炔丙基半胱氨酸吸收特性的研究
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摘要
炔丙基半胱氨酸(S-propargyl-cysteine,SPRC),是大蒜提取物烯丙基半胱氨酸的结构类似物。初步药理学研究表明,SPRC具有显著的心血管药理活性,对缺血缺氧心肌具有保护作用,非常有希望开发为一个具有良好市场前景的心血管药物。目前国际上关于SPRC的理化性质、胃肠吸收机制、体内药动学的研究还是空白。
在此背景下,本文首先测定了SPRC的溶解度、解离度和油水分配系数等与吸收相关的理化常数,然后在此基础上建立了大鼠在体原位胃肠灌注模型,研究了影响SPRC吸收的多种因素,探讨SPRC的小肠吸收机制,最后研究了SPRC的口服和静脉注射的体内药动学,探讨了SPRC的绝对生物利用度。
目的
通过对SPRC的理化性质(包括溶解度、解离度、油水分配系数),体内药动学,绝对生物利用度及在体胃肠吸收的研究,阐明炔丙基半胱氨酸的理化性质、最佳吸收部位、吸收影响因素、吸收机制等,为炔丙基半胱氨酸的处方设计优化提供依据。
方法
实验过程建立了各介质中(各种溶媒,胃肠灌注液,血浆等)SPRC测定的HPLC-UV和LC-MS/MS检测方法。所建立的相应介质中SPRC测定的HPLC-UV和LC-MS/MS检测方法,简单,灵敏,专属,稳定,准确度和精密度好,满足相应试验的分析检测要求。同时采用了经典摇瓶法测定溶解度和油水分配系数,利用电位滴定法测定滴定常数,建立了大鼠在体原位胃肠灌注模型研究胃肠吸收机制。
结果
体外理化性质研究表明,SPRC作为小分子水溶性化合物,在水及各介质中的溶解度良好；在正辛醇-水体系中logP为-2.16±0.030,脂溶性较差；在酸性环境中pKa=2.93±0.13,在碱性环境中pKa=7.73±0.33,PI=5.33±0.23；SPRC为两性化合物,SPRC的PI值在小肠的pH范围5.0～7.4内,靠近pH5.0。
大鼠在体原位胃灌注研究的实验结果表明SPRC在胃内吸收极少,同时大鼠在体原位肠灌注研究的实验结果表明在剂量分别为25,50,100 mg/kg时,SPRC的单位时间吸收转化率没有显著性差异(P>0.05)；在pH分别为5.0,6.0,7.4时,pH5.0时的单位时间吸收转化率与pH 6.0,7.4时的单位时间吸收转化率存在显著性差异(P<0.05)；SPRC十二指肠的单位时间吸收转化率与空肠、回肠的单位时间吸收转化率存在显著性差异(P<0.05)。本部分研究提示：SPRC主要在小肠被吸收,其中十二指肠吸收最好,SPRC吸收受pH影响显著,pH=5时吸收最好,主要为被动转运。
体内药动研究表明：大鼠i.g(口服)25、75和225mg/kg SPRC后,实测达峰时间分别为1.1±0.5、1.3±0.5和2.5±1.8 h, AUC与剂量间具有良好的线性关系。大鼠i.v.(静脉注射)SPRC后AUC与剂量及峰浓度与剂量关系表明AUC与剂量间同样具有良好的线性关系。口服不同剂量SPRC的绝对生物利用度分别为96.6%,97.0%,94.7%,提示该药在大鼠体内吸收良好。
结论
SPRC属于生物药剂学分类系统(BCS)Ⅰ类药物,具有高溶解度和高渗透性；其转运机制主要为膜孔转运,口服吸收良好,生物利用度高,适合开发为口服给药制剂。
S-propargyl-cysteine (SPRC) is a structural analog of S-allylcysteine (SAC) which is a sulfur-containing amino acid derived from garlic with antioxidant, anticancer, antihepatotoxic activities and cardioprotective effects. SPRC has cardioprotective effects in myocardial infarctions rats and could possibly be used to treat ischemic heart disease. Although the pharmacological effects of SPRC have been reported, the major physicochemical properties and absorption behaviors of SPRC have not been defined, which were the basis of developing SPRC oral dosage form.
OBJECTIVE
In this study, we determined equilibrium solubility, pKa, logP and logD of SPRC, investigated its absorption behaviors in rat gastrointestinal tract via three dosages, different pH environment and different intestine segments, it's pharmacokinetics characteristic and bioavailability. All these study are important, necessary and valuable to design and development of oral dosage form and rational clinical usage of SPRC.
METHOD
Saturation, electric titration and shake flask method were established to determine solubility, dissociation constant (pKa), true partition coefficient (logP) and apparent distribution coefficient (logD) of SPRC. Absorption mechanism of SPRC was investigated using rat in situ intestine perfusion model.
An HPLC method was established for the determination of S-propargylcysteine (SPRC) in the intestinal recirculating perfusate. Phenlsulfonphthalein (PP) was determined simultaneously for volumn correction. A C18 column was used with the mobile phase of water-acetonitrile by gradient elution at the detection wavelengths of 220 and 362 nm.
A simple, fast and sensitivemixed-mode reversed-phase and cation-exchange HPLC-MS/MS method has been developed and validated for the quantification of SPRC in the blood sample.
RESULT
The equilibrium solubility of SPRC in water was 31.91±0.46mg·mL-1; pKa of SPRC on acidic and basic region were roughly estimated to be 2.93±0.13 and 7.73±0.33, respectively, isoelectric point (PI) was 5.33±0.23; logP of SPRC was-2.16±0.027. Rat in situ perfusion trial indicated that hourly absorption percentage (P%/h) of SPRC showed significant difference (P<0.05) in different pH perfusion solutions and in different intestine segment, however, hourly absorption percentage (P%/h) of SPRC showed no significant difference (P>0.05) in different concentration perfusion solutions. After oral administration, SPRC was rapidly absorbed and the maximum plasma concentration (Cmax) appeared at 1.1±0.5,1.3±0.5, and 2.5±1.8 h following 25,75, and 225mg/kg administration, respectively. Log transformed Cmax/Dose and AUCO-t/Dosewere not different between dose levels after a single i.g. or i.v. administration (P>0.05), and the mean Tmax were also found to be independent of dose (P>0.05), indicating a kinetic linearity. The absolute bioavailability of oral SPRC was calculated to be 96.6%,97.0%, and 94.7%, respectively.
CONCLUSION
SPRC was categorized as biopharmaceutical classification system (BCS) class I drug, exhibited high solubility and permeability. Transport mechanism of SPRC was mainly membrane pore transport. Good absorption characteristic contribute to development of oral SPRC formulations.
在此背景下,本文首先测定了SPRC的溶解度、解离度和油水分配系数等与吸收相关的理化常数,然后在此基础上建立了大鼠在体原位胃肠灌注模型,研究了影响SPRC吸收的多种因素,探讨SPRC的小肠吸收机制,最后研究了SPRC的口服和静脉注射的体内药动学,探讨了SPRC的绝对生物利用度。
目的
通过对SPRC的理化性质(包括溶解度、解离度、油水分配系数),体内药动学,绝对生物利用度及在体胃肠吸收的研究,阐明炔丙基半胱氨酸的理化性质、最佳吸收部位、吸收影响因素、吸收机制等,为炔丙基半胱氨酸的处方设计优化提供依据。
方法
实验过程建立了各介质中(各种溶媒,胃肠灌注液,血浆等)SPRC测定的HPLC-UV和LC-MS/MS检测方法。所建立的相应介质中SPRC测定的HPLC-UV和LC-MS/MS检测方法,简单,灵敏,专属,稳定,准确度和精密度好,满足相应试验的分析检测要求。同时采用了经典摇瓶法测定溶解度和油水分配系数,利用电位滴定法测定滴定常数,建立了大鼠在体原位胃肠灌注模型研究胃肠吸收机制。
结果
体外理化性质研究表明,SPRC作为小分子水溶性化合物,在水及各介质中的溶解度良好；在正辛醇-水体系中logP为-2.16±0.030,脂溶性较差；在酸性环境中pKa=2.93±0.13,在碱性环境中pKa=7.73±0.33,PI=5.33±0.23；SPRC为两性化合物,SPRC的PI值在小肠的pH范围5.0～7.4内,靠近pH5.0。
大鼠在体原位胃灌注研究的实验结果表明SPRC在胃内吸收极少,同时大鼠在体原位肠灌注研究的实验结果表明在剂量分别为25,50,100 mg/kg时,SPRC的单位时间吸收转化率没有显著性差异(P>0.05)；在pH分别为5.0,6.0,7.4时,pH5.0时的单位时间吸收转化率与pH 6.0,7.4时的单位时间吸收转化率存在显著性差异(P<0.05)；SPRC十二指肠的单位时间吸收转化率与空肠、回肠的单位时间吸收转化率存在显著性差异(P<0.05)。本部分研究提示：SPRC主要在小肠被吸收,其中十二指肠吸收最好,SPRC吸收受pH影响显著,pH=5时吸收最好,主要为被动转运。
体内药动研究表明：大鼠i.g(口服)25、75和225mg/kg SPRC后,实测达峰时间分别为1.1±0.5、1.3±0.5和2.5±1.8 h, AUC与剂量间具有良好的线性关系。大鼠i.v.(静脉注射)SPRC后AUC与剂量及峰浓度与剂量关系表明AUC与剂量间同样具有良好的线性关系。口服不同剂量SPRC的绝对生物利用度分别为96.6%,97.0%,94.7%,提示该药在大鼠体内吸收良好。
结论
SPRC属于生物药剂学分类系统(BCS)Ⅰ类药物,具有高溶解度和高渗透性；其转运机制主要为膜孔转运,口服吸收良好,生物利用度高,适合开发为口服给药制剂。
S-propargyl-cysteine (SPRC) is a structural analog of S-allylcysteine (SAC) which is a sulfur-containing amino acid derived from garlic with antioxidant, anticancer, antihepatotoxic activities and cardioprotective effects. SPRC has cardioprotective effects in myocardial infarctions rats and could possibly be used to treat ischemic heart disease. Although the pharmacological effects of SPRC have been reported, the major physicochemical properties and absorption behaviors of SPRC have not been defined, which were the basis of developing SPRC oral dosage form.
OBJECTIVE
In this study, we determined equilibrium solubility, pKa, logP and logD of SPRC, investigated its absorption behaviors in rat gastrointestinal tract via three dosages, different pH environment and different intestine segments, it's pharmacokinetics characteristic and bioavailability. All these study are important, necessary and valuable to design and development of oral dosage form and rational clinical usage of SPRC.
METHOD
Saturation, electric titration and shake flask method were established to determine solubility, dissociation constant (pKa), true partition coefficient (logP) and apparent distribution coefficient (logD) of SPRC. Absorption mechanism of SPRC was investigated using rat in situ intestine perfusion model.
An HPLC method was established for the determination of S-propargylcysteine (SPRC) in the intestinal recirculating perfusate. Phenlsulfonphthalein (PP) was determined simultaneously for volumn correction. A C18 column was used with the mobile phase of water-acetonitrile by gradient elution at the detection wavelengths of 220 and 362 nm.
A simple, fast and sensitivemixed-mode reversed-phase and cation-exchange HPLC-MS/MS method has been developed and validated for the quantification of SPRC in the blood sample.
RESULT
The equilibrium solubility of SPRC in water was 31.91±0.46mg·mL-1; pKa of SPRC on acidic and basic region were roughly estimated to be 2.93±0.13 and 7.73±0.33, respectively, isoelectric point (PI) was 5.33±0.23; logP of SPRC was-2.16±0.027. Rat in situ perfusion trial indicated that hourly absorption percentage (P%/h) of SPRC showed significant difference (P<0.05) in different pH perfusion solutions and in different intestine segment, however, hourly absorption percentage (P%/h) of SPRC showed no significant difference (P>0.05) in different concentration perfusion solutions. After oral administration, SPRC was rapidly absorbed and the maximum plasma concentration (Cmax) appeared at 1.1±0.5,1.3±0.5, and 2.5±1.8 h following 25,75, and 225mg/kg administration, respectively. Log transformed Cmax/Dose and AUCO-t/Dosewere not different between dose levels after a single i.g. or i.v. administration (P>0.05), and the mean Tmax were also found to be independent of dose (P>0.05), indicating a kinetic linearity. The absolute bioavailability of oral SPRC was calculated to be 96.6%,97.0%, and 94.7%, respectively.
CONCLUSION
SPRC was categorized as biopharmaceutical classification system (BCS) class I drug, exhibited high solubility and permeability. Transport mechanism of SPRC was mainly membrane pore transport. Good absorption characteristic contribute to development of oral SPRC formulations.
引文
[1]Ariga T, Seki T. Antithrombotic and anticancer effects of garlic-derived sulfur compounds:a review [J]. Biofactors,2006,26(2):93-103.
[2]Goncagul G, Ayaz E, et al. Antimicrobial effect of garlic (Allium sativum) [J]. Recent Pat Antiinfect Drug Discov,2010,5(1):91-93.
[3]Alder R, Lookinland S, Berry JA, et al. A systematic review of the effectiveness of garlic as an anti-hyperlipidemic agent [J]. J Am Acad Nurse Pract,2003,15(3): 120-129.
[4]Rahman K, Lowe GM. Garlic and cardiovascular disease:a critical review [J]. J Nutr,2006,136(3 Suppl):736S-740S.
[5]Rivlin RS. Historical perspective on the use of garlic [J]. J Nutr,2001, 131:951S-954S.
[6]Block E. The chemistry of garlic and onions [J]. Sci Am,1985,252:114-119.
[7]Brace LD. Cardiovascular benefits of garlic (Allium sativum L) [J]. J Cardiovasc Nurs,2002,16:33-49.
[8]Gorinstein S, Jastrzebski Z, Namiesnik J, etc. The atherosclerotic heart disease and protecting properties of garlic:Contemporary data [J]. Mol Nutr Food Res,2007, 51:1365-1381.
[9]Amagase H. Clarifying the real bioactive constituents of garlic. [J]. J Nutr,2006, 136:716S-725S.
[10]Imai, J.; Ide, N.; Moriguchi, T.; Mastuura, H.; Itakura, Y. Antioxidant and scavenging effect of aged garlic extract and its constituents [J]. Planta Med.1994,60 (5),417-420.
[11]Yamasaki, T.; Lau, B. H. S. Garlic compounds protect vascular endothelial cells from hydrogen peroxide-induced oxidant injury [J]. Phytotherapy Res.1994,8, 408-412.
[12]Ide, N.; Lau, B. H. S. S-Allylcysteine attenuates oxidative stress in endothelial cells [J]. Drug De. Ind. Pharm.1999,25 (5),619-624.
[13]Numagami, Y.; Ohnishi, S. T. S Allylcysteine inhibits free radical production, lipid peroxidation and neuronal damage in rat brain ischemia [J], J. Nutr.2001,131 (3S),1100S-1105S.
[14]Welch, C.; Wuarin, L.; Sidell, N. Antiprolificative effect of the garlic compound S-allylcysteine on human neuroblastoma cell in vitro [J]. Cancer Lett,1992,63 (3), 211-219.
[15]Nakagawa, S.; Kasuga, S.; Matsuura, H. Prevention of liver damage by aged garlic extract and its components in mice [J].Phytotherapy Res,1989,3 (2),5053.
[16]Nishiyama, N.; Moriguchi, T.; Morihara, N.; Saito, H. Ameliora-tive effect of S-allylcysteine, a major thioallyl constituent in aged garlic extract, on learning deficits in senescence accelerated mice [J]. J Nutr,2001,131 (3S),1093S-1095S.
[17]Kodera Y, Suzuki A, Imada O, et al. Physical, chemical, and biological properties of S-allylcysteine, an amino acid derived from garlic [J]. J Agric Food Chem,2002, 50(3):622-632
[18]Rose P, Whiteman M, Moore PK, Zhu YZ. Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium:the chemistry of potential therapeutic agents [J]. Nat Prod Rep,2005,22:351-368.
[19]Chuah SC, Moore PK, Zhu YZ. S-allylcysteine mediates cardioprotection in an acute myocardial infarction rat model via a hydrogen sulfide-mediated pathway [J]. Am J Physiol Heart Circ Physiol,2007,293:H2629-H2630
[20]Wang Q, Liu HR, Mu Q, et al. S-propargyl-cysteine protects both adult rat hearts and neonatal cardiomyocytes from ischemia/hypoxia injury:the contribution of the hydrogen sulfide-mediated pathway [J]. J Cardiovasc Pharmacol,2009,54(2): 139-146.
[21]LENNERNAS H, ABRAHAMSSON B. The use of biopharmaceutic classification of drugs in drug discovery and development:current status and future extension[J]. JPharm Pharmacol,2005,57:273-285.
[22]RAEVSKY OA. Physicochemical descriptors in property-based drug design [J]. Mini-Rev Med Chem,2004,4(10):1041-1052.
[23]INGS RMJ, KING FD. Pharmacokinetics. In Medicinal Chemistry:Principles and Practice [M]. Cambridge,1994,67-85.
[24]S. Venkatesh, R.A. Lipper, Role of the development scientist incompound lead selection and optimization [J]. J. Pharm. Sci,2000,89 (2):145-154.
[25]E.H. Kerns, L. Di, Physicochemical profiling:overview of the screens [J]. Drug Discov Today Technol,2004,1 (4):343-348.
[26]E. Kerns, L. Di, Pharmaceutical profiling in drug discovery [J]. Drug Discov Technol,2003,8 (7):316-323.
[27]M. Stuart, K. Box, Chasing equilibrium:measuring the intrinsic solubility of weak acids and bases [J]. Anal. Chem,2005,77:983-990
[28]A. Avdeef, Pharmacokinetic Optimization in Drug Research:Biological, Physicochemical, and Computational Strategies, [LogP2000, Lipophilicity Symposium],2nd, Lausanne, Switzerland, Mar.5-9,2000,2001, pp.305-325.
[29]A. Avdeef, C. Berger, C. Brownell, pH-metric solubility.2:Correlation between the acid-base titration and the saturation shake-flask solubility-pH methods, [J]. Pharm. Res,2000,17 (1):85-89.
[30]C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings [J]. Adv Drug Deliv Rev,1997,23 (1-3):3-25.
[31]E.D. Carlson, P. Cong, W.H. Chandler Jr., etc. An integrated high throughput workflow for pre-formulations:polymorph and salt selection studies [J]. PharmaChem, 2003,2(7-8):10-15.
[32]王霄旸,谢人明,孙文基.朝藿定C平衡溶解度和表观油水分配系数的测定[J].药物分析杂志,2010,30(8)：1432-1434.
[33]张婷婷,徐文,胡生亮等.水飞蓟宾在不同介质中平衡溶解度和表观油水分配系数的测定[J].中国药学杂志2006,41(20)：1569-1571.
[34]Venkatarangan A, Taft D R, Bazil, et al. In vitro and in vivo intestinal absorption studies with a novel polypeptide:Correlation to its pKa and partitioning behavior [J]. Pharm Res,1997,14(11)Suppl:S647.
[35]ZhAI Y S, DU S Y, XU B, et al. O/W partition coefficient of PNS and absorption kinetics of it in rat intestine[J]. Zhongguo Zhong Yao Za Zhi,2010,35(8):984-988.
[36]SAKUMA S, OGURA R, MASAOKA Y, et al. Correlation between in vitro dissolution profiles from enteric-coated dosage forms and in vivo absorption in rats for high-solubility and high-permeability model drugs[J]. J Pharm Sci,2009,98(11): 4141-4152.
[37]HO HFH, PARK JY, MOROZOWICH W, et al. Physical model approach to the design of drugs with improved intestinal absorption [M]. Design of Biopharmaceutical Properties Through Prodrugs and Analogs. Vol 8. Washington DC: American Pharmaceutical Association,1977,136-227.
[38]Kin Y T, Alex A, Oksana T, et al. The permeation of amphoteric drugs through artificial membranes-an in combo absorption model based on paracellular and transmembrane permeability[J]. J Med Chem,2010,53(1):392-401
[39]Schanke LS, Tocco DJ, Brodie BB, et al. Absorption of drugs from the rat small intestine[J]. J Pharmacol Exp Ther,1958,123(1):81-88.
[40]Sutto SC, Rinald MT and Vukovinsky KE. Phenol red and comparison of the gravimetric 14c-Peg-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model [J]. AAPS Pharm Sci,2001,3(3):1-5.
[41]Tsuji A, Miyamoto E, Hashimoto N, Yamana T.1978. GI absorption of b-lactam antibiotics Ⅱ:Deviation from pH-Partition Hypothesis Penicillin absorption through in situ and in vitro lipoidal barriers [J]. J Pharm Sci,67:1705-1711.
[42]Zheng, Y.Q., Qiu, Y.H., Lu, F.M.Y., Daniel, H., Thomas, L.R.,1999. Permeability and absorption of leuprolide from various intestinal regions in rabbits and rats [J]. Int. J. Pharm,185:83-92.
[43]Anonymous.WHO expert committee on specifications for pharmaceutical preparations fortieth report; Annex 8:Proposal to waive in vivo bioequivalence requirements for WHO model list of essential medicine immediate release, solid oral dosageforms[EB/OL].[2007-12-15]http://whqlibdoc.who.int/trs/WHO_TRS_937_eng. pdf.
[44]Bakhtiar R, Ramos L, Tse FLS. High-throughput mass spectrometric analysis of xenobiotics in biological fluids [J]. Journal of liquid chromatography & related technologies,2002,25(4):507-540.
[45]Shou WZ, Zhang J. Recent development in high-throughput bioanalytical support for in vitro ADMET profiling [J]. Expert Opinion on Drug Metabolism and Toxicology,2010,6(3):321-336.
[46]Jemal M, Xia YQ. LC-MS development strategies for quantitative bioanalysis [J]. Current drug metabolism,2006,7(5):491-502.
[47]Hsieh YS, Korfmacher WA. Increasing speed and throughput when using HPLC-MS/MS systems for drug metabolism and pharmacokinetic screening [J]. Current drug metabolism,2006,7(5):479-489.
[48]梁文权.生物药剂学与药物动力学[M].北京：人民卫生出版社,2007.
[1]Wilson TH, Wiseman G, The use of sacs of everted of substances from the mucosal to the serosal surface [J]. Journal of Physiology,1954,123:116-121.
[2]Mossberg SM, Ammonia absorption in hamster ileum.Effect of PH and total CO2 on transport in everted sacs [J]. Journal of Physiology,1967,312(5):1327-1332.
[3]Carmona A. A simple in Vitro pefusion system to measure intestinal nutrient uptake [J]. The Journal of nutritional biochemistry,1998,9:52-57.
[4]Ussing HH, Zerahn K. Active transport of sodium as the source of electric current in the short-circuted isolated frog skin [J]. Acta Physiol Scand,1951,23:110-113
[5]L.Barthe, M.Bessouet, J.F.Woodly, et al. The improved everted gut sac:a simple method to study intestinal P-glycoprotein[J]. International Journal of Pharmaceutics,1998,173:255-258.
[6]Leppert PS, Fix JA. Use of evert ed intestinal rings for in vitro examination of oral absorption potential [J]. J Phar m Sci,1994,83:976.
[7]Uchiyama T, Sugiyama T, Quan YS, et al. Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers:their intestinal mucosal toxicity and absorption enhancing mechanism of nlauryl-betaD-maltopyranoside [J]. J Pharm Pharmacol 1999,51:1241.
[8]Artursson S, Epithelial Transport of drugs in cell culture.1:A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells [J]. Journal of Pharmaceutical Sciences,1990,79(7):476-482.
[9]梁桂贤,刘谦民.药物肠道吸收研究方法近况[J].国外医药合成药,生化药,制剂分册,1998,19(4)：251.
[10]Lo YL, Huang JD. Effects of sodium deoxycholat e and sodium caprate on the transport of epirubicin in human intestinal epithelial Caco-2 cell layers and evert ed gut sacs of rats [J]. Biochem Pharmacol,2000,59:665.
[11]Walle UK, Wal 1 e T. Transport of the cooked food mutagen 2-amino-l-methyl-6-phenylimidazo[4,5b]-pyridine (PhIP) across the human intestinal Caco-2 cell monolayer:role of efflux pumps[J]. Carcinogenesis,1999,20:2153.
[12]平其能,药剂学实验与指导方针,北京：中国医药科技出版社,1994,214-218.
[13]沈凯,王景田,药物肠吸收实验研究方法进展[J].中国新药杂志,2003,12(12)：988-991.
[2]Goncagul G, Ayaz E, et al. Antimicrobial effect of garlic (Allium sativum) [J]. Recent Pat Antiinfect Drug Discov,2010,5(1):91-93.
[3]Alder R, Lookinland S, Berry JA, et al. A systematic review of the effectiveness of garlic as an anti-hyperlipidemic agent [J]. J Am Acad Nurse Pract,2003,15(3): 120-129.
[4]Rahman K, Lowe GM. Garlic and cardiovascular disease:a critical review [J]. J Nutr,2006,136(3 Suppl):736S-740S.
[5]Rivlin RS. Historical perspective on the use of garlic [J]. J Nutr,2001, 131:951S-954S.
[6]Block E. The chemistry of garlic and onions [J]. Sci Am,1985,252:114-119.
[7]Brace LD. Cardiovascular benefits of garlic (Allium sativum L) [J]. J Cardiovasc Nurs,2002,16:33-49.
[8]Gorinstein S, Jastrzebski Z, Namiesnik J, etc. The atherosclerotic heart disease and protecting properties of garlic:Contemporary data [J]. Mol Nutr Food Res,2007, 51:1365-1381.
[9]Amagase H. Clarifying the real bioactive constituents of garlic. [J]. J Nutr,2006, 136:716S-725S.
[10]Imai, J.; Ide, N.; Moriguchi, T.; Mastuura, H.; Itakura, Y. Antioxidant and scavenging effect of aged garlic extract and its constituents [J]. Planta Med.1994,60 (5),417-420.
[11]Yamasaki, T.; Lau, B. H. S. Garlic compounds protect vascular endothelial cells from hydrogen peroxide-induced oxidant injury [J]. Phytotherapy Res.1994,8, 408-412.
[12]Ide, N.; Lau, B. H. S. S-Allylcysteine attenuates oxidative stress in endothelial cells [J]. Drug De. Ind. Pharm.1999,25 (5),619-624.
[13]Numagami, Y.; Ohnishi, S. T. S Allylcysteine inhibits free radical production, lipid peroxidation and neuronal damage in rat brain ischemia [J], J. Nutr.2001,131 (3S),1100S-1105S.
[14]Welch, C.; Wuarin, L.; Sidell, N. Antiprolificative effect of the garlic compound S-allylcysteine on human neuroblastoma cell in vitro [J]. Cancer Lett,1992,63 (3), 211-219.
[15]Nakagawa, S.; Kasuga, S.; Matsuura, H. Prevention of liver damage by aged garlic extract and its components in mice [J].Phytotherapy Res,1989,3 (2),5053.
[16]Nishiyama, N.; Moriguchi, T.; Morihara, N.; Saito, H. Ameliora-tive effect of S-allylcysteine, a major thioallyl constituent in aged garlic extract, on learning deficits in senescence accelerated mice [J]. J Nutr,2001,131 (3S),1093S-1095S.
[17]Kodera Y, Suzuki A, Imada O, et al. Physical, chemical, and biological properties of S-allylcysteine, an amino acid derived from garlic [J]. J Agric Food Chem,2002, 50(3):622-632
[18]Rose P, Whiteman M, Moore PK, Zhu YZ. Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium:the chemistry of potential therapeutic agents [J]. Nat Prod Rep,2005,22:351-368.
[19]Chuah SC, Moore PK, Zhu YZ. S-allylcysteine mediates cardioprotection in an acute myocardial infarction rat model via a hydrogen sulfide-mediated pathway [J]. Am J Physiol Heart Circ Physiol,2007,293:H2629-H2630
[20]Wang Q, Liu HR, Mu Q, et al. S-propargyl-cysteine protects both adult rat hearts and neonatal cardiomyocytes from ischemia/hypoxia injury:the contribution of the hydrogen sulfide-mediated pathway [J]. J Cardiovasc Pharmacol,2009,54(2): 139-146.
[21]LENNERNAS H, ABRAHAMSSON B. The use of biopharmaceutic classification of drugs in drug discovery and development:current status and future extension[J]. JPharm Pharmacol,2005,57:273-285.
[22]RAEVSKY OA. Physicochemical descriptors in property-based drug design [J]. Mini-Rev Med Chem,2004,4(10):1041-1052.
[23]INGS RMJ, KING FD. Pharmacokinetics. In Medicinal Chemistry:Principles and Practice [M]. Cambridge,1994,67-85.
[24]S. Venkatesh, R.A. Lipper, Role of the development scientist incompound lead selection and optimization [J]. J. Pharm. Sci,2000,89 (2):145-154.
[25]E.H. Kerns, L. Di, Physicochemical profiling:overview of the screens [J]. Drug Discov Today Technol,2004,1 (4):343-348.
[26]E. Kerns, L. Di, Pharmaceutical profiling in drug discovery [J]. Drug Discov Technol,2003,8 (7):316-323.
[27]M. Stuart, K. Box, Chasing equilibrium:measuring the intrinsic solubility of weak acids and bases [J]. Anal. Chem,2005,77:983-990
[28]A. Avdeef, Pharmacokinetic Optimization in Drug Research:Biological, Physicochemical, and Computational Strategies, [LogP2000, Lipophilicity Symposium],2nd, Lausanne, Switzerland, Mar.5-9,2000,2001, pp.305-325.
[29]A. Avdeef, C. Berger, C. Brownell, pH-metric solubility.2:Correlation between the acid-base titration and the saturation shake-flask solubility-pH methods, [J]. Pharm. Res,2000,17 (1):85-89.
[30]C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings [J]. Adv Drug Deliv Rev,1997,23 (1-3):3-25.
[31]E.D. Carlson, P. Cong, W.H. Chandler Jr., etc. An integrated high throughput workflow for pre-formulations:polymorph and salt selection studies [J]. PharmaChem, 2003,2(7-8):10-15.
[32]王霄旸,谢人明,孙文基.朝藿定C平衡溶解度和表观油水分配系数的测定[J].药物分析杂志,2010,30(8)：1432-1434.
[33]张婷婷,徐文,胡生亮等.水飞蓟宾在不同介质中平衡溶解度和表观油水分配系数的测定[J].中国药学杂志2006,41(20)：1569-1571.
[34]Venkatarangan A, Taft D R, Bazil, et al. In vitro and in vivo intestinal absorption studies with a novel polypeptide:Correlation to its pKa and partitioning behavior [J]. Pharm Res,1997,14(11)Suppl:S647.
[35]ZhAI Y S, DU S Y, XU B, et al. O/W partition coefficient of PNS and absorption kinetics of it in rat intestine[J]. Zhongguo Zhong Yao Za Zhi,2010,35(8):984-988.
[36]SAKUMA S, OGURA R, MASAOKA Y, et al. Correlation between in vitro dissolution profiles from enteric-coated dosage forms and in vivo absorption in rats for high-solubility and high-permeability model drugs[J]. J Pharm Sci,2009,98(11): 4141-4152.
[37]HO HFH, PARK JY, MOROZOWICH W, et al. Physical model approach to the design of drugs with improved intestinal absorption [M]. Design of Biopharmaceutical Properties Through Prodrugs and Analogs. Vol 8. Washington DC: American Pharmaceutical Association,1977,136-227.
[38]Kin Y T, Alex A, Oksana T, et al. The permeation of amphoteric drugs through artificial membranes-an in combo absorption model based on paracellular and transmembrane permeability[J]. J Med Chem,2010,53(1):392-401
[39]Schanke LS, Tocco DJ, Brodie BB, et al. Absorption of drugs from the rat small intestine[J]. J Pharmacol Exp Ther,1958,123(1):81-88.
[40]Sutto SC, Rinald MT and Vukovinsky KE. Phenol red and comparison of the gravimetric 14c-Peg-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model [J]. AAPS Pharm Sci,2001,3(3):1-5.
[41]Tsuji A, Miyamoto E, Hashimoto N, Yamana T.1978. GI absorption of b-lactam antibiotics Ⅱ:Deviation from pH-Partition Hypothesis Penicillin absorption through in situ and in vitro lipoidal barriers [J]. J Pharm Sci,67:1705-1711.
[42]Zheng, Y.Q., Qiu, Y.H., Lu, F.M.Y., Daniel, H., Thomas, L.R.,1999. Permeability and absorption of leuprolide from various intestinal regions in rabbits and rats [J]. Int. J. Pharm,185:83-92.
[43]Anonymous.WHO expert committee on specifications for pharmaceutical preparations fortieth report; Annex 8:Proposal to waive in vivo bioequivalence requirements for WHO model list of essential medicine immediate release, solid oral dosageforms[EB/OL].[2007-12-15]http://whqlibdoc.who.int/trs/WHO_TRS_937_eng. pdf.
[44]Bakhtiar R, Ramos L, Tse FLS. High-throughput mass spectrometric analysis of xenobiotics in biological fluids [J]. Journal of liquid chromatography & related technologies,2002,25(4):507-540.
[45]Shou WZ, Zhang J. Recent development in high-throughput bioanalytical support for in vitro ADMET profiling [J]. Expert Opinion on Drug Metabolism and Toxicology,2010,6(3):321-336.
[46]Jemal M, Xia YQ. LC-MS development strategies for quantitative bioanalysis [J]. Current drug metabolism,2006,7(5):491-502.
[47]Hsieh YS, Korfmacher WA. Increasing speed and throughput when using HPLC-MS/MS systems for drug metabolism and pharmacokinetic screening [J]. Current drug metabolism,2006,7(5):479-489.
[48]梁文权.生物药剂学与药物动力学[M].北京：人民卫生出版社,2007.
[1]Wilson TH, Wiseman G, The use of sacs of everted of substances from the mucosal to the serosal surface [J]. Journal of Physiology,1954,123:116-121.
[2]Mossberg SM, Ammonia absorption in hamster ileum.Effect of PH and total CO2 on transport in everted sacs [J]. Journal of Physiology,1967,312(5):1327-1332.
[3]Carmona A. A simple in Vitro pefusion system to measure intestinal nutrient uptake [J]. The Journal of nutritional biochemistry,1998,9:52-57.
[4]Ussing HH, Zerahn K. Active transport of sodium as the source of electric current in the short-circuted isolated frog skin [J]. Acta Physiol Scand,1951,23:110-113
[5]L.Barthe, M.Bessouet, J.F.Woodly, et al. The improved everted gut sac:a simple method to study intestinal P-glycoprotein[J]. International Journal of Pharmaceutics,1998,173:255-258.
[6]Leppert PS, Fix JA. Use of evert ed intestinal rings for in vitro examination of oral absorption potential [J]. J Phar m Sci,1994,83:976.
[7]Uchiyama T, Sugiyama T, Quan YS, et al. Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers:their intestinal mucosal toxicity and absorption enhancing mechanism of nlauryl-betaD-maltopyranoside [J]. J Pharm Pharmacol 1999,51:1241.
[8]Artursson S, Epithelial Transport of drugs in cell culture.1:A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells [J]. Journal of Pharmaceutical Sciences,1990,79(7):476-482.
[9]梁桂贤,刘谦民.药物肠道吸收研究方法近况[J].国外医药合成药,生化药,制剂分册,1998,19(4)：251.
[10]Lo YL, Huang JD. Effects of sodium deoxycholat e and sodium caprate on the transport of epirubicin in human intestinal epithelial Caco-2 cell layers and evert ed gut sacs of rats [J]. Biochem Pharmacol,2000,59:665.
[11]Walle UK, Wal 1 e T. Transport of the cooked food mutagen 2-amino-l-methyl-6-phenylimidazo[4,5b]-pyridine (PhIP) across the human intestinal Caco-2 cell monolayer:role of efflux pumps[J]. Carcinogenesis,1999,20:2153.
[12]平其能,药剂学实验与指导方针,北京：中国医药科技出版社,1994,214-218.
[13]沈凯,王景田,药物肠吸收实验研究方法进展[J].中国新药杂志,2003,12(12)：988-991.