| 1 Research background and purposeAlopecia areata(AA)is an inflammatory alopecia disease associated with an autoimmune disorder,which occurs in hair follicle and has a high incidence.At present,there is no standard therapy for alopecia areata,and the most commonly used hair growth-promoting drugs(such as minoxidil)are only effective for mild alopecia areata.Therefore,it is of great clinical value to develop a more effective treatment.JAK(Janus kinase)inhibitors(such as tofacitinib)can inhibit the immune inflammatory response by blocking the signal transduction of various key inflammatory factors in the pathogenesis of alopecia areata,and may be a potential therapeutic drug for alopecia areata.However,the available clinical evidences show that JAK inhibitors are not good at promoting hair growth,leading a long treatment cycle.In addition,the oral medication for long-term will increase the risk of infection.Considering that minoxidil and tofacitinib can act on different targets in the pathogenesis of alopecia areata,the combination of the two may have a synergistic effect and become a new treatment option for patients with moderate and severe alopecia areata.In this project,minoxidil and tofacitinib were combined into a compound preparation for the first time,and the gel was used as a carrier to explore the feasibility of formula and process,and to study the relationship between the composition of formula and drug release or transdermal behaviors.The quality control indicators of gels were initially determined.In alopecia areata model mice,it was investigated whether the combination of drugs had synergistic advantages.And the relationship between prescription ratio and curative effect was also explored.This project provided experimental evidences for the topical treatment of alopecia areata with minoxidil and tofacitinib,and provided a reference for the research and development of compound preparations.2 Methods and results2.1 Pre-studyThe detection methods of content and related substances have been established,which could achieve simultaneous detection for minoxidil and tofacitinib,and were verified by specificity,sensitivity and accuracy to meet the detection requirements.The release and transdermal test methods in vitro were established.The franz diffusion cell was used as the test device,and normal saline was selected as the receiving medium,which met the requirements of sink conditions and solution stability.The stability of minoxidil and tofacitinib under oxygen,acid,alkali,high temperature and other conditions was studied.It was found that acidic conditions could promote the degradation of minoxidil and tofacitinib.Tofacitinib was less stable under alkaline conditions,while minoxidil was stable under alkaline conditions but sensitive to oxygen.In the presence of oxygen,the color of the mixed solution of minoxidil and tofacitinib was darker than that of the unilateral solution.The above results showed that antioxidant selection,pH control system,temperature,etc.were the key factors in the formulation and process.2.2 Formulation and process studyThe main components of gels include active ingredients,solvent,gel matrix,antioxidant,and penetration enhancer,etc.By taking the dissolution and crystallization,formulation appearance,impurity growth,skin penetration and retention of APIs as the key indicators,it found that using the propylene glycol-water solution with a ratio of 7:3 as the mixed solvent,the gel could be kept without crystallization for a long time.With2.5% of CMC-Na as the matrix,the gel had a good formability and appearance,as well as a strong anti-deformation ability.With 0.1% of BHT as antioxidant,impurity growth could be effectively controlled.With 2.5% of L-menthol as penetration enhancer,the skin penetrations and retentions of minoxidil in gels were close to those of commercial formulation.On the basis that the loading of minoxidil was determined to be 2%,the stability of the preparation was good when the loading of tofacitinib was between 0.1% and 1.5%.Under the above conditions,the pH values of gels were in the range of 5.5 to 6.5,and decreased with the increase of tofacitinib loading.The gel formulation was determined based on the above results.Pre-swelling CMC-Na could save preparation time and avoid agglomeration.The identified technological process was short,and the preparation process had no obvious influence on the stability of the medicine.2.3 Quality and stability studyThe important indicators of gel quality include appearance,pH value,rheological properties,content and related substances,etc.,which reflect the external and internal properties and are the premise to ensure the safety and effectiveness of the preparation.According to the established formula and process,the gels were prepared for quality researches,and it was found that gels were colorless,clear and transparent,and easy to apply.The pH values were affected by the composition of formula.In the range of 5.5 to 6.5,the drug maintained a dissolved state in gels with no crystals precipitated,showing a good stability.The rheological properties of gels were related to the type and stength of the matrix,as well as temperature.The prepared gel was a plastic fluid based on 2.5% CMC-Na,which conformed to the Herschel-Bulkley model for the rheological curve,with good thixotropy and deformation resistance.The viscosity and modulus of the gel both decreased with the increase of temperature.According to the established content and related substances detection methods,the drug contents were 95% ~ 105% of the labeled amounts,and the total impurities were less than 1.5%.The above indicators were closely related to formula and process factors.The release and transdermal behaviors of drugs reflected the comprehensive performance of formula and directly affected the efficacy.The release profiles of APIs in gels were consistent with Higuchi equation.In different proportions,the release behaviors of minoxidil were consistent,and the release rates of tofacitinib were positively correlated with its strength.The transdermal law of drugs was the same as the law of release.The appearance,spreadability and rheological properties of gels did not change significantly after low temperature and freeze-thaw cycles.After being placed at 40 ℃/75% RH for 3 months,gels were colorless to light yellow,clear and transparent.The drug contents were 95% ~ 105% of the labeled amounts,and the total impurities were less than 1.5%.It showed a good stability.2.4 Efficacy exploration on alopecia areataThe animal models of alopecia areata were successfully established in C3H/HeN mice induced by imiquimod cream.After modeling,the weights of mice decreased significantly,and patchy alopecia areata areas were formed,with the decreased number of new hair follicles in the superficial dermis.There were seven groups in test,namely: blank gel group,minoxidil group 1(2%),minoxidil group 2(5%),tofacitinib group(0.5%),compound group 1(2%: 0.1%),compound group 2(2%: 0.5%),compound group 3(2%: 1.5%).According to different groups,drugs were administered at a frequency of 2 times a day,and the differences in drug effects were compared simultaneously with multiple indicators such as appearance,pathology,and biochemistry.The results showed that with administration for three weeks,the weights of mice in both minoxidil and compound groups had increased significantly.It was better for compound groups in promoting hair growth rate,promoting hair follicle regeneration and inhibiting inflammatory factors than the single drug groups with same strength,while had similar hair follicle regeneration effect,better IFN-γ/IL-4 inhibiting ability,and faster hair growth rate,compared with the high-strength minoxidil group,which showed that minoxidil and tofacitinib had synergistic advantages.Under the same loading of minoxidil,the compound groups with tofacitinib loadings of 0.5% and1.5% were more effective than the 0.1% compound group,but there was no significant difference between the two high drug loading compound groups.Based on safety considerations,2%:0.5% was selected as the ratio of minoxidil and tofacitinib.3 ConclusionThe minoxidil-tofacitinib gels prepared in this research showed good stability with reasonable prescription,feasible process,and strong drug loading ability,of which the rheological properties could meet the requirements of external application and storage.The gels were confirmed to be useful in the treatment of alopecia areata in C3H/HeN mice induced by imiquimod,and superior to single minoxidil and tofacitinib in promoting hair growth rate,promoting hair follicle regeneration and inhibiting inflammatory factors.After comprehensive consideration,2%:0.5% was selected as the ratio of minoxidil and tofacitinib.This subject provided key experimental evidences for the topical treatment of alopecia areata with minoxidil and tofacitinib,and also provided technical experience for the preparation and production of compound preparations.45 figures,42 tables,and 54 references... |
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