CN100404029C - Use of geldanamycin (C-3559)in preparing drug for herpesvirus genital tract affection - Google Patents
Use of geldanamycin (C-3559)in preparing drug for herpesvirus genital tract affection Download PDFInfo
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Abstract
The present invention relates to an application of geldanamycin in preparing a medicine for treating condyloma acuminata. The geldanamycin has no stimulation on the inner side skin of the thigh of a healthy calf and an effect on inhibiting the duplication of bovine papilloma virus DNA so that a papilloma body is atrophied, aged and lessened. The present invention also relates to an application of the geldanamycin in preparing a medicine for treating herpes virus genital tract affection. C-3559 has a broad-spectrum antivirus effect during the culture of cells and micromole level antivirus activity on a plurality of kinds of DNA and RNA viruses. Through 41 generation transmission in vitro, the medicine-resistant toxic strains of HSV-1 viruses on the geldanamycin are not separated. A 15th HSV-1 GA medicine-resistant training generation has the same aciclovir sensitivity with wild strains and geldanamycin without cell toxicity concentration can specially inhibit the synthesis of HSV-1 DNA. In a white mouse herpes virus type 2 genital tract affection model, the duplication of the type 2 of herpes viruses can be effectively inhibited, virus excretion quantity can be obviously reduced, the death rate of a white mouse is lowered, the average living day of an affected white mouse is prolonged, and an effect is superior to acyclovir with the same dosage. All dosage groups have no influences on the weight increase of the white mouse.
Description
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% (165748 example), is the 3rd; Herpesvirus genital tract affection accounts for 4.79% (35679 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
Y?e?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.
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:
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 38.039.5 ℃).
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 inside skin as the calf.
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
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
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, 37 ℃ of water-baths 20 minutes are got 10 μ g DNA and 1/6 volume, 6 * sample-loading buffer mixing, at 1.0% agarose gel electrophoresis, after electrophoresis finishes, it is forwarded on the nitrocellulose filter, do 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 dna .and, 50% Methanamide) middle prehybridization 4hrs, 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 1 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) in the medicine body that carries out for geldanamycin of the present invention Vaginitis drug effect examining report.
One. purpose: research C-3559 body is interior to the colpitic therapeutic action of small white mouse HSV-2: see Examine 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 middle traditional Chinese medicines of doing available from Products in China The secondary animal (unsoundness certificate) that product biological product determination rodent cultivation center provides, with The machine grouping.
Three. virus: herpes virus type 2 (HSV-2) SAV strain, this chamber preservation of going down to posterity, with sick Toxogen liquid or 1/30 virus stock solution used infect.
Four. medicine: C-3559 is provided by the chemical chamber of this institute, and purity is greater than 95%, 0.1% tells with a little Be added in the mortar after temperature-80200 μ l solutions, 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: with body weight be the female Kunming mouse of 13-15g be divided at random C-35595.72mg/kg, 2.86mg/kg, 1.43mg/kg, 0.5mg/ml the group and positive drug ACV2.86mg/kg control group, molten Agent (0.1% Tween-80) control group, the virus control group, every group of 10 small white mouses more than are infected group; With C-35595.72mg/kg, 2.86mg/kg, 1.43mg/kg, 072mg/ml group and positive drug ACV 2.86mg/kg control group, the solvent control group, Normal group, 6 every group, be non-infected group, Observe medicine to the toxicity of small white mouse.
2. administering mode: in the small white mouse vagina, inject every concentration 20 μ l medicines with micropipettor, The equal saline injection of virus control group and Normal group, every day three times.
3. mode of infection: it is rare to inject 20 μ l 1/30 with micro sample adding appliance in the infected group small white mouse vagina The virus liquid of releasing or virus stock solution used, non-infected group inject 20 μ l physiological saline and carry out identical processing, Begin then administration.
4. observation index:
Observe the death rate, average life day, observe every day, and record is dead, in totally 2 weeks, calculates death Rate and average life day.
5. viral separating experiment method: be that the female Kunming mouse of 13-15g is divided into C-35595.72mg/kg at random with body weight, 2.86mg/kg, 1.43mg/kg group and positive drug ACV 2.86mg/kg control group, the solvent control group, the virus control group, every group of 6 small white mouses, in the small white mouse vagina, inject the virus liquid infection that 20 μ l 1/30 dilute with micro sample adding appliance, at once administration after infecting, every day three times, after infection 48 and 96 hours, to insert in the small white mouse vagina with the wetting cotton swab of nutrient solution and wipe, then cotton is wiped away and put into the small test tube that fills the 1ml nutrient solution, extrude the liquid of cotton in wiping away and be sample, carry out TCID at 96 well culture plates that cover with individual layer Vero cell50Titration.
Six. the result:
The infection of HSV-2 cause small white mouse infection afterwards visible morbidity in about the 3rd day be the vagina redness also Have juice to flow out, also there is rear acroparalysis in the later stage, and is dead beginning in the 5th, 6 day.
First experiment is infected with virus stock solution used, and 2.86mg/ml group and ACV group can significantly increase Xiao Bai The average life day (P＜0.05) of mouse the results are shown in Table 4. Three dosage groups of C-3559 are to Mice Heavy increase is compared no significant difference with Normal group, the results are shown in Table 3.
The second batch experiment is infected with 1/30 virus liquid, and each dosage group and ACV group all can significant prolongations Infect the average life day of small white mouse, and can significantly improve the survival rate (P＜0.01) of infected group small white mouse. The results are shown in Table 5. The increase of administration group small white mouse body weight and the increase of Normal group small white mouse body weight do not have Difference on the statistics (P＞0.05), and normal control is compared in the increase of 1mg/ml dosage group small white mouse body weight The group small white mouse is many, and significant difference (P＜0.01) is arranged between the two. 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 TCID50Knot Fruit sees Table 8 and table 9, and the result shows that three dosage groups of C-3559 all can effectively reduce the small white mouse vagina Interior HSV-2 discharge rate has been compared marked difference with control group.
Table 4 C-3559 is to the curative effect (virus stock solution used infection) of HSV-2 vagina infection
| Group | Every group 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
| Group | Every group 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)
Annotate: 7 days, all administration groups were compared equal no significant difference, not statistically significant with the Normal group body weight in 14 days.
The non-infected group mouse of table 7. 0day, 7days, 14days body weight result (second batch experiment)
Annotate: 7 days, all C-3559 height, middle administration group and ACV administration group were compared equal no significant difference with the Normal group body weight in 14 days, not statistically significant, and the C-35591mg/ml group increases manyly than the Normal group body weight, the two significant difference
(7daas P＜0.05，14days P＜0.01)。
Vagina isolated viral result behind the table 8 HSV-2SAV strain infection small white mouse 48hrs
Vagina isolated viral result behind the table 9 HSV-2SAV strain infection small white mouse 96hrs
Conclusion:
C-3559 is in HSV-2 vagina infection model, but the copying of establishment HSV-2. Virus When stoste infects, prolong the average life day that HSV-2 infects small white mouse, statistical significance is arranged; 1/30 is rare When the virus liquid of releasing infects, can significantly reduce the death rate of small white mouse, prolong and infect the average of small white mouse There is statistical significance life day, and reduces HSV-2 discharge rate in the small white mouse vagina, and 48 is little after infecting The time 5.72mg/kg, 2.86mg/kg and 1.43mg/kg group is compared viral discharge rate with control group and is fallen respectively Low 794,158 and 100 times; Infect rear 96 hours 5.72mg/kg, 2.g6mg/kg and 1.43mg/kg Viral discharge rate reduces respectively 631,100 and 100 times compared with the control, and effect is better than same dose ACV. By the observation to the small white mouse body weight, show that all dosage groups of C-3559 are to Mice Heavily increasing does not have impact.
Claims (1)
1. the application of geldanamycin in preparation treatment herpesvirus II type reproductive tract infection medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4075339A (en) * | 1974-04-05 | 1978-02-21 | University Of Illinois Foundation | Biologically active compounds |
| US5387584A (en) * | 1993-04-07 | 1995-02-07 | Pfizer Inc. | Bicyclic ansamycins |
| CN1189340A (en) * | 1997-01-28 | 1998-08-05 | 陶佩珍 | Novel grand spectrum anti-virus application of Gerd mycomycin |
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| US4075339A (en) * | 1974-04-05 | 1978-02-21 | University Of Illinois Foundation | Biologically active compounds |
| US5387584A (en) * | 1993-04-07 | 1995-02-07 | Pfizer Inc. | Bicyclic ansamycins |
| CN1189340A (en) * | 1997-01-28 | 1998-08-05 | 陶佩珍 | Novel grand spectrum anti-virus application of Gerd mycomycin |
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