酮洛芬异丙酯在皮肤细胞中的代谢

目的：研究酮洛芬异丙酯在皮肤细胞中的代谢作用，为进一步研究利用酯类前体药物方法改善药物的经皮吸收特性提供实验依据。方法：将人包皮的第3代角质形成细胞或成纤维细胞超声破碎制成匀浆，加入不同量的酮洛芬异丙酯进行37℃温孵实验，通过高效液以谱法分别在不同时间测定细胞匀浆中酮洛芬异丙酯及酮洛芬的浓度。结果：酮洛芬异丙酯在表皮角质形成细胞和真皮成纤维细胞的匀浆中存在代谢现象，有前者的代谢能力明显大于后者。结论：酯类前体药物可在皮肤细胞中被代谢为活性母体药物本身。     

酮洛芬异丙酯在离体裸小鼠和猴皮肤中的渗透和代谢

目的　研究酮洛芬异丙酯在离体裸小鼠和猴皮肤中的渗透和代谢 ,阐明皮肤酯酶代谢的主要活性部位。方法　采用外科手术和酶消化方法制备离体皮肤 ,用水平双室扩散池进行体外渗透实验 ,HPLC方法测定样品中的药物浓度。结果　经全皮和去角质层皮肤渗透的酮洛芬异丙酯被代谢成酮洛芬 ,接受室中酮洛芬的浓度随时间延长而成线性增加。在裸小鼠皮肤中 ,酮洛芬全皮渗透的稳态速率是酮洛芬异丙酯全皮渗透的 2 5倍 ,而渗透实验结束后酮洛芬异丙酯及代谢物酮洛芬在皮肤中残留量是酮洛芬渗透实验结束后残留量的 2 2 2倍 ;酮洛芬异丙酯经真皮渗透时 ,代谢物酮洛芬的稳态形成速率大大小于经全皮渗透时的稳态形成速率。在猴皮肤中 ,酮洛芬异丙酯经全皮渗透时代谢物酮洛芬的稳态形成速率是经去角质层皮肤的 0 7倍 ,而渗透实验结束后皮肤中酮洛芬和酮洛芬异丙酯的残留量是去角质层皮肤的 2 0倍 ;酮洛芬异丙酯经真皮渗透时 ,代谢物酮洛芬的稳态形成速率小于经全皮和去角质层皮肤渗透时的稳态形成速率 ,渗透结束后皮肤中酮洛芬的残留量也小。结论　酮洛芬酯前体药物在皮肤原位能被代谢成活性母体酮洛芬本身 ,且在皮肤中能长时间驻留 ,有助于提高和延长酮洛芬的局部疗效。表皮层是皮肤酯酶代谢的主要活性部位     

组织工程皮肤替代物构建的实验研究

目的探索组织工程皮肤替代物的构建方法。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质,采用气—液界面方式进行培养,用HE染色观察培养物的结构。结果培养的组织工程皮肤替代物具有正常皮肤的形态结构,具有结构致密的真皮和分化良好的表皮。结论本方法可用于活性皮肤替代物的制备。     

体外构建新型化妆品检测用皮肤Inspectskin Ⅰ的初步研究

目的 探讨ES源表皮干细胞(ESC)与高分子材料构建人工皮肤Inspectskin Ⅰ的方法,为化妆品皮肤毒性检验替代模型奠定基础.方法 采用嵌入式培养法,先将3-羟基丁酸-co-3-羟基戊酸共聚物(PHBV)支架与原代小鼠成纤维细胞共培养5 d构建真皮,再于表面放置ESC细胞,气-液界面培养7～10 d,形态学和β1整合素、CK10、CK19免疫组化观察.结果成纤维细胞可在真皮支架内粘附,ESC细胞可在支架表面增殖分化形成含基底层、棘层和角化层的复层表皮结构,免疫组化表明基底层以上细胞中表达CK10明显增多,基底层有些细胞仍呈β1整合素和CK19阳性.结论 Inspectskin Ⅰ替代皮肤模型是由ESC细胞分化形成的角化复层表皮结构和PHBV真皮结构共同构成的全层皮肤模型.     

小型香猪复方壳多糖组织工程皮肤的构建及组织学特点

目的评价制备的小型香猪复方壳多糖组织工程皮肤在组织学方面的特性,为其修复皮肤缺损创面提供实验依据。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质。采用气—液界面方式进行培养,通过苏木精—伊红(HE)染色、比较小型香猪的体外构建组织工程皮肤与正常皮肤的组织学特征,包括表皮、真皮结构。结果制备的复方壳多糖组织工程皮肤表皮细胞增殖活跃,分层分化良好,厚约150μm,真皮层细胞生长正常,排列有序,与机体皮肤结构相似。结论复方壳多糖组织工程皮肤组织结构良好,符合新型皮肤替代物在治疗皮肤缺损时的组织学要求。     

P物质受体在人表皮角质形成细胞和真皮成纤维细胞中的表达调控

目的考察P物质受体(Neurokinin1R,NK1R)在正常人表皮角质形成细胞和真皮成纤维细胞中的表达调控特征。方法培养人表皮角质形成细胞株HaCaT细胞和真皮成纤维细胞,应用免疫组织化学法检测NK1R在两种细胞中的表达;采用RTPCR方法检测NK1R在两种细胞mRNA水平的表达特征,并采用流式细胞术定量检测在不同刺激因素及药物作用下两种细胞中NK1R的表达调控情况。结果NK1R在HaCaT细胞及真皮成纤维细胞中均有表达,阳性部位位于细胞膜及细胞质。NK1RmRNA在HaCaT细胞上的表达水平要高于在真皮成纤维细胞上的表达。P物质和IFNγ可以上调NK1R在两种细胞上的表达,而LPS抑制了NK1R的表达;抗组胺药盐酸西替利嗪和P物质受体特异性拮抗剂SpantideI可以降低两种细胞上NK1R的表达。结论皮肤角质形成细胞和真皮成纤维细胞在细胞、蛋白及mRNA水平均有NK1R的表达,而且这种表达可被过敏炎症因子调控,提示角质形成细胞和真皮成纤维细胞参与了皮肤免疫调控,神经肽P物质可能在皮肤过敏性炎症中起重要作用。     

盐酸西替利嗪对表皮角质形成细胞和真皮成纤维细胞P物质受体和细胞因子表达的影响

研究抗组胺药盐酸西替利嗪对表皮角质形成细胞系HaCaT细胞和真皮成纤维细胞P物质受体表达以及对P物质诱导两种细胞表达IFN-γ、IL-1β、IL-6及IL-8的影响。采用流式细胞术和Western blotting检测分析西替利嗪对两种皮肤细胞P物质受体表达的影响；以P物质刺激HaCaT细胞和真皮成纤维细胞，加入不同剂量的西替利嗪共孵育，采用ELISA方法测定西替利嗪对IFN-γ、IL-1β、IL-6及IL-8分泌的影响。结果表明，西替利嗪显著抑制HaCaT细胞和真皮成纤维细胞P物质受体的表达；P物质刺激可以显著增加HaCaT细胞和真皮成纤维细胞IFN-γ、IL-1β、IL-8的分泌量；1~100 μmol·L^-1的西替利嗪均可抑制皮肤细胞IL-1β、IL-8的分泌，但对IFN-γ的分泌无明显影响。P物质和西替利嗪对两种皮肤细胞IL-6的产生均无显著影响。     

在体猪耳静脉灌流经皮吸收模型的建立与应用

目的建立在体猪耳静脉灌流经皮吸收模型，为经皮吸收制剂研究提供新方法。方法建立在体猪耳静脉灌流经皮吸收模型。以葡萄糖利用试验及乳酸脱氢酶活性检测评价模型的生物学活性，以酮洛芬异丙酯和水杨酸甲酯为模型药物考察系统的应用。结果葡萄糖利用及乳酸脱氢酶活性检测表明系统7 h内保持良好生物学活性。酮洛芬异丙酯经皮渗透过程中被完全代谢为酮洛芬，稳态时酮洛芬累积形成量Q与时间t回归的方程为Q=-0.024+0.120t，形成速率为0.120　μg·cm^-2·h^-1。水杨酸甲酯经皮渗透过程中部分被代谢为水杨酸，稳态时水杨酸甲酯累积渗透量Q与时间t回归的方程为Q=-3.809+6.129　t，渗透速率为6.129　μg·cm^-2·h^-1；水杨酸累积形成量Q与时间t回归的方程为Q=-1.785+0.879　t，形成速率为0.879　μg·cm^-2·h^-1。结论该模型操作简便、价格经济，不仅可以考察药物的经皮吸收，而且能够用于研究药物的皮肤代谢。     

皮肤羧酸酯酶代谢的立体选择性

目的研究人皮肤羧酸酯酶代谢的立体选择性，为利用前体药物方法促进透皮吸收提供分子生物学基础。方法以酮洛芬乙酯为模型药物，利用皮肤匀浆代谢方法考察皮肤羧酸酯酶代谢的立体选择性。以人肝脏L02细胞株中羧酸酯酶的表达为对照，采用RT-PCR方法从mRNA水平研究皮肤组织及其细胞中羧酸酯酶的表达。结果 酮洛芬乙酯在人皮肤匀浆中的代谢产物以R-酮洛芬为主。人羧酸酯酶-2(hCE-2)在皮肤组织及其细胞中都有很强的表达，而人羧酸酯酶-1(hCE-1)在皮肤组织及其细胞中的表达量很小或不表达。结论皮肤表达的hCE-2是透皮吸收常用酯类前体药物的主要水解酶，其对手性酯类前体药物的水解具有立体选择性。     

西替利嗪对皮肤细胞炎症中单核趋化蛋白-1表达的干预作用

目的考察西替利嗪(CET)对皮肤细胞炎症模型中单核趋化蛋白-1(MCP-1)表达的干预作用。方法组胺(HIS)和IFN-γ刺激真皮成纤维细胞(DF)和人角质形成细胞株HaCaT细胞，采用RT-PCR和ELISA法考察两种细胞MCP-1 mRNA和蛋白表达水平。结果与对照组比较，HIS(10 μmol·L^-1)和IFN-γ(20 ng·mL^-1)组显著上调两种细胞(DF和HaCaT) MCP-1 mRNA表达，同时分别使DF的MCP-1蛋白分泌量增加3.5倍和8.4倍，对HaCaT细胞也有相似的影响趋势； CET (1和10 μmol·L^-1) 显著地抑制了它们对细胞MCP-1蛋白产生的增强作用。结论CET可能通过抑制MCP-1的表达而发挥抗皮肤炎症作用。     

Air-liquid interface culture of serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies

The objective of this study was to establish a drug transport study using human nasal epithelial (HNE) cell monolayers cultured by the air-liquid interface (ALI) method using serum-free medium (BEGM:DME/F12, 50:50). The cells were developed and characterized in comparison to those that have been previously cultured by the liquid-covered culture (LCC) method. The epithelial cell monolayer cultured by the ALI method resulted in a significantly higher transepithelial electrical resistance value (3,453 +/- 302 ohm x cm(2)) that was maintained (>1,000 ohm x cm(2)) for up to 20 days compared with that cultured by the LCC method. Observation by scanning electron microscopy revealed mature cilia after 2 weeks in the ALI culture, while flatten unhealthy ciliated cells were observed in the LCC method. After 21 days, higher level of MUC5AC and 8 mRNA were expressed in ALI culture which confirmed the secretory differentiation of HNE monolayers in vitro. No significant difference in the permeability coefficients of a model hydrophilic marker ((14)C-mannitol) and a lipophilic drug (budesonide) was observed between the two conditions on day 7. The passage 2-3 of the HNE monolayer using ALI condition retained the morphology and differentiated features of normal epithelium. Thus it would be a suitable model for in vitro nasal drug delivery studies.     

Metabolism of verapamil in cultures of rat alveolar epithelial cells and pharmacokinetics after administration by intravenous and inhalation routes.

Administration of therapeutic entities by inhalation opens new possibilities for drug entry into systemic circulation, but this requires passage through the alveolar epithelium. Little is known about the pulmonary metabolism of verapamil. Specifically, this cardiovascular drug suffers from extensive first pass metabolism. We therefore evaluated the metabolism of verapamil in cultured alveolar epithelium and compared findings with results after administration by inhalation and intravenous routes. Specifically, cell culture of alveolar epithelium was characterized by gene expression of surfactant proteins A, B, C, and D, by immunohistochemistry of surfactant protein C, by staining for laminar bodies, and by gene expression of cytochrome P450 monooxygenases. During 6 days of culture expression, all cellular differentiation markers were obvious, albeit at different levels. With testosterone as substrate, we found alveolar epithelial cells to produce several stereo- and site-specific hydroxylation products. This provided evidence for metabolic competence of cultured alveolar epithelial cells. With verapamil as substrate, only limited production of metabolites was observed in cell culture assays, and similar results were recorded after administration by inhalation and intravenous routes. Likewise, elimination of verapamil from lung tissue and plasma was similar by both routes of administration. In conclusion, administration of verapamil by inhalation-abrogated extensive first pass metabolism frequently seen after oral application, and this may well be extended to the development of drugs with similar pharmacokinetic defects.     

气液界面培养小鼠气管上皮细胞模型构建

目的构建小鼠气管上皮细胞原代培养模型,应用于外源化合物等的吸入暴露毒性评价。方法通过优化培养基配比成份,分离小鼠气管,蛋白酶低温消化得到上皮细胞,接种到预先包被胶原的Transwell半透膜中,通过细胞跨上皮电阻值与紧密连接蛋白的检测,评估细胞间紧密连接与细胞极化的形式。转换气液界面培养,标记纤毛蛋白,拍摄纤毛形态特征。结果MTEC分离分化培养后,MUC5AC蛋白,乙酰化α-微管蛋白(α-tubulin),β-微管蛋白-Ⅳ(β-tubulin-Ⅳ),闭合小环蛋白(ZO-1)的表达及紧密连接的形成与假复层上皮的形成均与小鼠体内气管组织结构类似,扫描电镜观察小鼠气管上皮细胞表面纤毛形态。结论本研究构建的小鼠气管上皮细胞培养方案,体外培养成功获得具有类似组织结构和生理功能的假复层纤毛柱状上皮,为气道上皮细胞的分离、分化和检测提供了稳定、可靠地方法。     

Evaluation of human nasal RPMI 2650 cells grown at an air-liquid interface as a model for nasal drug transport studies

This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air-liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air-liquid and liquid-liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins-differentiation markers-in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192 +/- 3 Omega . cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air-liquid interface for 10 days; a seeding density of 4 x 10(5)/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9-12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07 +/- 0.01 x 10(-6) cm/s and 16.1 +/- 0.1 x 10(-6) cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air-liquid interface than in cells grown at a liquid-liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air-liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates.     

The Metabolism of a Series of Ester Pro-drugs by NCTC 2544 Cells,Skin Homogenate and LDE Testskin

Abstract— The metabolism of a series of substituted pyrazolopyridine ester pro-drugs was investigated using NCTC 2544 cells, human skin homogenate and LDE Testskin as model systems. The compounds were incubated in each system and the disappearance of drug and the production of the major hydrolysis product was observed with time and quantitated using HPLC. The toxicity of the ester pro-drugs and the metabolites was examined in NCTC 2544 cells using a cell viability assay procedure. Hydrolytic activity was slightly higher in the cell culture model than in skin homogenate solution but the rank order of activity for each pro-drug was similar. The metabolic activity of LDE Testskin was much reduced compared with the other systems, but again the overall pattern of metabolism was not dissimilar. These findings indicate that NCTC 2544 cells provide a reasonable model for human skin ester hydrolysis both in terms of rate and in terms of substrate specificity.     

Air-Liquid Interface Culture of Serially Passaged Human Nasal Epithelial Cell Monolayer for In Vitro Drug Transport Studies

The objective of this study was to establish a drug transport study using human nasal epithelial (HNE) cell monolayers cultured by the air-liquid interface (ALI) method using serum-free medium (BEGM:DME/F12, 50:50). The cells were developed and characterized in comparison to those that have been previously cultured by the liquid-covered culture (LCC) method. The epithelial cell monolayer cultured by the ALI method resulted in a significantly higher transepithelial electrical resistance value (3,453 ± 302 ohm × cm²) that was maintained (>1,000 ohm × cm²) for up to 20 days compared with that cultured by the LCC method. Observation by scanning electron microscopy revealed mature cilia after 2 weeks in the ALI culture, while flatten unhealthy ciliated cells were observed in the LCC method. After 21 days, higher level of MUC5AC and 8 mRNA were expressed in ALI culture which confirmed the secretory differentiation of HNE monolayers in vitro. No significant difference in the permeability coefficients of a model hydrophilic marker (¹⁴C-mannitol) and a lipophilic drug (budesonide) was observed between the two conditions on day 7. The passage 2–3 of the HNE monolayer using ALI condition retained the morphology and differentiated features of normal epithelium. Thus it would be a suitable model for in vitro nasal drug delivery studies.     

乙醇-肉豆蔻酸异丙酯系统中有机胺对酮洛芬经皮渗透的促进作用

目的应用乙醇(E)-肉豆蔻酸异丙酯(IPM)2组分溶液系统,研究在EI系统中有机胺对酮洛芬(KP)的经皮渗透的影响,初步探讨其作用机理。方法用水平扩散池,以离体大鼠皮肤作为渗透屏障进行体外渗透实验。用HPLC测定样品中KP浓度。结果除二乙胺(DEtA)和三乙胺外,当EI系统[m(乙醇)∶m(肉豆蔻酸异丙酯)=90∶10]中加入乙醇胺、二乙醇胺、三乙醇胺、和N-(2-羟乙基)吡啶时,KP在溶液系统中溶解度均略有降低。所有加入有机胺的EI溶液系统均对KP产生明显的渗透促进作用。此外,在DEtA-EI系统中,KP的流量与DEtA的浓度成正比,而E的渗透流量恒定,与KP流量变化无关。结论结果表明,EI系统中有机胺对KP的经皮渗透有促进作用。这种渗透促进作用可能依赖于KP与胺之间所形成的离子对组成。     

The use of human nasal in vitro cell systems during drug discovery and development.

The nasal route is widely used for the administration of drugs for both topical and systemic action. At an early stage in drug discovery and during the development process, it is essential to gain a thorough insight of the nasal absorption potential, metabolism and toxicity of the active compound and the components of the drug formulation. Human nasal epithelial cell cultures may provide a reliable screening tool for pharmaco-toxicological assessment of potential nasal drug formulations. The aim of this review is to give an overview of the information relevant for the development of a human nasal epithelial cell culture model useful during drug discovery and development. A primary goal in the development of in vitro cell culture systems is to maintain differentiated morphology and biochemical features, resembling the original tissue as closely as possible. The potential and limitations of the existing in vitro human nasal models are summarized. The following topics related to cell culture methodology are discussed: (i) primary cultures versus cell lines; (ii) cell-support substrate; (iii) medium and medium supplements; and (iv) the air-liquid interface model versus liquid-liquid. Several considerations with respect to the use of in vitro systems for pharmaceutical applications (transport, metabolism, assessment of ciliary toxicity) are also discussed.