CN103142461A - Gel for vaginas and preparation method thereof - Google Patents

Abstract

The invention relates to the field of medicines and discloses a gel for vaginas and a preparation method thereof. The gel is prepared by an active ingredient cidofovir, water and carriers applicable to pharmacy, wherein the carriers comprise carbomer, ethylene diamine tetraacetic acid disodium, sodium hydroxide glycerol, benzyl alcohol, polyethylene glycol 400 and tween-80. The invention further provides application of the gel for preparing medicines for treating vagina human papilloma virus or herpes simplex virus infection. An accelerated stability test, a long-term stability test and an influencing factor test display that the gel is stable in quality. The cidofovir vagina gel is used in an inserted mode to treat the human papilloma virus (HPV) or herpes simplex virus (HSV) infection, and the gel has good clinical application prospects.

Description

A kind of vaginal gel and preparation method thereof

Technical field

The present invention relates to field of medicaments, particularly a kind of vaginal gel and preparation method thereof.

Background technology

Cidofovir (cidofovir) is the acyclic nucleoside phosphate ester derivative, is a kind of wide spectrum resisting DNA virus medicine.Studies show that its antivirus action mechanism is for suppressing viral dna polymerase, be converted into the addition product of active metabolite phosplate, bisphosphate and other phosphocholine under the effect of cell thymidine kinase, the cidofovir bisphosphate is by suppressing archaeal dna polymerase, suppress competitively the DNA that deoxycytidine-5-triguaiacyl phosphate is integrated into virus, slow down the synthetic of DNA, and make the viral DNA loss of stability, thus copying of virus suppressed, and the performance antivirus action.

The cidofovir clinical indication of external approval is mainly used in treating HIV sufferers cytomegalovirus (CMV) property retinitis at present.After cidofovir enters the cell of cmv infection, by two step phosphorylations, be converted into activated diphosphonic acid cidofovir.It and Deoxydization nucleotide (dNTP) are similar, can be incorporated into the CMV DNA chain of prolongation, stop the further prolongation of viral DNA as the competitive inhibitor of CMV archaeal dna polymerase.Mix if there is two continuous diphosphonic acid cidofovir, can cause the termination fully that viral DNA is synthetic.In addition, mixing the diphosphonic acid cidofovir of DNA chain can not be excised by 3 of viral dna polymerase ' to 5 ' end exonuclease activity, therefore can produce lasting antiviral effect, simultaneously, because its metabolic half life is very long, cidofovir forms HPMPCp, HPMPCpp and HPMPCp-choline, particularly HPMPCp-choline after by cellular uptake and can be used as the bank of cidofovir in cell in cell, its half-life in cell is grown (48h) especially.Due to the existence of phosphate group in the HPMPC molecule, make the extremely difficult excision cidofovir of repairase.

Cidofovir is clinical widely applicable in recent years, is all obtaining curative effect in various degree aspect treatment clinical multiple DNA virus infection, tumor and dermatosis.

Cervical cancer occupies the second in female tumor, account for 15% of cancer patient's sum.370000 new cases are approximately arranged every year, and approximately 200000 people are dead.Low and lack the factor such as cervix neoplasms generaI investigation means due to economic level, 80% new cases occur in developing country.The incidence rate of China's cervical cancer and mortality rate account for global 1/3.Now clear and definite high-risk human mammilla papillomavirus (HPV) infection is the basic paathogenic factor of cervical cancer, and most of cervical intraepithelial neoplasia (CIN)s (CIN) infect with HPV, and 90% cervical cancer specimen can detect HPV DNA.China's cervical cancer district occurred frequently examination data also shows, nearly all CIN III and cervical cancer patient HPV are all positive.Because the HPV infection rate is still rising, the cervical cancer caused Global prevalence of sexually transmitted disease (STD) substance one of the disease the most widely that has been considered to spread through sex intercourse.At present, also effectively do not treat cervix uteri high-risk HPV infection medicine, the HPV vaccine of development, also just go on the market in the world, can bring glad tidings to young woman.But, be Drug therapy for the unique method of the women who has infected.

The anti-human papilloma virus (anti-HPV) of cidofovir (HPV) activity has been that a plurality of Experiment and clinic presentations institutes confirm.Studies show that, HPV infection cell cell proliferation after cidofovir is processed is obviously suppressed, and cell cycle arrest was pointed out its DNA to synthesize and is blocked in the S phase; Do not infect the keratinocyte of HPV without changing.Further research is found, cidofovir can the time and the concentration dependent mode induce the HPV positive cell to occur that DNA is cracked and Caspase-3 is protease activated, and increasing with the levels such as p53 albumen, pRb albumen and Cyclin Dependent Kinase inhibitive factor p21/WAF-1 in cell.Cidofovir is expected to become HPV cervix uteri preventing and curing infection medicine.

Summary of the invention

The technical problem to be solved in the present invention is for providing a kind of vaginal gel that is applicable to treat HPV or HSV viral infection, but described gel vagina administration greatly reduce medicine the accumulating of kidney, and pharmacodynamics improves obviously.

Vaginal gel provided by the invention, by the active component cidofovir and pharmaceutically applicable carrier form, pharmaceutically applicable carrier comprises carbomer, disodiumedetate, sodium hydroxide glycerol, benzyl alcohol, PEG400, tween 80 and water.

Cidofovir is white or off-white color crystalline powder, odorless.Fusing point is 250.27 ℃ (decomposition), and uv absorption coefficient (E1cm%) is 463.62, is the chiral structure medicine, and its Enantiomeric excess is less than 3%, and specific optical rotation is-93 °; Dissolve in the 0.1mol/L sodium hydroxide solution; Slightly soluble in water and 0.1mol/L hydrochloric acid; Almost insoluble in methanol and acetonitrile, stable in the aqueous solution in the pH8-10 scope, easily degraded in acidic aqueous solution.Its structural formula is as follows:

As preferably, cidofovir content is 2-30g, and its agent viscosity is 5～80 (Pas).

The physicochemical property of cidofovir and biological characteristics and clinical practice show that it is fit to the preparation water soluble gel.The hydrophilic polymer of formation gel is commonly used to prepare the semi-solid preparation without fat, the polymer gel that some are synthetic such as poloxamer, carbomer gel, has highly transparent, its viscosity is subjected to the impact of pH and ionic strength, carbomer 934 and Acritamer 940 are used general in preparation is produced, carbomer (carbomer, Cb) be acrylic acid and alkyl sucrose crosslinked form one revolve acrylate copolymer, be to use at present more a kind of polymer substance.The acidoid solution that Cb formation pH value soluble in water is 3 left and right adds alkali can be neutralized into gel transparent, stiff, to people's safety, and nonirritant, anaphylaxis or allergy, and with skin, the substrate of good coupling is arranged.Adopt suitable pH value and concentration, to the equal nonirritant such as eye, nasal mucosa.Gel take carbomer as substrate has rheological properties preferably, Plastic Flow is arranged, and viscosity is stable, and release soon, easily is coated with exhibition, without greasy, to the tack of skin and mucosa nonirritant, medicine film and good uniformity, easy eluting, the advantage such as pollution clothes not.

Disodiumedetate is chelating agent commonly used, in vagina gel of the present invention as antioxidant and stabilizing agent.Glycerol is common medicinal supplementary material, and stable in properties is made wetting agent and penetrating agent.

PEG400: adjuvant commonly used, stable in properties is as diluent, penetrating agent and the release promoter of hydrogel adhesive.

Benzyl alcohol is Common Preservatives; Tween 80 is non-ionic surface active agent, and used as stabilizers in gel of the present invention increases release; Sodium hydroxide is as pH adjusting agent and thickening agent.

Through relatively all be better than the prescription take semisynthetic fibre elements such as sodium carboxymethyl cellulose and hydroxyethyl-celluloses as gel-type vehicle in character, aspect stable with the gel of carbomer substrate, gel take carbomer as substrate has rheological properties preferably, gel has Plastic Flow, and viscosity is stable, and release soon, easily is coated with exhibition, without greasy, to the tack of skin and mucosa nonirritant, medicine film and good uniformity, easy eluting, the advantage such as pollution clothes not.The optimization of therefore writing out a prescription take carbomer as substrate simultaneously, has been investigated the impact of pH value on gel stability.Result shows, pH value is larger, and catabolite is fewer, illustrates that the stability of medicine and pH value are inversely proportional to.Bibliographical information, the most stable pH of cidofovir aqueous solution is 8～10.Therefore, from multifactor considerations such as gel moulding process, viscosity, ductility, vitro release, the compatibility and drug compliance, the pH value of determining the cidofovir gel is 6.0～8.0.

In the specific embodiment, vaginal gel provided by the invention consists of:

Cidofovir 1-50g, carbomer 5-20g, disodiumedetate 0.1-0.5g, sodium hydroxide 0.6-30g, glycerol 50-200g, benzyl alcohol 5-20g, PEG400 50-200g, tween 80 1-5g, surplus is water, makes the 1000g gel.

Surplus is water, makes the 1000g gel

As preferably, cidofovir gel of the present invention consists of:

Cidofovir 2-30g, carbomer 8-15g, disodiumedetate 0.2g, sodium hydroxide 1.2-12.0g, glycerol 100g, benzyl alcohol 10.0g, PEG400 50-200g, tween 80 2.0g, surplus is water, makes the 1000g gel.

Cidofovir gel specification 4g:0.004-0.12g of the present invention, the content limit of determining according to rule is: this product contains cidofovir (C ₈H ₁₄N ₃O ₆P) be the 90.0-110.0% of labelled amount; This product character is stable, water white transparency semi-solid thing; Every weight of formulation is 4g, and viscosity is 50～80 (Pas), and the pH value of aqueous solution is 6.0～8.0.

Show the vagina gel stable in properties of described cidofovir by accelerated stability test, long-term stable experiment and influence factor's test.Animal mucosa irritation and anaphylaxis studies show that cidofovir hydrogel adhesive of the present invention possesses useful clinically biological characteristics.

The present invention also provides the application of described gel for the preparation of the medicine for the treatment of vagina human papillomavirus or herpesvirus infection.

The vagina gel of cidofovir of the present invention has following features: bioadhesive (along the property of medicine): prolong drug action time, play the effect of slow releasing preparation; Good biocompatibility; Good diffusion, medicine can stick on vaginal mucosa, slowly are diffused into mucosa from gel, thus the performance therapeutical effect; Absolute acid stability, the non-liquefaction of body temperature, lubricity is without oils and fats and nonirritant.

The present invention also provides the preparation method of described vagina gel, comprises following steps:

Step 1: disodiumedetate, the glycerol of recipe quantity is soluble in water, add recipe quantity carbomer fine powder evenly to be sprinkling upon on solution surface, immersion fully infiltrates carbomer, adds sodium hydroxide solution, stir, obtain pH and transfer to 6-8 carbomer glue;

Step 2: stir in the benzyl alcohol of recipe quantity and tween 80 and the carbomer glue that adds step 1 preparation after PEG400 mixes, add water and fully stir and make mixing, the centrifugal bubble of sloughing, the white gel substrate of having leisure;

Step 3: with the cidofovir rear dilute with water that sieves, add 5% sodium hydroxide solution to make the pH of cidofovir solution transfer to 6-8 again, be added to after the filtering with microporous membrane degerming in the blank gel-type vehicle of step 2 preparation, water complements to 1000g, stir, obtain the cidofovir gel.

It is low that cidofovir vagina gel of the present invention inserts administration artifact availability, enters the body circulation less, and less to the whole body systematic influence, medicine concentration in nephridial tissue is compared with intravenous administration, greatly reduces, and therefore can reduce medicine accumulating at kidney.Pharmacodynamics test shows that 0.2%, 0.5% compares with the excipient matched group with the virus control group and all has repeatably statistical significance the dead protective rate of HSV-2 infecting mouse vaginitis, average life day with 1% CDV gel; all can significantly reduce HSV-2 infecting mouse vagina virus load; effect is better than positive control drug 3% acyclovir ointment, has good potential applicability in clinical practice.

Description of drawings

Fig. 1 shows the variation of the 10 days impurity of crude drug room temperature of different pH;

Fig. 2 shows the variation of the gel preparation hot test impurity of different pH;

Fig. 3 is that injection and vagina give respectively Cidofovir injection and gel Rabbit Kidney Tissue-time diagram;

Fig. 4 is that vagina gives 1% cidofovir gel Rabbit Kidney, uterine cancer cell and plasma drug level-time diagram.

The specific embodiment

The invention discloses a kind of vaginal gel and preparation method thereof, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.Product of the present invention, method and application are described by preferred embodiment, the related personnel obviously can change methods and applications as herein described within not breaking away from content of the present invention, spirit and scope or suitably change and combination, realizes and use the technology of the present invention.

In order to make those skilled in the art understand better technical scheme of the present invention, the present invention is described in further detail below in conjunction with specific embodiment.

Embodiment 1: cidofovir gel prescription of the present invention is determined

1, commonly use at present the gel-type vehicle characteristics

1.1 to synthesize aggregation as gel-type vehicle

Polyacrylamide (PAAm) and polyvinyl alcohol also usually as gel-type vehicle, are a kind of hydrophilic polymeies, and stickiness descends after physically aged.

1.2 with semi synthetic polymer-cellulose derivative as gel-type vehicle:

Sodium carboxymethyl cellulose is stable to acid, relatively is fit to the preparation vagina gel, but easy breed bacteria; Hydroxypropyl cellulose aqueous solution is not suitable for vagina medicinal to the unstable easy generation hydrolysis of acid, also light degradation and biodegradation can occur; Hydroxyethyl-cellulose is as gel-type vehicle, relatively stable, bibliographical information with hydroxyethyl-cellulose as gel-type vehicle cidofovir preparation, its prescription is that 1% (w/w) cidofovir is as active therapeutic agent, 20% (w/w) propylene glycol, 2% (w/w) hydroxyethyl-cellulose, 0.18% Buddhist nun uncle tortoise beetle ester, 0.02% Ni Baijin propyl ester and 0.02% disodiumedetate.

1.3 polysaccharide is gel-type vehicle:

Polysaccharide commonly used has alginate, pectin, hyaluronic acid and starch etc., and various polysaccharide gels and uses thereof are as described in Table 1.

Table 1 polysaccharide is the gel of gel-type vehicle

2, gel-type vehicle screening in cidofovir gel prescription

on the basis of list of references according to the technical field of preparation, at first gel-type vehicle is screened, characteristic and route of administration according to gel-type vehicle, select carbomer, sodium carboxymethyl cellulose and hydroxyethyl-cellulose (cellulose family) are suitable as vagina administration cidofovir gel-type vehicle, set them and be the gel-type vehicle in prescription, carried out Formulation with drug level 1%, 2 class prescriptions have been screened, with identical evaluation index, the quality of each prescription of overall merit and each preparation process, filter out and be fit to be optimized after industrial prescription and the basic prescription of technique conduct.

Evaluation index adopts outward appearance, viscosity, ductility, high temperature and low-temperature stability, freeze-thaw stability, centrifugal stability to carry out overall merit for investigating index, screens optimum substrate prescription

With reference to domestic and international gel products, as follows through the designed 2 gellike substrate prescriptions of experimentation:

2-1, with the gel of cellulose derivative as gel-type vehicle

Prescription one (document prescription)

Hydroxyethyl-cellulose is that gel-type vehicle is by above-mentioned formula preparation cidofovir gel, be translucent gels, viscosity and ductility are that the cidofovir gel of gel-type vehicle preparation is poor than carbomer, solid antiseptic poorly soluble, and investigate at-15 ℃ of low-temperature stabilities and have medicine to separate out phenomenon.

Prescription two (improving prescription)

Be that gel-type vehicle improves to hydroxyethyl-cellulose on the basis of document prescription, prepared the cidofovir gel, the solid antiseptic changes liquid preservative into, and has improved the pH value of prescription drug, through centrifugal, high temperature and freezing-thawing test, meets the gel requirement.

Prescription three

Sodium carboxymethyl cellulose be gel-type vehicle by above-mentioned formula preparation cidofovir gel, be translucent light yellow gel, it is poor that viscosity and ductility are than carbomer that the gel base prepares the cidofovir gel, and fine particle shape fiber is arranged.

2-2, the gel take carbomer as gel-type vehicle

Prescription four (US2003007281A1 openly writes out a prescription)

It is as follows that US Patent No. 2003007281A1 discloses cidofovir gel preparation technology in an embodiment:

Carbomer940 and disodiumedetate are joined in 225mL water, and low rate mixing is even; Poloxamer 407 is dissolved in aqueous solution and the carbomer aqueous solution of 225mL; The solution that cidofovir is dissolved in 50mL water joins in said mixture, then the solution that potassium hydroxide is dissolved in 100mL water is added, and stirring at low speed is even; Separately polysorbate40 and phenethylamine butyl ester are added in Polyethylene Glycol, gained solution is heated to 65 ℃ and makes compound dissolution, adds benzyl alcohol after being cooled to room temperature, and the gained mixture is added in cidofovir solution, and stirring at low speed gets this gel preparation.

Prepare the cidofovir gel by external disclosed patent prescription and technique, be transparent semi-solid gel, but viscosity is relatively poor, through centrifugal, high temperature and freezing-thawing test discovery, lamination is arranged.May with in relevant with alkali number.

Prescription five (improving prescription)

Transparent semi-solid gel, viscosity and ductility are slightly poor, and after placing, poloxamer is separated out phenomenon, 60 ℃ of heating, lamination is arranged.

Prescription six (optimizing prescription)

Transparent semi-solid gel, viscosity and ductility are better, tack is strong, through centrifugal, high temperature and freezing-thawing test without lamination, good stability.

Prescription seven (relatively prescription)

Viscosity and ductility are better, and tack is strong, and Carbomer-940 sample transparency slightly is worse than carbomer-934p sample.

Gel phase related parameter index such as table 2 according to above each formula preparation:

The gel relevant parameter of each formula preparation of table 2 relatively

Each index with, with good, in, differ from three kinds of degree and estimate, draw optimizing prescriptions after overall merit.

Conclusion: through relatively all be better than the prescription take semisynthetic fibre elements such as sodium carboxymethyl cellulose and hydroxyethyl-celluloses as gel-type vehicle in character, aspect stable with the gel of carbomer substrate.

3, the selection of adjuvant in the prescription

The foundation (table 3) that the 3-1 adjuvant is selected

Screen according to the technology of document and this formulation art and adjuvant physicochemical property and the effect in prescription.

The 3-2 adjuvant is selected

Table 3 curative, adjuvant, source, lot number and effect

4, prescription screening and optimum preparation condition

Through relatively all be better than the prescription take semisynthetic fibre elements such as sodium carboxymethyl cellulose and hydroxyethyl-celluloses as gel-type vehicle, the optimization of therefore writing out a prescription take carbomer as substrate in character, aspect stable with the gel of carbomer substrate.Take the prescription 6 that preferably obtains as the basis, preparation 100g cidofovir gel screens each supplementary product consumption, obtains optimum prescription.

4-1 is take carbomer as substrate prescription screening and optimization

On prescription 6 basis, consumption, sodium hydroxide concentration, glycerol and the PEG-4000 consumption of carbomer is studied.Select 3 factors such as carbomer concentration (A), glycerol (B) and PEG400 (C) consumption to design respectively 3 levels, the cidofovir gel is optimized as investigating index take viscosity, stability and release.

(1) carbomer, glycerol and PEG400 use quantity research:

In prescription, naoh concentration is 0.5%, under the ceteris paribus condition, select 3 factors of carbomer concentration and glycerol and PEG400 consumption, design respectively 3 levels (table 4), preparation 100g cidofovir gel, the cidofovir gel is investigated as investigating index take viscosity, stability and release:

Table 4 factor level design table

Investigate index and assay method:

Outward appearance: visual, get the preparation sample appropriate, be encased in 10ml, transparent centrifuge tube, centrifugal 30 minutes of 3000rpm removes bubble and observes, and from the color and luster of gel, the aspects such as transparency consider;

Stability: carry out-15 ℃ of low temperature and 60 ℃ of high temperature and centrefuge experiment, investigate whether layering and sedimentary generation;

Adhesion: the mensuration of carrying out viscosity with the NDJ-8S rotational viscometer;

PH value of solution: get this product 2g, measure (Chinese Pharmacopoeia version appendix VI H in 2010) after adding water 25ml dissolving in accordance with the law

Release: (assay method is seen preparation data 10)

Get this product pack into (molecular weight Mw=8000) in the good semipermeable membrane of pre-treatment, the rear photograph (two appendix X D first methods of Chinese Pharmacopoeia version in 2010) of obturaging adopts phosphate buffer solution (0.2mol/L sodium dihydrogen phosphate with sodium hydroxide the transfer to pH5.0) 500ml of dissolution method first method device (two appendix X C of Chinese Pharmacopoeia version in 2010) take pH value as 5.0 as release medium, in 37 ℃, rotating speed is per minute 50 to turn, in accordance with the law operation.Then the 5ml that took a sample respectively at 1,4,8 hour replenishes isodose buffer solution.Sample solution is diluted suitable multiple, press ultraviolet spectrophotometry, measure ultraviolet absorptivity at the 280nm wavelength, use Standard reference, calculate each sample point drug accumulation and discharge percentage amounts.The following 5-6 of result.

Table 5L ₉3 ³Orthogonal experiment results

Table 6L ₉3 ³Orthogonal experiment release in vitro result

no matter from viscosity, pH, release and stability, influence factor's maximum be A (A〉B, C), be that the carbomer consumption is principal element, because under sodium hydrate content one stable condition, the principal element that affects preparation pH is the consumption of carbomer, show from the release that affects preparation and the result of viscosity, the consumption of carbomer is larger, the viscosity of preparation is larger, and release in vitro is just slower, therefore under the condition of preparation stabilization, reduce as far as possible the preparation pH value, to satisfy the needs of physiological environment (vaginal environment pH is as 5-6), from preparation viscosity and pH and the estimation of stability thereof that is fit to, the consumption of carbomer is 1%, glycerol is 10%, PEG400 is 10%.

(2) sodium hydroxide concentration:

Its solution ph of 1% of carbomer is about 3 left and right, add alkali can be neutralized into gel transparent, stiff, when forming hydrogel pH about 6～12 o'clock thickness the most, pH is gel stability between 5～11, according to vagina meta-acid environment, the cidofovir gel is the vaginal mucosa medication, should with the pH of environment consistent (vaginal environment pH is 5～6) as far as possible, but cidofovir belongs to acidic drug, can dissolve the minimum pH of cidofovir about 5～6, therefore the consumption minimum is advisable under the prerequisite that guarantees gel viscosity and preparation stabilization.Table 8 is that sodium hydroxide is respectively with 0.18%, 0.21%, 0.26%, 0.36%, 0.46%, 0.50%, 0.60%, 0.75% recipe quantity, other compositions and consumption thereof are fixing by prescription 6, carry out monofactorial prescription screening take viscosity, stability, pH, transparency as investigating index, evaluation result is listed table 7 in:

The impact of the content of different hydro sodium oxide on prescription in table 7 prescription

Intuitive analysis, the sample of 8 formula preparations of above-mentioned design is under room temperature, 4 ℃ and 60 ℃ of hot conditionss, and the gel moulding process all meets the requirements, and investigates and finds but carry out dosage form stability, and writing out a prescription in-15 ℃ of frozen process experiments 1,2,3 has a small amount of medicine to separate out.Result shows, in prescription, other compositions are fixedly the time, the critical selected amount of sodium hydroxide is 0.18%, this prescription pH is 5.01, although can guarantee that medicine dissolves fully, became muddy in one month but place at ambient temperature, in 0.21%～0.26% scope, stable and freezing-thawing test is undesirable under the preparation room temperature condition of preparation when sodium hydroxide.Therefore, select suitable viscosity, can guarantee again in the scope of the physiological environment of preparation pH value within being fit to the human vagina, the pH value of comparatively ideal drug gel prescription should be lower than 5.5.

(3) impact of cidofovir gel pH on preparation stability

Because carbomer and medicine all belong to acid, guaranteeing under the prerequisite that medicine can dissolve, pH is gel stability between 5.5～11, according to vagina meta-acid environment, the cidofovir gel is the vaginal mucosa medication, should try one's best consistent (vaginal environment PH is 5～6) with the PH of environment, but cidofovir belongs to acidic drug, can dissolve the minimum pH of cidofovir about 5～6, therefore the consumption minimum is advisable under the prerequisite that guarantees gel viscosity and preparation stabilization.Under room temperature condition sodium hydroxide is in 0.21%～0.75% scope, its with viscosity, preparation stability, pH, transparency for comparatively desirable.Therefore, select the gel preparation of different pH to carry out hot test in the influence factor on this basis, investigate content and the catabolite of cidofovir medicine, to determine the most stable pH, design six prescriptions for this reason and study, the results are shown in Table 8, impurity content and pH relation are referring to Fig. 1 and Fig. 2:

The different pH value gel prescription Chinese medicine content of table 8 and catabolite

In cidofovir raw material drug stabilisation Journal of Sex Research, medicine is dissolved with aqueous solution, and be deployed into the solution of different pH value with sodium hydroxide solution, investigate content and catabolite under room temperature condition, the results are shown in Table 9:

Content and catabolite in the different pH aqueous solutions of table 9 cidofovir medicine

Investigate discovery through cidofovir pharmaceutical aqueous solution and gel preparation under different condition, pH value is consistent to the stability influence of medicine, and namely pH value is larger, and catabolite is fewer.Can find out by test, between 7-8, the variation of catabolite tends towards stability substantially when the pH value of gel preparation.Therefore, consider from composite factors such as gel moulding process, viscosity, ductility, vitro release and medicine stabilities, the pH value of determining the cidofovir gel is 6.5-7.5.

This product is vagina medicinal, for the pH value of the physiological environment preparation that adapts to the normal person can not be too high; Simultaneously, the pH value of preparation has material impact to the stability of preparation, carries out strict control so the pH value of preparation should be important quality index.

Cidofovir gel pH assay method:

Get this product 2g, measure (Chinese Pharmacopoeia version appendix VI H in 2010) after adding water 25ml dissolving in accordance with the law, it is 6.5～7.5 that pH value should be.

Embodiment 2: prepare cidofovir gel of the present invention

(1) preparation 1% cidofovir gel

According to the form below is made 1000 gram prescriptions (being equivalent to 250 single doses)

Make the 1000g gel

Preparation process thereof of the present invention:

(1) disodiumedetate, the glycerol with recipe quantity is dissolved in 400ml water, adds recipe quantity carbomer fine powder evenly to be sprinkling upon on solution surface, soaks to be no less than 24 hours, and carbomer is fully infiltrated.Add 5% sodium hydroxide solution 70ml, stir, get the carbomer glue.The benzyl alcohol of recipe quantity and tween 80 are stirred with being added in the carbomer glue after PEG400 mixes, then supply weight to 750g with water for injection, fully stir and make mixing (but autoclaving), the centrifugal bubble of sloughing, the white gel substrate of having leisure.

(2) cidofovir being crossed 80 mesh sieves dilutes with 150ml water, add again 5% sodium hydroxide solution 50ml to make dissolving, obtain cidofovir solution, be added to after the filtering with microporous membrane degerming in the blank gel-type vehicle of method (1) preparation, complement to 1000g with water for injection, stir, obtain 1% cidofovir gel.

The 1% cidofovir gel that (3) will prepare according to the dosage packing of every 4g, and get final product.

(2) preparation 2% cidofovir gel

According to the form below is made 1000 gram prescriptions (being equivalent to 250 single doses)

Make the 1000g gel

Preparation process thereof of the present invention:

(1) disodiumedetate, the glycerol with recipe quantity is dissolved in 400ml water, adds recipe quantity carbomer fine powder evenly to be sprinkling upon on solution surface, soaks to be no less than 24 hours, and carbomer is fully infiltrated.Add 5% sodium hydroxide solution 70ml, stir, get the carbomer glue.The benzyl alcohol of recipe quantity and tween 80 are stirred with being added in the carbomer glue after PEG400 mixes, then supply weight to 750g with water for injection, fully stir and make mixing (but autoclaving), the centrifugal bubble of sloughing, the white gel substrate of having leisure.

(2) cidofovir being crossed 80 mesh sieves dilutes with 150ml water, add again 5% sodium hydroxide solution 100ml to make dissolving, obtain cidofovir solution, be added to after the filtering with microporous membrane degerming in the blank gel-type vehicle of method (1) preparation, complement to 1000g with water for injection, stir, obtain 2% cidofovir gel.

The 2% cidofovir gel that (3) will prepare according to the dosage packing of every 4g, and get final product.

(3) preparation 0.5% cidofovir gel

According to the form below is made 1000 gram prescriptions (being equivalent to 250 single doses)

Make the 1000g gel

Preparation process thereof of the present invention:

(1) disodiumedetate, the glycerol with recipe quantity is dissolved in 400ml water, adds recipe quantity carbomer fine powder evenly to be sprinkling upon on solution surface, soaks to be no less than 24 hours, and carbomer is fully infiltrated.Add 5% sodium hydroxide solution 70ml, stir, get the carbomer glue.The benzyl alcohol of recipe quantity and tween 80 are stirred with being added in the carbomer glue after PEG400 mixes, then supply weight to 750g with water for injection, fully stir and make mixing (but autoclaving), the centrifugal bubble of sloughing, the white gel substrate of having leisure.

(2) cidofovir being crossed 80 mesh sieves dilutes with 150ml water, add again 5% sodium hydroxide solution 25ml to make dissolving, obtain cidofovir solution, be added to after the filtering with microporous membrane degerming in the blank gel-type vehicle of method (1) preparation, complement to 1000g with water for injection, stir, obtain 0.5% cidofovir gel.

The 0.5% cidofovir gel that (3) will prepare according to the dosage packing of every 4g, and get final product.

(1) preparation 0.2% cidofovir gel

According to the form below is made 1000 gram prescriptions (being equivalent to 250 single doses)

Make the 1000g gel

Preparation process thereof of the present invention:

(1) disodiumedetate, the glycerol with recipe quantity is dissolved in 400ml water, adds recipe quantity carbomer fine powder evenly to be sprinkling upon on solution surface, soaks to be no less than 24 hours, and carbomer is fully infiltrated.Add 5% sodium hydroxide solution 70ml, stir, get the carbomer glue.The benzyl alcohol of recipe quantity and tween 80 are stirred with being added in the carbomer glue after PEG400 mixes, then supply weight to 750g with water for injection, fully stir and make mixing (but autoclaving), the centrifugal bubble of sloughing, the white gel substrate of having leisure.

(2) cidofovir being crossed 80 mesh sieves dilutes with 150ml water, add again 5% sodium hydroxide solution 10ml to make dissolving, obtain cidofovir solution, be added to after the filtering with microporous membrane degerming in the blank gel-type vehicle of method (1) preparation, complement to 1000g with water for injection, stir, obtain 0.2% cidofovir gel.

The 0.2% cidofovir gel that (3) will prepare according to the dosage packing of every 4g, and get final product.

Embodiment 3: cidofovir gel relevant parameter of the present invention is measured

(1) gel preparation viscosity:

Instrument: rotating cylinder viscometer: NDJ-8s(Shanghai Ji Chang geological instrument company limited)

Viscosity measurement method: sample is loaded on diameter＞5cm, in the hydrostatic column of highly＞15cm, be positioned in centrifuge with 3000rpm centrifugal 10 minutes after the appropriate gel of packing into, remove all bubbles, after selecting No. 4 rotors to equip, regulate rotor and require height in sample, sample is put into 37 ℃ of waters bath with thermostatic control, after 20 minutes, carry out viscosimetric analysis with 6 rev/mins of rotating speeds, each sample in measurement three times is averaged, and viscosity is at 50～75Pas.

(2) stability of gel preparation:

Centrifugal test: get cidofovir gel 10g, in the centrifuge tube with scale of packing into, rotating speed 3000rpm, centrifugal 30 minutes, without the layering denaturalization phenomenon;

High-temperature stability and low-temperature stability test: sample thief 20g, pack in airtight package, part is put in 60 ℃ of thermostatic drying chambers, take out after 6 hours, another part is put in-15 ℃ of refrigerators, takes out after 24 hours, after returning to room temperature, observe gel without lamination, and the viscosity of measuring is also without significant change.

(3) gel preparation release:

Measure according to two (appendix X D) drug release determination methods of Chinese Pharmacopoeia version in 2010.

Get this product pack into (molecular weight Mw=8000) in the good semipermeable membrane of pre-treatment, obturage rear according to phosphate buffer solution (0.2mol/L sodium dihydrogen phosphate with sodium hydroxide the transfer to pH5.0) 500ml of dissolution method first method device (two appendix X C of Chinese Pharmacopoeia version in 2010) take pH value as 5.0 as release medium, in 37 ℃, rotating speed is per minute 50 to turn, in accordance with the law operation.At 1 hour, 4 hours, the 8 hours 5ml that take a sample respectively, then replenish isodose buffer solution.With the centrifugal (3000rpm of sample solution, 10min), get the suitable multiple of supernatant dilution and (get respectively 1 hour, 4 hours, 8 hours sampling 3ml, 2ml, 2ml, put the 10ml measuring bottle, buffer is diluted to scale, shake up), press ultraviolet spectrophotometry, measure ultraviolet absorptivity at the 280nm wavelength, use the standard control method, calculate each sample point drug accumulation and discharge percentage amounts, every stripping quantity at 1 hour, 4 hours, 8 hours of this product should be 20%～35%, 55%～75%, 80%～95% of expression amount.

Embodiment 4: cidofovir gel pharmacodynamic study of the present invention

The cidofovir gel is used for the treatment of the safety of the double-blind placebo-controlled contrast that people HPV infects and the II phase clinical research of effectiveness shows, the cidofovir gel not have to occur and the report of Cidofovir related nephrotoxicity, 30 routine MethodsThe cases enrolled; Wherein 19 examples are for accepting the cidofovir treatment group, 11 examples are for accepting the placebo treatment group, the intermediate value and the wart baseline intermediate value that compare two groups of wart quantity, cidofovir treatment group 9/19(47%) response fully (the overall healing) is arranged, under comparing, placebo group is 0 response (p=0.006).The cidofovir treatment group does not have a routine patient the state of an illness that development is arranged, and under comparing, placebo group has 5/11 example (45%) patient that development is arranged, and two groups of side effect reporting have comparability.It is serious side effect that grade appears in 3 routine patients, and wherein 2 examples occur in placebo group, and 1 example occurs in medication therapy groups.Accept cidofovir treatment patient and labium majus and nympholabial ulcer only occur, think relevant with Drug therapy.The cidofovir treatment group has the non-because completed treatment of 4 routine patients or PD and leaves too early treatment: wherein 2 examples lose and follow up a case by regular visits to, and another 2 examples require stopped treatment.The modal side effect of reporting is pain, pruritus and erythra to occur at the medicinal application position; Report that in the cidofovir treatment group such reaction has 13 examples (68%), by contrast placebo group be 7 examples (64%) (p=1.0), it is reversible stopping after medicinal application.(6 examples are accepted cidofovir to have in 20 routine patients of application site reaction 11 examples corrosion or ulcer occur; 5 examples are accepted placebo).The cidofovir treatment group patient who ulcer occurs confirms that by PCR 1 example thinks the HSV infection of laboratory toxicity.Neutropenia appears in the routine patient of placebo group 1.

The toxicity test result that CDV gel single vagina administration gives rabbit shows, after administration the 7th day, soft stool, crissum filth, lassitude, the prostrate and symptom of becoming thin appearred in 3% group of 1 animal of test sample, lasting 2 days, and death in the 9th day after administration.Soft stool appearred on the 12nd day in 1% group of 1 animal of test sample after administration, continue two days.Other animal is showed no Novel presentation.Under this experimental condition, the safe dose that CDV gel single vagina gives rabbit is 129.6mg/ (containing CDV), is equivalent to clinical plan with 72～90 times of dosage.

Cidofovir vagina gel vagina repeatedly injects and to give the toxicity test result in 13 weeks of rat and show, duration of test, and all animals are euthanasia according to plan all, does not find animal dead.Clinical observation as seen, 1.62mg/ only and 4.86mg/ dosage group Some Animals external genital one cross slightly redness of property, other animal has no other abnormal clinical responses relevant to drug toxicity.The indexs such as the body weight of each dosage treated animal, appetite, cytometry, coagulation function, blood biochemical, ophthalmology, urine, organ weights, organ coefficient change there are no the regularity of toxicology meaning.The pathological examination result shows, 1.62 with vagina and the visible reaction relevant to test sample of cervix uteri of 4.86mg/ dosage treated animal, mainly comprise the necrosis of vaginal mucosa epithelium and vaginal wall, inflammation and cervix uteri hypertrophy; This medicine is relevant with the time that continues medication with dosage to the toxicity damage effect of vagina; Convalescent period, this medicine was obviously alleviated the toxicity damage effect of vagina, and the pathological examination of other histoorgan has no the change relevant to the test sample toxic pathology.1% and 3% cidofovir vagina gel vagina injection repeatedly gives the Wistar rat, and vagina and cervix uteri are had certain stimulation; Under 4.86mg/ dosage only (being equal to 16.2mg/kg), have no general toxic reaction.

Embodiment 5: cidofovir gel pharmacokinetic of the present invention

Employing parallel test design, the pharmacokinetic that has carried out the administration of cidofovir vagina gel rabbit vagina.Pharmacokinetic is divided into single-dose and multiple dosing (continuous 7 days) dual mode; Single-dose adopts 10mg/kg injection group as a control group, and the cidofovir vagina gel is divided into 1% and 3% two dosage group; Multiple dosing adopts the administration in continuous 7 days of 1% cidofovir vagina gel, administration every day 1 time; Each dosage group is carried out separately.Measure the blood drug level data of the medicine of drug administration by injection and vaginal jellies administration dual mode, drugs is in the situation of accumulating of toxicity target organ (kidney) and site of action (uterus).Adopt the HPLC/MS/MS method to measure, calculate blood plasma and organize Chinese medicine concentration with internal standard method, according to the drug level time data in animal body, calculate the pharmacokinetic parameter of cidofovir and estimate its safety.

In single-dose research, compare rabbit single intravaginal and used 1% and the Pharmacokinetic Characteristics of 3% two kind of concentration cidofovir vagina gel and cidofovir intravenously administrable, result shows (table 10), the cidofovir vagina gel is by after vagina administration, medicine enters blood less (1% or 3% concentration cidofovir vagina gel absolute bioavailability is respectively 3.99% and 2.18%), mainly concentrate on medicine-feeding part, 1% or 3% concentration cidofovir vagina gel C _maxBe respectively 1362 ± 146 μ gL ^-1With 2979 ± 1461 μ gL ^-1T _max(h) be respectively 0.43 ± 0.27 and 0.58 ± 0.17; Increase AUC with dosage _0-tnCorresponding increase, but not with the proportional relation of dosage, illustrate that the absorption of cidofovir gel does not have dose dependent.Cidofovir intravenously administrable mean residence time MRT(1.17h), the mean residence time MRT that 1% and 3% concentration cidofovir vaginal jellies vagina inserts after administration is respectively 1.74h and 1.80h, show that vaginal jellies inserts administration, drug absorption is also very fast by metabolism rapidly.

By measuring the concentration (table 11) of medicine in toxicity target organ (kidney), find 1% cidofovir gel vagina administration, the nephridial tissue drug level reaches maximum concentration 35424 ± 26709.8 μ gL at 2h ^-1The Cidofovir injection intravenous administration, medicine namely reaches Cmax 525600 ± 40047.47 μ gL at nephridial tissue 20min ^-1, both maximum concentrations differ 14 times.To 12h, to give and 1% cidofovir vaginal jellies, kidney Chinese medicine concentration is 1701.6 ± 2049.9 μ gL ^-1And the Cidofovir injection intravenous administration, kidney Chinese medicine concentration is 87780 ± 22591.41 μ gL ^-1, be 51 times of vaginal jellies administering mode; To 24h, the Cidofovir injection intravenous administration, kidney Chinese medicine concentration is appointed up to 50940 ± 7593.62 μ gL ^-1Illustrate that cidofovir gel vagina administration is significantly less than the drug administration by injection mode in the accumulation of nephridial tissue Chinese medicine.In the minute scope, the nephridial tissue Chinese medicine exposes as the 3.6%(AUC ratio that adopts with dosage drug administration by injection mode), illustrate that the administration of cidofovir vaginal jellies is less owing to entering the blood circulation medicine, compare with drug administration by injection, can reduce in kidney and accumulate.By contrasting 1% cidofovir gel vagina administration different time points, medicine is in the concentration (table 12 and Fig. 3-4) of site of action (uterus) and toxicity organ (kidney), find that medicine concentration in the uterus is much higher than kidney, mainly concentrate on the uterus after drug administration is described, but lowering of concentration is rapid than kidney.

In multiple dosing research, adopt the administration in continuous 7 days of 1% cidofovir gel, contrast the 1st day with the 7th day Related Drug for parameter (table 13), on average C _maxBe respectively 972 ± 1461 and 909 ± 817 μ gL ^-1μ gL ^-1AUC _0-tnMeansigma methods is respectively 939.10 ± 1359.28 and 593.92 ± 405.17 μ ghL ^-1Renal clearance CL is respectively 0.76 ± 0.75 and 0.45 ± 0.26Lkg ^-1H ^-1, slightly descended to the 7th day.With 1% cidofovir vaginal jellies single-dose renal clearance CL0.42 ± 0.41Lkg before ^-1H ^-1Compare, no significant difference illustrates that bioavailability is low due to the administration of cidofovir vagina gel, and it is less that medicine enters blood, lower in the nephridial tissue accumulation, and 7 days successive administrations are in vivo without accumulating, and are less on the nephridial tissue impact, and occur without bad event.

The product of cidofovir listing at present is injection, and clinical dosage is the 375mg/ person-portion, and cidofovir vagina gel clinical dosage of the present invention is the 40mg/ person-portion, is significantly less than the dosage of listing product.It is low that the cidofovir vaginal jellies inserts administration artifact availability, enters the body circulation less, and less to the whole body systematic influence, medicine concentration in nephridial tissue is compared with intravenous administration, greatly reduces, and therefore can reduce medicine accumulating at kidney.After administration, drug main will concentrate on medicine-feeding part, mainly brings into play the topical antiviral effect, so drug safety.

Table 10 rabbit single gives average pharmacokinetic parameter after Cidofovir injection and high-concentration and low-concentration gel

Table 11 injection and vagina give Cidofovir injection and gel Rabbit Kidney and plasma drug level-time data

Table 12 vagina gives 1% cidofovir gel Rabbit Kidney, uterine cancer cell and plasma drug level-time data

Table 13 rabbit single gives gel-filled dose of cidofovir and contrasts with the later on average pharmacokinetic parameter of multiple dosing

Embodiment 6: gel-filled dose of test of pesticide effectiveness of cidofovir of the present invention

For reagent: according to gel-filled dose of the cidofovir of embodiment 2 preparations: 0.05%CDV gel, 0.2%CDV gel, 0.5%CDV gel, 1%CDV gel, excipient (blank gel)

Specification: 0.05%:4g.0.2%：4g。0.5%：4g。1%：4g。

1, HSV-2 goes down to posterity and mouse vagina infection toxicity test

Virus is 10 with the maintenance medium dilution ^-3Inoculation grows up to the Vero cell of monolayer, and 37 ℃ of 5%CO2 incubators were cultivated after 2 days, cytopathy occurred, collected virus liquid, and is centrifugal after freezing 3 times.Supernatant is made virus stock solution used, is diluted to 1:15,1:30,1:60,1:120,1:240 with culture fluid.Inoculation female mice vagina, every Mus 20 μ l, 6 mices of every concentration are observed morbidity, record death, calculate mortality rate, on average life number of days.

2, CDV gel therapeutic test:

Mouse vagina infected virus after 2 hours, and vagina is local with syringe (syringe needle adds sleeve pipe) administration, and each every mice administration 25 μ l, record death condition every day at totally 7 days every day 2 times.Continuous Observation 14 days, still survival do calculating in 14 days in 14 days, calculating mortality rate and average the life day are compared with virus control group and excipient matched group, calculate protective rate and extending life rate.Every group of 10 mices.

First experiment:

CDV gel dosage: 0.2%, 0.5%, 1%, 2 hours vagina topicals after viral infection, each every mice administration 25 μ l, every day 2 times, continuous 7 days.Virus control group and Normal group give normal saline, positive control drug acyclovir ointment (3%), and excipient gives blank gel, and all matched group administration times are with CDV gel treatment group.

The second batch experiment:

CDV gel dosage: 0.05%, 0.2%, 0.5%, 1%, 2 hours vagina topicals after viral infection, each every mice administration 25 μ l, every day 2 times, continuous 7 days.Virus control group and Normal group give normal saline, positive control drug acyclovir ointment (3%), and excipient gives blank gel, and all matched group administration times are with CDV gel treatment group.

The 3rd batch of experiment:

CDV gel dosage: 0.05%, 0.2%, 1%, 2 hours vagina topicals after viral infection, each every mice administration 25 μ l, every day 2 times, continuous 7 days.Virus control group and Normal group give normal saline, positive control drug acyclovir ointment (3%), and excipient gives blank gel, and all matched group administration times are with CDV gel treatment group.

3, viral separation test:

Got mouse vagina secretions with the sterile cotton swab in rear 72 hours in infection, insert in the 1ml maintenance medium as virus stock solution used, then take 10 times of dilutions of maintenance medium as 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, inoculation 96 well culture plate Vero cell monolayers, 5%CO ₂TCID is calculated in 37 ℃ of cultivations of incubator 144 hours ₅₀, measure Drug therapy impact on local viral secretory amount after 72 hours.Every group of 6 mices.

First experiment:

CDV gel dosage: 0.05%, 0.2%, 1%, 2 hours vagina topicals after viral infection, each every mice administration 25 μ l, every day 2 times, for three days on end.Virus control group and Normal group give normal saline, positive control drug acyclovir ointment (3%), and excipient gives blank gel, and all matched group administration times are with CDV gel treatment group.

The second batch experiment:

CDV gel dosage: 0.05%, 0.2%, 1%, 2 hours vagina topicals after viral infection, each every mice administration 25 μ l, every day 2 times, for three days on end.Virus control group and Normal group give normal saline, positive control drug acyclovir ointment (3%), and excipient gives blank gel, and all matched group administration times are with CDV gel treatment group.

4 results are calculated and are analyzed

Dead protective rate= (virus control group mortality rate-experimental group mortality rate)* 100%

The extending life rate= (experimental group on average life number of days-Virus Matched group is life number of days on average)

The virus control group is life number of days * 100% on average

11.4.3 median effective dose ED ₅₀, the Reed-Muench method is calculated.

${\mathrm{ED}}_{50}=\mathrm{Anti}\log (A+\frac{50-B}{C-B}\×D)$

The cumulative suppression ratio C=of the drug level B of the cumulative suppression ratio of A=log＜50%＜50%〉50% cumulative suppression ratio, the D=log extension rate

5, statistical procedures

Adopt statistical procedure, compare mortality rate with X 2 method, card steps (Kaplan-Meier) method and more on average lives day.

6, result of the test

6.1HSV-2 infecting mouse vagina toxicity test

HSV-2 333 strains, 2 times of dilutions are 5 dilution factors (1/15-1/240), every group of 6 animals, vaginal infection 20 μ l/ are only.Observed for two weeks, press Reed ﹠amp; The Muench method is calculated median infective dose LD ₅₀, record LD ₅₀=10 ^-2.78The viral infection dosage of mice is: 1/30, be equivalent to 20 LD ₅₀

Three dosage groups 0.2% of first test CDV gel, 0.5% and 1% with the comparison of virus control group; the dead protective rate of HSV-2 infecting mouse vaginitis is respectively 100%, 100% and 100%; the extending life rate is respectively 137.3%, 137.3% and 137.3%, and dead protective rate and extending life rate all have statistical significance.Positive control drug 3% acyclovir ointment is 90% to the dead protective rate of HSV-2 infecting mouse vaginitis, and the extending life rate is 128.8%, and dead protective rate and extending life rate all have statistical significance.Three dosage groups 0.2% of CDV gel, 0.5% and 1% and the excipient matched group relatively; the dead protective rate of HSV-2 infecting mouse vaginitis is respectively 100%, 100% and 100%; the extending life rate is respectively 154.5%, 154.5% and 154.5%, and dead protective rate and extending life rate all have statistical significance.Positive control drug 3% acyclovir ointment is 90% to the dead protective rate of HSV-2 infecting mouse vaginitis, and the extending life rate is 145.5%, and dead protective rate and extending life rate all have statistical significance.Three dosage groups 0.2%, 0.5% and 2% of this batch of test CDV gel all are better than positive control drug 3% acyclovir ointment, and data result sees Table 14.

Four dosage groups 0.05% of second batch test CDV gel, 0.2%, 0.5% and 1% with the comparison of virus control group; the dead protective rate of HSV-2 infecting mouse vaginitis is respectively 90%, 100%, 100% and 100%; the extending life rate is respectively 131.0%, 141.4%, 141.4% and 141.4%, and dead protective rate and extending life rate all have statistical significance is all arranged.Positive control drug 3% acyclovir ointment is 60% to the dead protective rate of HSV-2 infecting mouse vaginitis, and the extending life rate is 119.0%, and dead protective rate and extending life rate all have statistical significance.Four dosage groups 0.05% of CDV gel, 0.2%, 0.5% and 1% and the excipient matched group relatively; dead protection is respectively 90%, 100%, 100% and 100% to HSV-2 infecting mouse vaginitis; the extending life rate is respectively 123.3%, 133.3%, 133.3% and 133.3%, and dead protective rate and extending life rate all have statistical significance.Positive control drug 3% acyclovir ointment is 90% to the dead protective rate of HSV-2 infecting mouse vaginitis, and the extending life rate is 111.7%, and dead protective rate and extending life rate all have statistical significance.Four dosage groups 0.05%, 0.2%, 0.5% and 1% of this batch of test CDV gel all are better than positive control drug 3% acyclovir ointment.Data result sees Table 14.

Three dosage groups 0.05% of the 3rd batch of test CDV gel, 0.2% and 1% with the comparison of virus control group; the dead protective rate of HSV-2 infecting mouse vaginitis is respectively 20%, 100% and 100%; the extending life rate is respectively 92.5%, 164.2% and 164.2%; the dead protective rate of 0.05% CDV gel is without statistical significance; the extending life rate has statistical significance, and the dead protective rate of 0.2% and 1% CDV gel and extending life rate all have statistical significance.Dead protection is 70% to positive control drug 3% acyclovir ointment to HSV-2 infecting mouse vaginitis, and the extending life rate is 141.5%, and dead protective rate and extending life rate all have statistical significance.Three dosage groups 0.05% of CDV gel, 0.2% and 1% and the excipient matched group relatively; the dead protective rate of HSV-2 infecting mouse vaginitis is respectively 20%, 100% and 100%; the extending life rate is respectively 75.9%, 141.4% and 141.4%; the dead protective rate of 0.05% CDV gel is without statistical significance; the extending life rate has statistical significance, and the dead protective rate of 0.2% and 1% CDV gel and extending life rate all have statistical significance.Positive control drug 3% acyclovir ointment is 70% to the dead protective rate of HSV-2 infecting mouse vaginitis, and the extending life rate is 120.7%, and dead protective rate and extending life rate all have statistical significance.This batch of test positive control drug 3% acyclovir ointment is better than 0.05% CDV gel, and 0.2% and 1% CDV gel all is better than positive control drug 3% acyclovir ointment.Data result sees Table 14.

The therapeutic effect of table 14.CDV gel to the HSV-2333 infecting mouse

Do the check of Kaplan-Meier method, * p＜0.05 with virus control; * p＜0.001

Do X 2 test with virus control, #p＜0.05; ##p＜0.001

2, the inhibitory action of CDV gel of the present invention to HSV-2333 infecting mouse vaginal secretion virus.

HSV-2 infects vaginitis mice local application 3 days (72 hours), gets mouse vagina secretions, is placed in culture fluid, and 10 times of dilutions are measured TCID in the Vero cell ₅₀

First test is compared with the virus control group: 0.05% CDV gel reduces by 213.8 times of mouse vagina virus loads, 0.2% CDV gel reduces the mouse vagina virus load greater than 1778.3 times, 1% CDV gel reduces the mouse vagina virus load greater than 1778.3 times, and positive control drug 3% acyclovir ointment reduces by 63.1 times of mouse vagina virus loads; Compare with the excipient matched group: 0.05% CDV gel reduces by 2630.3 times of mouse vagina virus loads, 0.2% CDV gel reduces the mouse vagina virus load greater than 21877.6 times, 1% CDV gel reduces the mouse vagina virus load greater than 21877.6 times, and positive control drug 3% acyclovir ointment reduces by 776.2 times of mouse vagina virus loads.Three dosage groups 0.05%, 0.2% and 1% of this batch of test CDV gel all are better than positive control drug 3% acyclovir ointment.The results are shown in Table 15.

The second batch test is compared with the virus control group: 0.05% CDV gel reduces by 3162.3 times of mouse vagina virus loads, 0.2% CDV gel reduces the mouse vagina virus load greater than 26302.7 times, 1% CDV gel reduces the mouse vagina virus load greater than 26302.7 times, and positive control drug 3% acyclovir ointment reduces by 100 times of mouse vagina virus loads; Compare with the excipient matched group: 0.05% CDV gel reduces by 416.9 times of mouse vagina virus loads, 0.2% CDV gel reduces the mouse vagina virus load greater than 3467.4 times, 1% CDV gel reduces the mouse vagina virus load greater than 3467.4 times, and positive control drug 3% acyclovir ointment reduces by 13.2 times of mouse vagina virus loads.Three dosage groups 0.05%, 0.2% and 1% of this batch of test CDV gel all are better than positive control drug 3% acyclovir ointment.Three dosage groups 0.05%, 0.2% and 1% of this batch of test CDV gel all are better than positive control drug 3% acyclovir ointment.The results are shown in Table 15.

The effect of table 15.CDV gel to HSV-2333 infecting mouse vaginal secretion virus

7, sum up

Under this experimental condition, 0.2%, 0.5% compares with the excipient matched group with the virus control group and all has statistical significance the dead protective rate of HSV-2 infecting mouse vaginitis, average life day with 1% CDV gel.0.05%, 0.2% and 1% CDV gel all can significantly reduce HSV-2 infecting mouse vagina virus load.

Positive control drug 3% acyclovir ointment reduces to compare with the excipient matched group with the virus control group to the dead protective rate of HSV-2 infecting mouse vaginitis, average life day, virus load and all has statistical significance, and the illustrative experiment model is set up.

8, conclusion and analysis

0.2%, 0.5% with 1% CDV gel, the dead protective rate of HSV-2 infecting mouse vaginitis, average life day are compared with the excipient matched group with the virus control group and all have repeatably statistical significance.The present invention 0.05%, 0.2% and 1% CDV gel all can significantly reduce HSV-2 infecting mouse vagina virus load, all are better than positive control drug 3% acyclovir ointment.

The above is only the preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. vaginal gel, by the active component cidofovir and pharmaceutically applicable carrier form, pharmaceutically applicable carrier comprises carbomer, disodiumedetate, sodium hydroxide glycerol, benzyl alcohol, PEG400, tween 80 and water.

2. vaginal gel according to claim 1, is characterized in that, cidofovir content is 2-30g.

3. vaginal gel according to claim 1, is characterized in that, its agent viscosity is 5～80 (Pas).

4. vaginal gel according to claim 1, is characterized in that, it consists of:

Cidofovir 0.5-50g

Carbomer 5-20g

Disodiumedetate 0.1-0.5g

Sodium hydroxide 3-8g

Glycerol 50-200g

Benzyl alcohol 5-20g

PEG400 50-500g

Tween 80 1-5g

Surplus is water, makes the 1000g gel.

5. vaginal gel according to claim 1, is characterized in that, it consists of:

Cidofovir 2-20g

Carbomer 8-15g

Disodiumedetate 0.2g

Sodium hydroxide 6.0g

Glycerol 100g

Benzyl alcohol 10.0g

PEG400 50-200g

Tween 80 2.0g

Surplus is water, makes the 1000g gel.

6. vaginal gel according to claim 1, is characterized in that, it consists of:

Cidofovir 5-20

Carbomer 8-10g

Disodiumedetate 0.2g

Sodium hydroxide 6.0g

Glycerol 100g

Benzyl alcohol 10.0g

PEG400 100g

Tween 80 2.0g

Surplus is water, makes the 1000g gel.

7. vaginal gel according to claim 1, is characterized in that, it consists of:

Cidofovir 10.0g

Carbomer 10.0g

Disodiumedetate 0.2g

Sodium hydroxide 6.0g

Glycerol 100g

Benzyl alcohol 10.0g

PEG400 100g

Tween 80 2.0g

Surplus is water, makes the 1000g gel.

8. the described vaginal gel of claim 1-7 any one is for the preparation of the application of the medicine for the treatment of vagina human papilloma virus infection.

9. the described vaginal gel of claim 1-7 any one is for the preparation of the application of the medicine for the treatment of intravaginal herpesvirus infection.

10. the preparation method of the described vaginal gel of claim 1-7 any one, is characterized in that, comprises following steps:

Step 1: disodiumedetate, the glycerol of recipe quantity is soluble in water, add recipe quantity carbomer fine powder evenly to be sprinkling upon on solution surface, immersion fully infiltrates carbomer, adds sodium hydroxide solution, stir, obtain pH and transfer to 6-8 carbomer glue;

Step 2: stir in the benzyl alcohol of recipe quantity and tween 80 and the carbomer glue that adds step 1 preparation after PEG400 mixes, add water and fully stir and make mixing, the centrifugal bubble of sloughing, the white gel substrate of having leisure;

Step 3: with the cidofovir rear dilute with water that sieves, add 5% sodium hydroxide solution to make the pH of cidofovir solution transfer to 6-8 again, be added to after the filtering with microporous membrane degerming in the blank gel-type vehicle of step 2 preparation, water complements to 1000g, stir, obtain the cidofovir gel.

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