JPH0789862A - Anti-inflammatory, immunosuppressive and analgesic composition and method for treating autoimmune disease using the same - Google Patents

Abstract

PURPOSE:To obtain a medicine composition useful for autoimmune diseases, containing the same medicine carrier as that of anti-inflammatory, immunosuppressive and analgesic compositions derived from ant poison, ammonium chloride and L-arginine. CONSTITUTION:A pharmaceutically acceptable carrier equal to a medicine composition containing anti-inflammatory, immunosuppressive and analgesic compositions derived from ant poison is mixed with ammonium chloride and L-arginine. Further the mixture is blended with a compound selected from choline chloride, ethanolamine, putrescine, magnesium chloride, calcium chloride and L-lysine. The added amount of ammonium chloride is 1,500-2,500mug/ml and that of L-arginine is 1,200-2,200mug/ml. The blend is pharmaceutically manufactured into a dosage form such a tablet, capsule, solution, peppermint drop, syrup, elixin or powder. Swelling and inflammation of articular rheumatism are alleviated and ache is eliminated by administration of 0.5-1.5ml of the preparation per day for 10 days.

Description

Detailed Description of the Invention

[0001]

The present invention relates to anti-inflammatory properties derived from ant venom,
The present invention relates to a pharmaceutical composition equivalent to a pharmaceutical composition containing an immunosuppressive and analgesic composition and a method for treating an autoimmune disease using these compositions.

[0002]

[Prior art]

(Toxic Extract) The biological activity of the crude extract of poisonous insects of poisonous needle insects has been studied for over 100 years. In fact, the first chemical elucidation of ant venom was part of the 17th century. In the same study, formic acid was distilled from the ants of Formica rufa. Treatments with crude venom extracts (eg bee venom) have been reported for the treatment of rheumatoid arthritis. 1935 March
UK Patent No. 425543 issued on March 30, US Patent No. 21112828 issued on August 5, 1938, and 19
U.S. Pat. No. 2,154,934, issued Apr. 18, 39, reports the use of ant venom for the treatment of rheumatism. However, in reality, these treatments do not have a marked and certain effect due to insufficient purity of the venom extract or insufficient reproducibility of the venom extract composition. In addition, since individual insects have only a very small amount of poison, such a venom extract treatment method is expensive and cannot be adopted.

The biological activity of ant species has also been reported in the literature.
However, there are over 7,600 ants worldwide,
Moreover, the properties of most of the ant venoms, including 9 or more subfamilies in the ant family (formicidae), have not been identified or isolated. Even if the characteristics of ant venom have been elucidated, they are at most partially elucidated or partially purified. In fact, most ants have a maximum of 10μ per ant.
Since it has only g of poison, there is a practical problem in obtaining an ant venom of sufficient purity and sufficient purity for biochemical or clinical research. This is one of the reasons why there is not enough information about ant venom. It is known that various proteins, peptides, highly volatile organic compounds, etc. are contained in ant venom, and it is also known that the content ratio of these components varies depending on the ant species. For example, fire ant, Soi
enopsis invite and S. ric
htri) poison is rich in piperidine alkaloids. S. S. invicta fire ants (red fire ants) contain a much higher proportion of certain types of alkaloids than do North American fire ants. Fire ant venom also contains small amounts of protein. In contrast, the African solenosis, S.
Pink Tachicipes (Solenopsis, S. Pin
poisonous is the new world fire ant (new wo)
RLd fire ant) alkaloid compositions. When stabbed with this kind of ant's poison, the effect on humans is much less than that of a red fire ant. It was also reported that some other ant venoms contained nitrogen compounds. It is also known that some ants contain terpinenes in their poison. There are also reports that there are various poisons that are mostly composed of various proteins whose characteristics have not yet been clarified. For example, the Australian Bulldog Ant [(australianBu
ll Dog ants), Myrmccia pyr
lf ormis and M.F. Rulosn] is known to have significant pharmacological activity. These ant venoms include histamine, phospholipase A, peptide fractions (which are involved in smooth muscle stimulation), hemolytic factors and histamine releasing activity. Other subspecies of bulldog ant venom have also been reported to contain different proteinaceous venom components that have various biological effects on humans. New World Accumulation Ant [(New w
ord harvesterant), Pogono
myrmex badius] is at least four kinds of enzymes, namely, hyaluronidase, phosphorivase A,
It is known to have acid phosphotase and esterase.

In light of the above, it is not surprising that these crude ant venom extracts have a variety of in vivo activities. It is clear that many of the in vivo effects of applying ant venom needles and ant venom extracts to humans are bad and often very painful and uncomfortable. In fact, it has been reported that the ant venom causes anaphylaxis in humans and neurotoxicity in mammals. On the other hand, crude preparations of ant venom have been attempted to be used for treatment of human diseases such as rheumatoid arthritis [Schultz, D.R.].
Et al., Clinical Research
each), 26 (1), 1978; Schulz (Sc
Hultz, D.H. R. ) Et al., The Journal of Immunology (the Journal of Immu)
noology), 126, 1994-1998 (198).
1)]. Furthermore, the effect on human serum complement activation pathway (pathway) has also been studied by a polysaccharide derived from the venom of tropical ant Pseudomyrmex [Dicminger, L., Z.・ The Journal of Immunology
of Immunology), 123 (5), 197.
9]. Polysaccharides in Pseudomilmex ant venom have been isolated and characterized [Schütz
ltz, D.I. R. ) Et al., Molecular Immunology, 1
6, 253-264, 1979]. It has been reported that a crude preparation of ant venom obtained from Pseudomilmex sp. Has some therapeutic effect on some symptoms of rheumatoid arthritis [Altman, RD].
Et al, Arithritis and Rheumatism (Art
chris and and Rheumatims), 2
6, (4 Supplement), S55, 1983: Altman (RD) et al., Earthlights and Rheumatism, 27, 3 (1984)]. However, in vivo studies with these crude formulations have shown only limited improvement.
Sixty months of venom-treated patients showed minimal disease activity at 6 months of follow-up, while 40% of the patients had minimal or no sickness. Sensitivity to poisons was not perfect for all parameters, for active illness walking time,
It has also been reported that several measurements such as grip strength, ESR, rheumatoid factor and consumption of non-steroidal anti-inflammatory drug did not show significant improvement or reduction.

It has been reported that the crude ant venom filtrate of the genus Pseudomilmex contains active anti-inflammatory agents with apparent molecular weight factors below 1000. This substance was said to functionally inactivate C3 in normal human serum in vitro regardless of inflammation [Pyrnes (Byrnes, JJ) et al., Clinical Research, No. 33
Vol. 2, No. 1985). Further, it has been reported that an anti-inflammatory and analgesic compound was purified from the ant venom of the genus Pseudomilmex (JP-A-2-149594). Finally, a purified polysaccharide product from the genus Pseudomilmex is reported to have activity as an antirheumatic agent (US Pat. No. 42475 issued Jan. 27, 1981).
No. 40).

Autoimmune Disease-Rheumatoid Arthritis Autoimmune disease is generally defined as a disorder in which the immune system produces autoantibodies against endogenous antigens, resulting in damage to endogenous tissues. A wide variety of diseases are believed to be the result of autoimmune disorders. Although the identity of the factors that cause or initiate these abnormalities has not been clarified, it is believed that genetic factors play an important role. Examples of diseases considered to be the result of autoimmune disorder include systemic lupus erythematosus, Graves' disease, myasthenia gravis, mixed connective tissue disease, and rheumatoid arthritis. Autoimmune diseases
It is largely common in that connective tissue undergoes similar immune and inflammatory changes. Rheumatoid arthritis (hereinafter abbreviated as RA) is, for example, chronic systemic inflammation that mainly affects the synovium of peripheral joints. It is characterized by the proliferation of synovial cells and the progressive destruction of joints and periarticular structures. The cause is still unknown. Deposition of aggregated IgG-rheumatoid factor (RF) -complement complexes in synovial fluid occurs in RA. Rheumatoid factor (RF) is mostly self IgG
I with determinant specificity in certain regions of the derived heavy chain
gM globulin (sometimes IgG or IgA).
IgG-RF complement aggregates are also found in neutrophils, where they trigger the release of lysosomal enzymes involved in the inflamed joint response. Serum cells are also present in large numbers in the joint and synthesize anti-IgG antibodies. Pleural-derived (T) cells and lymphokines are also found in rheumatoid joints and are involved in inflammatory complex reactions.

The American College of Rheumatology
"Probably RA", "deterministic RA" and "typical RA"
Has established diagnostic criteria for RA [Bulletin on R
(heumatic Diseases), Volume 9, Volume 4
No., pp. 175-176, December 1958, and Primer on the Rheumatic Days (P
rimer on the Rheumatic Di
, JAMA, Volume 224, Page 799,
April 30, 1973, American Medical Association (American
Medical Association)].
These criteria are primarily intended for interviews in clinical studies, but also serve as a guide for clinical diagnosis of RA. For example, some of the criteria below are used. 1. Stiffness in the morning 2. 2. Movement or tenderness in at least one joint. Swelling of at least one joint (not only due to hypertrophy or fluidization of soft tissue, overgrowth of bone) (observed by a physician) Swelling of at least one joint other than 4.2 and 3 items (observed by a physician) ( (Do not leave an interval of 3 months or more with the confirmation of a few items). Symmetrical joint swelling with simultaneous symptoms on the same joint on both sides of the body (as observed by a physician) 6. Subcutaneous nodules (on the observation of a physician) on the protrusions and extensor surfaces of bones, or in areas near joints X-ray changes specific to rheumatoid arthritis 8. Positive agglutination test-RF is positive measured at 2 facilities showing a test result with a positive rate of 5% or less in the normal group. 9. Deposition of a small amount of mucin precipitate from synovial fluid (with fragments and cloudy solution) 10. Changes in characteristic pathological histology of synovial tissue 11. Changes in characteristic histopathological features of subcutaneous nodules showing granuloma lesions in which cell necrosis appears in the central part and peripherally-disseminated proliferating mononuclear cells and peripheral fibrosis and chronic inflammatory cell infiltration appear

(Therapeutic Method for Rheumatoid Arthritis) Although the underlying cause of RA has not been clarified, various therapeutic methods have been used to reduce symptoms such as pain, inflammation and swelling associated with this disease. Conservative treatment methods include resting, warming and cooling, and, in some cases, physical treatment and exercise. Other treatments include non-steroidal anti-inflammatory drugs for patients such as aspirin (trade name: salicylate), sibuprofen (trade name: motrin), indomethacin, fenoprofen (trade name: Nalphon), naproxen (trade name). : Naprosin), tolmethine (trade name: tectin), salinduck (trade name: clinorun), sodium meclofenamate (trade name: mechromene), piroxicam (trade name: Felden), and the like. However, many of these drugs cause serious gastrointestinal disorders and the like. Other anti-inflammatory agents used for the treatment of RA include hydroxychloroquine sulfate (trade name: plaqueenyl), which is an antimalarial agent. However, this drug still has severe visual
It causes skeletal and muscular disorders. Gold salts have also been used to treat RA. It has also been shown that these agents slow the bone erosion of rheumatoid arthritis. In fact, many patients have benefited from gold treatment. However,
The toxic reaction of gold salt also appears. Penicillamine has also been used in patients with severe RA, while its toxicity is significant. Corticosteroids have also been used as anti-inflammatory agents in the treatment of RA, but they may cause serious problems when used for a long period of time. In short, most of the drugs accepted for the treatment of RA have serious side effects and are mostly effective only during the treatment period. It has been found that many of the above compounds, with the exception of gold salts, provide little or no RA symptoms in patients once their use is discontinued.

[0009]

Accordingly, it is an object of the present invention to provide a safe and durable drug for RA and its related conditions. With in vivo administration,
It is also an object of the present invention to provide a highly pure anti-inflammatory, immunosuppressive and analgesic composition which exhibits higher activity in the treatment of autoimmune diseases such as RA while maintaining a low level of side effects as compared with a crude preparation. Is the purpose of. It is yet another object of the invention to provide a pharmaceutical composition comprising said anti-inflammatory, immunosuppressive and analgesic composition. It is still another object of the present invention to provide a method for treating an autoimmune disease, which uses a compound having a high purity and a high activity and a related pharmaceutical composition, which has a long-lasting effect and has no side effect.

[0010]

SUMMARY OF THE INVENTION Broadly stated, the invention disclosed and described herein provides an anti-inflammatory, immunosuppressive and analgesic equivalent natural composition derived from ant venom. Included are inflammatory, immunosuppressive and analgesic pharmaceutical compositions and methods of treating autoimmune diseases using these compositions. A particularly useful pharmaceutical composition for use in the present invention is the genus Pseudomyrme.
x), preferably a composition having the same effect as a composition obtained from an ant of the genus Tripularinus of the genus Pseudomilmex. The ant-derived anti-inflammatory, immunosuppressive and analgesic composition is about 3120c.
m ^-1 , 3020 cm ^-1 , 1480 cm ^-1 , 1400 cm
^-1 and 950 cm ^-1 with characteristic absorption, about 1640
The cm ^-1 and 1540 cm ^-1 with an infrared spectrum does not show the characteristic absorption. The present invention provides an anti-inflammatory, immunosuppressive and analgesic pharmaceutical composition having a composition elucidated by further analyzing this anti-inflammatory, immunosuppressive and analgesic compound derived from ant venom. The present invention also provides an anti-inflammatory, immunosuppressive and analgesic pharmaceutical composition containing a compound selected from choline chloride, L-arginine, putrescine, ethanolamine, ammonium chloride, calcium chloride, magnesium chloride and L-lysine. Also provide.

One of the autoimmune diseases that can be treated by this method is rheumatoid arthritis. The method has been shown to reduce swelling and inflammation and eliminate the pain caused by autoimmune disease in patients with autoimmune disease. In addition, the method of applying the pharmaceutical composition to a patient of about 0.5 ml to 1.0 ml per day for about 10 days reduces pain and reduces swelling for at least about 4 weeks after the treatment. I know. In fact, it appears that this treatment alleviates rheumatoid arthritis symptoms for some period of time, although it is not yet clear for some months or years, or for the life of the patient, for some time. Hereinafter, the present invention will be described in more detail. The present invention relates to a pharmaceutical composition having anti-inflammatory, immunosuppressive and analgesic effects equivalent to those of an anti-inflammatory, immunosuppressive and analgesic composition obtained from an ant of the genus Pseudomilmex triplarinas, and discloses them. To do. More specifically,
This pharmaceutical composition is characterized by containing a compound selected from choline chloride, L-arginine, putrescine, ethanolamine, ammonium chloride, calcium chloride, magnesium chloride and L-lysine. The pharmaceutical composition of the present invention is prepared by containing a pharmaceutically acceptable carrier, and examples of such a carrier include 0.25M NaCl and physiological saline, and further contains albumin and the like as a stabilizer. May be done. The pharmaceutical composition of the present invention includes 2500-
3500 μg / ml choline chloride, 1200-2200
μg / ml L-arginine HCl, 700-1700
μg / ml putrescine / HCl, 500-1500μ
g / ml ethanolamine, 1500-2500 μg
/ Ml ammonium chloride, 5-200 μg / ml calcium chloride, 5-200 μg / ml magnesium chloride, 5-200 μg / ml L-lysine.2HCl at a concentration of a compound selected from these compounds Is preferred.

Assay Method The following assay was used to confirm the characteristics of the ant venom extract disclosed in Japanese Patent Application Laid-Open No. 2-149594 used as a control of the pharmaceutical composition disclosed herein. Was given. 1. Inhibitory activity against third component of normal human serum complement (C3) After adding 20 μl of fresh serum and 80 μl of sample (poisonous aqueous solution) collected from 10 normal volunteers to a polypropylene tube, 37 ° C. for 4 hours Incubate with shaking. The obtained serum is 0.1% (w
/ V) Gelatin, 2.5% (w / v) glucose and 2.5 mM of Peronal buffer (pH 7.3, hereinafter referred to as GGVB) containing 0.15 mM CaCl ₂ and 1 mM MgCl ₂ and diluted in this order. , 50-12800
Prepare a 2-fold diluted sample. 200 μl of each of the diluted serum samples thus obtained was added to 1 × 10 ⁸ / ml.
Hemolytic intermediate, EACl ^gp 4 ^hu [EA excess guinea pig serum complement first component (Cl ^gp ) and excess human serum complement fourth
Obtained by reacting with component (C4 ^hu )] 100μ
1, 100 CH50 unit / ml, human complement second component (C2) 100 μl and 100 CH50 unit / ml
Human complement fifth component (C5), sixth component (C6) and seventh component (C7), each containing ml, are mixed with 100 μl each and then incubated at 30 ° C. for 30 minutes. 1
100 μl each of the eighth component (C8) and the ninth component (C9), each containing 00 CH50 units / ml, are added to the mixture, which is then incubated for another 30 minutes at 37 ° C. 1 ml of ice-cold CCVB is added to the resulting reaction mixture, followed by centrifugation at 3000 rpm for 10 minutes. Collect the supernatant and measure its absorbance. The degree of hemolysis in each diluted sample was determined using distilled water instead of GGVB. At that time, the absorbance at 415 _nm obtained during hemolysis of the EACl ^gp 4 ^hu hemolytic intermediate was set to 100%. The dilution ratio (titer) of serum showing 50% hemolysis (CH50 unit) was determined. The C3 inhibitory activity of the sample was calculated by the following formula. Inhibitory activity (%) = {(AB) / A} × 100 (In the formula, A represents the C3 titer of normal human serum treated with distilled water or 0.25M NaCl at 37 ° C. for 4 hours, and B Represents the C3 titer of normal human serum treated with the sample at 37 ° C. for 4 hours.)

2. Inhibitory activity against mouse spleen cell growth 100 μl of RPM1-1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 5% fetal bovine serum in which 2 × 10 ⁶ / ml BALB / c strain mouse spleen cells were suspended was mixed with 10 μl of concanavalin A.
A known ant venom extract sample at a concentration of g / ml and 50 μl is mixed with 50 μl of the same lysed medium. 5%
After incubating at 37 ° C. for 48 hours in air containing CO ₂ , 0.5 μCl / 25 μl of ³ H-thymidine was added dropwise, followed by further incubation for 18 hours under the same conditions. Whole mouse spleen cells were collected by a cell harvester, and the intensity of beta ray was measured by a liquid scintillation counter. About 30,000 spleen cells activated by Concanavalin A in the control without sample addition
It contained 0 dpm ³ H-thymidine. Growth inhibitory activity was calculated by the following formula. Growth inhibitory activity (%) = {(A−B) / A} × 100 (In the formula, A is the amount of ³ H-thymidine (dpm) in the control, and B is ² H-when the sample is added. The amount of thymidine (dpm) is shown.)

3. Coloring Test Using Periodic Acid-Schiff Reagent (PAS) 0.2 ml of ant venom extract sample and 0.02 ml of 2% aqueous periodic acid solution are placed in a glass test tube and mixed well. After standing at room temperature, add 1.0 ml of Schiff reagent and mix well. After further standing at room temperature for 30 minutes, it exhibited a reddish purple color as compared with a control using water.

4. Color test by anthrone method 100 to a mixture of 50 ml of 95% sulfuric acid and 10 ml of distilled water.
mg of Anthrone was dissolved. 3 ml of the anthrone reagent prepared in this way was added to 0.5 m of the ant venom extract sample solution
was added to a glass test tube with 1. This test tube was immersed in boiling water for 10 minutes, cooled to room temperature, and then subjected to a colorimetric method. As a result, a blue-green color was exhibited in comparison with a control using water.

5. Coloring Test by Carbazole Sulfate Method 0.5 ml of the ant venom extract sample is added to a glass test tube, 3 ml of concentrated sulfuric acid is added to the glass tube under ice cooling, and immersed in boiling water for 20 minutes. After cooling to room temperature, 0.1 ml of 0.1 carbazole in 95% ethanol is added to this mixture, which is then mixed well. After the test tube was left at room temperature for 2 hours, it exhibited a reddish purple color as compared with a control using water.

6. Adsorption test with Concanavalin-bound Sepharose A Concanavalin-bound Sepharose A (Pharmacia Fine Chemicals) 10 mm in diameter x 100 mm in length
Column and 50 mM NaCl pH 7.0 was passed at a flow rate of 1 ml / min. 1 ml of ant venom extract sample solution
Was collected and the elution fractions were collected while monitoring A ₂₁₄ . The inhibitory activity of the sample on normal human serum C3 was measured before and after passing through a concanavalin A column.

7. Infrared Absorption Spectra Analysis After freeze-drying the ant venom extract sample, the sample was further vacuum dried at room temperature for 20 hours. The infrared absorption spectrum of the sample was measured by the diffuse reflection method using a Fourier transform infrared spectrophotometer FTS-15C type (Digilabo).

8. Nuclear Magnetic Resonance Spectroscopy After lyophilizing the ant venom extract sample, the sample was further vacuum dried at room temperature for 20 hours. The sample was dissolved in D ₂ O and measured by Varian XL-300.

Reference Example Reference 1 Preparation of crude ant venom extract The preparation of an anti-inflammatory / analgesic compound using the control ant venom of the present invention is described below by reference example. further,
The properties, activity and usefulness of clinical efficacy against autoimmune diseases of these compounds are also described.

1. Water extract of ant venom Poisonous needles and venom sacs were separated from 150 ants of the genus Tripramulinus sp. 5 ml of distilled water was added to the poison needle and the poison sac, and then shredded. The shredded mixture was No. After filtering with a glass filter of No. 3, washing the filtrate with a small amount of water, combining the obtained filtrates,
Distilled water was added to bring the total volume to 15 ml. Then, the toxic water extract was obtained by first filtering with Amicon YM10 (molecular weight cutoff 10,000) and then with Amicon YM2 (molecular weight cutoff 1000). The poisonous water extract obtained is pale yellow, transparent and
_{280 nm} = 0.485, A _{260 nm} = 0.649, A _{214 nm}
= 2.60 and pH was 5.7. The solid content after freeze-drying was 0.89 mg / ml. The inhibitory activity against normal human serum C3 was 64%.

2. Preparation of fraction EP-1 by pretreatment of ant venom (AV) water extract 100 ml of the ant venom water extract obtained in the above 1 was 4
It was concentrated to 10 ml in 30 minutes while heating at 5 ° C.
0.22 μm GV filter for water-insoluble matter (Millipore)
After removal with, the concentrate was frozen once and thawed at room temperature.
A small amount of insoluble material was removed by filtration with a 0.22 μm CV filter. 1 ml of the obtained toxic water extract was subjected to FPLC liquid chromatography (Pharmacia Fine Chemicals) at room temperature using a mono QHR 10/10 column and distilled water as a mobile phase. A. Elution was performed with distilled water at a flow rate of 2 ml / min while monitoring at _{214 nm} . First non-adsorbed fraction, EP-1, 12m
1 was collected to give a total of 120 ml. The other fractions were eluted with a maximum concentration of 0.25M NaCl. Anion exchange FP
The result of LC chromatography is shown in FIG.

[0023]

FIG. 1 120 ml of the obtained fraction EP-1 was concentrated to 12.5 ml by freeze-drying. The nearly colorless transparent fraction EP-1 showed 99% inhibitory activity against normal human serum C3, but the other fractions did not show C3 inhibitory activity. Furthermore, the fraction EP-1 was subjected to such pretreatment to inhibit the growth of mouse spleen cells stimulated with concanavalin A (even at a 1 / 8-fold dilution) by 80%. Further, the fraction EP-1 was positive in the PAS staining test, the anthrone color test and the carbazole sulfate color test.

3. Liquid Chromatographic Fractionation of Fraction 26 4 ml of the fraction EP-1 obtained as described above was applied to a GS320P column (Asahi Kasei) equilibrated with distilled water at room temperature. A. The material in the 5-10 main fractions was eluted with distilled water at a flow rate of 1 ml / cm ² per minute while monitoring at _{214 nm} . Then 50 mM N at pH 4-8
Fractions immediately eluted with aCl (fraction 26) were collected for a total of 24 ml. The 24 ml sample was concentrated to 4.8 ml by freeze-drying. The chromatography of this adsorbed liquid is shown in FIG.

[0025]

FIG. 2 shows the infrared absorption spectrum of the obtained fraction 26 in FIG.

[0026]

FIG. 3 shows the results obtained in various characteristic tests of Fraction 26.

[0027]

[Table 1]

4. Inactivation of C3 by ant venom raw material Sabant Speedback, 200 ml sample of ant venom extract (molecular weight 1000), (Savant Instrument)
To about 18 ml and adjusted to 20 ml with distilled water. 0.22 μm Milex GV (Millipore)
In filtered, after removing the precipitate was subjected to the 1/10 concentrate Mono QHR _^10/10 by 2ml minutes. Fraction EP-
80 ml of 1 was subjected to FPLC 10 times, and the collected fractions were 2 times.
Concentrated to 5 ml. In the next step, add 4 m of this fraction EP-1
Each l was subjected to adsorption FPLC. The active substance is 50 mM N
Elution with aCl gave fraction 26 (total volume 75 ml).
About 98% of impurities were removed from the 1000 molecular weight AV extract by two FPLC methods (depending on the peak area at A ₂₁₄ ). 75 ml of fraction 26 was concentrated to 15 ml and filtered through a 0.22 μm Milex GV for sterilization. Dispense the AV preparation into sterile vials,
Used for evaluation in rheumatoid arthritis patients (4 ml, 3 vials). The inactivity of normal human serum C3 by these AV fractions is shown in Table 2.

[0029]

[Table 2]

Reference Example 2 Clinical Assay Below, fraction 26 of the ant venom extract obtained by the method described above is used.
Described below are clinical data supporting that the drug is used for treating patients with autoimmune diseases such as rheumatoid arthritis. The clinical data obtained with Fraction 26 was compared to the clinical data obtained with Fraction 1 (designated EP-1). From the results below, in contrast to EP-1, it was revealed that in the case of fraction 26, there are some signs that administration of them led to improvement of the patient's symptoms. The clinical data described below and summarized in 3-14 were obtained according to the following therapeutic protocol. Treatment Procedure Item Content 1. Treatment method 0.5 to 1.0 ml per day for a minimum of 10 days per patient 1. Dose 10 days (depending on the amount of fraction 26 available). Where indicated, albumin was added as a stabilizer. 3. Administration schedule Daily injections excluding weekends, injections for a total of 10 days 4. Dosage form Subarm subcutaneous injection 5. The data were based on the standards approved by the FDA and the American College of Rheumatism listed in the table. Altman et al., Note to Arthritis Andrew-Matism 1984 (the contents of this reference are incorporated herein by reference) In Tables 3-14, each numbered letter (eg, N
o. 82. J. A) shows the patient's initials. "EP
-1 "is FPLC system and Mono Q column (anion exchanger)
Outflow peak no. 1 (effluent pea
k no. 1) is shown. Also, "HA" is a headache.
Ache) is shown. "NSAID" means a non-steroidal anti-inflammatory drug (eg, aspirin). The following two criteria were used to establish the pain index. That is, a. Determine the total number of painful joints, b. Determine the degree of severity of each joint with 1 = gradual, 2 = moderate, 3 = terrible, and multiply the degree of severity by 1.
Find the findings of each joint and add them as the severity of all joints. Therefore, pain index = (total number of joints experiencing pain / total severity of all joints) “Swelling index” was determined in the same manner as the pain index. "Sed rate" means the sedimentation rate of red blood cells, "grip strength" was measured three times for each hand, and the average was taken. "Walking" is the time required to walk 50 steps per second. Is expressed as “age”, and “age” indicates age.
"ne)" represents the condition of the patient before treatment with fraction 26 or EP-1 and represents the time from when the first clinical effect appears. "Week of diagnosing symptoms" is 10 days. Shows the condition of patients who were seen one week after the first injection of the treatment of 1. The subsequent examinations, unless indicated otherwise, in turn indicate that they were performed one week after the previous injection. (For example, medical examination No. 2 shows that the medical examination was performed two weeks after the second injection, and medical examination No. 3 showed three weeks after the third injection.) Each measurement of rheumatic patients was
American Journal of Medical Science (the Am.J.Med.Sci.). 232: 3
00-310 (1956) and Arthritis
Rheumatism (Arthritis Rheum.). 1:
550-522 (1958) according to Lansbury criteria.

[0031]

[Table 3]

Patient No. At 90, improvements were seen for all parameters. Over 50% pain relief over 2 weeks with 2/3 of swelling, or about 66
% Has been reduced. After 4 weeks there was again a 50% reduction in both pain and swelling. Examination No. It was reported that the sore throat at the time of 2 lasted for 24 hours. Fourth medical examination No. At 4, headache and itching of the torso for 2 days were reported.

[0033]

[Table 4] The patient responded dramatically within a week of being injected. In the week following the third week of injection (week 3), the pain increased slightly but swelling continued. Pain increased and swelling increased slightly at 5 weeks. Improvement was seen in each parameter at 8 weeks. Injection site pain was reported. The walking time increased and the erythrocyte sedimentation rate decreased.

[0034]

[Table 5] Improvement was seen in all parameters. At Week 2, there was a 2/3 reduction in both joint pain and swelling. Sensation lasted until the most recent assessment, week 4. Reported fever, sore throat, and faint voice were the results of the examination. There was 3
This lasted 12 days. Purulent sore throat was also seen. This was dealt with antibiotics.

[0035]

[Table 6]

The patient was dramatically sensitized in the first week (day 6) and had mostly recovered by the second week. Discontinue injection (4 weeks)
The number of joints with pain and swelling increased in the 1st week, but still only half of the pretreatment number. The patient remained at the same level until 8 weeks, but the pain increased thereafter. Swelling is the standard amount of 5
It remained below 0%. Local reaction included injection site pain.

[0037]

[Table 7] Joint swelling showed minimal to moderate improvement, but slight improvement in pain. 10 treatments for patients
It was discontinued due to lack of efficacy at week 1. A local reaction of injection occurred at the 12-14th injection.

[0038]

[Table 8] Improvement was seen in all parameters. Pain and swelling were significantly reduced by 1 week. The effect continued until 12 weeks. Pain increased at week 12 and treatment for this patient was discontinued. Sympathy-like symptoms were present at the time of the last injection and continued for 2 days thereafter.

[0039]

[Table 9] At the end of the second week, joint swelling and pain improved to a moderate degree. At the 6th week, each parameter deteriorated, so the study of this patient was stopped due to lack of efficacy. Sensho-like symptom was reported in the first week, which spontaneously healed in 2-3 days.

[0040]

[Table 10]

There was minimal improvement in pain and moderate improvement in swelling. The minimal improvement did not continue and this patient also lacked efficacy, so the study was terminated at 3 weeks. It was reported that itching of the lips lasted for only one day. An upper respiratory tract symptom and headache were seen a week later, but it subsided in 4 days. This patient is patient No. The same person as 83, and the same patient requested retreatment with another fraction.

[0042]

[Table 11] Injections were given every other day, but the patient showed minimal improvement in pain and swelling. Swelling 5 at the second week
I got 0%. Examination No. At 8 the number of swollen joints was further reduced. There were also improvements in grip strength and walking time. The erythrocyte sedimentation rate was determined by examination No. It gradually dropped from 75 to 60 at 8. Examination No. 2, loss of appetite, nausea, bloating,
Diarrhea was reported, which lasted for 2 days. Although the swelling did not worsen, the pain increased, so the administration of motrin was restarted in 3 weeks.

[0043]

[Table 12] Patients showed> 50% improvement from baseline to 14 weeks. The patient continued to improve, with pain and swelling reduced by more than 90%.

[0044]

[Table 13] Patient No. 100 showed a moderate reduction in joint pain and swelling and the effect was maintained up to 10 weeks. Patients continue to show improvement from longitude to moderate.

[0045]

[Table 14] Patient No. 102 showed moderate improvement to remarkable improvement in the joint within 2 weeks, and the effect continued until 8 weeks. Second treatment of venom extract 9 months later, 2 of the first result
It was twice as effective. After 22 weeks, it continues to show remarkable improvement.

In summary, three patients injected with the ant venom extract fraction 26 were clearly responsive, with 2 of them having a dramatic improvement. Therefore, from the results of the above patients, the fraction 26
Is concluded to contain an effective anti-inflammatory and analgesic compound.

[0047]

【Example】

Example 1 Characterization of Fraction 26 As a result of comparison of the NMR peak of Fraction 26 with a standard of known material, active fractions were found to be choline chloride, arginine, putrescine, ethanolamine, ammonium chloride, calcium chloride, chloride. It was identified as magnesium and lysine.
Based on the amount of each component found in several different samples of fraction 26, the following concentrations of the "average" pharmaceutical composition were prepared in 0.25M NaCl. Choline chloride 2890 μg / ml L-arginine · HCl 1398 μg / ml Putrescine · HCl 880 μg / ml Ethanolamine 608 μg / ml Ammonium chloride 1722 μg / ml Calcium chloride 122 μg / ml Magnesium chloride 168 μg / ml L-lysine · 2HCl 10 μg / ml

In clinical trials, this pharmaceutical composition was shown to be as effective in alleviating arthritic conditions as natural ant venom (fraction 26). These clinical trials and results are detailed in Example 2. In order to determine which component of fraction 26 was required for its activity, a pharmaceutical composition with one component removed was prepared. Concanavalin A-induced lymphocyte proliferation inhibitory activity was compared. This growth inhibitory activity serves as an index of immunosuppressive activity and anti-inflammatory activity. University of Miami Medical School (University of Maiami Med)
Blood was obtained from randomly selected individuals on the I.S. Lymphocytes were separated by Ficoll-Hypaquen and centrifugation. Lymphocytes from individual donors were used as received. The lymphocyte count was adjusted to 1 × 10 ⁶ cells / ml. 1
00 μl of cells were added to microtiter plates. Two concanavalin A mixtures were prepared and 100 μl was added per well to give concentrations of either 5 or 10 μg / treatment. 25 μl of each pharmaceutical composition was added. The plates were incubated at 37 ° C. under CO ₂ air for 5 days. 18 hours after adding ³ H-thymidine to each well, cells were harvested. ^The rate of incorporation of ³ H-thymidine was measured with a scintillation counter. The results are shown in Table 15.

[0049]

[Table 15] The excluded pharmaceutical composition of ammonium chloride showed the greatest reduction, followed by arginine and choline chloride. The reduction in inhibition by the composition excluding putrescine or ethanolamine is relatively small. Compositions with the exclusion of calcium chloride, magnesium chloride or lysine did not significantly affect the inhibitory activity, especially at low concentrations of concanavalin A.

Example 2 Pharmaceutical Composition and Immunosuppressive Activity In order to see the equivalence of activity between the pharmaceutical composition of the present application and natural ant venom (fraction 26), a certified rheumatologist (Certif).
ied Rheumatologist) 8
The two patients were compared in a double-blind study. The pharmaceutical composition of the present application used has the composition described in Example 1 above. Administration is as per the test for natural ant venom (fraction 26). 4 patients (115, 116, 119 and 120)
The present invention was administered with the pharmaceutical composition of the present invention, and the remaining four were administered with natural ant venom (fraction 26). Table 1 shows the treatment progress of each patient
6 to 23.

[0051]

[Table 16]

[0052]

[Table 17]

[0053]

[Table 18]

[0054]

[Table 19]

[0055]

[Table 20]

[0056]

[Table 21]

[0057]

[Table 22]

[0058]

[Table 23]

At least 3 out of 4 patients receiving the pharmaceutical composition of the present invention had a therapeutic effect. Patient No. 11
Including the 9 delayed therapeutic effects, all 4 patients receiving the pharmaceutical composition showed therapeutic effects. Table 24 shows a summary of the therapeutic effects by doctors and patients. From these results, the pharmaceutical composition of the present invention was equivalent in activity to natural ant venom.

[0060]

[Therapeutic method] As shown in the above examples, the anti-inflammatory property,
One aspect of this invention is that the immunosuppressive and analgesic compositions may be used to treat patients with autoimmune diseases such as rheumatoid arthritis. Generally speaking, the pharmaceutical composition used in the present therapeutic method comprises an anti-inflammatory drug containing a compound selected from choline chloride, L-arginine, putrescine, ethanolamine, ammonium chloride, calcium chloride, magnesium chloride and L-lysine. It contains a sex, immunosuppressive and analgesic composition and a pharmaceutically acceptable carrier.

Generally speaking, the present method for treating patients with autoimmune diseases comprises a compound selected from choline chloride, L-arginine, putrescine, ethanolamine, ammonium chloride, calcium chloride, magnesium chloride, L-lysine. An effective amount of a pharmaceutical composition containing the anti-inflammatory, immunosuppressive and analgesic composition described above and a pharmaceutically acceptable carrier is administered to a patient with autoimmune disease. In a preferred method of treatment, the patient may be administered the pharmaceutical composition described above for 10 days. However, depending on the professional judgment of the patient's response to this treatment, the doctor can significantly shorten it (2 or 3 days) or prolong it (months or years), or It may be temporarily interrupted. However, based on the above clinical results, it is possible to administer pain to an autoimmune disease patient by administering the pharmaceutical composition at about 0.5 to 1.0 ml / day for about 10 days,
From inflammation to swelling for at least 1 to 4 weeks, 2
It has been found to reduce by the second week or in some cases considerably longer.

Since the composition is effective in small amounts, it can be administered to patients in many dosage forms. The pharmaceutical composition is
It may be administered in a suitable oral dosage form (eg, tablet, capsule, solution, mint drop, syrup, elixir, suspension, gel, powder, etc.). It is preferably formulated so as to avoid destruction by digestive juices. Further, the present anti-inflammatory, immunosuppressive and analgesic composition may be administered in a parenteral dosage form as shown in the above clinical examples. In this type of dosage form, the active ingredient may be in solution or suspension. Local administration of the present anti-inflammatory, immunosuppressive and analgesic compositions to a patient is also within the scope of the invention. In this dosage form, the active ingredient can be formulated in dosage forms such as ointments, creams, pastes, plates, powders, aerosols, lotions, bandages, solutions and the like. The pharmaceutical composition of the present invention can be administered to patients in need of treatment for autoimmune diseases such as rheumatoid arthritis. The dose, dose rate, and administration method are almost the same as those currently employed in clinical studies on human-derived substances, and, for example, preferably about 0.5 to 1.
It is recommended to administer 0 ml parenterally. However, much lower, eg about 0.01 ml per day
The range of doses determined by the doctor is considered to be an effective amount for treating patients with autoimmune diseases, from the dose of 1 to about 10 ml per day. In determining the dose, the physician took into account other patients and various other factors based on their own experience with this composition and other autoimmune drugs as well as how the patients responded. In addition, you should make a judgment from a professional standpoint as a doctor.

The pharmaceutical composition of the present invention is prepared by mixing the anti-inflammatory, immunosuppressive and analgesic composition with a pharmaceutically acceptable carrier, excipient, etc. according to a known method for producing a pharmaceutical composition.
It can be formulated. Suitable excipients and their formulation are
For example, the description by Remington Pharmaceutical Sciences (EW Martin) is incorporated herein by this reference. Such compositions will contain an effective amount of ant venom extract and an appropriate amount of excipients to prepare a pharmaceutically acceptable formulation suitable for effective administration to the subject. Although the present anti-inflammatory, immunosuppressive and analgesic compositions have been described in detail, the present invention provides
It is needless to say that the formulations and treatment methods disclosed herein should not be limited to the above, and that the formulations and treatment manufacturing methods may be changed within the spirit and scope of the present invention.

[0064]

EFFECTS OF THE INVENTION The anti-inflammatory, immunosuppressive and analgesic composition of the present invention has a long-lasting effect, especially against autoimmune diseases, and is a highly pure and highly active pharmaceutical composition with no side effects. Can be provided.

[0065]

[Brief description of drawings]

[0066]

FIG. 1 shows anion exchange chromatography, in which the arrow indicates the elution peak of the first free extract fraction EP-1.

[0067]

FIG. 2 shows adsorption chromatography, in which the arrow indicates the elution peak of the ant venom (ie AV) fraction 26.

[0068]

FIG. 3 is an infrared absorption spectrum of the AV fraction 26 (absorption is Kubelka-Mun
k) method has been corrected; SAMF is sample No.
RES means split; SCANS means serial number of measurement).

[Table 24]

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 13, 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]

FIG. 1 shows anion exchange chromatography, in which the arrow indicates the elution peak of the first free extract fraction EP-1.

FIG. 2 shows adsorption chromatography, in which the arrow indicates the elution peak of the ant venom (ie AV) fraction 26.

FIG. 3 is an infrared absorption spectrum of the AV fraction 26 (absorption is Kubelka-Mun
k) method has been corrected; SAMF is sample No.
RES means split; SCANS means serial number of measurement).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location A61K 47/18 E // A61K 35/64 AAH 7431-4C ABC 7431-4C ABE 7431-4C ABG 7431 -4C

Claims (26)

[Claims] 1. An anti-inflammatory, immunosuppressive and analgesic composition comprising an effective amount of ammonium chloride and L-arginine in combination with a pharmaceutically acceptable carrier. 2. The composition of claim 1 further comprising an effective amount of choline chloride. 3. The composition of claim 1 further comprising an effective amount of ethanolamine. 4. The composition according to claim 1, further comprising an effective amount of putrescine. 5. The composition of claim 1, further comprising an effective amount of ethanolamine and putrescine. 6. The composition according to claim 2, further comprising an effective amount of at least one of magnesium chloride, calcium chloride and L-lysine. 7. A composition according to claim 1 which comprises an effective amount of ammonium chloride, L-arginine, choline chloride, ethanolamine, putrescine, magnesium chloride, calcium chloride and L-lysine. 8. 1500 to 2500 μg / ml ammonium chloride and 1200 to 2200 μg / ml L-
The composition according to claim 1, which comprises arginine. 9. The composition according to claim 8, which contains 2500 to 3500 μg / ml of choline chloride. 10. The composition according to claim 9, which comprises 500 to 1500 μg / ml of ethanolamine. 11. The composition according to claim 9, which comprises 700 to 1700 μg / ml putrescine. 12. The composition according to claim 9, which comprises 500 to 1500 μg / ml of ethanolamine and 700 to 1700 μg / ml of putrescine. 13. The composition according to claim 8, which contains at least one or more of magnesium chloride, calcium chloride and L-lysine in an amount of at least 5 to 200 μg / ml. 14. A composition according to claims 1-7, wherein the composition is in a form suitable for oral administration. 15. The composition according to claim 1, which is in a form suitable for parenteral administration. 16. The composition of claims 1-7, wherein the composition is in a form suitable for topical administration. 17. The composition according to claim 1, wherein the composition form is selected from tablets, capsules, solutions, mint drops, syrups, elixirs, suspensions, gels and powders. 18. The composition according to claim 1, wherein the form of the composition is selected from a solution and a suspending agent. 19. The composition according to claim 1, wherein the composition form is selected from an ointment, a cream, a paste, a plater, a powder, an aerosol, a lotion, a plaster and a solution. 20. The solution of the composition is aqueous.
7. The composition according to 7. 21. An effective amount of ammonium chloride and L-
A method for treating an autoimmune disease, which comprises administering a composition containing arginine. 22. The method of treatment of claim 21, which comprises administering a composition further comprising an effective amount of choline chloride. 23. The method of treatment according to claim 22, which comprises administering a composition further comprising an effective amount of ethanolamine. 24. The method of treatment according to claim 22, which comprises administering a composition further containing an effective amount of putrescine. 25. The method of treatment according to claim 22, which comprises administering a composition further comprising an effective amount of ethanolamine and putrescine. 26. The method of treatment according to claim 25, which comprises administering a composition containing an effective amount of magnesium chloride, calcium chloride and L-lysine.