Application of Rat In Situ Single-pass Intestinal Perfusion in the Evaluation of Presystemic Extraction of Indinavir Under Different Perfusion Rates

Background/PurposeFirst-pass effect has been an important concern for oral pharmaceuticals. An in vivo system was developed for measuring different concentrations of pharmaceuticals in the portal vein and hepatic vein (via the inferior vena cava) for delineating presystemic metabolism under different perfusion rates by using indinavir as an exemplary agent.MethodsAn in situ single-pass intestinal perfusion technique was modified from previous studies to concomitantly obtain portal and hepatic venous bloods. Portal and hepatic venous samples were simultaneously taken from rats at appropriate time points using the perfusion model of 1 mg/mL indinavir at flow rates of 0.05, 0.1, 0.5 and 1.0 mL/min. The indinavir concentrations were assayed by binary-gradient high-pressure liquid chromatography with UV detection.ResultsThe mean indinavir concentrations in portal vein concentration−time profiles at different perfusion times under various flow rates were all higher than those obtained for hepatic veins. At flow rates of 0.5 and 1.0 mL/min, in particular, the area under the curve (AUC) and maximal concentration (C max) of indinavir absorption were significantly different between portal veins and hepatic veins (p < 0.05), indicating considerable hepatic involvement in the presystemic extraction of indinavir. The system also has potential for use when estimating the hepatic extraction ratio (E H) and hepatic clearance (Cl H).ConclusionThis in vivo approach could provide another useful tool for improving our basic understanding of the absorption kinetics and hepatic metabolism of pharmaceuticals under development and facilitating the clinical application of such

Detection of EBV Infection and Gene Expression in Oral Cancer from Patients in Taiwan by Microarray Analysis

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis

Association of ORAI1 Haplotypes with the Risk of HLA-B27 Positive Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The aetiology of ankylosing spondylitis is still unclear. Previous studies have indicated that genetics factors such as human leukocyte antigen HLA-B27 associates to AS susceptibility. We carried out a case-control study to determine whether the genetic polymorphisms of ORAI1 gene, a major component of store-operated calcium channels that involved the regulation of immune system, is a susceptibility factor to AS in a Taiwanese population. We enrolled 361 AS patients fulfilled the modified New York criteria and 379 controls from community. Five tagging single nucleotides polymorphisms (tSNPs) at ORAI1 were selected from the data of Han Chinese population in HapMap project. Clinical statuses of AS were assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global Index (BAS-G). Our results indicated that subjects carrying the minor allele homozygote (CC) of the promoter SNP rs12313273 or TT homozygote of the SNP rs7135617 had an increased risk of HLA-B27 positive AS. The minor allele C of 3′UTR SNP rs712853 exerted a protective effect to HLA-B27 positive AS. Furthermore, the rs12313273/rs7135617 pairwise allele analysis found that C-G (OR 1.69, 95% CI 1.27, 2.25; p = 0.0003) and T-T (OR 1.75, 95% CI 1.36, 2.27; p<0.0001) haplotypes had a significantly association with the risk of HLA-B27-positive AS in comparison with the T-G carriers. This is the first study that indicate haplotypes of ORAI1 (rs12313273 and rs7135617) are associated with the risk of HLA-B27 positive AS

Effects of St. John’s wort on the Cytochrome P450 Activities of Rat Intestine and Liver

St. John’s wort（Hypericum perforatum L.）萃取物是目前普遍使用的草藥之一，主要是因為它們有治療輕度至中度憂鬱症活性的報告。然而，在近年的研究中發現，St. John’s wort有影響特定生體轉化第一期單氧酵素cytochrome P450（CYP）的作用，分為長期使用後的CYP促進作用與短期使用後的CYP抑制作用。CYP在藥物代謝中扮演很重要的角色，因此，如對CYP造成促進作用的物質，會造成藥物血中濃度降低，而造成治療失敗，如HIV蛋白酶抑制劑indinavir。 本研究使用週齡14 ± 2（10-20）週以及59 ± 2（54-62）週分別代表年輕與年長的Wistar大鼠，連續餵食低劑量（150 mg/day）或高劑量（300 mg/day）St. John’s wort 15天。藉由分離出肝臟及小腸的微粒體，測定CYP1A的7-ethoxyresorufin O-deethylation（EROD）、CYP2E1的p-nitrophenol hydroxylation（PNPH）及CYP3A的erythromycin N-demethylation（EMND）活性，探討St. John’s wort對CYP活性的作用。實驗發現大鼠肝臟CYP1A的活性在年輕低劑量組有被促進（58.8 ± 11.2 vs. 37.2 ± 12.7 pmol/mg protein/min，p &lt; 0.05），在年長高劑量組也有被促進（109.3 ± 6.8 vs. 57.8 ± 28.8 pmol/mg protein/min，p &lt; 0.05）。肝臟CYP2E1的活性只有在年輕低劑量組會被促進（1.98 ± 0.34 vs. 1.20 ± 0.40 nmol/mg protein/min，p &lt; 0.05）。而大鼠肝臟CYP3A的活性在年輕低劑量組會被促進（0.72 ± 0.11 vs. 0.31 ± 0.07 nmol/mg protein/min，p &lt; 0.001），小腸CYP3A的活性卻不會。本研究進而發現St. John’s wort之促進大鼠肝臟CYP2E1及CYP3A的作用，會受大鼠週齡不同而影響，僅在年輕的大鼠中才有發現促進作用。 以上結果顯示，St. John’s wort確實有促進大鼠肝臟特定CYP的作用，而沒有促進大鼠小腸CYP3A的作用。而在不同週齡與不同劑量時，其促進作用之程度並不相等。綜合本實驗室近年研究St. John’s wort與indinavir的交互作用，推測除了CYP之外還有其他重要因素及機轉，影響此交互作用。St. John’s wort (Hypericum perforatum L.) extract is one of the most commonly used herbal medications, mainly because of their activity in treating mild to moderate depression. In the recent study, St. John’s wort has the long-term inductive and short-term inhibitive effects of regulating some of the biotransformation phase I monooxygenase, cytochrome P450 (CYP). CYP plays an important role in drug metabolism. Therefore, the matter which has the inductive effects of CYP is able to reduce plasma concentrations of certain drugs, like HIV protease inhibiter, indinavir, to make the treatment fail. Oral administration of either low dose (150 mg/day) or high dose (300 mg/day) St. John’s wort extracts for 15 days was given to the 14±2 (10-20) and 59±2 (54-62) weeks old (representing the young-adult and aged group, respectively) Wistar rats in our research. The activities of 7-ethoxyresorufin O-deethylation (EROD) of CYP1A, p-nitrophenol hydroxylation (PNPH) of CYP2E1, and erythromycin N-demethylation (EMND) of CYP3A in isolated hepatic microsome and intestinal microsome were determined for investigating the effect of St. John’s wort on CYP. We found that the hepatic CYP1A activities were induced in the young-adult low dose group (58.8 ± 11.2 vs. 37.2 ± 12.7 pmol/mg protein/min，p &lt; 0.05) and the aged high dose group (109.3 ± 6.8 vs. 57.8 ± 28.8 pmol/mg protein/min，p &lt; 0.05). The induction of hepatic CYP2E1 activity was only found in young-adult low dose group (1.98 ± 0.34 vs. 1.20 ± 0.40 nmol/mg protein/min，p &lt; 0.05). CYP3A in liver, not intestine, could be induced in young-adult low dose group (0.72 ± 0.11 vs. 0.31 ± 0.07 nmol/mg protein/min，p &lt; 0.001). However, when we discover that St. John’s wort only induces the hepatic CYP in young-adult rats, there are some differences between the inductive effects of St. John’s wort on hepatic CYP, especially CYP2E1 and CYP3A, in young-adult rats and aged rats. According to the results, it demonstrated that St. John’s wort can induce the activity of hepatic CYP but not intestinal CYP3A in rats. Either the age of rats or dosage of St. John’s wort might vary the inductive effect of rat hepatic CYP. We combine the results with the recent research in drug interaction of St. John’s wort and indinavir in our lab. Besides CYP, there might be other important factors and mechanisms in this interaction.中文摘要..................................................i 英文摘要................................................iii 第一章 文獻探討...........................................1 1.1 前言..................................................1 1.2 研究背景..............................................3 1.2.1 Cytochrome P450.....................................3 1.2.2 經影響Cytochrome P450之中草藥藥品交互作用研究方法...8 1.2.3 St. John’s wort研究綜述...........................16 第二章 研究目的..........................................27 第三章 實驗材料與方法....................................28 3.1 實驗材料.............................................28 3.1.1 實驗動物...........................................28 3.1.2 實驗藥品與試劑.....................................28 3.1.3 實驗儀器...........................................30 3.2 實驗方法.............................................31 3.2.1 實驗動物處理.......................................31 3.2.2 大鼠肝臟微粒體製備.................................32 3.2.3 大鼠小腸微粒體製備.................................34 3.2.4 7-Ethoxyresorufin O-Deethylation（EROD）反應檢測...37 3.2.5 p-Nitrophenol Hydroxylation（PNPH）反應檢測........37 3.2.6 Erythromycin N-Demethylation（EMND）反應檢測.......38 3.2.7 反應檢測定量方法之確認.............................39 3.2.8 蛋白質定量.........................................40 3.2.9 統計分析...........................................40 第四章 實驗結果..........................................42 4.1 大鼠體重、肝臟重量及St. John’s wort餵食劑量換算.....42 4.2 大鼠肝臟微粒體蛋白質含量.............................42 4.3 酵素活性檢測標準迴歸直線與確認.......................46 4.4 St. John’s wort對年輕大鼠肝臟酵素活性的影響.........46 4.4.1 年輕大鼠肝臟CYP1A活性.............................46 4.4.2 年輕大鼠肝臟CYP2E1活性............................51 4.4.3 年輕大鼠肝臟CYP3A活性.............................51 4.5 St. John’s wort對年長大鼠肝臟酵素活性的影響.........54 4.5.1 年長大鼠肝臟CYP1A活性.............................54 4.5.2 年長大鼠肝臟CYP2E1活性............................54 4.5.3 年長大鼠肝臟CYP3A活性.............................57 4.6 大鼠成年組與年長組肝臟CYP活性之比較..................57 4.7 St. John’s wort對大鼠小腸CYP3A活性的影響............59 第五章 討論..............................................61 5.1 實驗方法討論.........................................61 5.1.1 實驗動物處理.......................................61 5.1.2 微粒體製備.........................................61 5.1.3 大鼠小腸微粒體製備問題探討.........................62 5.1.4 蛋白質測定方法.....................................63 5.1.5 活性測定方法.......................................64 5.2 實驗結果討論.........................................67 5.2.1 肝臟重量與體重的關係...............................67 5.2.2 大鼠肝臟微粒體蛋白質含量...........................67 5.2.3 St. John’s wort對大鼠肝臟CYP1A活性影響之探討......69 5.2.4 St. John’s wort劑量對年輕組大鼠肝臟CYP活性之影響..70 5.2.5 年輕大鼠與年長大鼠肝臟CYP活性測定結果之探討........72 5.2.6 St. John’s wort對大鼠小腸CYP3A活性影響之探討......73 5.3 綜合討論：St. John’s wort對大鼠CYP的影響以及與indinavir的藥品交互作....................................75 第六章 結論與未來方向....................................77 參考文獻.................................................7

Application of Rat In Situ Single-pass Intestinal Perfusion in the Evaluation of Presystemic Extraction of Indinavir Under Different Perfusion Rates

First-pass effect has been an important concern for oral pharmaceuticals. An in vivo system was developed for measuring different concentrations of pharmaceuticals in the portal vein and hepatic vein (via the inferior vena cava) for delineating presystemic metabolism under different perfusion rates by using indinavir as an exemplary agent. Methods: An in situ single-pass intestinal perfusion technique was modified from previous studies to concomitantly obtain portal and hepatic venous bloods. Portal and hepatic venous samples were simultaneously taken from rats at appropriate time points using the perfusion model of 1 mg/mL indinavir at flow rates of 0.05, 0.1, 0.5 and 1.0 mL/min. The indinavir concentrations were assayed by binary-gradient high-pressure liquid chromatography with UV detection. Results: The mean indinavir concentrations in portal vein concentration−time profiles at different perfusion times under various flow rates were all higher than those obtained for hepatic veins. At flow rates of 0.5 and 1.0 mL/min, in particular, the area under the curve (AUC) and maximal concentration (C max) of indinavir absorption were significantly different between portal veins and hepatic veins (p < 0.05), indicating considerable hepatic involvement in the presystemic extraction of indinavir. The system also has potential for use when estimating the hepatic extraction ratio (E H) and hepatic clearance (Cl H). Conclusion: This in vivo approach could provide another useful tool for improving our basic understanding of the absorption kinetics and hepatic metabolism of pharmaceuticals under development and facilitating the clinical application of such

Single gyroid-structured metallic nanoporous spheres fabricated from double gyroid-forming block copolymers via templated electroless plating

Herein, we suggest a methodology for the fabrication of well-defined metallic nanoporous spheres with single gyroid (SG) structure by simply using self-assembled diblock copolymer with double gyroid (DG) structure as a template for electroless plating. Note that owing to the consideration of thermodynamic stability, the self-assembly of diblock copolymers gives rise to a DG phase instead of an SG phase. By controlling the nucleus density for the reduction of Pd ions within the diblock copolymer template, SG-structured Ni can be easily fabricated through the nucleation and growth processes. Consequently, nanoporous Ni spheres with uniform pore sizes and high specific surface areas can be fabricated. Moreover, nanoporous Ni spheres with controlled microscale particle sizes can thus be obtained by controlling the reduction time for the growth of Ni, which enables the feasibility of recyclability via magnetic fields. The combination of structural and morphological characteristics of the fabricated nanoporous Ni spheres make them appealing for use in a wide variety of applications, such as high-efficiency and well selectivity hydrogenation catalysts with recyclability due to their narrow pore size distributions, high specific surface areas, 3D curved surfaces, and controlled microscale particle sizes

Phenethyl isothiocyanate triggers apoptosis in human malignant melanoma A375.S101 cells through reactive oxygen species and the mitochondria-dependent pathways

[[abstract]]We have reported previously that phenethyl isothiocyanate (PEITC) induces apoptosis in human osteosarcoma U-2 OS cells. Cytotoxic activity of PEITC towards other cancer cells such as human malignant melanoma and skin cancer cells has not been reported. In this study, the anticancer activity of PEITC towards human malignant melanoma cancer A375.S2 cells was investigated. To determine the mechanisms of PEITC inhibition of cell growth, the following end points were determined in A375.S2 cells: cell morphological changes, cell cycle arrest, DNA damage and fragmentation assays and morphological assessment of nuclear change, reactive oxygen species (ROS) and Ca(2+) generations, mitochondrial membrane potential disruption, and nitric oxide and 10-N-nonyl acridine orange productions, expression and activation of caspase-3 and -9, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, poly (adenosine diphosphate-ribose) polymerase, and cytochrome c release, apoptosis-inducing factor and endonuclease G. PEITC induced morphological changes in time- and dose-dependent manner. PEITC induced G2/M phase arrest and induced apoptosis via endoplasmic reticulum stress-mediated mitochondria-dependent pathway. Western blot analysis showed that PEITC promoted Bax expression and inhibited Bcl-2 expression associated with the disintegration of the outer mitochondrial membrane causing cytochrome c release, and activation of caspase-9 and -3 cascade leading to apoptosis. We conclude that PEITC-triggered apoptotic death in A375.S2 cells occurs through ROS-mediated mitochondria-dependent pathways