JPS6215529B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anti-inflammatory agent whose active ingredient is tetrahydroesterastin, which is chemically derived from elastin obtained by culturing specific microorganisms. Esterastin is a substance isolated and collected by Umezawa et al. from actinomycete culture solution (Japanese Patent Application Laid-open No. 53-98901).
No.), has esterase inhibitory activity, and in toxicity tests on mice, no toxicity was observed at a dose of 250 mg/Kg (ip). The chemical structure of esterastine is as follows: It was recognized that this was the case. Furthermore, by treating esterastin with a catalytic reduction reaction, a new and useful anti-esterase active substance, tetrahydroesterastin, which has antiesterase activity similar to esterastin and has a broader group of inhibitory enzymes than esterastin, is produced. can get. (Special Patent Application No. 1983-61725, i.e., Special Publication No. 1983-61-
(Refer to Publication No. 45620). The chemical structure of this tetrahydroesterastine is as follows: It was recognized that this was the case. Esterastin, for example, is produced by Streptomyces lavendrei strain MD4-C1 (Feikoken Bibori No. 3723), which was isolated from a soil sample within the Institute of Microbial Chemistry.
This is a substance with anti-esterase activity isolated from the culture obtained by culturing. Esterastine and tetrahydroesterastine reduce humoral antibody-producing cells and also have a suppressive effect on cell-mediated immunity, and are effective against many intractable diseases induced by immune reactions, such as arthritis, lithium, and multiple sclerosis. The present inventors further investigated the pharmacological effects of esterastine and tetrahydroesterastine, and experimentally confirmed that these compounds have strong anti-inflammatory effects. The gist of the present invention is an anti-inflammatory agent containing as an active ingredient at least one of tetrahydroesterastine and its acid addition salt. Tetrahydroesterastine used as an active ingredient in the anti-inflammatory agent of the present invention also includes its pharmacologically acceptable salts. Representative salts include acid addition salts at the amino group, such as hydrochloride, sulfate, methanesulfonate, trichloroacetate, and the like. Hereinafter, these will be collectively referred to as the present active ingredient substances. The anti-inflammatory agent of the present invention is used in an appropriate administration method, and in the case of an injection, a PH adjuster, a buffer, a stabilizer, an excipient, etc. may be added to the active ingredient. Freeze-dried injections can be made by freeze-drying by a conventional method, and by adding pH adjusters, buffers, stabilizers, isotonic agents, topical narcotics, etc. to this active ingredient. It is prepared as a solution or suspension for subcutaneous, intramuscular, or intravenous injection. When preparing a solid preparation, the active ingredient is combined with conventional excipients, stabilizers, and if necessary, binders,
By adding a disintegrant, a lubricant, a coloring agent, a flavoring agent, a flavoring agent, etc., it can be made into tablets, granules, powders, capsules, ointments for external application, etc. by conventional methods. The dosage of this active ingredient varies depending on the administration method and symptoms, but the usual dosage for adults is 0.5 mg/Kg of tetrahydroesterastine.
200mg/Kg is appropriate and should be administered 3 times a day or more than 3 times a day depending on the symptoms. Regarding the production of tetrahydroesterastine,
Esterastine is dissolved in methanol, a known hydrogenation catalyst such as platinum oxide or palladium is added, and reduction is carried out for 2 hours to overnight in a hydrogen stream or using a Parr reduction apparatus. The catalyst is removed by filtration and the liquid is concentrated to obtain tetrahydroesterastin. Further, if necessary, it can be further purified by silica gel column chromatography. As the elution solvent, a mixed solvent such as ethyl acetate and chloroform is used, and a mixture ratio of 1:1 is particularly suitable. Tetrahydroesterastin is obtained as a white powder and has the following physicochemical properties. That is, melting point 102.5-104℃, molecular weight measured by mass spectrometry 510, elemental analysis value: C65.84%,
H9.71%, _N4.93 %, _O18.12 % _{and C28H50N2O6}
It has a molecular formula of Infrared absorption spectrum (potassium bromide tablets): 3320, 2920, 1830, 1720, 1650,
It has specific absorption bands at 1610, 1545, 1185, 1120, 885, and 720, and the nuclear magnetic resonance absorption spectrum (deuterium chloroform solution, δppm) is 3.2 (CH at 2nd position) and 4.34.
(3rd place CH), ~2.1 (4th place CH ₂ ), 5.02 (5th place CH),
1.26 (6th to 15th CH ₂ , 2' to 5' CH ₂ ), 0.88 (16th
CH ₃ , 6′ position CH ₃ ), ~1.76 (1′ position CH ₂ ), 4.72 (2″ position
CH), 2.76 and 2.98 (3″ position CH ₂ ), 6.78 (2″ position
NH), 2.03 (2″ CDCH ₃ ), 5.44 and 5.80 (3″
_CONH2 ) exhibits absorption. Next, a test example showing the anti-inflammatory effect of tetrahydroesterastine will be explained. In addition, the edema intensity and suppression intensity in the test example was measured by measuring the volume of the footpad using Fujihira's device 1, 3, 5, and 6 hours after the onset of inflammation, and calculating the edema intensity using the following formula. This was done by calculating the inhibition rate of the edema intensity in the gum arabic solution administration group (the same applies to the following test examples). Edema intensity (%) = B-A/A x 100 A: Footpad volume before inflammatory agent injection B: Footpad volume after inflammatory agent injection Suppression rate (%) = Ic-It/Ic x 100 Ic: Edema intensity It of vehicle administration group: Edema intensity test example of drug administration group 1 Wistar male rats weighing 130 ± 5 g were placed in 1 group of 6 rats.
Used as a pet. The sample tetrahydroesterastin (used as a similar suspension in the following test examples) suspended in a 0.5% gum arabic solution was dissolved by adding 0.5 ml of methyl alcohol, and then dissolved as a surfactant.
One drop of Tween 80 was added, and a 0.5% gum arabic solution was added to form a suspension. Tetrahydroesterastine 50 and 100mg/
One hour later, 0.05 ml of 1% carrageenan saline was subcutaneously injected into the right foot pad of the rat, causing inflammation. After measuring the volume of the footpad 2, 3, 5, and 6 hours after the onset of inflammation, and calculating the edema intensity, the suppression rate for the edema intensity in the 0.5% gum arabic solution administration group was calculated.
The effect of tetrahydroesterastine on carrageenan paw edema was calculated (Table 1). As a result, it was found that oral administration of 50 and 100 mg/Kg of tetrahydroesterastine significantly suppressed edema.

[Table] Test Example 2 One group of 5 male Wistar rats weighing 170±10g.
They were used as animals. Tetrahydroesterastine
The rats were orally administered at doses of 10, 50 and 100 mg/Kg, and one hour later, 0.05 ml of 8% rabbit anti-rat serum was subcutaneously injected into the right footpad of the rats to induce inflammation. After measuring the volume of the footpad 1, 3, 5, and 6 hours after the onset of inflammation and calculating the edema intensity, we calculated the suppression rate of the edema intensity in the 0.5% gum arabic solution administration group and determined whether the allergy caused by the tetrahydroesterastine antiserum was measured. The effect on foot edema was investigated (Table 2). As you can see from the table, tetrahydroesterastine
Oral administration of 10, 50 and 100 mg/Kg suppresses allergic edema, albeit mildly.

[Table] Production examples of esterastine and tetrahydroesterastine are shown below. Reference example 1 Glycerin 1.5%, cottonseed flour (Falma Media) 1.5%, salt 0.3%, L-asparagine 0.2%,
Medium 300 consisting of antifoaming agent (adecanol) 0.005%
is charged into a 570 stainless steel tank, and 120
After sterilizing at ℃ for 20 minutes, a seed culture of Streptomyces MD4-C1 strain (Feikoken Bibori No. 3723) (2 days of aeration and agitation culture) was inoculated, with stirring at 200 revolutions per minute and aeration rate of Culture was carried out at 27° C. for 48 hours at 300 rpm. This culture solution is passed through to solid matter containing bacterial cells34.2
Got Kg. This solid was extracted twice with 100% ethanol, and the extract was concentrated under reduced pressure to a volume of 6. This was extracted twice with butyl acetate 6, and the extract was concentrated under reduced pressure to obtain 128.2 g of esterastine crude powder (ID ₅₀ =0.08 mcg/ml). The obtained crude powder was purified by the following method. In other words, 128.2 g of coarse powder is mixed with 500 g of chloroform.
ml of silica gel (Wako Gel C-100)
Esterastine was adsorbed through a column packed with 1.5 kg. Then, after washing the tower with 10 parts of chloroform and 10 parts of chloroform-methanol (100:1),
Elution was performed with chloroform-methanol (80:1). 2500ml of the active fraction was concentrated to dryness under reduced pressure.
4.83 g of brown coarse powder was obtained (ID ₅₀ =
0.002mcg/ml). Next, mix this coarse powder with methanol.
The solution was dissolved in 20 ml of Sephadex LH-20 swollen with methanol, passed through a column of 2, and developed and eluted with 4 of methanol. The active fraction was concentrated to dryness under reduced pressure to obtain 656 mg of pale yellow powder (ID ₅₀ =0.0004 mcg/ml). This powder was dissolved in 5 ml of ethyl acetate, and silica gel (Wakogel C-
300) Esterastine was adsorbed through a 250 g column. Furthermore, the column was developed with ethyl acetate, and the active fraction was separated.
1000 ml was concentrated to dryness under reduced pressure to obtain 351 mg of esterastine as a white powder (ID ₅₀ =0.0002 mcg/ml). Reference example 2 Dissolve 95ml of esterastine in 10ml of methanol, add 20mg of platinum oxide, and dissolve under the pressure of 20LBS of hydrogen.
I gave back my time. The platinum oxide was removed by filtration, and the solution was concentrated to obtain 96 mg of a white powder of tetrahydroesterastine, which showed a single spot on thin layer chromatography. Reference Example 3 10 mg of esterastine was dissolved in 0.5 ml of 99% methanol, 2 mg of platinum oxide was added, and reduction was carried out under a pressure of 20 LBS of hydrogen for 2 hours. The platinum oxide was removed by filtration, and the liquid was concentrated to obtain 9.7 mg of white powder. This was then applied to a silica gel column and eluted with a mixed solvent of ethyl acetate and chloroform (1:1) to obtain 5.9 mg of a white powder of tetrahydroesterastine, which was homogeneous in thin layer chromatography.

Claims (1)

[Claims] Linear formula An anti-inflammatory agent comprising at least one of the following tetrahydroesterastine and its acid addition salt as an active ingredient.