CN1099870C - Novel grand spectrum anti-virus application of Gerd mycomycin - Google Patents

Abstract

The present invention relates to a novel broad-spectrum antiviral application of geldanamycin. The geldanamycin has an inhibiting effect on a plurality of kinds of viral replications in vitro and a curative effect on different herpesvirus infectious models in vivo. Through tests in vivo, the geldanamycin has exact curative activity on mouse encephalitis infected by herpes simplex viruses I and herpes simplex viruses II, rabbit keratitis infected by the herpes simplex viruses I and guinea pig skin models infected by the herpes simplex viruses I and is negative through Ames tests. The geldanamycin acts on the early stage of the viral replications, does not influence the absorption and the release of the herpes simplex viruses I, and does not have a direct virus inactivation effect.

Description

The purposes of geldanamycin in preparation New-type wide-spectrum antiviral drugs

The invention belongs to the purposes of geldanamycin (Geldanamycin) in the preparation antiviral drugs.

Geldanamycin is the anthelmintic antibiotic that the American scholar actinomycetes of delivering in 1970 produce, and applies for a patent respectively in 1971 and 1978, and the patent No. is 3595955 and 4075339.

Among the present invention, in Yunnan Province's soil, be separated to geldanamycin by the inventor and produce bacterium (actinomycetes 17997), and find that it has the broad-spectrum disease resistance cytotoxic activity, and do not see the report that the research of geldanamycin antiviral is arranged at present.The present invention confirms that geldanamycin has duplicated inhibitory action external to multiple DNA and RNA viruses, in the body the different models of herpesvirus infection is had definite therapeutic effect.At present, the antiviral drugs overwhelming majority of clinical practice is a nucleoside derivates, be viral dna polymerase or reverse transcriptase inhibitors, and be narrow spectrum, geldanamycin is a non-nucleoside, wide spectrum, non-viral enzyme inhibitor, can become antiviral drugs through further research and development, treat the human diseases due to various herpesvirus infections and other DNA viruses and the RNA viruses.

Actinomycetes 17997 belong to the suction monoid of streptomycete, pitch-dark streptomycete, S.atrolaccus Yen1957.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, does not form sclerotium and conidium device, and spore surface is smooth.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.

Extraction separation goes out single active princlple from the fermentation liquid of 17997 bacterium number, has carried out physical and chemical property determining, and is identical with geldanamycin through differentiating.

I extracts, 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 crude product.

2. above-mentioned 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%, and purity reaches more than 95%.

The mensuration and the discriminating of II.17997 active constituent physicochemical property

Active constituent is carried out stability, fusing point, dissolubility, elementary analysis, UV, IR, MS reach ¹HNMR reaches ¹³The 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:

Table A

Character	17997	Geldanamycin
			Proterties solubility stability fusing point elementary analysis weight molecule formula UV (nm) IR by1HNMR, 13CNMR and DEPT spectrum	Yellow acicular crystal is soluble in chloroform, organic solvents such as dichloromethane, be dissolved in acetone, methanol etc. slightly, be slightly soluble in water.Dry product is stable, and solution is to heat, acid, alkali, photo-labile.253 ℃--256 ℃ of C 62.12H 7.08N 4.98(O25.87) 560 (FABM) C 29H 40N 2O 9304 256 (methanol) 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. 13The 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. and is dissolved in acetone, ethyl acetate etc. slightly, is slightly soluble in water.Dry product is stable, and solution is to hot acid alkali instability.252 ℃--255 ℃ of value of calculation: C 61.69H 7.16N 5.15(O25.65) measured value: C 62.13H 7.26N 5.0(O25.68) 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 17997 should have the structure identical with geldanamycin.

During the New-type wide-spectrum antiviral of geldanamycin provided by the present invention is used the classification of geldanamycin generation bacterium is identified; Chemical extraction and structure to geldanamycin are studied; To the mensuration of geldanamycin inside and outside antiviral activity, comprise 1. geldanamycin antiviral IC in cell culture ₅₀Mensuration, the 2. IC of geldanamycin anti human immune deficiency virus 1 type (H1V-1) in different cell culture ₅₀Reach synergism with AZT, 3. geldanamycin in cell with acyclovir (ACV-anti-herpesvirus drug of first choice) the active comparison of anti-HSV-1,4. geldanamycin is to the toxicity of different cells and to the influence of VERO cell DNA, RNA, protein synthesis, 5. geldanamycin is to the therapeutical effect of HSV-1 mice encephalitis, 6. geldanamycin is to HSV-2 mice encephalitis therapeutical effect, 7. geldanamycin is to the therapeutical effect of HSV-1 rabbit corneal inflammation, 8. geldanamycin infects the sick therapeutical effect that decreases of guinea pig skin, the 9. Salmonella reversion test of geldanamycin to HSV-1.Geldanamycin antivirus action mechanism has been carried out preliminary study.

Experimental result confirms that geldanamycin (17997) does not have direct deactivation to HSV-1, do not influence the absorption of HSV-1, infected preceding 12 hours or 4 hours dosing unrestraint HSV-1 activity, infect administration in back 3 hours the HSV-1 of inhibition activity is arranged, infect the back 6 hours anti-HSV-1 loss of activity of dosing, illustrate that geldanamycin (17997) acts on HSV-1 and duplicates in early days, it is synthetic to suppress HSV-1DNA, geldanamycin (17997) discharges HSV-1 does not also have influence, and geldanamycin (17977) is to HIV-1 reverse transcriptase and the equal unrestraint effect of DHV replication complex archaeal dna polymerase.

The present invention relates to the purposes of geldanamycin in preparation New-type wide-spectrum antiviral drugs.The purposes of geldanamycin in the preparation anti-herpesvirus medicament.The purposes of geldanamycin in the preparation anti-herpes simplex virus medicament.The purposes of geldanamycin in the preparation anti-AIDS drug.The purposes of geldanamycin in preparation AIDS virus resisting medicine.The purposes of geldanamycin in the anti-Coxsackie virus medicine of preparation.The purposes of geldanamycin in the anti-vesicular stomatitis virus medicine of preparation.

Illustrated below by table 1-14 and accompanying drawing 1, accompanying drawing 2 and 3 pairs of experimental results of the present invention of accompanying drawing:

Fig. 1 is 17997, ACV and the average pathological changes curve of normal saline group rabbit corneal.

Fig. 2 is 17997, ACV and normal saline group rabbit corneal virus are separated titre-time graph.

Fig. 3 is 17997 treatment HSV-1 Cavia porcellus skin lesion effects (arrow is represented the medicine tid to the district).

Table 1:17997 antiviral activity in cell culture

Virus	+Cell culture	The cytopathy political reform	*IC 50μ M/L erosion class method
				Herpes simplex virus I-type	VERO	0.62±0.39	0.052±0.02
Herpes simplex virus I-type	VERO	4.00±1.80	0.024
				Vesicular stomatitis virus	VERO	0.84±0.74	0.38
Coxsackie B virus 3	VERO	1.36	1.13±0.59
				Human immunodeficiency virus I type	C8166 H 9	0.036(P24) ** 0.048(P24) *
Coxsackie B virus 6	VERO	-
				Influenza virus A type	MDCK	-
Influenza virus is B-mode	MDCK	-

IC ₅₀=drug level+following English name of suppressing 50% cytopathy or erosion class form is that the cell strain title commonly used is to human immunodeficiency virus I type, with measuring viral nuclear antigen-P24 method table 2 17997 antiviral activity in cell culture

^*TD ₅₀μM/L

Virocyte culture experiment method mtt assay IC ₅₀μ M/L TD ₅₀/ IC ₅₀The herpes simplex virus type 1 VERO erosion method 182 0.052 3500 herpes simplex virus type 2 VERO of the class erosion method 182 0.024 7583 human immunodeficiency virus type 1 C8166 P24 40 of class ^#0.036 1111

H ₉P24 3.2 ^#0.048 66.7 vesicular stomatitis virus VERO erosion class method 182 0.38 479 Coxsackie B virus ₃VERO erosion class method 182 1.13 161 ^*TD ₅₀-to the virose drug level of 50% cell, MTT=tetramethyl azo azoles salt ^#To the human immunodeficiency virus type 1, use ³The H thymidine mixes the function cells Strain of method table 3 17997 anti human immune deficiency virus 1 type in different cell culture ^△Experimental technique IC ₅₀μ M IC ₅₀μ M therapeutic index C8166 IIIB P24 0.036 40 1111H9 IIIB P24 0.048 3.2 66.7 ^☆H9 IIIB P24 0.15 0.71 4.7PBMC 018a P24 0.018 0.018 1PRMC ⁺It is that viral chronic infection cell strain+018C is collaborative anti human immune deficiency virus 1 type drugs with function cell virus experimental technique 17997 IC of AZT multidrug resistant disease strain table 4 cell culture interior 17997 and azidothymidine AZT (AZT) that 018C P24 0.022 0.018 0.83 △ measures viral nuclear antigen-this H9 of P24 ☆ ₅₀μ M A27 therapeutic index 17997 PBMC 018a P24 0.046 0.69AZT PBMC 018a P24 0.0045 0.5517997+AZT PBMC 018a P24 0.035 0.00056 1.1 has synergism, (individual 1.3 times), (individual 80 times)

Can see by table 3, table 4 result, 17997 in cell culture to H1V-1 sensitive strain (018a) or AZT (anti-AIDS drug of first choice) persister (018C) all have suppress active and 17997 and AZT synergism is arranged.Table 5 is comparison 17997 and acyclovir (ACV) anti-herpes simplex virus 1 type active medicine cell culture experiments method IC in cell culture ₅₀μ M/L ^*The therapeutic index remarks 17997 VERO erosion method 0.052 3500ACV VERO of the class erosion method 8.48＞519ACV VERO of the class erosion method 0.089-6.7 of class document ^*Therapeutic index=TD ₅₀/ IC ₅₀The toxicity of 17997 pairs of different cell strains of table 6

Cell strain experimental technique TD ₅₀μ M/L (48 hours)

VERO cytopathy 35.4

VERO tetramethyl azo azoles salt 182.0

MDCK methyl-azoles salt 18.0

C8166 ³H thymidine 40.0

H ₉ ³H thymidine 17997 pairs of VERO cells of 3.2 tables 7 (akinete)

The influence of nucleic acid and protein synthesis

Biomacromolecule is synthetic	Labelled compound	TC 50μM/L
			The synthetic RNA synthetic protein of DNA is synthetic	3HT dR( 3The H thymidine) 3The H uracil 3The H leucine	68.07±2.27 130.9±19.5 144.2±17.1

The therapeutic effect of 17997 pairs of herpes simplex virus type 1 infecting mouses of table 8 encephalitis

Dosage viral infection group mg/kg amount, LD ₅₀ ^#The average survival of survival rate (%) day sky 17997, bid * 7 0.370 5.6 14/16 (87.5) ^{* *}14.25 ^{* *}Lumbar injection 0.185 5.6 14/16 (87.5) ^{* *}14.50 ^{* *}

0.093 5.6 15/15 (100.0) ^{* *}15.00 ^{* *}17997 toxicity are shone 0.370 0 5/5 (100.0), bid * 7,0.093 0 5/5 (100.0) 17997, qd * 7 0.370 5.6 13/16 (81.2) are injected in abdomen 0.185 0 5/5 (100.0) chamber ^{* *}13.9 ^{* *}Lumbar injection 0.185 5.6 12/16 (75.0) ^{* *}13.7 ^{* *}

0.093 5.6 10/15 (67.0) ^*13.2 ^{* *}17997 toxicity are to 0.370 0 5/5 (100.0) photograph qd * 7. 0.093 0 5/5 (100.0) 17997, bid * 7 0.370 5.6 7/16 (43.8) are injected in abdominal cavity 0.185 0 5/5 (100.0) ^*10.94 ^*The subcutaneous injection acyclovir, bid 88.8 5.6 5/17 (29.4) 10.65 ^** 7, oral virus control 10% Tween 80 0.2ml/ Mus 5.6 1/16 (6.3) 7.75bid * 7, lumbar injection

#LD ₅₀=virus virulence unit, 1LD ₅₀For making the viral dilution degree of 50% dead mouse

^*p＜0.05， ^**p＜0.01， ^***p＜0.001

Table 8 result shows, mice (Kunming kind, 12-15 gram) abdominal cavity infection 5.6LD ₅₀Herpes simplex virus type 1 infects and used 17997 various dose in back 1 hour, and every group of mice survival rate and average life day are observed in different approaches administration 7 days.Intraperitoneal administration bid * 7 group, the survival rate of 0.37mg/kg, 0.185mg/kg, 0.093mg/kg is respectively 87.5%, 87.5% and 100%, compares with matched group current deposit rate (6.3%), and three dosage groups all have the difference of statistical significance.Three groups average life day is 14.25,14.5,15.0 to compare with matched group 7.75, and the difference of statistical significance is also arranged.The average life that intraperitoneal administration qd * 7 group, the survival rate of three dosage groups are respectively 81.2%, 75.0% and 67.0%, three dosage group day is respectively 13.9,13.7 and 13.2, and the difference of statistical significance is also all arranged compared with the control.Subcutaneous administration group bid * 7,0.37mg/kg current deposit rate be 43.8%, average life day is 10.94, the difference that statistical significance is arranged compared with the control, the equal avirulence of used 17997 dosage, 17997 pairs of HSV-1 infecting mouse encephalitis of experimental result explanation have definite therapeutic effect, than positive control drug acyclovir (acycloguanosine, ACV) effective.

The therapeutic effect of 17997 pairs of herpes simplex virus type 2 infecting mouses of table 9 encephalitis

Dosage viral infection amount group mg/kg LD ₅₀ Average survival day 17997 of survival rate (%), bid * 7 0.370 2.96 13/17 (76.5) ^*12.24 ^*Lumbar injection 0.185 2.96 12/17 (70.6) ^*12.29 ^*

0.093 2.96 8/17 (47.1) 11.24 ^*17997 toxicity are injected 0.093 0 5/5 (100) acyclovir to 0.370 0 5/5 (100) photograph bid * 7 abdomens, 0.185 0 5/5 (100) chamber, 100.0 2.96 3/16 (18.8) 9.9bid * 7 oral virus control 10% 0.2ml/ Mus 2.96 3/16 (18.8) 8.8 Tween 80s, bid * 7 lumbar injections

Table 9 result shows, mice (Kunming kind, 12-15 gram), abdominal cavity infection 2.96LD ₅₀Herpes simplex virus type 2 infects back 1 hour with 17997 various dose intraperitoneal administrations, and bid * 7 are observed and respectively organized mice survival rate and average life day.The survival rate of three dosage group 0.37mg/kg, 0.185mg/kg and 0.093mg/kg is respectively 76.5%, 70.6% and 47.1%; Average life day is respectively 12.24,12.29 and 11.24, compare except that the survival rate of minimum dose group (0.093mg/kg) with matched group survival rate (18.8%) and average life day (8.8 days), other data all have statistical significant difference, the survival rate of positive control acyclovir group is identical with matched group, though average life day prolongs 1.1 days, does not have statistical significance.17997 pairs of HSV-2 infecting mouses of presentation of results encephalitis has definite therapeutic effect, and is more effective than positive control drug acyclovir.Table 10 17997.ACV ^☆And normal saline matched group rabbit corneal pathological changes scoring

Days post inoculation	17997	ACV	Normal saline	The P value
					1	1.12	1.12	1.25	＞0.05
2	1.12	1.25	1.88	＞0.05
					4	1.25	1.63	2.5	＜0.05
6	1.25	1.88	3.14(7)	＜0.001
					8	0.83(6)	1.71(7)	2.85(6)	＜0.001
10	0.33(6)	1.14(7)	2.4(5)	＜0.001
					Average pathological changes	1.02	1.46	2.23	-
The average healing day	9.6	11.5	＞12	-

-capable statistical test

* bracket inner digital is represented an eye number, surplusly is all 8 average results.

☆ ACV=acyclovir

Three groups of sickness rate of suffering from conjunctivitis, iritis and eyelid herpes of table 11 compare

	17997	ACV	Normal saline
				Conjunctivitis
	2/8(25％)	3/8(37.5％)	8/8(100％)
				Iritis	1/8(12.5％)	3/8(37.5％)	6/8(75％)
The eyelid herpes	0/8(0％)	0/8(0％)	2/8(25％)

Table 10, table 11, Fig. 1 and Fig. 2 have shown the effect of 17997 treatment HSV-1 rabbit corneal inflammation.12 of the healthy no ophthalmic white rabbits in Beijing after the 0.5%Dicaine local anaesthesia, infect HSV-1 at the cornea cut, isolate and feed, lagophthalmos divides 3 groups at random, 8 every group, (17997 group of 20 μ g/ml, ACV organize 0.1% eye drop and physiological saline solution matched group), virus inoculation administration after 18 hours, five times on the one (9AM-5PM), 2 hours at interval, each two, totally 12 days, Fig. 1 i.e. the interior data of table 10, and Fig. 2 is each group, the isolating result of different time virus.Experimental result points out that 17997 (20 μ g/ml) have therapeutic effect to HSV-1 rabbit corneal inflammation, slightly are better than the therapeutic effect of ACV.Each group scoring statistical analysis of table 12 17997 Cavia porcelluss experiment

Each group incrustation of (P value) (rank test) ACV=acyclovir table 13 17997 Cavia porcelluss experiment and cure time are relatively

Group	Average natural law
			Incrustation	Recovery from illness
	The blank white virus control of 17997 ACV	4(P＜0.01) 3.9(P＜0.01) 4.7(P＞0.05) 5.4			7.1(P＜0.01) 6.3(P＜0.01 8.6(P≤0.05) 9.9

Table 12, table 13 and Fig. 3 have shown the effect that 17997 treatment HSV-1 infect the Cavia porcellus skin lesion.Healthy albino guinea-pig, body weight 250-300 gram, female, the back depilation, after stabbing skin with percussopunctator, infect with the HSV-1 friction, each Cavia porcellus back of the body has 6 skin lesion, random packet, every group of 8 skin lesion (17997 group of 0.3% cream, ACV organizes 3% cream, blank frost and virus control group).Infect beginning administration in back 48 hours, morning every day is given the sick scoring of decreasing in tid * 5, and the sick progress curve that decreases of each group is listed in Fig. 3, and table 12 is the statistical analysis of Fig. 3 data.Presentation of results 17,997 0.3% cream have treatment HSV-1 guinea pig skin infectious effect, and the ACV effect is better than 17997.

The Salmonella reversion test result of table 14 17997 (return and become clump count/ware) group dosage ☆ TA97 ☆ TA98 ☆ TA100 ☆ TA102

(μ g/ ware)-S9+S9-S9+S9-S9+S9-S9+S9

0.0 185.0±9.0 180.0±2.0 48.5±2.5 32.5±7.5 197.0±5.0 162.0±6.0 281.5±0.5 247.0±4.0

1 117.0±12.0 99.5±15.5 45.0±1.0 28.5±2.5 194.0±5.0 179.0±3.0 320.5±33.5 221.5±21.5

10 104.7±12.0 108.0±1.0 41.0±3.0 27.5±1.5 176.0±0.0 138.5±32.5 260.0±58.0 252.0±2.017997 100 128.5±37.5 132.5±22.5 38.5±0.5 34.0±9.0 106.0±10.0 94.0±2.0 260.0±25.0 243.0±9.0

1000 111.0±1.0 85.0±2.0 32.5±1.5 29.5±1.5 92.5±8.5 79.5±4.5 201.5±32.5 188.5±8.5

2500 67.0±15.0 50.0±3.0 31.5±1.5 23.5±1.5 78.0±2.0 83.5±7.5 218.0±11.5 167.5±4.5

5,000 101.0 ± 3.0 60.0 ± 8.0 37.0 ± 1.0 25.0 ± 2.0 92.0 ± 17.0 72.5 ± 8.5 231.5 ± 6.5 170.0 ± 16.09-Fluorenones 0.2 983 ± 102,7-2AF 20.0 508.5 ± 17.52-AF 10.0 1736 ± 509 1358.5 ± 39.5DMC 20.0 1440.5 ± 2.5MMS 2.0 6740 ± 370 carboplatins 100.0 2310.5 ± 65.5 1270 ± 37 ☆ strain name

The result judges: 17997 Salmonella reversion tests the results are shown in Table 14, as can be seen from the table, 17997 each dosage become clump count (activation system and disactivation system) all in normal range to returning of each bacterial strain, and positive mutagenic agent is returned change bacterium colony number average obviously above 2 times, so judge that 17997 Salmonella reversion test result is negative.

Claims (7)

1. the purposes of geldanamycin in preparation New-type wide-spectrum antiviral drugs.

2. geldanamycin is in the purposes of preparation in the anti-herpesvirus medicament.

3. geldanamycin is in the purposes of preparation in the anti-herpes simplex virus medicament.

4. geldanamycin is in the purposes of preparation in the anti-AIDS drug.

5. the purposes of geldanamycin in preparation AIDS virus resisting medicine.

6. geldanamycin is in the purposes of preparation in the anti-Coxsackie virus medicine.

7. geldanamycin is in the purposes of preparation in the anti-vesicular stomatitis virus medicine.

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