摘要
目的：本文以水难溶性药物美洛昔康为模型药物，为避免口服给药引起的对胃肠道的刺激，减少系统毒性，分别制备美洛昔康凝胶剂和微乳经皮给药制剂。考察美洛昔康凝胶在大鼠、比格犬和人体内的药动学及局部组织分布，一方面验证美洛昔康在靶组织的富集程度及药物进入全身血液循环的量，另一方面研究该药物的经皮渗透机制。
方法：在处方前研究中，采用高效液相色谱法(HPLC)建立了美洛昔康的体外分析方法；测定了药物的溶解度及油水分配系数；为满足漏槽条件对接收介质进行选择；考察了不同促渗剂的促渗作用，筛选出最佳的促渗剂。在处方前研究的基础上，以Carbopol 940作为凝胶基质，在单因素考察的基础上，利用正交设计对凝胶剂的处方进行优化。通过假三元相图和体外经皮渗透实验考察各因素对微乳形成的影响，以药物在微乳中的溶解度和经皮渗透速率为指标，从而筛选出最优处方。以微乳的电导、粘度、粒径等物理化学性质为指标，对微乳的结构进行推测，并对微乳的物理化学性质与其透皮速率之间的相关性进行了评价。
以扶他林(双氯芬酸软膏)为阳性对照药，采用醋酸致小鼠扭体实验、二甲苯致小鼠耳肿胀实验、蛋清致大鼠足跖肿胀实验及棉球致大鼠肉芽肿实验，分别对美洛昔康凝胶剂和微乳剂外用的抗炎作用和炎性镇痛作用进行药效学研究。
采用高效液相色谱法和液相色谱-串联质谱法(LC／MS／MS)测定美洛昔康凝胶剂经皮用药后大鼠、犬和人体内的血浆、肌肉和滑液等组织中的药物浓度，进而探讨美洛昔康经皮给药后的药动学行为和经皮渗透机制。
结果：在处方前研究中，测得美洛昔康溶解度具有pH依赖性，当pH＞7.4时，溶解度迅速增加。以蒸馏水为水相时，美洛昔康的油水分配系数为1.84±0.10。体外经皮渗透实验选择20％乙醇-pH7.4磷酸盐缓冲液作为接收介质。促渗剂选择为卡必醇(Transcutol)。
美洛昔康凝胶剂的处方确定为1.0％的卡波姆(Carbopol)、20％的丙二醇(PG)、10％的Transcutol。稳定性实验结果表明高温对凝胶剂的含量影响较大，凝胶剂应常温密封保存。
美洛昔康微乳透皮给药传递系统，最终处方为美洛昔康0.375％，肉豆蔻酸异丙酯(IPM)5％，Tween 85／乙醇(1:1)50％和水，计算得到其经皮渗透速率为5.40μg／cm~2／h。测得微乳的粒径为36.9 nm，由透射电镜形态学考察显示微乳乳滴为圆球形，外观圆整。核磁共振波谱(NMR)法确证表明药物分子结构中的羟基易与表面活性剂聚氧乙烯链上的氧结合成氢键。
美洛昔康凝胶剂和微乳剂局部外用具有良好的炎性镇痛效应，可以控制急性炎症并能降低炎症晚期的增生性反应。
大鼠分别灌胃和经皮给予7.5 mg／kg美洛昔康后，经皮给药血浆中药物浓度远低于灌胃给药，AUC_(0-t)值分别为54.9μg·h／mL和480.1μg·h／mL；肌肉中药物浓度相当，经皮给药的AUC_(0-t)值(25.2μg·h／mL)略高于灌胃给药的AUC_(0-t)值(17.2μg·h／mL)。说明经皮给药血药浓度低，靶部位药物浓度高，从而减少了高血药浓度所导致的系统毒副作用。
犬分别经皮和口服给予1.25mg／kg和0.31 mg／kg美洛昔康后，结果显示经皮和口服给药的C_(max)分别为22±9 ng／mL和780±136 ng／mL，AUC_(0-t)分别1.00±0.43μg·h／mL和23.87±8.26μg·h／mL；12 h经皮给药的关节滑液与血药浓度比值(7.68)远远的高于口服给药的滑液与血药浓度的比值(0.87)，表明经皮给药后药物有在滑液浓集的特点；给药部位滑液的药物浓度(C_(max)为167.36±60.17)远高于未给药部位滑液的浓度(C_(max)为20.60±12.34)，说明经皮给药部位滑液中的药物除了少量由血液循环再分布外，主要是由于药物经皮直接渗透进入滑液的结果。
美洛昔康分别以口服(7.5 mg)和经皮(30 mg)给予10名健康受试者，计算得到美洛昔康凝胶经皮给药后的AUC_(0-t)仅为口服给药的1.02％，表明药物进入系统循环的量很小。经皮给药后的t_(1／2)(60.4±47.7 h)延长，大约是口服给药t_(1／2)(27.9±11.3h)的2.16倍，这是因为皮肤起到了储库的作用，药物缓慢地渗透到靶组织。每位患者分别口服和经皮给予7.5 mg和15 mg的美洛昔康，结果表明达峰时刻的滑液与血浆中药物浓度的比分别为0.68和9.6，经皮给药后药物有在滑液浓集的特征。
结论：制备了美洛昔康凝胶和美洛昔康微乳两种经皮给药制剂，建立的HPLC和LC／MS／MS法快速、灵敏、可靠，可以应用于美洛昔康在动物和人体内的药动学研究。美洛昔康在大鼠、比格犬和人体内的血浆及肌肉、滑液等局部组织的药物浓度测定结果表明，该药经皮给药进入系统循环的量少，靶组织的浓度相对较高。美洛昔康局部给药的经皮渗透机制主要是通过直接渗透进入皮下靶组织，而血液循环再分布是次要的。为美洛昔康经皮给药新剂型的开发和临床应用提供了科学依据。
OBJECTIVES: Poorly water-soluble meloxicam (Mel) was considered as a model drug in this paper. In order to avoid gastrointestinal irritation and reduce systemic toxicity via oral administration, meloxicam gels and microemulsion transdermal system were prepared, respectively. The pharmacokinetics and topical tissues distribution of meloxicam gels in rats, beagle dogs and human were investigated. On one hand, the degree of meloxicam enriched in target tissues and the amount of meloxicam entered into the circulation of blood were investigated. On the other hand, the transdermal permeation mechanisms were studied in detail.
METHODS: During the preformulation study, HPLC method was established for analysis of meloxicam in vitro. The solubility and oil/water partition coefficient of meloxicam were determined. The receptor medium was chosen to maintain the sink condition. Different permeation enhancers were investigated to screen the optimal permeation enhancers of meloxicam. From the result of preformulation, Carbopol 940 was decided to act as the gel base. The orthogonal experiment was applied to optimize the formulation of Mel gel by single factor experiments. The effects of various factors on the formation of microemulsion were investigated through pseudo-ternary phase diagrams and transdermal permeation rate of meloxicam. The optimal formulation was screened using the solubility of meloxicam in microemulsion and and transdermal permeation rate as index. The structures of microemulsion were presumed by electric conductance, viscosity, particle size as main evaluating indexes, and the correlation between the physicochemical properties and transdermal permeation rate of meloxicam microemulsion was evaluated.
The acetic acid-induced body writhing test in mice, the dimethylbenxene-induced ear edema test in mice, the ovalbumin-induced rat hind paw edema and the cotton-induced granuloma test in rat were carried out to investigate the analgetic and anti-inflammatory effects of both meloxicam microemulsion and meloxicam gel with the votalin (diclofenac ointment) as positive control.
High-performance liquid chromatography (HPLC) method and high-performance liquid chromatography-tandem mass spectrometric (LC/MS/MS) method were applied for the quantification of meloxicam in plasma, muscle and synovial fluid in rats, dogs and human after transdermal or oral administration of meloxicam in order to study the pharmacokinetic behavior and skin permeation mechanism.
RESULTS: During the preformulation study, the solubility of meloxicam was pH-dependent and the solubility increased quickly when pH was higher than 7.4. The oil/water partition coefficient of meloxicam was 1.84±0.10 when distilled water was used as aqueous phase. 20% Ethanol- phosphate buffer (pH 7.4) was chosen as the receptor medium in transdermal permeation experiments and transcutol was chosen as penetration enhancer.
The preparation of meloxicam gel was 1.0% Carbopol, 20% propylene glycol and 10% transcutol. The results of stability experiments showed that high temperature had great effect on the content of Meloxicam in gel, so gel should be enclosed and stored at common temperature.
The final formulation of meloxicam microemulsion was obtained, consisting of 0.375% meloxicam, 5% IPM, 50% Tween 85/Ethanol (1:1) and water and the transdermal permeation rate was 5.40μg/cm~2/h. The droplet size of microemulsion was 36.9 nm and phase transition temperature was 90℃. The morphology of microemulsion by transmission electron microscope was globe and regular. NMR indicated that hydroxy group of meloxicam was easy to bind with oxygen of polyoxyethylene of surfactant and form hydrogen bond.
Local administration of either meloxicam gel or microemusion could inhibit both acute inflammation and the proliferation reaction in the late period of inflammation.
After meloxicam was administered to rats at a dose of 7.5 mg/kg by intragastric and transdermal administration, respectively, the results showed that drug concentration in blood was very low after transdermal administration. AUC_(0-t) was 54.9μg·h/mL and 480.1μg·h/mL, respectively. Drug concentration in muscle was similar, and AUC_(0-t) (25.2μg·h/mL) in muscle after transdermal administration was a little higher than that of oral administration (17.2μg·h/mL). It indicated that transdermal administration could produce low plasma concentration and relatively high concentration in target tissues, which reduced the system toxicant and side effects.
After meloxicam was administered to dogs at a dose of 1.25 mg/kg and 0.31 mg/kg by transdermal and oral administration, the results showed that maximum concentration in plasma was 22±9 ng/mL and 780±136 ng/mL, AUC_(0-t) was 1.00±0.43μg·h/mL and 23.87±8.26μg·h/mL, respectively.
The concentration ratio of synovial fluid to plasma (7.68) at 12 h after transdermal administration was much higher than that after oral administration (0.87), which indicated that Meloxicam could concentrate in synovial fluid after transdermal administration. The drug concentration in synovial fluid from the treated side (C_(max)= 167.36±60.17) was higher than that (C_(max)= 20.60±12.34) from the untreated side, which indicated that the drug in the treated site was mainly reached by direct penetration into synovial fluid besides small amounts which was redistributied by systemic circulation, thereby transdermal penetration mechanism was illuminated.
After meloxicam was administered to 10 health volunteers at a dose of 30 mg/kg and 7.5 mg/kg by transdermal and oral administration, respectively, the results showed AUC_(0-t) after transdermal administration was only 1.02% of that after oral administration, which indicated that the amount entered into systemic circulation was very small. Drug concentration in blood was very low after transdermal administration. The long half-life (t_(1/2), 60.4 h) after transdermal administration was 2.16 times as long as that after oral administration (27.9 h). This was because skin acted as a storeroom and drug penetrated slowly into target tissues. After meloxicam was administered to each patient at a dose of 7.5 mg and 15 mg/kg by oral and transdermal administration, respectively, the results showed the concentration ratio of synovial fluid to plasma at peak moment was 0.68 and 9.6, respectively, and drug could concentrate in synovial fluid by transdermal administration.
CONCLUSION: In this study, meloxicam gel and meloxicam microemulsion of transdermal delivery were prepared. Fast, sensitive and robust HPLC and LC/MS/MS methods were developed, and successfully applied to the pharmacokinetic study of meloxicam after an oral or transdermal administration. The drug concentration of meloxicam in plasma, muscle and synovial fluid of rats, dogs and humans were investigated, the results showed that transdermal administration could produce low plasma concentration and high concentration in target tissues. Moreover, a transdermal permeation mechanisms of meloxicam after transdermal administration were proposed. It can be concluded that the drug in target tissue was mainly by direct penetration, while redistribution by systemic circulation was minor. Reliable scientific basis was provided for the new transdermal dosage form development and clinical application of meloxicam.
引文
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