CN1179723C - Application of geldanamycin in preparation of medicine for curing SARS - Google Patents

Abstract

The present invention relates to an application of geldanamycin on preparing medicine for treating severe acute respiratory syndrome. The inhibition test of geldanamycin to SARS coronavirus with the cytopathy method indicates that geldanamycin has obvious inhibiting effect on the duplication of SARS coronavirus, IC50 is from 0.34 to 1.57 mumol/L, and the therapeutic index is from 19 to 88.

Description

The application of geldanamycin in preparation treatment SARS (Severe Acute Respiratory Syndrome) (SARS) medicine

Technical field

The present invention relates to the application of geldanamycin (Geldanamycin) in preparation treatment SARS (Severe Acute Respiratory Syndrome) (SARS) medicine.

Background technology

Geldanamycin is to have a broad-spectrum disease resistance toxic action secondary metabolite of (comprising DNA and RNA viruses) by what Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences's isolating geldanamycin in the soil of Luojia Mountain, Wuhan, Hubei Province area produced separation and purification in the fermentation liquid of bacterium (actinomycetes C-3559).

The first half of the year in 2003, a country has met with the attack of paroxysmal SARS (Severe Acute Respiratory Syndrome) (severe acute respiratory syndrome, english abbreviation SARS) (hereinafter to be referred as SARS) surplus the China and the world 20, and this disease is by due to the sars coronavirus, infectiousness is extremely strong, infect soon, incubation period is short, only has 2～14 days, number of the infected is many, the death rate of the onset height, very harmful, and still do not have the specially good effect antiviral drugs.

Summary of the invention

The purpose of this invention is to provide the application of a kind of geldanamycin in preparation treatment SARS (Severe Acute Respiratory Syndrome) medicine.

Geldanamycin is to have a broad-spectrum disease resistance toxic action secondary metabolite of (comprising DNA and RNA viruses) by what Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences's isolating geldanamycin in the soil of Luojia Mountain, Wuhan, Hubei Province area produced separation and purification in the fermentation liquid of bacterium (actinomycetes C-3559).

Actinomycetes C-3559 belongs to the suction monoid of streptomycete, pitch-dark streptomycete, S.atrolaccus Ye n 1957.Its morphological characteristic comprises: the base silk does not rupture, and has only the gas silk to form spore chain, and fibrillae of spores is tight spiral type, do not form sclerotium and conidium device, spore surface is smooth, and cultural characteristic comprises: after the spore maturation, the fibrillae of spores self-dissolving makes the bacterium colony surface form the wet speckle (suction phenomenon) of black viscosity.

Shellfish Nat culture medium			Grape sugar asparagine culture medium
						The gas silk	The base silk	Pigment	The gas silk	The base silk	Pigment
Greyish black	The gold leaf Huang	Trace gold leaf Huang	Greyish black	Milky white	Do not have

Physiological feature comprises: 1. utilization of carbon source: at the culture medium well-grown of carbon sources such as glucose, fructose, sucrose, xylose, mannose, arabinose, rhamnose, maltose and inositol.2. other physiological feature: cellulose does not utilize, and milk peptonizes, the nitrate reduction feminine gender.The cell wall chemical feature comprises: contain LL-DAP and belong to cell wall I type, sugared type C does not contain characteristic sugar.

The extraction of geldanamycin, separation method

1. X5 macroporous adsorptive resins on the filtering fermentation liquor rear filtrate, absorption finish after water dashes, and wash live part with 80% acetone, and removing acetone, yellow mercury oxide is arranged is the geldanamycin crude product.

2. above-mentioned geldanamycin crude product, reuse macroporous absorption post carries out the chromatography second time, washes live part with 80% acetone, and purity is placed on and separates out yellow acicular crystal in the refrigerator more than 90%, is purity and reaches geldanamycin more than 95%.Carry out secondary recrystallization again, purity can reach the geldanamycin more than 98%.Extraction recovery can reach 30%～35%.

To adopting the single active component of said method extraction separation in the fermentation liquid of actinomycetes C-3559, the i.e. mensuration of C-3559 active constituent physicochemical property and discriminating

Active constituent is carried out stability, fusing point, dissolubility, elementary analysis, UV, IR, MS reach ¹HNMR reaches ¹⁸The mensuration of wave spectrums such as CNMR and DEPT spectrum, and carried out the retrieval of documents and materials, find that it is identical with the geldanamycin of benzene Ansamycin apoplexy due to endogenous wind.

Both property lists are compared to following table:

Character	C-3559	Geldanamycin
			Proterties solubility stability fusing point elementary analysis weight molecule formula UV (nm) IR by1NHMR， 18CNMR and DEPT spectrum	Yellow acicular crystal is soluble in chloroform, organic solvents such as dichloromethane, be dissolved in acetone slightly, methanol etc., it is stable to be slightly soluble in the water dry product, and solution is to warm, acid, alkali, 253 ℃ of-256 ℃ of C of photo-labile 62.12H 7.08N 4.93(025.87) 560(FABM) C 29H 40N 2O 9304 256 (methyl alcohol) KBr sheet, 3,510 3,446 3,350 3,315 1,734 1,705 1,675 1,655 1,615 1,595 1,510 1108 1057CM-11. 18The CNMR spectrum is measured as 29 carbon 2. and finds out that by DEPT 2 CH of 11-CH of 9 quaternary carbons are arranged 27 CH 3	Yellow acicular crystal is soluble in chloroform, dichloroethanes etc., be dissolved in acetone slightly, ethyl acetate etc., it is stable to be slightly soluble in the water dry product, and solution is to warm, unstable 252 ℃ of soda acid--255 ℃ of value of calculation: C 61.69H 7.16N 5.15(025.65) measured value: C 62.13H 7.26N 5.0(025.65) 560 C 29H 40N 2O 9304 255 (methyl alcohol) nujol mull, 3,510 3,445 3,350 3,315 1,734 1,700 1,676 1,655 1,635 1,608 1,590 1,510 1108 1057CM-12 CH of 11-CH of 9 quaternary carbons of 29 carbon are arranged 27 CH 3

According to above-mentioned data, we infer that active component C-3559 should have the structure identical with geldanamycin.

With the sick epidemic period of in February, 2003 Beijing SARS by Viral Laboratory, Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research center from isolating viral BJ 01 strain of Beijing patient's lung tissue and responsive Vero-E thereof ₆Cell, the regulation by extracorporeal antivirus effect test of pesticide effectiveness part in " new drug (Western medicine) preclinical study guideline " detects geldanamycin (hereinafter to be referred as C-3559) to Vero-E ₆The toxicity of cell and at Vero-E ₆The interior inhibition effect of cell to sars coronavirus.Repeated experiments shows: geldanamycin (C-3559) is at Vero-E ₆The IC that suppresses sars coronavirus in the cell ₅₀Be 0.34～1.57umol/L, the IC of positive control drug ribavirin ₅₀Be 52.0ug/ml.Illustrate that geldanamycin (C-3559) can obviously suppress sars coronavirus and duplicate.

Be the external SARS coronary virus resistant drug effect of the medicine examining report that carries out at geldanamycin of the present invention below.

One. test objective: detect the effect of the external SARS coronary virus resistant of C-3559.Detect unit: Viral Laboratory, Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C Micro biological Tests research center.

Two. material

1. be subjected to reagent:

C-3559 (purity＞98%) is provided by Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences.

2. positive control medicine:

Ribavirin (virazole) injection, 0.1g/ml, Shandong XinHua Pharmacy stock Co., Ltd produces, authentication code: H19993063, lot number: 02100020.Clinical application: be used for viral pneumonia and bronchitis that respiratory syncytial virus causes.Clinical Coming-of-Age Day, consumption was a 0.5g, 2 times on the one.

3. cell:

The Vero-E6 cell, preserve in this ventricular cell storehouse.With the DMEM culture fluid that contains 10% hyclone, at 37 ℃ of 5%CO ₂Incubator was cultivated 2-3 days, went down to posterity once.

4. Strain:

Sars coronavirus, in February, 2003 the sars coronavirus epidemic period, by the isolated viral BJ01 strain from the patient's lung tissue of Beijing of this chamber, the 5th generation.

5. reagent:

DMEM culture medium and hyclone, Gibco BRL company product, lot number is respectively 31600-026 and 16000-36.

6. instrument:

96 porocyte culture plates, Corning company.Biological inverted microscope XDS-1B type, the optical instrument factory, Chongqing.

Three. method and result

(1) compound method of medicine

Be subjected to reagent: C-3559, yellow powder is mixed with the 10mmol/L mother solution with DMSO, 0.22 μ m disposable aspiration needle formula filter filtration sterilization, 4 ℃ of preservations.The medicinal pure water of positive control is mixed with the 10mg/ml mother solution.Be mixed with desired concn with cell growth medium during use.

2.C-3559 toxic action to the Vero-E6 cell

Every milliliter 30～400,000 Vero-E6 cell inoculation % porocyte culture plates, every hole 0.1ml puts 37 ℃ of 5%CO ₂Incubator is cultivated 24h, the test medicine that adds variable concentrations, test medicine doubling dilution is 80～5 μ mol/L, each concentration 4 hole, every hole 0.1ml establishes no drug cell matched group simultaneously, cultivated 5 days, with observation of cell metamorphosis (CPE) is index, and be inverted observed and recorded CPE under the apparent mirror every day, and Gestalt is changed to 4; 75% is 3; 50% is 2; 25% is 1; No form is changed to 0.According to the REED-MUENCH method, calculate the maximal non-toxic concentration (TD of this medicine to the Vero-E6 cell ₀) be 20 μ mol/L and median toxic concentration (TD ₅₀) be 30 μ mol/L.The TD of positive control drug ribavirin ₀And TD ₅₀For greater than 6000 μ g/ml.Test repeats secondary, unanimity as a result.

3.SARS coronavirus toxicity test (micromethod)

Sars coronavirus BJ01 strain the 5th generation virus liquid is kept the continuous 10 times of dilutions of liquid with DMEM: 10 ^-1～10 ^-10The viral liquid of variable concentrations of dilution is inoculated in the Vero-E6 cell 96 well culture plate holes that grow up to monolayer, each concentration 4 hole, every hole 0.1ml establishes the normal cell contrast simultaneously, puts 37 ℃ of 5%CO ₂Cultivate in the incubator, observed CPE continuously 5～7 days, the record result.Calculate 50 3nfective dose (TCID according to the REED-MUENCH method ₅₀).

4.C-3559 inhibition test (cellular morphology method of changing) to sars coronavirus

With every milliliter 30～400,000 Vero-E6 cell inoculations to 96 well culture plates, 37 ℃ of 5%CO ₂Cultivate 24h in the incubator, discard growth-promoting media, with sars coronavirus BJ01 strain the 5th generation virus liquid, keeping the liquid dilution with DMEM is 10～100 TCID ₅₀/ 0.1ml, every hole 0.1ml inoculation Vero-E6 cell adsorbed 2 hours, discarded viral liquid.Add the variable concentrations medicinal liquid, test medicine doubling dilution is 20～0.3 μ mol/L, every hole 0.1ml.Set up normal cell contrast, virus control and positive drug matched group simultaneously.37 ℃ of cultivations, every day the observation of cell metamorphosis.Test is final observing time when changing with virus control hole Gestalt, and record CPE. presses the medium effective concentration (IC that the REED-MUENCH method is calculated C-3559 ₅₀). test repeats secondary, the results are shown in following table.

C-3559 anti-SARS virus IC ₅₀

The experiment batch	The experiment date	Drug level (μ mol/L)	The CPE/ hole	Infective virus amount TCID 50	IC50 (μmol/L)
						I	2003.5.12- 2003.5.14	20.0	----	10	0.86
10.0	----
		5.0	----
2.5	----
		1.3	+++-
0.6	3+3+3+3+
		0.3	4+4+4+4+
0	4+4+4+4+
		II	2003.5.20- 2003.5.22	20.0	----			100	1.57
10.0	----
				5.0	----
2.5	+-++
				1.3	3+3+3+3+
0.6	3+4+3+4+
				0.3	4+4+3+4+
0	4+4+3+4+
				20.0	----	10	0.34
10.0	----
				5.0	----
2.5	----
				1.3	+---
0.6	+---
				0.3	2+2+2+2+
0	4+4+3+3+
				Ribavirin				400.0	++-+	100	52.0 (μg/ml)
200.0	++++
		100.0	2++++
50.0	2+2+2+2+
		25.0	2+2+3+3+
12.5	2+3+2+4+

Brief summary: C-3559 presses down the IC of sars coronavirus in the VERO-E6 cell ₅₀Be 0.34～1.57 μ mol/L, the IC of positive control drug ribavirin ₅₀Be 52.0 μ g/ml.

4. conclusion:

(1) C-3559 is changed to index with cellular morphology, the TD of C-3559 to cultivating 5 days in the toxicity of Vero-E6 cell: the C-3559 adding Vero-E6 cell ₀Be 20 μ mol/L, TD ₅₀Be 30 μ mol/L.

(2) C-3559 in the Vero-E6 cell to the inhibitory action of sars coronavirus: C-3559 suppresses the IC of sars coronavirus ₅₀Be 0.34～1.57 μ mol/L; Test shows that C-3559 can obviously suppress sars coronavirus and duplicate; Average IC ₅₀Be 0.96 μ mol/L.

(3) therapeutic index of C-3559 (TI) is 19～88.

(4) IC of positive control drug ribavirin ₅₀Be 52.0 μ g/ml, TI＞115.Annotate: IC ₅₀The drug level that=inhibition 50% cytopathy or plaque form.

Therapeutic index=TD ₅₀/ IC ₅₀

Claims (1)

1. the application of geldanamycin in preparation treatment SARS (Severe Acute Respiratory Syndrome) SARS medicine.