| Objective: CA was used as model drug to do research of external dermal drug praeparatum——ointment. CA is immunosuppression, whose oral administration is for treating rejection, atopic dermatitis and so on. The large dosage of oral administration can easily cause untoward effects, so CA is made topical external preparation to treat atopic dermatitis. As permeability barrier, skin tissue can avoid the adsorption of CA to the systemic circulation, so CA could be delivered directly to the focus of infection, enhance therapeutic effect of atopic dermatitis and reduce untoward effects. How CA can be effectually absorbed by skin, and less or not enter systemic circulation is the key of making topical external preparation.MethodsOintment content method: The RP-HPLC was developed to determine ointment content. Absolute recovery, relative recovery, precision and sensitivity were investigated.Optical condition of preparing: Basing on single factor trial results, Fusion method was used to make ointment whose inner phase liquid droplet diameter, relative material content and uniformity was used as evaluation indexes. Design 3 factors and 3 levels orthgonal experiment to investigate the best preparing condition, and factors and levels liked that:Factor A was water-bathed temperature—60, 70 and 80℃;Factor B was rotation time—5, 10 and 15 min;Factor C was rotation speed—4000, 7000 and 10000 rpm.Selection of penetration enhancer: In condition of 40℃and RH75%, accelerative test of ointment was conducted with 5% different penetration enhancers . Drug content was determined on the 0, 15th, 30th day in order to investigate the stability of drug in each penetration enhancer.According to"cutaneous irritation reaction value"standard score and"cutaneous irritation intensity value", single and multiple irritant test on rabbit was conducted to select penetration enhancer among the primarily—selected formulas. Compared with imported ointment, franz-type diffusion cell system was used to monitor the cumulative release amounts from ointment of in vitro permeation test by RP-HPLC.Cumulative release and permeation rate was calculated in in vitro permeation test with nylon membrane and mice's skin as permeability barriers, respectively. Permeation activity of ointments was investigated which had the same viscosity with a range of penetration enhancer content. It should be fitted equations and determined optimal penetration enhancer content.Ointment quality and stability test: The drug content and uniformity was determined. Appearance and drug content were investigated in stress test, accelerative test, long—term test and the physical stability by eye-observing. The chemical stability of ointment was explored by HPLC.Acute toxicity test: As trial animal, albinos guinea-pigs were painted large dose sample—conducting acute toxicity test on the skin. According to weight and sex, animals were divided into 3 groups, randomly: drug group, intact base group, blank group to investigate self-made ointment and intact base's acute toxicity to animals'skin, respectively. The weight, breath, hairs, limbs activity, eyes, MM, CNS and record death ratio of each group animals were observed.Sensitization test: As trial animal, albino guinea-pigs were divided into 3 groups, randomly according to weight abserved, drug group, intact base group and control group. DNCB acetone solution was used to paint positive control group animals', 7% solution was for sensitizing and 0.5% for irritating. 7% solution was painted on the first, 7th, 14th day, and 0.5% solution was painted on the 28th day. Sensitization phenomenon was observed on animals in positive control group. Operations on animals in drug group and intact base group were the same as that mentioned above. Sensitization effect and sensitization ratio by comparing those two groups with group positive control were observed.Pharmacodynamics: Albino guinea-pigs were used as trial animal to set up atopic dermatitis model. DNCB was used to treat animal's skin so as to sensitize, irritate and encrust. Finally, atopic dermatitis was get induced. According to weight and sex and crust area, animals were divided into 4 groups, randomly: imported ointment (A), self—made ointment(B), intact base(C) and blank(D). Animals of group ABC were painted twice daily, once per 12h, which lasted 12d. Dermatitis state and measure area and evaluate the therapeutic effect of self-made ointment were observed.ResultsOintment content method: The standard curve of high performance liquid phase assay to determine ointment content: A=12336C-7837.9 (r=0.9999, n=7). The recovery of low, middle and high concentration: 100.2, 100.4, 99.67%. The RSD of inter-day and intra-day: 1.5, 1.4, 2.1% and 2.4, 1.9, 2.3%.Preparing Condition: The results of orthogonal experiment showed that the optical preparing condition was: water-bathed temperature was 70℃, rotation time was 10 min, rotation speed was 10000rpm.Penetration enhancer selection: In condition of 40℃and RH75%, CA content of ointment 1, 3 and 4 were more stable than CA content of others decreased sharp. The stimulus reaction of penetration enhancer 1 was slightly, penetration enhancer 3 and 4 had no stimulus effect. A RP-HPLC method was set up to investigate CA concentration in receptor medium of the release experiment. The standard curve: A=13.479C-373.4 (r=o.9998, n=7); the recovery of low, middle, high concentration: 104.6, 101.1, 98.42%; The RSD of inter-day and intra-day: 4.0, 1.4, 1.8% and 4.5, 1.9, 2.0%.The cumulative release profile in vitro could be fit in Higuch equation by doing permeation test in vitro which was used to select penetration with nylon membrane and mice's skin as permeability barriers.. The release tendency of ointment 3 and ointment 4 was the same that the penetration rate decreased as the penetration enhancer concentration increased, respectively. Imported ointment was used as control article to select the formulation. Then 7.7% ointment and 10.9% ointment 4 had been chosen with middle viscosity. According to cutaneous irritation test, the optimal formula was ointment 3.Ointment quality and stability test: Prepare 3 batches of formula ointment 3, average CA content is 0.996mg/g, uniformity′s RSD were all below 3% with RH 92.5%, CA content decrease sharp. With 60℃, CA content decreased sharp, the relative substance was increased rapidly and ointment became soft and liquidize turbidly. With 40℃, CA content slightly changed on the 5th day, and decreased on the 10th day, ointment was soft. With high light 4500±500lx, CA content decreased sharp. Long term test with 25℃,RH 60%, the results of 0, 3, 6, 9month indicated CA content in ointment was stable.Acute toxicity test: After removing ointment, animals were observed for 7days successively: animals'weight increased normally and the breath, hairs, limbs activity, eyes membrane mucosa and CNS were normal and there was no death.Sensitization test: By sensitization and irritation of DNCB, animals in group positive control all reacted positively on the 28th day whose sensitized skin showed purple erythema which did not dismiss after 72h. Skins of animals in group drug and group blank base were normal and maintain negative after 72h, which indicated that ointments had no sensitization.Pharmacodynamics: The atopical dermatitis model was induced by DNCB. The inflammation skin showed cardinal red crust and the result indicated that the concrete speed of animals in imported-ointment group was faster than animals′in the other group after 12 days painting. The residue area ratio of group drug and group imported ointment were 0 and 2%, and the residue area of animals in group intact base and group blank are 46% and 44%, respectively. The effect on animals had no difference between imported-ointment group and self-made-ointment group. The effect on animals had significant difference between intact-base group and imported-ointment group. The effect on animals had significant difference between blank group and imported-ointment group.Conclusion: All of the tests indicated that self-made 0.1% CA ointment had good permeation effect and stability, and had no cutaneous irritation, sensitization, acute toxicity, and was consistent with the requirement of topically external ointment. It had no significant difference on therapuetic effect of atopic dermatitis—compared with marketed imported CA ointment, and the results had practical reference meaning.
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