比阿培南在人肝微粒体、人肾S9和体外人血浆中的稳定性及其代谢产物鉴定

目的:考察比阿培南在人肝微粒体、人肾S9和体外人空白血浆中的代谢稳定性,并推测代谢产物的结构及可能的代谢途径.方法:采用高效液相色谱-串联质谱联用仪(HPLC-MS/MS)检测比阿培南分别与人肝微粒体、人肾S9和人空白血浆孵育后孵育液中剩余的比阿培南含量,比较代谢稳定性.此外,利用快速液相色谱-离子阱-飞行时间质谱联用...     

Production and selection of antigen-specific fully human monoclonal antibodies from mice engineered with human Ig loci

The ability to produce highly specific fully human monoclonal antibodies to human antigens has potential significant applications to human therapy. This review describes the creation of novel mouse strains engineered to produce a diverse repertoire of fully human antibodies in the absence of mouse antibodies. These mouse strains have been generated by introducing megabase-sized human immunoglobulin loci, containing the majority of the human antibody gene repertoire, in nearly germline configuration, into mice deficient in mouse antibody production. The mice produce high levels of human IgMkappa and IgGkappa antibodies with a diverse adult-like repertoire. Upon immunization with multiple human antigens the mice generate high affinity, antigen-specific fully human monoclonal antibodies with neutralization activity. Comparison of these mice to other strains containing limited human antibody gene repertoire underscores the importance of the large number of variable genes for faithful reproduction of functional and diverse human antibody response in mice.     

The long-awaited magic bullets: therapeutic human monoclonal antibodies from transgenic mice

The ability to produce a diverse repertoire of fully human monoclonal antibodies (mAbs) may have significant applications to human therapy. This update describes the creation of a novel tool for the production of therapeutic human mAbs: a mouse strain engineered to produce a large range of human antibodies in the absence of mouse antibodies. This strain, XenoMouse, has been generated by the introduction of large segments of human immunoglobulin loci, containing the majority of the human antibody gene repertoire, into mice deficient in mouse antibody production. The mice produce a diverse array of authentic fully human IgGkappa antibodies. Upon immunisation with multiple human antigens the mice generate large panels of high affinity, antigen-specific fully human mAbs with therapeutic activities. XenoMouse-derived hybridomas were shown to be stable, producing significant levels of human mAbs. XenoMouse technology represents an efficient and reliable tool for the production of therapeutic human mAbs, which can accelerate the evaluation and validation of antibody therapy in human disease.     

Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors

The purpose of this study was to elucidate the role of human organic anion transporters (human OATs) in the induction of drug-induced skeletal muscle abnormalities. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been clinically used for lowering plasma cholesterol levels, and are known to induce various forms of skeletal muscle abnormalities including myopathy and rhabdomyolysis. Immunohistochemical analysis revealed that human OAT1 and human OAT3 are localized in the cytoplasmic membrane of the human skeletal muscles. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs. Human OAT3, but not human OAT1, mediates the transport of pravastatin. Fluvastatin inhibited organic anion uptake mediated by human OAT1 in a mixture of competitive and noncompetitive manner, whereas simvastatin and fluvastatin noncompetitively inhibited the organic anion uptake mediated by human OAT3. In conclusion, the organic anion transporters OAT1 and OAT3 are localized in the cytoplasmic membrane of human skeletal muscles. Pravastatin, simvasatin, and fluvasatin inhibit human OATs activity. These results suggest that muscle organic anion transporters play a role in the muscular side effects of HMG-CoA reductase inhibitors.     

Species difference in stereoselective involvement of CYP3A in the mono-N-dealkylation of disopyramide.

1. To determine which CYP isoenzyme is involved in the N-dealkylation of disopyramide (DP) metabolism in human and dog, and to determine the stereoselectivity of DP metabolism with human CYP and dog CYP isoenzymes, the following in vitro metabolism studies of DP were conducted: correlation between human CYP isoenzyme activities and DP metabolism with human liver microsomes; inhibition of DP metabolism in human and dog liver microsomes with chemical inhibitors of CYP isoenzymes; inhibition of DP metabolism in human microsomes with human CYP antibodies; inhibition of DP metabolism in dog liver microsomes with human and dog CYP antibodies; metabolism of DP with human (CYP3A4) and dog (CYP3A12) cDNA-expressed isoenzymes; determination of Km and Vmax of DP enantiomers by using cDNA-expressed CYP3A4 and CYP3A12. 2. In human liver microsomes, the formation of the mono-N-dealkylated disopyramide (MNDP) metabolite was best correlated with CYP3A4 activities. DP metabolism was substantially inhibited by ketoconazole, troleandomycin (TA) and human CYP3A4 antibody. DP was metabolized by cDNA-expressed CYP3A isoenzymes. In dog liver microsomes, DP metabolism was inhibited by ketoconazole, TA and dog anti-CYP3A12. DP was also metabolized by cDNA-expressed CYP3A12. 3. CYP3A4 and CYP3A12 are the principal isoenzymes involved in DP metabolism in human and dog respectively. There was no stereoselectivity in N-dealkylation of DP by human CYP3A4. However, there was notable stereoselectivity in the N-dealkylation by dog CYP3A12.     

肿瘤坏死因子相关凋亡诱导配体的体内体外抗人肝细胞癌的作用

目的：研究Bcl-2蛋白的过表达对Trail诱导肝癌细胞凋亡的影响,以及Trail蛋白体内和体外对人肝癌细胞的杀伤作用。方法：克隆Trail基因并在大肠杆菌中表达Trail重组蛋白。检测Trail重组蛋白在体内和体外对肿瘤的杀伤作用。用台盼蓝拒染法检测细胞的凋亡率。将真核细胞表达质粒pcDNA3-Bcl-2转染到人肝癌细胞BEL－7404细胞中,并通过G418 400mg/L筛选得到Bcl-2蛋白稳定表达的细胞株。结果：重组蛋白Trail能够有效地杀死人肝癌细胞（包括BEL－7404,SMMC－7721,和BEL－7402等细胞）。在体外,过量表达的Bcl-2蛋白能抑制Trail重组蛋白对人肝癌细胞BEL－7404的杀伤作用。重组蛋白Trail能够有效地抑制人肝癌细胞BEL－7404在裸鼠中形成实体瘤。结论：在体内和体外,Trail重组蛋白能够杀死肝癌细胞,Bcl-2蛋白过表达可以抑制Trail重组蛋白对肝癌细胞BEL－7404的杀伤作用。Trail蛋白是一种潜在的肝癌治疗剂。     

异氟醚通过激活Ryanodine受体对ReNcell CX人神经干细胞损伤和存活发挥双向调节作用

目的研究Ryanodine受体在吸入性麻醉剂异氟醚对ReNcell CX人神经干细胞损伤和存活的影响中的作用。方法将培养的ReNcell CX人神经干细胞应用2.6%异氟醚分别处理1、4、8、12 h后,测量LDH和MTT,研究异氟醚对ReNcell CX人神经干细胞存活的时间效应;在异氟醚处理前加入Ryanodine受体特异性阻滞剂100 nM丹曲林,再测量LDH和MTT,研究阻断Ryanodine受体能否消除异氟醚对ReNcell CX人神经干细胞存活的影响。结果 2.6%异氟醚处理1 h,LDH低于对照组,MTT高于对照组(P<0.05),说明促进ReNcell CX人神经干细胞存活;2.6%异氟醚处理12 h,LDH高于对照组,MTT低于对照组(P<0.05),说明抑制ReNcell CX人神经干细胞存活;加入Ryanodine受体阻滞剂丹曲林后,再用2.6%异氟醚处理12 h,LDH和MTT与对照组差异无统计学意义(P>0.05),说明异氟醚通过激活Ryanodine受体影响ReNcell CX人神经干细胞存活。结论异氟醚对ReNcell CX人神经干细胞损伤和存活具有双向作用,这种作用通过内质网膜上Ryanodine受体介导。     

Implications of the lack of accuracy of the lifetime rodent bioassay for predicting human carcinogenicity

The NTP lifetime rodent bioassay (LRB) is the "gold standard" for predicting human carcinogenicity. Unfortunately, little attempt has been made to validate it against human carcinogenicity. Here we show that the extremely limited data available do not support either of the two common interpretations of LRB results. If a risk-avoidance interpretation is used where any positive result in a sex/species combination is considered positive, 9 of the 10 known human carcinogens tested are positive, but an implausible 22% of all chemicals are positive. If a less risk averse interpretation is used where only chemicals positive in both rats and mice are considered positive, only 3 of the 6 known human carcinogens tested are positive. In either interpretation, some known human carcinogens are not positive in the LRB, potentially allowing widespread human exposure to misidentified chemicals. Improving the predictive accuracy of the LRB and other tests for human carcinogenicity requires that test results be validated against the known human carcinogenicity of chemicals. This will require redirecting available resources from screening chemicals to validating carcinogenicity tests as well as a substantial investment in epidemiology to identify more known human carcinogens and presumed human non-carcinogens.     

尿液荧光定量聚合酶链法测定人巨细胞病毒的临床价值

目的 探讨荧光定量聚合酶链法在婴幼儿尿液人巨细胞病毒检测中的临床价值.方法 选择患者40例,所有患者入院后进行抗病毒及对症支持治疗,应用荧光定量聚合酶链法测定患者治疗前后的人巨细胞病毒-DNA、人巨细胞病毒-IgM,并比较治疗前后不同时间段尿液中的人巨细胞病毒-DNA水平.结果 治疗后,人巨细胞病毒-DNA、人巨细胞病毒-IgM阳性率均低于治疗前（P＜0.05）,荧光定量聚合酶链法测定其阳性率均＞90％.治疗后晨尿、上午随机尿液及下午随机尿液中人巨细胞病毒-DNA水平低于治疗前同时段（P＜0.05）.结论 荧光定量聚合酶链法在早期诊断婴幼儿尿液人巨细胞病毒感染中具有重要价值.     

Gain of function mutation of the alpha7 nicotinic receptor: distinct pharmacology of the human alpha7V274T variant

In the human alpha7 nicotinic receptor, valine-274 in the pore-lining transmembrane-2 region was mutated to threonine to produce the variant human alpha7V274T, which was evaluated electrophysiologically following expression in Xenopus laevis oocytes. Inward current rectification was strong in human alpha7V274T as in the human alpha7 wild type nicotinic receptor. However, human alpha7V274T was 100-fold more sensitive to the nicotinic receptor agonists acetylcholine, (-)-nicotine and 1,1-dimethyl-4-phenylpiperazinium. Choline also activated human alpha7V274T (EC50 = 12 microM) and was 82-fold more potent than at human alpha7 wild type nicotinic receptor. (-)-Cotinine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine (ABT-089), weak partial agonists at human alpha7 wild type, were much stronger agonists at human alpha7V274T with EC50 values of 70 microM, 4 microM and 28 microM and fractional activation values of 93%, 96% and 40%, respectively. However, (-)-lobeline, a human alpha7 wild type nicotinic receptor antagonist, and dihydro-beta-erythroidine, which activates chick mutagenized alpha7 nicotinic receptors, had only weak agonist-like activity at human alpha7V274T (< or = 4% of the maximal acetylcholine response). Methyllycaconitine, mecamylamine, d-tubocurarine and dihydro-beta-erythroidine retained antagonist activity and, indeed, appeared to be at least as potent at human alpha7V274T as at human alpha7 wild type. These results support and extend the concept that human nicotinic receptor pharmacology can be profoundly altered by single amino acid changes in the pore-lining segment.     

人精子染色体分析方法及其在遗传和毒理学中的应用

环境化学物质对人类生殖细胞的诱变作用研究甚少,但其意义重大,因为生殖细胞的遗传损伤有可能对下一代产生不良影响。1978年Rudak等人利用去透明带金黄地鼠卵与人精子在体外异种受精,获得人精子染色体中期相标未,建立了人精子染色体分析方法。十年来共分析100多位正常人21390组精子染色体核型,平均结构染色体畸变率为11.5％;非整倍体率为1.4%。人精子在体外经辐射线(X线等)照射后,染色体畸变率明显升高,与剂量呈线性关系。martin等人观察了18名经放疗/化疗以及经化疗后的肿瘤患者,治疗后精子染色体畸变率亦明显升高,且在数年至十多年后仍高于正常人。人精子染色体分析方法可用于环境化学物质对人类生殖细胞(精子)诱变作用的监测。     

S-oxidation of S-methyl-esonarimod by flavin-containing monooxygenases in human liver microsomes

1. Studies using human liver microsomes and recombinant human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the metabolism of S-methyl-esonarimod (M2), an active metabolite of esonarimod (KE-298, a novel antirheumatic drug). 2. S-oxidative activities of M2 significantly correlated with those of methyl p-tolyl sulfide, a specific substrate of FMOs, as tested using 10 different human liver microsomes (r(2) = 0.539, p<0.05). Thermal treatment of microsomes reduced the S-oxidative activity in the absence of the NADPH-generating system at 45 degrees C for 5 min. However, methimazole, a known competitive substrate of FMOs, was a weak inhibitor of the S-oxidation in liver microsomes. 3. Recombinant human FMO1 and FMO5 produced M3 in greater quantities than recombinant human FMO3. The S-oxidation of M2 by recombinant human FMO5 was not appreciably inhibited in the presence of methimazole. In contrast, methimazole was effective in suppressing the catalytic activity of recombinant human FMO1 and FMO3. 4. The apparent K(m) (K(m app)) for the S-oxidation of M2 in human recombinant FMO5 (2.71 microM) was similar to that obtained using human liver microsomes (2.43 microM). 5. The present results suggest that the S-oxidation of S-methyl esonarimod reflects FMO5 activity in the human liver because the recombinant FMO5 data match well with the human liver microsomal experiments.     

Selection of Suitable Prodrug Candidates for in vivo Studies via in vitro Studies; The Correlation of Prodrug Stability in Between Cell Culture Homogenates and Human Tissue Homogenates

Purpose. To determine the correlations/discrepancies of drug stabilities between in the homogenates of human culture cells and of human tissues. Methods. Amino acid/dipeptide monoester prodrugs of floxuridine were chosen as the model drugs. The stabilities (half-lives) of floxuridine prodrugs in human tissues (pancreas, liver, and small intestine) homogenates were obtained and compared with ones in cell culture homogenates (AcPC-1, Capan-2, and Caco-2 cells) as well as human liver microsomes. The correlations of prodrug stability in human small bowel tissue homogenate vs. Caco-2 cell homogenate, human liver tissue homogenate vs. human liver microsomes, and human pancreatic tissue homogenate vs. pancreatic cell, AsPC-1 and Capan-2, homogenates were examined. Results. The stabilities of floxuridine prodrugs in human small bowel homogenate exhibited the great correlation to ones in Caco-2 cell homogenate (slope = 1.0-1.3, r2 = 0.79-0.98). The stability of those prodrugs in human pancreas tissue homogenate also exhibited the good correlations to ones in AsPC-1 and Capan-2 cells homogenates (slope = 0.5-0.8, r2 = 0.58-0.79). However, the correlations of prodrug stabilities between in human liver tissue homogenates and in human liver microsomes were weaker than others (slope = 1.3-1.9, r2 = 0.07-0.24). Conclusions. The correlations of drug stabilities in cultured cell homogenates and in human tissue homogenates were compared. Those results exhibited wide range of correlations between in cell homogenate and in human tissue homogenate (r2 = 0.07 - 0.98). Those in vitro studies in cell homogenates would be good tools to predict drug stabilities in vivo and to select drug candidates for further developments. In the series of experiments, 5'-O-D-valyl-floxuridine and 5'-O-L-phenylalanyl-L-tyrosyl-floxuridine would be selected as candidates of oral drug targeting delivery for cancer chemotherapy due to their relatively good stabilities compared to other tested prodrugs. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.     

Genotoxicity of carcinogens in human hepatocytes: application in hazard assessment

Evaluation of chemical genotoxicity has been used in assessing human cancer hazard, based on the observation that most human carcinogens are known to be DNA-reactive. The availability of data on the DNA-reactivity of compounds in metabolically competent human cells would assist hazard assessment by providing direct information of human genotoxicity. To evaluate the reliability of human hepatocytes for this purpose, the induction of DNA repair by DNA-reactive carcinogens of several structural classes and related noncarcinogens was studied. All the carcinogens elicited DNA repair synthesis, whereas the noncarcinogens did not. These studies provide additional support for the use of human hepatocytes in a DNA repair test in the investigation of genotoxicity. The demonstration of genotoxicity in human cells is suggested to provide important information for hazard assessment.     

The nonlinear pharmacokinetics of prednisone and prednisolone. II. Plasma protein binding of prednisone and prednisolone in rabbit and human plasma

The protein binding characteristics of prednisone and prednisolone were determined in human and rabbit plasma and in a 4.7 per cent human serum albumin (HSA) solution. The influence of prednisolone on prednisone binding in human plasma was also examined. Prednisolone exhibited nonlinear binding and prednisone linear binding characteristics in both human and rabbit plasma. Prednisone binding was not influenced by the presence of prednisolone. Prednisone binding to HSA was linear but to a degree substantially lower than observed in human plasma, suggesting the possibility that prednisone binds to other proteins in human plasma. The results support the hypothesis that the protein binding characteristics of prednisone and prednisolone do not explain the reported nonlinear pharmacokinetics of prednisone.     

Dermal absorption of mucopolysaccharide polysulfate (heparinoid) in human and minipig

Dermal absorption of mucopolysaccharide polysulfate (MPS, the active ingredient of Hirudoid") in human and minipig was investigated by using 14C-labeled MPS. Three types of human and minipig skin samples were used: intact, dried and tape-stripped. At 24 h after application of 14C-MPS to intact human skin on a Franz cell in vitro, the radioactivity was detected in 0.98, 1.34, and 0.08% of the applied dose in stratum corneum, epidermal-dermal skin, and receptor fluid, respectively. In dried human skin, the amount of radioactivity detected was similar to that in intact human skin. By contrast, in tape-stripped human skin, higher radioactivity was detected in epidermal-dermal skin and receptor fluid (2.85 and 0.33% of the applied dose, respectively) than in intact or dried skin. Minipig skin showed 1.5 to 4.5 times greater dermal absorption of 14C-MPS, as compared with human skin. In an in vivo study with minipig, radioactivity was detected at the dosing skin site after dermal administration of 14C-MPS. The stability of 14C-MPS in human skin after dermal application was evaluated by agarose gel electrophoresis and ion-exchange chromatography. It was suggested that 14C-MPS absorbed into human skin would be stable because the chromatogram behaviors of the radioactivity on the two types of method were not shifted. Microautoradiography of human and minipig skins after 14C-MPS dosing showed that radioactivity was widely distributed in the epidermis in the area near hair follicles. The present results clearly demonstrate that MPS is stable and that a small fraction of it is percutaneously absorbed by human and minipig skin.     

Regional haemodynamic effects of human and rat adrenomedullin in conscious rats.

1. Male, Long Evans rats were chronically instrumented with pulsed Doppler flow probes and intravascular catheters to permit assessment of the regional haemodynamic responses to human and rat adrenomedullin, to compare the responses to human adrenomedullin to those of human alpha-CGRP in the absence and presence of the CGRP1-receptor antagonist, human alpha-CGRP [8-37], and to determine the involvement of nitric oxide (NO)-mediated mechanisms in the responses to human adrenomedullin, relative to human alpha-CGRP. 2. Human and rat adrenomedullin (0.3, 1, and 3 nmol kg-1, i.v.) caused dose-dependent hypotension and tachycardia, accompanied by increases in renal, mesenteric and hindquarters flows and vascular conductances. At the lowest dose only, the hypotensive and mesenteric vasodilator effects of rat adrenomedullin were significantly greater than those of human adrenomedullin. 3. Human alpha-CGRP at a dose of 1 nmol kg-1 caused hypotension, tachycardia and increases in hindquarters flow and vascular conductance, but reduction in renal and mesenteric flows, and only transient vasodilatations in these vascular beds. These effects were substantially inhibited by human alpha-CGRP [8-37] (100 nmol kg-1 min-1), but those of human adrenomedullin (1 nmol kg-1) were not; indeed, the mesenteric haemodynamic effects of the latter peptide were enhanced by the CGRP1-receptor antagonist. 4. In the presence of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 183 nmol kg-1 min-1), there was only a slight, but significant, inhibition of the hindquarters hyperaemic vasodilator effect of human adrenomedullin, but not that of human alpha-CGRP.(ABSTRACT TRUNCATED AT 250 WORDS)