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。     

酮洛芬及其异丙酯对HaCaT细胞构建的组织工程皮肤的渗透作用

目的体外构建适用于经皮给药研究的组织工程皮肤。方法以表皮角质形成细胞系HaCaT细胞及人真皮成纤维细胞为细胞来源，用I型牛胶原蛋白为真皮基质包埋成纤维细胞，其上接种HaCaT细胞，采用气-液界面方式培养，观察不同的培养介质对组织工程皮肤的影响，HE染色切片观察培养皮肤结构形态。以酮洛芬及其异丙酯为模型药物研究经皮渗透和代谢特性。结果HaCaT细胞经气-液界面培养可形成类表皮层，但不能形成完整的角质层。维生素C可明显促进细胞增殖，维生素D₃可促进细胞分化，雌二醇对此组织工程皮肤没有明显的影响。同皮肤细胞匀浆代谢相似，酮洛芬异丙酯被代谢成原药酮洛芬。结论HaCaT细胞在气-液界面培养条件下可形成多层分化不完全的表皮层，保留了一定的酶活性，可用于药物的经皮渗透和代谢等研究。     

酮洛芬异丙酯在离体裸小鼠和猴皮肤中的渗透和代谢

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     

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。     

氮酮对喃氟啶透皮吸收的影响

本文研究了喃氟啶软膏剂对小白鼠和裸鼠离体皮肤的透皮吸收。比较了小白鼠和裸鼠皮肤渗透性、药物浓度和透皮吸收促进剂对喃氟啶透皮吸收的影响。实验结果表明，小白鼠皮肤对喃氟啶渗透性大于裸鼠皮肤，采用透皮吸收促进剂和提高药物浓度可大大提高药物的透皮吸收速率。本研究为临床用药和配制喃氟啶软膏剂选择适宜药物浓度和促进剂浓度提供了依据。     

酮洛芬贴片体外透皮释放方法学研究

目的:建立酮洛芬贴片体外透皮释放方法学。方法 :采用HPLC法测定释放液中酮洛芬的含量,测定条件:DiamonsilC18色谱柱(150 mm×4.6 mm,5μm),流动相为pH 3.5磷酸盐缓冲液-乙腈-水(2∶53∶45),检测波长为253 nm,流速为1.0mL.min-1,柱温25℃。以裸鼠皮肤为实验皮肤,采用Frans扩散池方法进行三批酮洛芬贴片样品的体外透皮实验。结果:在该HPLC条件下,酮洛芬与其他杂质分离良好,进样量在0.509~40.72μg.mL-1时,酮洛芬浓度与峰面积呈良好的线性关系(r=0.999 9),回收率为101.09%,RSD为1.23%。三批酮洛芬贴片样品的透皮释放速率分别为18.157,17.973,20.001μg.cm-2.h-1,药物透皮释放符合零级动力学过程。结论:本文建立的酮洛芬贴片体外透皮释放方法简便,重现性好,可以用于控制产品质量。     

酮洛芬醇质体体外经皮渗透研究

目的:考察酮洛芬醇质体的体外经皮渗透特性。方法:乙醇注入法制备酮洛芬醇质体,采用Franz扩散池,以离体小鼠皮为皮肤屏障,对酮洛芬醇质体的体外经皮渗透量、稳态透皮速率进行研究,并测定24 h时皮肤中的滞留量。结果:酮洛芬醇质体体外24 h累积渗透量Q为(720.88±2.04)μg.cm-2,稳态透皮速率J为(28.15±0.20)μg.cm-2.h-1,24 h皮肤中的滞留量(50.86±1.44)μg.cm-2,与同剂量酮洛芬脂质体及其30%醇溶液相比,均有明显提高(P<0.05)。结论:醇质体可显著增加酮洛芬的体外经皮渗透,值得进一步研发。     

羧酸酯酶与酯类药物代谢

［摘要］哺乳动物羧酸酯酶（Carboxylesterases, CESs, EC3.1.1.1）属于B族酯酶，组成一个多基因家族，其基因产物定位于多种组织的内质网中。CESs参与机体内源性和外源性化合物的水解代谢，并与其他代谢酶或运载体共同作用，极大地影响酯类药物的体内代谢。近年来对于CESs的研究逐步增多，人们将其分为5个亚族, 分别为CES1、CES2、CES3、CES4和CES5。人体内参与酯类药物代谢的CESs主要为hCE 1（人类CES1家族同工酶）和hCE 2（人类CES2家族同工酶）。此两种酶在组织分布和对酯类药物代谢特征上存在较大差异，如小肠上皮由于CES2表达会对部分酯类药物的口服吸收产生重要影响。因此在进行酯类药物尤其是酯类前药合成时，必须考虑该类药物的物理化学和生物学性质，结合体内CESs表达特征和活性综合评价才能获得成功。     

美国透皮吸收研究近况

近年来美国对透皮吸收的研究正在广泛深入地开展,每年在药学年会上都发表大量的文章。1986～1988年美国药学科学学术年会中就有二百余篇有关透皮吸收的研究报告,内容涉及新的透皮给药系统的研究、药物理化性质与透皮特性的关系、介质对药物透皮速率的影响、药物在表皮内的代谢、透皮的前体药物研究、透皮吸收实验方法的研究、透皮吸收促进剂研究与离子透入研究等。现将美国透皮吸收研究近况介绍如下。透皮给药系统的研究目前成为商品的透皮给药系统有东莨菪     

重组人干扰素α-2b软膏透皮试验研究

目的：考察不同透皮吸收促进剂对重组人干扰素α-2b软膏透皮吸收的促进效果。方法：采用单室水平扩散池，计时2h后从接受室内取样，用细胞病变抑制法测定透过Swiss小鼠腹部皮肤的干扰素的活性。结果：在软膏中加入透皮吸收促进剂能明显改善透皮吸收．且以2％水溶性氮酮和2％1，2-丙二醇联合应用的效果最好。结论：2％水溶性氮酮和2％1，2-丙二醇联用，提高了药物生物利用度和疗效。     

Stereoselective characteristics and mechanisms of epidermal carboxylesterase metabolism observed in HaCaT keratinocytes

There is increasing evidence that epidermal carboxylesterase may be involved in the stereoselective hydrolysis of prodrugs in percutaneous absorption. The present study was designed to evaluate the stereoselective characteristics and mechanisms of ketoprofen ethyl ester hydrolysis by epidermal carboxylesterase expressed in HaCaT keratinocytes. Ketoprofen ethyl ester was mainly hydrolyzed to R-ketoprofen by carboxylesterase of human HaCaT keratinocytes. Human carboxylesterase-1 (hCE-1) and human carboxylesterase-2 (hCE-2) were intensively detected in L02 hepatocytes, hCE-2 was also intensively detected in HaCaT keratinocytes, but hCE-1 was not detected in HaCaT keratinocytes. hCE-2 is thus an abundant carboxylesterase in HaCaT keratinocytes which may be responsible for stereoselective hydrolysis of ketoprofen ethyl ester.     

The distribution of esterases in the skin of the minipig

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.     

Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture

Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases in paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig.     

Design, synthesis and characterization of captopril prodrugs for enhanced percutaneous absorption

Most drugs are designed primarily for oral administration, but the activity and stability profiles desirable for this route often make them unsuitable for transdermal delivery. We were therefore interested in designing analogues of captopril, a model drug with poor percutaneous penetration, for which the sustained steady-state blood plasma level associated with transdermal delivery (and which is unattainable orally) would be particularly beneficial. Quantitative structure-permeability relationships (QSPRs) predicted that ester and thiol prodrug derivatives of captopril would have lower maximal transdermal flux (J(m)) than the parent drug, since the increases in permeability coefficient (k(p)) of prodrugs would be outweighed by the reductions in aqueous solubility. Therefore, the aim of this study was to synthesize a series of prodrugs of captopril and to determine if a QSPR model could be used to design therapeutically viable prodrugs. Molecules with the highest predicted k(p) values were synthesized and characterized, and J(m) measured in Franz diffusion cells from saturated aqueous donor across porcine skin (fresh and frozen). In-vitro metabolism was also measured. Captopril and the prodrugs crossed the skin relatively freely, with J(m) being highest for ethyl to butyl esters. Substantial first-order metabolism of the prodrugs was observed, suggesting that their enhanced percutaneous absorption was complemented by their metabolic performance. The results suggested that QSPR models provided excellent enhancements in drug delivery. This was not seen at higher lipophilicities, suggesting that issues of solubility need to be considered in conjunction with any such use of a QSPR model.     

Diacyl glyceryl ester prodrugs for slow release in the skin: synthesis and in vitro degradation and absorption studies for naproxen derivatives

Diacyl glyceryl ester derivatives of naproxen were synthesized and tested for transdermal and dermal administration. Diacyl derivatives of aliphatic acids of various chain length were compared. The pharmaceutical properties of these compounds, such as lipophilicity, hydrolysis in a buffer solution at various pH values and degradation in human serum and hairless mouse skin homogenate, were investigated. All the diacyl derivatives were relatively stable in a neutral buffer solution, but were rapidly degraded to release naproxen in human serum and hairless mouse skin homogenate. The diacyl compounds could not penetrate hairless mouse skin in vitro. However, significant absorption into the skin could be measured, and this increased with increasing lipophilicity. A more than 100-fold difference in absorption was observed. The prodrugs were slowly hydrolyzed to naproxen inside the skin. The release of naproxen to the receptor compartment of diffusion cells showed that this type of prodrug could be used for controlled drug delivery.     

Simultaneous transport and metabolism of nicotinic acid derivatives in hairless mouse skin

In vitro simultaneous transport and metabolism of three ester prodrugs of nicotinic acid (NA), methyl nicotinate (MN), ethyl nicotinate (EN) and butyl nicotinate (BN) were studied using excised skin from hairless mouse. Hydrolysis studies of these esters with and without skin homogenate were also done at 37 degrees C. Both the ester and NA were detected in all receiver solutions in permeation studies, and no chemical hydrolysis of the esters was found, indicating that the esters were hydrolyzed during the skin permeation process. The total (ester+NA) flux from a saturated solution of ester prodrugs was higher than that of NA and was highest for MN, followed by EN and BN, whereas the total permeability coefficient of ester prodrugs increased from MN to BN. A difference in the NA/total flux ratio was found among these prodrugs; thus, esterase activity was also dependent on the alkyl chain length of the esters. The total flux from each ester solution increased linearly with the donor concentration. NA flux from MN and EN solutions increased with an increase in the donor concentration and reached a plateau at the high concentration range, suggesting that metabolic saturation occurred. NA fluxes at the plateau were similar among ester prodrugs and corresponded to the Vmax estimated from the hydrolysis experiment. The order of donor concentration at which NA reached a plateau also corresponded to the order of Km. It was confirmed that a difference in alkyl chain length of the ester prodrugs affected not only permeability but also metabolism in the skin permeation process.     

Synthesis and evaluation of Ketorolac ester prodrugs for transdermal delivery

Alkyl esters of ketorolac were synthesized as potential prodrugs for transdermal delivery and evaluated to determine the relationship between their skin permeation characteristics and their physicochemical properties. Solubility of the prodrugs in various vehicles was determined at room temperature while lipophilicity was obtained as 1-octanol/water partition coefficients (logP) and capacity factors (k') using HPLC. Metabolism of the prodrugs to ketorolac was studied both in rat skin homogenate and in plasma. Rat skin permeation characteristics of the prodrugs saturated in propylene glycol were investigated using the Keshary-Chien permeation system at 37 degrees C. An increase in logP and capacity factor values of the prodrugs were observed in proportion to their alkyl chain length. Good linear relationship between the logP values and capacity factor was observed (r(2) = 0.92). Prodrugs were rapidly degraded to ketorolac both in the skin homogenate and in plasma following a first-order kinetics. To determine accurate amounts of prodrug permeated, both the prodrug and parent drug concentration in the receptor solution were determined in mole units. The skin permeation rate of the alkyl ester prodrugs was significantly higher with a shorter lag time than that of ketorolac. The permeation rate of ketorolac reached maximum in its 1-propyl ester form as 46.61 nmol/cm(2)/h, and a parabolic relationship was observed between the permeation rate and the logP values of the prodrugs. Alkyl ester prodrugs of ketorolac having optimum lipophilicity could improve the transdermal delivery of ketorolac.     

Human carboxylesterase isozymes: catalytic properties and rational drug design

Human carboxylesterase 1 (hCE-1, CES1A1, HU1) and carboxylesterase 2 (hCE-2, hiCE, HU3) are a serine esterase involved in both drug metabolism and activation. Although both hCE-1 and hCE-2 are present in several organs, the hydrolase activity of liver and small intestine is predominantly attributed to hCE-1 and hCE-2, respectively. The substrate specificity of hCE-1 and hCE-2 is significantly different. hCE-1 mainly hydrolyzes a substrate with a small alcohol group and large acyl group, but its wide active pocket sometimes allows it to act on structurally distinct compounds of either large or small alcohol moiety. In contrast, hCE-2 recognizes a substrate with a large alcohol group and small acyl group, and its substrate specificity may be restricted by a capability of acyl-hCE-2 conjugate formation due to the presence of conformational interference in the active pocket. Furthermore, hCE-1 shows high transesterification activity, especially with hydrophobic alcohol, but negligible for hCE-2. Transesterification may be a reason for the substrate specificity of hCE-1 that hardly hydrolyzes a substrate with hydrophobic alcohol group, because transesterification can progress at the same time when a compound is hydrolyzed by hCE-1. From the standpoint of drug absorption, the intestinal hydrolysis by CES during drug absorption is evaluated in rat intestine and Caco2-cell line. The rat in situ single-pass perfusion shows markedly extensive hydrolysis in the intestinal mucosa. Since the hydrolyzed products are present at higher concentration in the epithelial cells rather than blood vessels and intestinal lumen, hydrolysates are transported by a specific efflux transporter and passive diffusion according to pH-partition. The expression pattern of CES in Caco-2 cell monolayer, a useful in vitro model for rapid screening of human intestinal drug absorption, is completely different from that in human small intestine but very similar to human liver that expresses a much higher level of hCE-1 and lower level of hCE-2. Therefore, the prediction of human intestinal absorption using Caco-2 cell monolayers should be carefully monitored in the case of ester and amide-containing drugs such as prodrugs. Further experimentation for an understanding of detailed substrate specificity for CES and development of in vitro evaluation systems for absorption of prodrug and its hydrolysates will help us to design the ideal prodrug.     

Metabolism of captopril carboxyl ester derivatives for percutaneous absorption

Objectives To determine the metabolism of captopril n‐carboxyl derivatives and how this may impact on their use as transdermal prodrugs. The pharmacological activity of the ester derivatives was also characterised in order to compare the angiotensin converting enzyme inhibitory potency of the derivatives compared with the parent drug, captopril. Methods The metabolism rates of the ester derivatives were determined in vitro (using porcine liver esterase and porcine ear skin) and in silico (using molecular modelling to investigate the potential to predict metabolism). Key findings Relatively slow pseudo first‐order metabolism of the prodrugs was observed, with the ethyl ester displaying the highest rate of metabolism. A strong relationship was established between in‐vitro methods, while in‐silico methods support the use of in‐vitro methods and highlight the potential of in‐silico techniques to predict metabolism. All the prodrugs behaved as angiotensin converting enzyme inhibitors, with the methyl ester displaying optimum inhibition. Conclusions In‐vitro porcine liver esterase metabolism rates inform in‐vitro skin rates well, and in‐silico interaction energies relate well to both. Thus, in‐silico methods may be developed that include interaction energies to predict metabolism rates.     

Stability and in vitro metabolism of dipeptide model prodrugs with affinity for the oligopeptide transporter.

One approach to increase drug stability and to facilitate oral absorption of low bioavailability drugs may be to design oligopeptide ester prodrugs which are stable in the gastrointestinal tract, are transported via the oligopeptide transporter, and finally release the parent drug molecule into the blood circulation and/or by its site of action. In these kinds of prodrugs the ester linkage may be broken by pH dependent and/or enzyme catalyzed hydrolysis. The objective of the present study was to investigate the degradation mechanism and rate of the model compounds Glu(OBzl)-Sar, D-Glu(OBzl)-Ala and Asp(OBzl)-Sar in aqueous solution and in relevant biological media and to compare these results with those of our previous study of D-Asp(OBzl)-Ala. Furthermore, the resulting aqueous stability and in vitro metabolism data are related to our previous affinity data to evaluate if Glu-Sar, D-Glu-Ala, and Asp-Sar have potential as pro-moieties in these kinds of prodrugs. The degradation rates follow first-order kinetics, show maximun stability at pH 4-5 with maximum half-lives for Asp(OBzl)-Sar, Glu(OBzl)-Sar, and D-Glu(OBzl)-Ala of 115 h, 30 days and 152 days, respectively. The stability was dependent on buffer concentration, temperature, pH, and ionic strength. In biological media such as 80% human plasma, human gastric juice and intestinal fluid, and 10% rat jejunal homogenate at 37 degrees C, the half-lives were greater than 1 h except for the hydrolysis of Glu(OBzl)-Sar in 10% rat jejunal homogenate, where the half-life was approximately 16 min. All the stabilized dipeptides may have potential as drug carriers targeting hPepT1.