100
0 to eoooo centiboise is preferred.

Preferred crosslinking agents include, for example, polyfunctional epoxy compounds.

Here, the polyfunctional epoxy compound is a compound having at least one epoxy group, and a compound having one or more functional groups suitable for crosslinking HA, including the epoxy group. say.

Examples of such compounds include helomethyloxalane compounds and bisepoxy compounds. Examples of the halomethyloxirane compound include epichlorohydrin, epibromohydrin 1β-methylepichlorohydrin, and β-methylepibromohydrin.

As a bisepoxy compound, 1,2-bis(2,3-
Epoxypropoxy)ethane, 1,4-bis(2,3-
epoxypropoxy)butane, 1,6-bis(2,3-
Examples include epoxypropoxy)hexane and diglycidyl ether of bisphenol A or bisphenol F.

Among the crosslinked HA used in the present invention, one using a polyfunctional epoxy compound as a crosslinking agent and its manufacturing method are detailed in Japanese Patent Application No. 1988-88440.

Usually, HA or its salt with several thousand to several hundred molecules of imitation is 0.
．． Dissolve in an alkaline aqueous solution to a concentration of 5% or more, preferably 1.0% or more, and add a water-soluble organic solvent to a concentration of 30% of the total liquid volume.
It is preferably added in an amount of 50% or more. The alkaline aqueous solution preferably has a pH of 8 to 14, and has a pH of 8 to 14.
More preferably H12-14, the alkali usually includes metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, and sodium carbonate;
Gold Ji such as potassium carbonate! Examples include carbonates. Examples of water-soluble organic solvents include methanol, ethanol, impropatol, acetone, dioxane, etc.
These may be used alone or as a mixture.

By adding these water-soluble organic solvents, the reaction can be carried out effectively, and the decomposition (low molecularization) of HA by alkali can also be suppressed.

Next, one or more of the above polyfunctional epoxy compounds is added to the obtained solution, and the temperature is 0 to 100°C, preferably 10 to 6°C.
The reaction time, which is carried out at 0°C, more preferably 15 to 40°C, varies depending on the reaction temperature, but around 20°C, the reaction time is 20°C.
~48 hours is preferred, and around 40°C, 2 to 3 hours is preferred.

Further, the crosslinked HA used in the present invention can also be prepared as follows.

That is, adding the water-soluble organic solvent to the alkaline solution of HA or its salt, separating the obtained starch syrup precipitate, and adding a polyfunctional epoxy compound to the precipitate,
The reaction can be carried out very efficiently at a temperature of 50°C or lower. To separate the starch syrup-like precipitate 1 For example, the supernatant may be removed by decantation 0 The reaction temperature is usually 10 to 50°C, most preferably 20 to 50°C.
The temperature is 40°C. The higher the temperature, the faster it is necessary to complete the reaction. Generally, the temperature is around 2 ℃ around 40℃.

At around 20°C, 24 to 48 hours is preferable.

In the preparation of crosslinked HA, the solubility and viscosity of the resulting crosslinked HA or salt thereof can be adjusted by changing the molar ratio of HA or its salt and the polyfunctional epoxy compound, that is, the degree of crosslinking.

For IA (with a molecular weight of around 1 million), if the number of moles of the polyfunctional epoxy compound used is 1 to 10 moles per 1 mole of HA, it will be water-soluble and soluble in a 1% physiological saline solution. Viscosity (20℃, shear rate 1sec
-') can be 1000-80000 centibore.

When applying the therapeutic agent for arthropathy of the present invention to various arthropathy, crosslinking 1 (A) is usually dissolved in physiological saline and has a viscosity that can pass through an injection needle, that is, 500 centibore (20
°C, sliding speed 1 sec') or less, preferably 5000
~30,000 centipoise (20℃, shear rate 11F)
1 sec-').

[Effects of the Invention] According to the present invention, it is possible to provide a therapeutic agent for arthropathy that is highly safe and exhibits resistance to tissue enzymes such as hyaluronidase. Such arthropathy therapeutic agent of the present invention is
When dissolved in physiological saline, etc., the solution becomes a solution with properties very similar to those of normal human synovial fluid, and is useful as a therapeutic agent for various arthropathy such as osteoarthritis and rheumatoid arthritis.

[Examples of the Invention] Hereinafter, the present invention will be explained in more detail with reference to Preparation Examples, Test Examples, and Examples, but these are not intended to limit the scope of the present invention in any way.

Preparation example Marujaku B and] 1 (+) HA sl/lium salt (molecular weight 730000)
l Og IN sodium hydroxide aqueous solution (p) + 13
．． After dissolving it in 7) to a concentration of 2%, it was sterilized with a 0.22μ membrane and ethanol 50011!2. and 6.0 tsl of epichlorohydrin, and the mixture was reacted at 20°C for 24 hours. After adding acetic acid to adjust the pH of the reaction solution to 6.4, 3,000 layers of ethanol were added to precipitate the 5-crosslinked H
A (hereinafter referred to as [cross-linked) IA-IJ] was prepared.

Collection et al.
8.5g shear rate 1sec-') Element analysis value C: 42.0%
, H: 4.87%.

N: 3.29%, Na: 5.81% (2) Except for changing the amount of epichlorohydrin, which is a crosslinking agent, (1
), three types of crosslinked HA shown in Table 1 were prepared.

Table 1.
Dissolved at a concentration of 1% and measured (20°C, rubbing speed 15ec)
-1) The results were as follows.

Crosslinked HA-245000 centipores MAHA-3
27,000 centiboes Cross-linked HA-48,000 centiboes HA sodium salt 1,500 centiboes Ushibimaru hyaluronidase was added to these solutions at a concentration of 0.09°C and reacted at 50°C. 15.35.55.
The viscosity was measured after 70 minutes and the ratio to the viscosity before one reaction was calculated.

The results are shown in Figure 1. In FIG. 1, the square, Δ, O, and φ marks respectively represent the ratio of the viscosity of crosslinked HA-2, 3, 4 and HA sodium salt to the viscosity before the reaction between the reaction surfaces of the acetic acid solution.

From FIG. 1, it can be seen that the cross-linked HA used in the present invention has the following characteristics compared to HA:
High resistance to hyaluronidase, the degree of which is
It can be seen that the higher the degree of crosslinking, the more remarkable it is.

Test example 1 -LL! 9 minutes 7
- 100 mg of HA sodium salt of 3,700 σO and 730,000, respectively, 5.0 mg of IN sodium hydroxide; O
Add 5mM of ethanol and 25,50,100,200p of epichlorohydrin, respectively, to the solution dissolved in JL, and react at 40 °C for 2 hours.After the reaction, post-treatment was performed according to Preparation Example (1). Ta.

Also, molecules 11,700,000 (7) HA sodium t
lj75mg IN sodium hydroxide? , ethanol in a solution dissolved in 5ral? , 5+ai and 40 ml or 80 ml of epichlorohydrin were added and reacted at 40° C. for 2 hours. Furthermore, on the same plane and under the same conditions as the reaction described in L, [2-μ
C) A reaction was carried out using epichlorohydrin (obtained from Amajam Japan), and the degree of crosslinking was calculated from the radioactivity of this labeled compound. Table 2 shows the relationship between crosslinking degree and viscosity.
Shown below.

Table 2 shows that in crosslinked HA, there is a proportional relationship between the degree of crosslinking and the viscosity.

Test Example 2 Rotational viscometer (■Tokyo Keiki E-type viscometer) for 1% physiological saline solution of HA two-ton rack @HA-1 and HA used for synthesis
The viscosity was measured at 37° C. by changing the rubbing speed, and the non-Newtonian index (m − T) was calculated. In addition, the viscosity of the joint fluid of normal people and the joint fluid of osteoarthritis patients was similarly measured, and the non-Newtonian index was calculated. The results are shown in Figure 2.

In FIG. 2, the 0 mark, the φ mark 1 mark, and the ■ symbol represent cross-linked HA-1 and 1% saline solution of HA, joint fluid of a normal person, and synovial fluid of a patient with mild osteoarthritis, respectively. Represents viscosity at shear rate.

From FIG. 2, it can be seen that the 1% physiological saline solution of cross-linked HA-1 used in the present invention exhibits a physical pattern very similar to that of the synovial fluid of a normal person.

Test Example 3 The stringability of 11-dish double intermediately crosslinked HA-1 and IA (used for synthesis) was measured using a Watanabe-type stringability measuring device (Hiroshi Ikeuchi 1 Journal of the Japanese Orthopedic Society,
Measurements were made using a device modeled after 34.175 (198G). The results are shown in FIG.
0.5% physiological saline solution, 1% physiological saline solution,
The stringiness is shown at various pulling speeds of a 1% physiological saline solution of HA and a dog's normal joint fluid.

It can be seen from FIG. 3 that the crosslinked HA-1 used in the present invention has stringiness similar to synovial fluid.

Test Example 4 i11-1 4-week-old ddY male mice were preliminarily bred for one week. The body hair of the mice used in the experiment at the beginning of the test was 21-2
It was 7g.

The mice were divided into 15 mice in each group, and each group was treated with 1 layer of 0.5% physiological saline solution of cross-linked H^-1 (cross-linked HA-1 soomg/Kg) and 1 m of the same 1% physiological saline solution. /10g body lid (crosslinked HA-, 11100
1) s/Kg) or physiological saline (1 mJL/10 g body weight) was administered intraperitoneally.

General symptoms were observed at the same time every day according to the Irwin method. On the 7th day after administration, 5 mice in each group were sacrificed, and on the 144th day, the remaining surviving mice were sacrificed, and the injection site and major organs were macroscopically observed.

The results are shown below.

(1) There were no deaths in either group.

1ii+ 500 ragl Kg throw! Group F! 00
There were no differences in general symptoms, weight changes, water intake, or water intake between the 0+sg/Kg administration group and the control group.

G11l At autopsy on day 7 and day 14, control group and 5
There was no difference between the 00mg/Kg administration groups, but +00C
Only in the 1 mg/Kg administration group, on day 7 and day 144, the amount of turmeric in the residual fluid in the LTL cavity was higher than in the control group.

On the 144th day, it was estimated that crosslinked HA-1 of 366 to 3.8 lipid gems remained.

~) There was almost no effect on organs in each group.

Example 1 - HA' The analgesic effect of mHA-1 was investigated in the following manner.

Peagle dogs of both sexes were used, and 20 pg or 2 mg of bradykinin or acetylcholine, respectively, was administered as a pain substance into the knee joint cavity of one hind leg of cross-linked HA-12,5+.
wg10.5 ■ Administration at the same time as normal saline, injection rj,
Changes in lateral hindlimb load were measured over time. In addition, as a control, H used as a raw material in the preparation example instead of crosslinked HA-1
Physiological saline containing 5 liters of AI and lithium salt and 10.5 m of it was used. The analgesic effect was compared based on the normal 50% load recovery time. The results are shown in Table 3.

Table 3 Table 3 shows that the arthropathy therapeutic agent of the present invention has an analgesic effect equivalent to twice the amount of HA sodium salt.

Example 2 Ovalbumin physiological saline solution (10 g/all) 0
．． 5+JL and Freund's complete adjuvant physiological saline solution (10+sg/intestinal J1) 0.5L mixed solution was administered subcutaneously to 12 rabbits (average weight 3kg) at 10-day intervals.
The animals were sensitized, and on the 9th day after the final sensitization, 0.2 g of an ovalbumin physiological saline solution (5 sg/m) was administered into the knee joint cavity as an antigen to induce allergic arthritis. Crosslinked HA-1 saline solution (20IIg 1 layer IL) 0.1
The layer structure was added to an ovalbumin physiological saline solution (10 layer g/la
n) It was administered into the knee joint cavity together with a 0.1-layer solution, and the number of exudate cells in the joint cavity was measured 3.7 and 144 days after administration. The results are shown in Table 4.

Table 4 From Table 4, it can be seen that the number of exudate cells was clearly reduced by administration of cross-linked HA-1.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between viscosity reduction and time when various types of crosslinked HA and HA are treated with hyaluronidase. Figure 2 is a diagram showing the viscosity measurement results of cross-linked HA-1, HA, and joint fluid of normal people and osteoarthritis patients. It is a figure which shows the stringiness measurement result of. 1 thought time (cow) Running speed without pulling up (C starvation/sec)

Claims (1)

[Claims] A therapeutic agent for arthrosis characterized by comprising cross-linked hyaluronic acid.