JPH07278176A - Anti-inflammatory agent - Google Patents

Abstract

PURPOSE:To provide a harmless and safe anti-inflammatory agent containing a sulfatide as an active component and useful for the treatment of inflammatory diseases such as hepatitis and chronic nephritis. CONSTITUTION:This ainti-inflammatory agent contains a sulfatide of formula (R-CO is a fatty acid residue), i.e., cerebroside sulfate as an active component. The daily dose of the sulfatide for adult is 0.1-1g for oral administration, 10-100mg for intravenous injection, 50-300mg for intramuscular injection and 30-150mg for subcutaneous or intradermic injection. The concentration of the sulfatide in an external agent is usually 0.1-1%(W/W). The sulfatide can be produced e.g. by extracting bovine cerebral cinerea with chloroform/methanol, etc., and purifying the extract by Folchs partition method and a Q-Sepharose column.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an invention for supplying a safe therapeutic agent capable of going to a hospital to patients with inflammatory diseases including hepatitis such as viral and drug-induced hepatitis and chronic nephritis.

[0002]

Background Art There are various inflammatory diseases, but hepatitis and nephritis are clinically common, and development of highly safe therapeutic agents against them is expected. Although hepatitis includes viral and drug-type, most of them are viral. As therapeutic agents for hepatitis, biological response modifiers such as interferon and interleukin 2 (biological response modi) are used.
fier; BRM), neominophagen C, which is an intravenous injection of glycyrrhizin, and the like are used. However, since side effects such as fever are observed in BRM, there is a problem in long-term administration. When glycyrrhizin is used for a long period of time, it is necessary to control blood pressure and electrolyte concentration.
In addition to this, herbal medicine ingredients saikosaponin, saponin,
Gomisin and the like are known, but none have been clinically applied. In addition, there is no effective therapeutic drug for nephritis so far, but a steroid drug and an antiplatelet drug dipyridamole are often used as a therapeutic drug for nephrosis which is a pre-stage of chronic nephritis. However, these must be used for a long period of time, especially when given to young people, full moon face, dizziness, headache, vomiting, etc., in severe cases, infections, gastrointestinal bleeding, metabolic disorders, There are concerns about side effects such as osteoporosis and thrombosis.

Recently, attention has been paid to the relationship between inflammation and the selectin family that recognizes sugar chains among cell adhesion factors. E-selectin (Beviracqua, MP et al.
(1987) Proc. Natl. Acad. Sci.
USA. 84, 9238-9243), P-selectin present in platelets (Geng, JG et al.
(1990) Nature. 343, 757-76
0), L-selectin present in leukocytes (Ley, K .;
et al. (1991) Blood. 77,2553
-2555), these ligand sugar chains are sialyl Le ^x or sialyl Le ^a (Lowe, J. B.
et al. (1990) Cell. 53,475-4
84; Philipis, M .; L. et al. (19
90) Science. 250, 1130-1132;
Magnani, J .; L. (1991) Glycoco
njugate J.N. 1, 318-320; Takad.
a, A. et al. (1991) Biochem. B
iophys, Res. Commun. 179,713
-719). In addition, since it is considered that the adhesion between leukocytes and vascular endothelial cells via selectin and these sugar chains triggers the inflammatory reaction, the ligands sialyl Le ^X and sialyl Le ^a were directly administered to the body. Attempts have been made to suppress inflammation. The present inventors have previously shown that a glycolipid sulfatide having a sulfate group binds to L-selectin more strongly than sialyl Le ^x and Le ^a by an in vitro experiment, which is different from sialyl Le ^x and Le ^a. It was found that the binding to L-selectin is calcium independent (Suzuki, Y. et al. (1993) B.
iochem. Biophys, Res. Commu
n. 190, 426-434. ). However, it is not known how it affects inflammation in vivo.

[0004]

As described above, hepatitis is roughly classified into viral ones and those caused by drugs such as alcohol, but hepatitis B and C viruses are overwhelmingly predominant. In recent years, it has been reported that interferon is particularly effective as a therapeutic drug for hepatitis C,
The frequency of use is also increasing (Shiro Iino et al .: Basic and clinical, 26: 339, 1992; Hiroshi Suzuki et al .: Liver / gall / pancreas,
23: 1065, 1991). However, short-term administration causes influenza-like symptoms such as fever, and long-term administration causes side effects such as weight loss, diarrhea, vomiting, arrhythmia, hair loss and autoimmune abnormality. Therefore, it is desired to develop a new safer therapeutic drug or an auxiliary drug that can be used in combination with interferon or the like. In addition, nephritis is roughly classified into primary glomerular origin and secondary origin due to other diseases. The former occupies about 80% and is more common in younger age groups. Pathologically, it is classified into a minimal change group, monoglomerular sclerosis, membranous nephropathy, mesangial proliferative nephropathy, and membranous proliferative glomerulonephritis. Proteinuria is a common clinical finding and, in severe cases, is diagnosed as nephrotic syndrome. Further, in recent years, renal diseases are increasing not only in the elderly but also in the younger generation, and therefore development of therapeutic agents with few side effects is desired.

[0005]

[Means for Solving the Problems] As a result of diligent studies by the present inventors using a carbon tetrachloride-induced hepatitis model and a reflux nephritis model prepared by ureteral ligation, sulfatide is effective in both models. It was found that the substance can be used as an anti-inflammatory agent.

The present invention is based on this new finding, and is an anti-inflammatory agent, an agent for improving hepatitis or renal inflammation, which contains sulfatide as an active ingredient.

Sulfatide used in the present invention, that is, cerebroside sulfate is a known glycolipid specified by the following general formula (1), is present in a relatively large amount in brain white matter, and its importance as a myelin-constituting lipid is also pointed out. Has been done.

[0008]

[Chemical 1] (In the formula, R-CO- represents a fatty acid residue)

The sulfatide of the present invention can be extracted and purified according to a conventional method, for example, a method of extracting and purifying from bovine brain (Lipids, 22 (9) P. 588-598.
(1985); Biochemistry Experiment Course, "Lipid Chemistry", Vol. 368-388, Tokyo Kagaku Dojinsha) and the like. Sulfatide can also be produced by chemical synthesis. An example of a method for preparing a purified product from bovine brain is shown in Experimental Example 1.

In the present invention, sulfatide is administered to patients for the purpose of anti-inflammatory, amelioration of hepatitis symptoms and amelioration of renal inflammation. Since sulfatide is a myelin-constituting lipid of the brain and has little toxicity, the administration method is not particularly limited, and it can be administered internally, externally or by injection. The injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, or the like. The daily dose for an adult is about 0.1 g to 1 g for oral administration, about 10 mg to 100 mg for intravenous administration, and 50 for intramuscular administration.
mg to 300 mg, subcutaneous or intradermal about 30 mg to 15
It is administered at 0 mg once daily or in several divided doses.
As an external use, for example, as an ointment, topically applied to the outer skin,
Further, it can be applied to the outer skin, throat, nasal cavity in the form of a spray, or can be instilled as an eye drop. The preferred concentration of sulfatide in the external preparation varies depending on the dosage form and application site, but is usually about 0.1 to 1% (w / w).

Hereinafter, in order to further clarify the effect of the anti-inflammatory agent sulfatide of the present invention, it will be described in detail with reference to experimental examples and examples, but the present invention is not limited thereto.

[0012]

【Example】

Experimental Example 1 (Method for preparing sulfatide) 1 kg of fresh bovine brain gray matter was homogenized by adding 5 volumes of acetone, and then filtered. Lyophilize the extract and add 5 volumes of chloroform /
Methanol (2: 1, v / v), then chloroform /
After extraction with methanol (1: 1, v / v), evaporation was performed. The dried product was extracted with 3 volumes of acetone and then treated with 3 volumes of diethyl ether to remove glycerophospholipids. Then 37 with 0.5N NaOH / methanol
The mixture was treated at ℃ for 3 hours, dialyzed against distilled water, and then freeze-dried. This crude fraction was subjected to the Forch partition method (Folchs pa).
and then the lower layer was collected and applied to a Q-Sepharose column equilibrated with chloroform / methanol / water (30: 60: 8, v / v / v), and eluted with a linear gradient of sodium acetate 0-4M. . Sulfate was eluted in the 1M to 1.5M sodium acetate elution fraction. After confirming comparison of each fraction with a sulfatide standard substance by thin-layer chromatography, the solvent was evaporated. The residue was dissolved in distilled water, dialyzed against distilled water, and then freeze-dried. A white powder of 99% unusual purity of sulfatide was obtained.

Example 1 Evaluation of sulfatide by carbon tetrachloride-induced hepatitis model 1) Preparation of liver damage model: Wistar female rats (9-11 weeks old, body weight about 160 g) were mixed with olive oil at an equal ratio. It was prepared by once administering 1.5 ml / kg of the carbon tetrachloride (manufactured by Wako Pure Chemical Industries, Ltd.) solution into the abdominal cavity. (Inflammation book: Inflammatory animal experimental method, experimental liver fibrosis, published by Igakushoin, 253-277) 2) Administration of sample solution: sulfatide 0.3, 1.0, 3. 15 hours before carbon tetrachloride administration. 0 mg / kg each 1
Each dose was administered intramuscularly. The control group was given the same amount of galactosylceramide having no sulfate group, and the negative control group was given water for injection. 3) Serum biochemical test: Twenty-four hours after administration of carbon tetrachloride, thoracotomy was performed under ether anesthesia, 2 ml of blood was collected from the heart, allowed to stand at room temperature for 1 hour, and centrifuged to obtain serum. Serum glutamate oxaloacetate transaminase (GO
T: glutamic oxaloacetic tr
and serum glutamate pyruvate transaminase (GPT: glutamate py).
The measurement of the ruvic transaminease activity was performed using a Wako Pure Chemical Industries kit. 4) Statistical analysis: All measured data are average ±
S. It was shown by E and statistically analyzed by Student's t-test.

The results are shown in Table 1, and the inhibitory effect on the elevation of serum GOT and GPT was observed depending on the concentration of sulfatide, and a remarkable effect was observed especially at 1.0 mg / kg or more. No such effect was observed with the control galactosylceramide.

Table 1 Effect of sulfatide on serum transaminase ────────────────────────────────── Transaminase treatment group ── ───────────────────── GOT (U / L) (% inhibition) GPT (U / L) (% inhibition) ──────── ─────────────────────────── Control 53 ± 3 29 ± 2 CCl ₄ 2787 ± 491 (0) 1430 ± 338 (0) CCl ₄ + Sulfatide 2520 ± 635 (10) 1160 ± 420 (19) (0.3 mg / kg) CCl ₄ + Sulfatide 1013 ± 483 ^* (64) 624 ± 236 ^* (56) (1.0 mg / kg) CCl ₄ + Sulfatide 855 ± 273 ^* (69) 440 ± 197 ^* (69) (3.0 mg / kg) ────────────────────────────────── ── control _{52 ± 3 28 ± 2 CCl 4} 3 16 ± 542 (0) 2016 ± 290 (0) CCl 4 + galactosyl 3280 ± 745 (14) 1760 ± 390 (13) ceramide (1.0mg / kg) ─────────────── ─────────────────── ^* P <0.05, ^** P <0.005

Example 2 Sulfatide 1.0 mg / kg was intramuscularly administered 15 hours before the administration of carbon tetrachloride to hepatitis rats prepared by the above method, and the liver tissue was observed with a microscope. Only carbon tetrachloride was administered. Compared with the case, remarkable suppression of hepatocellular injury was confirmed. (Fig. 1) Fig. 1-1 is a normal rat, Fig. 1-
2 shows the rat histology of carbon tetrachloride only rats, and FIGS. 1-3 show the liver tissue images of carbon tetrachloride and sulfatide 1.0 mg / kg rats.

Example 3 Evaluation of Sulfatide Using Reflux Nephritis Model Sulfatide (1) was added to 5 4-week-old female Wistar rats.
(mg / body) and right ureteral ligation was performed immediately thereafter. As a control group, galactosylceramide (1 mg / body) was administered to 5 animals, and immediately after that, right ureteral ligation was performed. All were sacrificed 24 hours later, and the right kidney was removed.
The obtained renal tissue was rapidly frozen to prepare a frozen section of 4 μm, and then monocyte infiltration was performed by an enzyme antibody method using anti-rat monocyte / macrophage monoclonal antibody (ED-1) and biotin-labeled anti-rat IgG antibody. It was investigated. The number of monocytes was 400 by an optical microscope for the sections stained with the enzyme antibody method using ED-1 for 5 animals in each group.
20 fields of view were observed at double magnification, and the number of monocytes stained with ED-1 was counted. Statistical processing was performed by Wilcoxon test.

The results are shown in FIG. 2. In the galactosylceramide administration group, a large number of monocytes (159.2 ±) were found in the interstitium of the renal medulla.
14.5 / 20 fields of view, n = 5) were recognized, while
The number of monocytes was significantly lower in the sulfatide group (8
6.4 ± 24.4 / 20 fields of view, n = 5). Statistically, a significant difference was recognized at P <0.0001. Sulfatide is mainly present in distal renal tubular epithelial cells in the kidney, but is believed to be redistributed in interstitial and peritubular small blood vessels and promote leukocyte infiltration in association with renal tubular disorder of reflux renal inflammation .
The administration of sulfatide significantly suppressed leukocyte infiltration in this model, indicating that sulfatide is effective in inhibiting leukocyte infiltration into the interstitium in renal disease.

[0019]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a harmless anti-inflammatory agent which can be administered by various methods such as internal use, external use and injection.

[Brief description of drawings]

1 to 3 are photographs showing the results in Example 2, and FIG.
Shows a normal rat, FIG. 2 shows a liver tissue image of a rat administered with carbon tetrachloride only, and FIG. 3 shows a liver tissue image of a rat administered with carbon tetrachloride and sulfatide. FIG. 4 is a graph showing the results in Example 3, in which a large number of monocytes (ED-1 positive) were observed in the interstitium of renal cells in the galactosylceramide administration group, but the number of monocytes was much higher in the sulfatide administration group. Very few.

[Procedure amendment]

[Submission date] July 12, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

1 is a micrograph showing a liver tissue image of a normal rat in Example 2,

FIG. 2 is a micrograph showing a liver tissue image of a rat to which only carbon tetrachloride was administered in the same Example,

FIG. 3 is a micrograph showing a liver tissue image of a rat to which carbon tetrachloride and sulfatide were administered in the same Example.

FIG. 4 is a graph showing the results in Example 3, where a large number of monocytes (ED-1 positive) were observed in the interstitium of renal cells in the galactosylceramide administration group, but the number of monocytes in the sulfatide administration group was Far less.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location A61K 31/715 ACV A61K 37/20 ACV (72) Inventor Kano Kuni Suma Ward, Suma-ku, Kobe Hyogo Prefecture 1-Chome 4-26 Royal Itayajuku 501 (72) Inventor Junichi Kajiwara 5-2 Kashinodai, Nishi-ku, Kobe-shi, Hyogo D- 810 (72) Inventor Kazuo Kato 6-10-3 Momoyamadai, Tarumi-ku, Kobe-shi, Hyogo

Claims (3)

[Claims] 1. An anti-inflammatory agent containing sulfatide as an active ingredient. 2. A hepatitis symptom-improving agent containing sulfatide as an active ingredient. 3. A renal inflammatory condition improving agent comprising sulfatide as an active ingredient.