CN1788724A - Use of geldanamycin (C-3559)in preparing drug for herpesvirus genital tract affection - Google Patents

Abstract

The present invention is application of geldanamycin (C-3559) in preparing medicine for treating condyloma acuminata and genital tract infection of herpesvirus. Geldanamycin has no stimulation on medial femoral skin of health calf and can inhibit ox papilloma virus DNA copy and make tumor atrophy, age and reduce. C-3559 has broad spectrum antivirus effect and micromole level antivirus activity on several kinds of DNA and RNA viruses. The 41st extracorporeal passage generation has no geldanamycin resisting HSV-1 virus strain separated, and the 15th HSV-1 GA drug-resisting training generation has the same aciclovir sensitivity as the wild strain. Geldanamycin in no cell toxicity concentration can inhibit the synthesis of HSV-1 DNA specifically. Experiment on mouse shows that geldanamycin can inhibit type-II herpesvirus copy in the effect higher than that of aciclovir.

Description

The application of geldanamycin (C-3559) in preparation herpesvirus genital tract affection medicine

Technical field

The application is that application number is to be JIUYUE in 2003 25 days 03134665.0 applying date, and invention and created name is the dividing an application of patent application of the application of geldanamycin (C-3559) in preparation treatment condyloma acuminatum, herpesvirus genital tract affection medicine and preparation poliomyelitis virus drugs.

The present invention relates to geldanamycin (Geldanamycin, GA) application in preparation treatment condyloma acuminatum, herpesvirus genital tract affection medicine and preparation poliomyelitis virus drugs.

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).

Condyloma acuminatum and herpesvirus (being mainly herpes virus type 2) reproductive tract infection is the whole nation and global spreading disease, and is popular extensive, outbreak repeatedly, treatment difficulty.31 the province report of infectious disease systems in the whole nation in 2002 report, and 7 kinds of sexually transmitted disease (STD) are 744 848 examples except that acquired immune deficiency syndrome (AIDS), and report incidence is 58.15/10 ten thousand.Wherein condyloma acuminatum accounts for 22.25% (165 748 example), is the 3rd; Herpesvirus genital tract affection accounts for 4.79% (35 679 example), is the 5th, and case load rose to some extent than calendar year 2001.Owing to fail to report, the actual numeral that infects has report China women's condyloma acuminatum to infect the peak between 20～29 years old much larger than the report numeral, and infection rate is 1606.1/10 ten thousand, and women's infection rate descends gradually more than 35 years old.(human papillomavirus HPV) belongs to papovaviridae to the human papillomavirus, is nonencapsulated DNA viruses.Condyloma acuminatum is one of modal sexually transmitted disease (STD) that is caused by the human papillomavirus, and human papilloma virus infection is relevant with cervical cancer, the infection that multiple vertebrates all has human papillomavirus to cause.HPV In vitro culture difficulty does not also have suitable animal model, so lack special antiviral drugs.The condyloma acuminatum Therapeutic Method is more, and chemotherapy and naturopathy are arranged, and the former has podophyllin, podophyllotoxin, trichloroacetic acid, interferon etc.; The latter has liquid nitrogen freezing, laser, excision etc.But the cure rate of these methods only is 18.5%～40.1%, and relapse rate extra-high-speed (22%～67%).These therapies are nonspecific treatment, and some medicine corrosivity is strong, easy damaged healthy skin, the special anti-papillomatosis cytotoxic drug of urgent clinical needs.Though herpesvirus genital tract affection has acyclovir medicines such as (ACV), because existing medicine easily produces drug resistance, make the treatment delay, outbreak repeatedly, the patient suffering can't bear.

Summary of the invention

The purpose of this invention is to provide a kind of geldanamycin in preparation treatment condyloma acuminatum, the application in herpesvirus genital tract affection and the poliovirus infection 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

Yen 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 You1HNMR， 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-1 1. 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, acid, unstable 252 ℃ of-255 ℃ of value of calculation: the C of alkali 61.69H 7.16N 5.15(025.65) measured value: C 62.13H 7.25N 5.0(025.68) 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 C-3559 should have the structure identical with geldanamycin.

The application of geldanamycin in preparation treatment condyloma acuminatum medicine.

The application of geldanamycin in preparation treatment herpesvirus genital tract affection medicine.

The application of geldanamycin in preparation poliomyelitis virus drugs.

Geldanamycin antivirus action mechanism is different with existing antiviral drugs, and it is synthetic to suppress viral DNA, is difficult for producing drug resistance, does not have crossing drug resistant with acyclovir.The geldanamycin herpesvirus resisting activity is strong, and characteristics are arranged.

The present invention has done the experiment that anti-herpesvirus 2 types (HSV-2) vaginitis drug effect in the fibromatous therapeutical effect of milk cattle skin of nipple, medicine extracorporeal antivirus effect activity, the medicine body is detected three aspects to geldanamycin, and experimental result shows:

1. the medicine that geldanamycin is carried out is to the fibromatous therapeutical effect of milk cattle skin of nipple aspect:

In the stimulation experiment of geldanamycin, when geldanamycin concentration is 0.5mg/ml and 0.25mg/ml, calf thigh inside skin is had no stimulation to calf thigh inside skin.

At the medicine geldanamycin milk cattle skin of nipple fibroma is treated in the experiment, because geldanamycin has the effect of the viral dna replication of inhibition, so the cow teats dermatofibroma is presented excellent curative, can make the tumor body that atrophy, aging, smaller volume took place in a week; 6 all interior these aged tumor bodies still can continue to wear out after the drug withdrawal, have aged tumor body to come off in the 4th, 5 weeks respectively; Simultaneously do not find that geldanamycin damages skin of nipple.

2. the active aspect of medicine extracorporeal antivirus effect:

Geldanamycin has the broad-spectrum disease resistance toxic action in cell culture, to multiple DNA, RNA viruses all has the antiviral activity in micromole's level, wherein the therapeutic index to herpes simplex virus type 1 (HSV-1) is 3763, therapeutic index to herpes simplex virus type 2 (HSV-2) is 230, therapeutic index to vesicular stomatitis virus (VSV) is 833, therapeutic index to poliovirus 1 type (Polio-1) is 2820, therapeutic index to Coxsackie virus (Cox B3) is 248, therapeutic index to sars coronavirus is 88, is 1111 to human immunodeficiency virus type 1's (HIV-1) therapeutic index.

In the training of external HSV-1 geldanamycin drug resistance strain, use the cell experiment method, HSV-1 was passed for 41 generations continuously in African green monkey kidney cell (VERO) and under the finite concentration geldanamycin condition, do not see the drug resistance strain that produces geldanamycin, HSV-1 geldanamycin drug resistance trains the 15th generation and wild strain to have identical acyclovir sensitivity.Pair cell non-toxic concn geldanamycin can synthesize by special inhibition HSV-1DNA.

3. geldanamycin anti-herpesvirus 2 types (HSV-2) vaginitis drug effect context of detection in the mice body:

Geldanamycin can effectively suppress duplicating of HSV-2 in HSV-2 vaginal infection model.When herpesvirus stock solution infected, geldanamycin can prolong the average life day that HSV-2 infects white mice; When the herpesvirus liquid inductance of 1/30 dilution dyes, can significantly reduce the mortality rate of white mice, prolong the average life day of infecting white mice, and reduce white mice intravaginal HSV-2 output, compared with the control, significant difference, effect is better than the acycloguanosine (ACV) of same dose; And by to the observation of white mice body weight, show that all dosage groups of geldanamycin do not have influence to the white mice body weight gain.

Be at the mould relevant experiment of Ge Erde of the present invention below.

(1) medicine that carries out at geldanamycin of the present invention is to the fibromatous therapeutical effect of milk cattle skin of nipple

On addressed human papillomavirus's In vitro culture difficulty, also do not have suitable animal model, can only adopt milk cattle skin of nipple fibroma that similar viral bovine papilloma virus causes as model evaluation geldanamycin treatment parillomarvirus infections curative effect.

Observe the stimulation of medicine geldanamycin (C-3559) to calf thigh inside skin

One. animal: Beijing Black is spent 1 of milk cattle 6 age in days bull calf in vain, cattle numbers 97026.

Two. medicine: the C-3559 medicinal liquid is provided by the development of Institute of Medicinal Biological Technique, the Academy of Medical Sciences.Detect unit: animal medicine institute of China Agricultural University.

Three. experimental technique: select the Beijing Black of clinical health to spend 1 of bull calf cattle in vain.On right hind thigh inside skin, choose 3 * 3cm ²Two of areas, respectively be coated with the C-3559 medicinal liquid that with concentration be respectively 0.5mg/ml and 0.25mg/ml twice every day on every skin; The left hind corresponding site is chosen onesize skin area, the solvent (10% Tween 80 that does not contain medicinal liquid) that is coated with C-3559 for twice every day, in contrast.Experiment was carried out 7 days continuously.

Four. observation item: experiment beginning is preceding 3 days certainly, and measure and tried Niu Tiwen twice of every day (morning 8:00, afternoon 3:00), observes agents area skin and has or not red, swollen, hot, bitterly and malfunction; Have or not blister, pathological changes such as ulceration, necrosis, incrustation, carry out record item by item.To determine the biological action of C-3559 medicinal liquid to calf skin.

Five. experimental result

1. skin color and variation: by experiment in 7 days, though left hind thigh inboard contrast skin or change color does not all take place the skin of right hind corresponding site do not blister, ulcer etc. yet, with coating position skin indistinction not.

2. in experiment periods, calf searches for food, and the drinking-water and the mental status are normal, body temperature fluctuate between 38.8-39.5 ℃ (normal body temperature is 38.0-39.5 ℃).

Six. brief summary:

1. smeared the C-3559 medicinal liquid of 0.5mg/ml and 0.25mg/ml at calf thigh inside skin in continuous 7 days, and do not see that any pathological changes took place skin, illustrate that this drug level has no stimulation to calf thigh inside skin.

2. in experiment periods, tried that calf searches for food, drinking-water, body temperature, breathing and the mental status be all normal.

Medicine geldanamycin (C-3559) is to milk cattle skin of nipple fibroma treatment experiment

One. animal: clinical ill cattle is divided into 2 groups at random, and 6 every group, wherein one group as experimental group, and another group is group in contrast.

Two. medicine: C-3559 medicinal liquid and solvent thereof (10% Tween 80), by the development of Institute of Medicinal Biological Technique, the Academy of Medical Sciences, it is 0.5mg/ml that concentration is provided, solvent does not contain the medicine composition for the solvent of preparation C-3559 medicinal liquid.

Three. experimental technique: the milk cattle of natural occurrence in the cows is accessed from original teams and groups, concentrate and raise, make experimental group and matched group term harmonization, that is: the quantity of feeding environment, feeding manner, forage grass, feedstuff and quality basically identical.Two groups of milch cows are in charge of by same poultry raiser, the person of milking, implementation manual milking, milk cattle go up groove three times every day, when searching for food, last groove milks, immediately medicine and solvent are applied in respectively on the tumor of experiment cattle and contrast cow teats skin after milking at every turn, it is all discarded for safety to milk, and experiment was carried out 7 days continuously.

Four. observe and record: preceding 3 days of experiment beginning, two groups of Niu Tiwen of twice measurement every day finished until experiment.Day by day statistics is tried cattle and the change of experiment cattle disease situation---tumorigenic position, tumor body size (using kind of calliper).Character, color have or not the sign etc. that comes off.Perform record.

Five. experimental result

The effect of table 1 C-3559 treatment milk cattle papilla fiber tumor

^*Refer to the tumor base diameter.

Experiment shows that the C-3559 medicinal liquid has the effect of control papilla fiber tumor growth, can impel the tumor body aging, and area dwindles.In the experiment that lasts 7 days, the fibroma that is in growth stage does not only increase, and average diameter has dwindled 12.5% on the contrary, and color is become brown by original skin-color, illustrates and has taken place to wear out.Matched group tumor body color no change, average diameter has increased by 4.1%, illustrates that the tumor body is in growth stage, does not take place aging.

In order to determine that the C-3559 medicinal liquid is to the fibromatous late result of cow papillary skin, on the basis that early stage, (in back 7 days of the experiment beginning) observed, we have only carried out the observation in continuous 6 weeks to experimental group and matched group cattle, method is weekly the size of tumor to be measured, color and situation such as whether come off are carried out record, and the result is as follows:

The tumor body of No. 93084 of experimental group (the tumor body has been brownish black before the experiment), No. 92,002 two cow heads is respectively at the 4th week and came off in the 5th week, though the tumor body of other 4 cow head does not come off, but the diameter summation reduces to 12.7mm by original 14.6mm, reduce 12.3%, wherein the tumor body of No. 93070 milch cows narrows down to 2.4mm by original 3.1mm, and color is by the original brown brownish black that becomes.In the matched group except that the tumor body of No. 93046 milch cows (being brownish black before the experiment) came off in the 5th week, the tumor body color of other 5 cow head does not all change, it still is skin-color, and the summation of tumor body diameter is increased to 26.4mm by original 20.8mm, wherein the tumor body diameter of No. 93073 milch cows still is outside the 6.3mm, other tumor body of 4 all has increase in various degree, illustrates that these tumor bodies still are in growth stage.

Above presentation of results, the C-3559 medicinal liquid except that can make in the continuous use phase tumor body take place aging; 6 all these aged tumor bodies still can continue wear out (reduced) after the drug withdrawal, except each milk cattle of experimental group (No. 93084) and matched group (No. 93046) has been taken place before experiment aged tumor body (brownish black) comes off, the tumor body of experimental group other end milk cattle (No. 92002) also comes off, and the tumor body of other milk cattle of matched group is not only unaged, still continued growth (it is big that diameter becomes).

Six. discuss

It is a kind of benign tumor that is caused by bovine papilloma virus (Bovine papilloma Virus) that Corii Bovis seu Bubali skin fibropapilloma disease (Fibropapillomatosis) has another name called wart (Warts).The genome of virus is bifilar cyclic DNA.This disease is commonly encountered diseases, the frequently-occurring disease of milk cattle, and the sickness rate of indivedual cows can reach more than 30%.Grow in the tumor body of skin of nipple, especially be finger-like and give birth to the elder, its length often can reach 1.5-2cm, milch cow discomfort when milking, even pain.The tumor body is suffering under the situation of secondary infection ulcer to take place.This disease has the tumor body nature stage such as to take place, grow, wear out, come off, and this process often needed 10 months or the longer time.

Seven. brief summary

Because the C-3559 medicinal liquid has the effect of the viral dna replication of inhibition, so the cow teats dermatofibroma is presented excellent curative, can make the tumor body in a week, atrophy take place, aging, smaller volume.6 all these aged tumor bodies still can continue wear out (reduced) after the drug withdrawal, except each milk cattle of experimental group (No. 93084) and matched group (No. 93046) has been taken place before experiment aged tumor body (brownish black) comes off, the tumor body of experimental group other end milk cattle (No. 92002) also comes off, and the tumor body of other milk cattle of matched group is not only unaged, still continued growth (it is big that diameter becomes).

Do not find in the experiment that this kind medicine damages skin of nipple.

(2) the medicine extracorporeal antivirus effect activity of carrying out at geldanamycin of the present invention.

One. the antiviral spectrum research C-3559 of geldanamycin in cell to the inhibitory action of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human immunodeficiency virus type 1, vesicular stomatitis virus (VSV), Coxsackie virus (Cox B3), poliovirus 1 type Polio-1, sars coronavirus SARS-coronavirus, influenza virus A type Influenza A and the B-mode Influenza B of influenza virus.

Material:

(1) Strain: herpes simplex virus type 1 (HSV-1) SM44 strain, herpes simplex virus type 2 (HSV-2) 333 strains, human immunodeficiency virus type 1 (HIV-1) IIIB strain, vesicular stomatitis virus (VSV), Coxsackie virus (Cox B3), anti-/ 243/72 strain in influenza first type virus of A/Guangdong, the anti-97-13 strain of the B-mode Ji of influenza virus are this chamber and preserve strain; Poliovirus 1 type Polio-1 is available from Chinese biological goods calibrating institute; Coronavirus SARS-coronavirus is the isolating epidemic strain in Beijing during the SARS.

(2) cell: African green monkey kidney cell (Vero), Testis et Pentis Canis passage cell (MDCK) is preserved by Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences; African green monkey kidney cell (Vero-E6) is so kind as to give by institute of section of army five.

Method:

(1) seed culture of viruses preparation:

1. take out the frozen test virus seed culture of viruses of low temperature, do 10 times of dilutions, be inoculated in then and covered with in the cell monolayer bottle, put in 37 ℃ of incubators and cultivate, observe pathological changes day by day with cell maintenance medium, when treating that pathological changes appears in 3/4 cell, results virus ,-20 ℃ are frozen.

2. with cell multigelation three times, releasing virus.It is centrifugal then that (3000rpm 10min) removes cell debris, and supernatant is required viral suspension.Packing, frozen standby in-80 ℃.

(2) passage: in covering with the culture bottle of cell, add 0.25% pancreatin 0.2ml, 0.02%EDTA5ml, 37 ℃ digested 20-25 minute, and the sucking-off Digestive system adds culture fluid piping and druming, goes down to posterity at 1: 3, puts in 37 ℃ of incubators and cultivates.

(3) C-3559 tests the inhibition of virus:

Ten thousand cell inoculations of every milliliter of 20-30,96 porocyte culture plates, every hole 0.1ml, 37 ℃ of 5%CO ₂Cultivated 24 hours, and abandoned culture fluid, add 100TCID ₅₀37 ℃ of 5%CO of virus ₂Adsorbed 1 hour, and discarded and add the continuous 2 times of diluents of C-3559 behind the viral liquid, establish the cell contrast simultaneously, virus control, positive drug contrast.Culture plate is placed 37 ℃ of 5%CO ₂Incubator is cultivated, and treats that virus control group cytopathy reaches at 4 o'clock (about 32 hours), the observation of cell pathological changes, and destroying fully is 4; 75% is 3; 50% is 2; 25% is 1; Anosisly become 0.Press Reed and Muench method and calculate IC ₅₀

The result:

Table 2 geldanamycin is at the IC of intracellular antiviral ₅₀ ^*Value

Virus	cell	IC 50(μM)			Therapeutic index TD 50#/ IC 50
						CPE	PFU	p24
		HSV-1 HSV-2 VSV Polio-1 Cox B3 SARS HIV-1 Influ.A Influ.B	Vero Vero Vero Vero-E6 Vero Vero-E6 MDCK MDCK	1.52 0.42 0.03 1.41 0.96 - -					0.093	0.036	3763 230 833 2820 248 88 1111

IC ₅₀ ^*: suppress 50% cytopathic drug dose

TD ₅₀#: cause 50% Cytotoxic drug dose

Two. the training method and the result of external HSV-1 geldanamycin drug resistance strain:

With above-mentioned cell experiment method, HSV-1 was passed for 41 generations continuously in the VERO cell and under the finite concentration GA condition, do not see the drug resistance strain (table 3) that produces GA.

Table 3 training HSV-1 geldanamycin drug resistance strain situation

Drug resistance training algebraically		IC 50(μM)
			4 10 15 20 31 36 41	GA resistance training Zhu 0.79 1.19 2.67 1.50 0.69 0.78 2.22	Wild Zhu 0.79 1.17 2.23 2.00 1.23 1.33 1.24

The HSV-1GA drug resistance trains the 15th generation and wild strain to have identical acyclovir sensitivity.

Three. geldanamycin is to inhibitory action (Southern blot the detects viral dna replication) method of HSV-1DNA:

The Vero cell monolayer infects with HSV-1 (1 plaque unit/cell), and the virus of not adsorbing with ice-cold PBS flush away behind the absorption 1h adds the culture medium that contains 4 μ M geldanamycins (GA) or do not contain GA, puts 37 ℃, cultivates in the 5%CO2 incubator.Establish the infection matched group of cell matched group and solvent 1% dimethyl sulfoxide simultaneously.12h collecting cell after infection is with the cell after the drug treating about 2 * 10 ⁷Individual, wash twice with PBS, add 500 μ l cell pyrolysis liquid (50mM Tris-HCl, pH7.5,150mM NaCl, 5mM EDTA, 1%SDS, 500 μ g/ml E.C. 3.4.21.64s) 56 ℃ digested 2 hours, and add 500 μ l phenol extractings once, water phenol: chloroform: isoamyl alcohol (50: 48: 2) extracting once, water places new centrifuge tube, add 50 μ l 3M NaAc (pH 5.2) and 1ml pre-cooling dehydrated alcohol ,-20 ℃ of precipitations 2 hours, centrifugal 20 minutes of 4 ℃ of 10000g, pre-cooling 70% ethanol is washed once, the centrifugal supernatant that goes, air-dry, be dissolved among the 100 μ l TE.It is the RNA enzyme that 100 μ g/ml do not have the DNA enzyme that every pipe adds final concentration, and 10 μ gDNA and 1/6 volume, 6 * sample-loading buffer mixing are got in 37 ℃ of water-baths 20 minutes, at 1.0% agarose gel electrophoresis, electrophoresis forwards it on nitrocellulose filter to after finishing, and does roasting after 1 hour for 80 ℃, at 68 ℃ of prehybridization solution (5 * SSC, 0.1%SDS, 5 * Denhardt ' liquid, 100 μ g/ml degeneration salmon sperm dnas, and 50% Methanamide) prehybridization 4hrs in is containing 5 * 10 then ⁷Cpm ³²42 ℃ of hybridization 24hrs in the prehybridization solution of HSV-1 thymidine kinase (tk) DNA of P deoxy cytidine triphosphate labelling.Hybridization finishes, and (0.1 * SSC, 0.1%SDS) give a baby a bath on the third day after its birth time, washes twice for 65 ℃ ,-20 ℃ of autoradiography behind the X-ray sheet on the folder by room temperature with film washing liquid for nitrocellulose filter.Development, photographic fixing according to a conventional method, the ultimate analysis band.The results are shown in Figure l is the contrast of 1 cell, and 2HSV-1 infects contrast, 3DMSO contrast, 4GA 4 μ M;

Fig. 1 explanation is the same with the cell contrast through the infection cell that 4 μ M GA handle, virus-free DNA band.

(3) anti-herpesvirus 2 types (HSV-2) vaginitis drug effect examining report in Zhen the medicine body that geldanamycin of the present invention is carried out.

One. purpose: research C-3559 body is interior to the colpitic therapeutic action of small white mouse HSV-2: observe average life day and the death rate of small white mouse, and carry out virus and separate.

Two. animal: Kunming mouse is female, and 13-15g examines and determine the secondary animal (unsoundness Zheng book) that the Zhong state medicine biological product determination rodent cultivation Zhong heart of doing provides, random packet available from Chinese biological Zhi product.

Three. virus: blister sore poison 2 types (HSV-2) SAV Zhu, Zhe go down to posterity the chamber preservations, Yong virus stock solution used or the infection of 1/30 virus stock solution used.

Four. medicine: the chemical chamber of this institute of C-3559 You provides, and purity is added to mortar Zhong after greater than a little 0.1% Tween-80,200 μ l solution of 95%, Yong, and the solubilizer while grinding is made into 4mg/ml, 2mg/ml, 1mg/ml, the suspension of 0.5mg/ml, 4 ℃ of preservations.

Five. method:

1. grouping: be that the female Kunming mouse of 13-15g is divided into C-35595.72mg/kg, 2.86mg/kg, 1.43mg/kg, 0.5mg/ml Zu and positive drug ACV2.86mg/kg control group at random with body weight, solvent (0.1% Tween-80) control group, virus control Zu, every Zu of 10 Zhi small white mouses more than are infected group; With C-35595.72mf/kg, 2.86mg/g, 1.43mg/kg, 0.72mg/ml Zu and positive drug ACV 2.86mg/kg control group, solvent control Zu, Normal group, every Zu of 6 Zhi are non-infected group, observe medicine to the toxicity of small white mouse.

2. administering mode: Yong micropipettor Zhu in the small white mouse vagina enters every concentration 20 μ l medicines, the equal saline injection of virus control Zu and Normal group, every day three times.

3. mode of infection: Yong micro sample adding appliance Zhu in the infected group small white mouse vagina enters virus liquid or the virus stock solution used of 20 μ l, 1/30 dilution, and non-infected group Zhu enters 20 μ l physiological saline and carries out identical processing, begins then administration.

4. observation index:

Observe the death rate, average life day, observe every day, and record is dead, and totally 2 Zhou calculate the death rate and average life day.

5. viral separating experiment method: be that the female Kunming mouse of 13-15g is divided into C-3559 5.72mg/kg at random with body weight, 2.86mg/kg, 1.43mg/kg Zu and positive drug ACV 2.86mg/kg control group, solvent control Zu, virus control Zu, every Zu of 6 Zhi small white mouses, the virus liquid that Yong micro sample adding appliance Zhu in the small white mouse vagina enters 20 μ l, 1/30 dilution infects, at once administration after Yu infects, every day three times, after the Zai infection 48 and 96 hours, the cotton swab that the Yong nutrient solution is wetting inserts in the small white mouse vagina and wipes, then cotton is wiped away and put into the small test tube that fills the 1ml nutrient solution, extrude the liquid that cotton wipes away Zhong and be sample, 96 well culture plates that Zai covers with individual layer Vero cell carry out TCID₅₀Titration.

Six. the result:

The infection of HSV-2 cause small white mouse Zai infection afterwards visible morbidity in about the 3rd day be that the red and swollen and You juice of vagina flows out, also You hind leg paralysis of later stage, Zai the 5th, beginning in 6 days are dead.

First experiment Yong virus stock solution used infects, and 2.86mg/ml Zu and ACV Zu significantly Zeng add average life day (P＜0.05) of small white mouse, the results are shown in Table 4. Three dosage Zu of C-3559 add Yu Normal group the Zeng of small white mouse body weight and compare no significant difference, the results are shown in Table 3.

The virus liquid that second batch is tested Yong 1/30 infects, and each dosage Zu and ACV Zu all can infect the average life day of small white mouse by significant prolongation, and can significantly improve the survival rate (P＜0.01) of infected group small white mouse. The results are shown in Table 5. The Zeng that the Zeng of administration Zu small white mouse body weight adds Yu Normal group small white mouse body weight adds the difference (P＞0.05) on the no statistics, and 1mg/ml dosage Zu small white mouse body weight Zeng adds more than the Normal group small white mouse, You significant difference between Zhi the two (P＜0.01). The results are shown in Table 6 and table 7.

HSV-2 infected rear 48 and 96 hours, separated small white mouse vagina virus, titration TCID₅₀The results are shown in Table 8 and table 9, but the result show three dosage Zu of C-3559 all the You effect reduce HSV-2 discharge rate in the small white mouse vagina, compared marked difference Yu control group.

Table 4 C-3559 is to the curative effect (virus stock solution used infection) of HSV-2 vagina infection

Zu is other	Every Zu of mouse number	Survive number (%)	Average life day
				Virus control	10	0	5.8
Solvent control	10	0	6.2
				0.72mg/kg	10	0	6.5
1.43mg/kg	10	0	7.1
				2.86mg/kg	10	0	7.8 *
ACV 2.86mg/kg	10	0	7.8 *

^*P＜0.05

Table 5 C-3559 is to the curative effect (virus liquid infected in 1: 30) of HSV-2 vagina infection

Zu is other	Every Zu of mouse number	Survive number (%)	Average life day
				Virus control	10	2(20)	7.6
Solvent control	10	1(10)	7.5
				1.43mg/kg	10	8(80) **	12.8 **
2.86mg/kg	10	8(80) **	12.7 **
				5.72mg/kg	10	10(100) **	14 **
ACV 2.86mg/kg	10	6(60)	11.7 *

^*P＜0.05， ^**P＜0.001

The non-infected group mouse of table 6. 0day, 7days, 14days body weight result (first test)

Zu is other	Fate	Body weight (g)					On average
								Zheng often contrast	0days	15.05	13.28	15.2	14.76	14.96	14.65±0.7
7days	19.48	18.68	21.47	20.92	20.16	20.14±1.0
							14days		24.97	26.28	27.76	25.42	28.02	26.49±1.2
ACV	0days	16.15	15.94	13.53	15.95	14.38									15.19±1.1
							7days	22.58	18.84	22.23	20.43	20.28	20.87±1.4
	14days	24.03	28.03	27.39	24.27	28.46								26.44±1.9
							C-3559 0.72   mg/kg	0days	12.55	12.46	12.4	13.47	13.91		12.96±0.6
7days	19.87	20.32	20.97	21.35	21.33	20.77±0.6
								14days	28.9	29.88	27.35	24.82	29.86	28.16±1.9
C-3559 1.43   mg/kg	0days	16.03	14.84	15.86	16.65	15.61									15.80±0.6
							7days	21.16	22.31	20.93	21.84	20.13	21.27±0.8
	14days	29.28	30.78	24.72	26.9	27.96								27.93±2.1
							C-3559 2.86   mg/kg	0days	15.89	14.7	16.03	15.06	16.84		15.70±0.8
7days	20.52	22.85	21.97	21.7	18.83	21.17±1.4
								14days	26.67	28.12	30.2	26.32	29.17	28.10±1.5

Zhu: 7 days, all administration Zu compared equal no significant difference, not statistically significant Yu the Normal group body weight in 14 days.

The non-infected group mouse of table 7. 0day, 7days, 14days body weight result (the 3rd batch of experiment)

Zu is other	Fate	Body weight (g)						On average
									Zheng often contrast	0days	16.26	14.3	15.22	14.48	14.86	14.84	14.99±0.6
7days	20.92	21.72	22.68	21.05	20.2	20.67	21.21±0.8
								14days		27.92	27.16	26.1	29.08	27.63	26.13	27.34±1.0
ACV	0days	14.6	15.6	16.21	17.31	16.96	13.3										15.66±1.4
								7days	21.8	22.3	22.41	24.98	20.75	23.35	22.60±1.3
	14days	27.17	30.76	24.47	27.67	25.7	26.79									27.09±1.9
								C-3559 1.43   mg/kg	0days	15	15.93	14.43	16.74	16.53	15.26		15.65±0.8
7days	20.62	22.66	25.43	22.34	25.16	24.92	23.52±1.8 *
									14days	29.54	29.28	31.08	32.25	28.62	28.22	29.83±1.4 **
C-3559 2.86   mg/kg	0days	16.54	13.9	15.48	16.97	15.17	14.17										15.37±1.1
								7days	24.43	23.62	21.92	21.14	24.04	21.07	22.70±1.4
	14days	28.91	26.2	28.22	27.67	29.02	24.8									27.47±1.5
								C-3559 5.72   mg/kg	0days	15.86	16.45	17.27	15.83	14.56	14.75		15.79±0.9
7days	20.82	22.97	21.56	25.84	24.29	20.79	22.71±1.9
									14days	27.91	30.38	30.05	26.17	29.73	28.75	28.83±1.5

Zhu: 7 days, all C-3559 height, Zhong administration Zu and ACV administration Zu compared equal no significant difference Yu the Normal group body weight in 14 days, not statistically significant, and C-3559 1mg/ml Zu adds manyly than Normal group body weight Zeng, the two significant difference

(7days P＜0.05，14days P＜0.01)。

Vagina isolated viral result behind the table 8HSV-2SAV Zhu infection small white mouse 48hrs

Zu is other	Virus stock solution used	-1	-2	-3	-4	-5	TCID 50	On average
									Virus control 1	4	4	4	4			10 -3.5	10 -3.3
Virus control 2	4	4	4	4	2		10 -4
								Virus control 3	4	4	4	4			10 -3.5
Virus control 4	4	4	4	4	2		10 -3.5
								Virus control 5	4	4	4	2			10 -3
Virus control 6	4	4	4				10 -2.5
								Solvent control 1	4	4	4				10 -2.5	10 -2.9
Solvent control 2	4	4	2	2			10 -2.5
								Solvent control 3	4	4	4	4			10 -3.5
Solvent control 4	4	4	4	4			10 -2.5
								Solvent control 5	4	4	4	4	4		10 -4.5
Solvent control 6	4	4	2				10 -2
								ACV 2.86mg/kg 1#	4	4	4				10 -2.5		10 -2.5
ACV 2.86mg/kg 2#	4	4	4				10 -2.5
								ACV 2.86mg/kg 3#	4	4	2				10 -2
ACV 2.86mg/kg 4#	4	4	4	2			10 -3
								ACV 2.86mg/kg 5#	4	4					10 -1.5
ACV 2.86mg-/kg 6#	4	4	4	4			10 -3.5
								C-3559 1.43mg/kg 1#	4	4	4				10 -2.5	10 -1.3
C-3559 1.43mg/kg 2#	0
								C-3559 1.43mg/kg 3#	4	4	4	4			10 -3.5
C-3559 1.43mg/kg 4#	0

C-3559 1.43mg/kg 5#	4	4					10 -1.5
									C-3559 1.43mg/kg 6#	0
C-3559 2.86mg/kg 1#	0																10 -1.1
								C-3559 2.86mg/kg 2#	4	4	2				10 -2
C-3559 2.86mg/kg 3#	4	4	2				10 -2
								C-3559 2.86mg/kg 4#	4	4	2				10 -2
C-3559 2.86mg/kg 5#	4						10 -0.5
								C-3559 2.86mg/kg 6#	0
C-3559 5.72mg/kg 1#	0															10 -0.4
								C-3559 5.72mg/kg 2#	0
C-3559 5.72mg/kg 3#	0
								C-3559 5.72mg/kg 4#	4	4	4				10 -2.5
C-3559 5.72mg/kg 5#	0
								C-3559 5.72mg/kg 6#	0

Vagina isolated viral result behind the table 9 HSV-2 SAV Zhu infection small white mouse 96hrs

Zu is other	Virus stock solution used	-1	-2	-3	-4	-5	TCID 50	On average
									Virus control 1	4	4	4	4			10 -3.5	10 -3.3
Virus control 2	4	4	4	2			10 -3
								Virus control 3	4	4	4	4			10 -3.5
Virus control 4	4	4	4				10 -2.5
								Virus control 5	4	4	4	4			10 -3.5
Virus control 6	4	4	4	4			10 -3.5
								Solvent control 1	4	4	4	2			10 -3	10 -3.4
Solvent control 2	4	4	4	4			10 -3.5
								Solvent control 3	4	4	4	4	4		10 -4.5
Solvent control 4	4	4	4	4			10 -2.5
								Solvent control 5	4	4	4	4			10 -3.5
Solvent control 6	4	4	4	4			10 -3.5
								ACV 2.86mg/kg 1#	4	4					10 -1.5		10 -1.9
ACV 2.86mg/kg 2#	4	4	4				10 -2.5
								ACV 2.86mg/kg 3#	4	4					10 -1.5
ACV 2.86mg/kg 4#	4	4					10 -1.5
								ACV 2.86mg/kg 5#	4	4	2				10 -2
ACV 2.86mg/kg 6#	4	4	4				10 -2.5
								C-3559 1.43mg/kg 1#	4	4					10 -1.5	10 -1.3
C-3559 1.43mg/kg 2#	0
								C-3559 1.43mg/kg 3#	4	4	4	4	2		10 -4
C-3559 1.43mg/kg 4#	0
								C-3559 1.43mg/kg 5#	4	4	4				10 -2.5
C-3559 1.43mg/kg 6#	0
								C-3559 2.86mg/kg 1#	0								10 -1.3
C-3559 2.86mg/kg 2#	4	4	4				10 -2.5
								C-3559 2.86mg/kg 3#	4	4	4				10 -2.5
C-3559 2.86mg/kg 4#	4	4	4	2			10 -3
								C-3559 2.86mg/kg 5#	0
C-3559 2.86mg/kg 6#	0
								C-3559 5.72mg/kg 1#	0							10 -0.5
C-3559 5.72mg/kg 2#	0
								C-3559 5.72mg/kg 3#	0
C-3559 5.72mg/kg 4#	4	4	4	2			10 -3
								C-3559 5.72mg/kg 5#	0
C-3559 5.72mg/kg 6#	0

Conclusion:

In the C-3559 Zai HSV-2 vagina infection model, but the copying of establishment HSV-2. When virus stock solution used infects, prolong the average life day that HSV-2 infects small white mouse, the You statistical significance; When the virus liquid of 1/30 dilution infects, can significantly reduce the death rate of small white mouse, prolong the average life day of infecting small white mouse, the You statistical significance, and HSV-2 discharge rate in the reduction small white mouse vagina, infect rear 48 hours 5.72mg/kg, 2.86mg/kg and 1.43mg/kg Zu compare viral discharge rate and reduce respectively 794,158 and 100 times Yu control group; Infect rear 96 hours 5.72mg/kg, 2.86mg/kg and 1.43mg/kg compare viral discharge rate and reduce respectively 631,100 and 100 times Yu contrast, the ACV of effect You Yu same dose. By the observation to the small white mouse body weight, show that all dosage Zu of C-3559 are to the long nothing impact of small white mouse body weight Zeng.

Claims (1)

1. the application of geldanamycin in preparation treatment herpesvirus genital tract affection medicine.