| Herpes simplex virus is alpha type of herpes virus, it is mainly affects human’sskin,mouth, lips and mucous membranes of genital organs, which is a great threat to human health.HSV has two serum types, namely, herpes simplex virus type I(HSV-1) and herpes simplex virus type II(HSV-2).HSV-2 genital infection is a common sexually transmitted disease,in recent years, some studies have indicated that HSV-2 infection may be related to the occurrence of cervical cancer and other diseases, but also significantly increase the spread of HIV pathogens. Therefore HSV-2 vaginal infection disease has been widely valued.Brincidofovir,also known as CMX001 is a new broad-spectrum anti DNA virus medicine, is a phosphate derivative of cidofovir, have good antiviral effect of herpes virus, adenovirus, smallpox virus,cytomegalovirus,EBvirus, JCvirusand some other DNA virus.Compared with cidofovir, it has the advantages of stronger antiviral effect,lower toxicity, moreroutes of administration and so on. It is a promising antiviral drug.The gel is a new type of preparation developed in recent years, it is refers to the thick liquid latex or semi-solid formulations which is uniform, suspension or emulsion made up with drug and suitable auxiliary materials. The gel is often used in local administration and the one used in the vagina if often Hydrogels.The gelplays a direct role in the treatment of HSV-2 infection in vaginal lesions,it has some characteristics such as easy to use, good compliance, good adhesion,not easy to flow out and so on,which is suitable for vaginal administration.At present, there is no literature report on CMX001 gel at home and abroad.CMX001 gel was studiedon treatment of viral infections of the skin or mucous membranesin this paper, the main contents are as follows:Objective:Prepare the safe,effective,stable,controllable CMX001 gelfor the treatment of viral infections in the skin or mucous membranes through formulation screening and preparation process optimization. The quality and stability was studied and preliminary pharmacodynamic was evaluated after the gel was prepared.Methods:(1) The study of preformulation:Firstly, the physical and chemical properties of CMX001 were studied, including the properties, melting point, solubility and the saturated solubility of CMX001 in different solvents and different p H phosphate buffer solutions.(2)The study of formulation and preparationprocess:Gelpreparationusuallyconsistsofthegelmatrix,humectants,antiosi dants,preservatives,emulsifiers and other excipients can be added in according to the actual situation. Therefore, three steps were used to study the prescription and process.The first step: determine the composition of the gel,the gel matix was screened in the following 4 kinds of hydrogel matrix: natural gum, cellulose derivatives, ethylene polymers and acrylic resin. Select 1-2 representative matrix of each kind above to paticipent the matrix screening, preparedifferent kinds of blank gel matrix,select the appropriate one with appearance, viscosity, Coatability and preparation process as the evaluation index.Then the other components were also screened to determine the composition of the prescription.The second step: to optimize the prescription,with the appearance, viscosity and release rate of the gel as the evaluation index,the orthogonal design was carried out on the basis of the preliminary gel preparationdetermination, and the optimal prescription was selected by the orthogonal design method(3)The study on the quality of CMX001 gel: The study on 3 batches of CMX001 gel including appearance, viscosity, p H value, load difference, identification, related substances, content and release.To test and measure according to the general principles of the 2015 edition of the Chinese Pharmacopoeia,to establish and validate the methodology of the related substances, content and release rate.(4) The study on the stability of CMX001 gel:The content mainly includes gel appearance stability test,influence factor experiment, accelerated stable test and long-term stable test.The study on the appearance of stability is mainly to observe whether CMX001 gel appearance changes in centrifugal, high temperature and low temperature conditions.Influence factor experiment, accelerated stable test and long-term stable test werebased on the 2015 edition of Chinese pharmacopoeia.(5)The study of preliminary pharmacodynamic:In vitro pharmacodynamic studies taking Vero cells as the host to establish the HSV-1 and HSV-2 virus infection model, taking acyclovir as the positive control, to determine the IC50 of CMX001.In vivo pharmacodynamic study taking BALB / c mice infected with HSV-2 as animal model,taking3% acyclovir ointment as positive control drug, to determine the efficacy of 0.1%, 0.2% and 0.5%CMX001 gel.Give a markby observing the appearance of the experimental animals, vaginal infection symptoms and systemic symptoms, the titer of the virus was determined 3 days after infection as well.Evaluateon the therapeutic effect of CMX001 gel cmprehensively.Results:(1) The study of preformulation: The chemical name of CMX001 is P-[[(1s)- 2-(4- amino- 2- oxo- 1(2H)- pyrimidin-2-yl)- 1-(hydroxymethyl) ethoxy] methyl]- [3-(hexadecane oxygen) propyl] phosphate, the product is a white crystalline powder, melting point is 191~194 ℃, the molecular weight is 561.7, themolecular formula is C27H52N3O7 P.CMX001 is soluble in 0.1M sodium hydroxide solution, slightly soluble in methanol, very slightly soluble in ethanol, in ethyl acetate, chloroform, waterand 0.1M hydrochloric acid solution are not dissolved. The saturated solubility of CMX001 in p H4.5, 5.5, 6.5 and 7.5 phosphate buffer was 0.06mg/ml, 0.11 mg/ml, 4.91mg/ml, 24.94 mg/ml respectively.(2)The study of formulation and preparation process:The final formulation and preparation process were determined through the formulation screening, optimization and preparation process research.The preparation process of CMX001gel(total 1000g):weighing propylene glycol 50 g, glycerin 75 g, disodium ethylenediaminetetraacetate 0.2g in 300 ml of water, and mix well to dissolve;weighing carbomer 974 p 10g, sprinkle the powder uniformly dispersed in the mixture liquid surface,close the mouth of the container, swelling 24h;weighing sodium hydroxide 4g into 200 ml water, dissolved and mixed, formulated into 2% solution of sodium hydroxide; weighing CMX001 10 g into2% sodium hydroxide solution,then dissolveand mix well, add it in the mixture slowly with stirring constantly;add in 10 g benzyl alcohol, 2g Tween-80,75 g absolute ethanol and stir well; Addlwater to a total weight of 1000 g.(3)The study on the quality of CMX001 gel:The three batches of gelwere all transparent and homogeneous semi solid gel;p H values were in the range of 6.5-7.5.viscosity were 50-60 Pa ? S;the actuel load of gel were more than 93% inin theory; UV Vis spectrophotometric identification in the wavelength of 275±2nm has the maximum absorption, with minimal absorption at the wavelength of 250±2nm,the peak time of CMX001 gel was the same as the reference using high performance liquid chromatography to identify.The relative substances of the three batches of samples were determined by high performance liquid chromatography, the methodology was established before the determination, membrane adsorption test confirmed membrane on related substances did not affect the determination, the RSD value of sampling precision was 1.78% and the RSD value of repeatability was1.37%, test solution was stable in 24 hours, average rate of recovery and RSD values were 99.97% and 1.35% respectivily, linear equation was y = 6.6886 x- 0.2700(r = 1.0000),there was a good linear relationship between concentration and peak area within the scope of 0.30ug/ml-11.98ug/ml,theyall met the requirements;the content of the three were 0.170%, 0.172%, 0.168%, respectively. The contents of the three batches of samples determined by the same instrument,the methodology was established before the determination, membrane adsorption test confirmed membrane had no effect on Determination of content, the RSD value of sampling precision was 0.27% and the RSD value of repeatability was 0.14%, test solution was stable in 24 hours, average rate of recovery and RSD values were 98.26% and 0.39% respectivily, linear equation wasy = 7370.7486 x + 69.1168(r = 0.9999), there was a good linear relationship between concentration and peak area within the scope of 0.24-0.84mg/ml,theyall met the requirements;the contents of the three batches of samples were 98.61%, 98.70%, 98.35%, respectively.The release rate of three batches of samples were determined by UV visible spectrophotometry. Prior to the determination, used p H7.4 phosphate buffer as the release of liquid, standard curve equation was A=0.013C- 0.001(r = 1), the concentration and absorbance value showed a good linear relationship with in the range of 4μg/ml~80μg/ml, test solution was stable in 72 hours, the RSD value of testing precision was 0.72% and average rate of recovery and RSD values were 100.19% and 0.73%respectively.The release rate of 1h, 6h, 12 h, 24 h and 48 h were about 10%, 31%, 45%, 55%, 65%, respectively.(4) The study on the stability of CMX001 gel:The appearance of the three batches of gel were not changed in centrifugal and high-temperature conditions, but some white agent was separated out at low temperature;there was no significant change in content and related substances of three batches of samples which were placed for 10 days under the conditions of influence factors conditions;the appearance, viscosity, p H value, content, related substances and release of the three batches of samples were not significantly changed in the accelerated and long-term experimental conditionsfor 6 months.(5)The study of preliminary pharmacodynamic:Themedian inhibition concentration(IC50) of CMX001 to HSV-1 and HSV-2 was 0.073μM and 0.070μMrespectively.;inthein vivo test, CMX001 gel preparation was more effective on the treatment against HSV-2 in mice vaginal than the positive control group,among them, 0.5% of the CMX001 gel has the best therapeutic effect.Conclusion: CMX001 gel preparation was prepared,the formulation and preparation process of the gel was reasonable.Quality gels controlled by detecting the appearance, viscosity, load difference, p H value, content, related substances and release are qualified.The properties were stable when the gel was transported and stored for 6 months in the conditions of closed, dark, room temperature,pay attention to antifreeze, avoid contact with strong bases, strong acids, strong oxidizing substances. |
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