三种茶饮料对小鼠肠道首过效应及肝脏Cyp3a、Cyp2e1的影响

目的:研究市售罐装绿茶、冰红茶及茉莉清茶饮料对小鼠肠道首过效应和肝脏细胞色素氧化酶(cytochrome P450,CYP)Cyp3a、Cyp2e1的影响。方法:SPF级小鼠随机分为6组,其中绿茶组、冰红茶组、茉莉清茶组小鼠分别自由饮用绿茶、冰红茶、茉莉清茶7 d。用血中扑热息痛(ac-etaminophen,APAP)的浓度推断肠道首过效应。采用差速离心法分离肝/肠微粒体,以Bradford法定量蛋白,紫外分光光度法定量Cyp3a、Cyp2e1及血中APAP浓度。结果:绿茶组血中APAP浓度较空白组明显升高(P<0.01),而冰红茶组与空白组相比则显著降低(P<0.01);绿茶组和冰红茶组小肠Cyp3a酶活性较空白组显著升高(P<0.05或P<0.01)。各供试物组肝脏Cyp3a酶活性较空白组明显升高(P<0.01),各供试物组肝脏Cyp2e1酶活性较空白组显著降低(P<0.01)。结论:绿茶、冰红茶能显著改变肠道首过效应(绿茶抑制肠道P-gp,诱导肠道Cyp3a;冰红茶则诱导肠道P-gp及肠道Cyp3a;而茉莉清茶对肠道首过效应无影响)。各供试物均诱导肝脏Cyp3a的酶活性,抑制肝脏Cyp2e1的酶活性。故在服用经CYP3A、CYP2E1和P-gp代谢/转运的药物期间,大量或经常饮用上述茶可能影响这些药物的临床疗效和/或不良反应。     

柴胡总皂苷对小鼠肠道首过效应和肝脏Cyp3a、Cyp2e1的影响

目的:研究柴胡总皂苷对小鼠肠道首过效应(Cyp3a,P-糖蛋白)和肝脏细胞色素氧化酶(Cyp3a,Cyp2e1)的影响。方法:供试物灌胃给小鼠2次/d,连续3 d。实验当日,对乙酰氨基酚(Acetaminophen,APAP;P-gp底物)以50 mg/kg经口投予后60 min断头采血,并摘取肝脏和全段小肠。以分光光度法测定血中APAP浓度;用梯度离心法分离小鼠肝/肠微粒体;以分光光度法检测微粒体中Cyp3a/Cyp2e1活性;以实时荧光定量法测定Cyp3a11/Cyp2e1 mRNA在小鼠肝脏中的表达。结果:血中APAP浓度测定结果和P-糖蛋白偶联的ATP酶活性测定结果显示,柴胡总皂苷各剂量组与对照组之间均无统计学差异(P>0.05);在肝脏和肠道微粒体实验中不论以氨基吡啉还是以红霉素为底物测定Cyp3a,仅有柴胡总皂苷高剂量组(150 mg/kg)的Cyp3a活性显著高于对照组(P<0.05);仅有柴胡总皂苷中剂量组(75 mg/kg)的Cyp2e1活性显著低于对照组(P<0.05);RT-PCR结果显示,仅有柴胡总皂苷高剂量(150 mg/kg)时能够诱导Cyp3a11在肝脏中的表达。结论:柴胡总皂苷对小鼠肝脏和肠道中的Cyp3a具有一定的诱导作用,对肝脏中的Cyp2e1具有一定的抑制作用,对小鼠肠道P-糖蛋白的转运活性无影响。     

Intestinal flora induces the expression of Cyp3a in the mouse liver

1. In order to determine the effects of intestinal flora on the expression of cytochrome P450 (CYP), the mRNA expression of CYP was compared between specific pathogen-free (SPF) and germ-free (GF) mice.

2. Most of the major CYP isozymes showed higher expression in the livers of SPF mice compared with GF mice.

3. Nuclear factors such as pregnane ? receptor (PXR) and constitutive androstane receptor (CAR), as well as transporters and conjugation enzymes involved in the detoxification of lithocholic acid (LCA), also showed higher expression in SPF mice.

4. The findings suggest that in the livers of SPF mice, LCA produced by intestinal flora increases the expression of CYPs via activation of PXR and CAR.

5. Drugs such as antibiotics, some diseases and ageing, etc. are known to alter intestinal flora. The present findings suggest that such changes also affect CYP and are one of the factors responsible for individual differences in pharmacokinetics.

     

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver

Abstract

1.?Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans.

2.?Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice.

3.?Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans.

4.?Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans.

5.?The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.     

Differences in the pharmacokinetics of Cyp3a substrates in TSOD and streptozotocin-induced diabetic mice

1. The pharmacokinetics of drugs can change in diabetes mellitus and even among diabetics. They may differ between type I diabetes (T1DM) and type 2 diabetes (T2DM).

2. As triazolam was administered orally to Tsumura, Suzuki, obese, diabetes (TSOD) mice and streptozotocin (STZ) mice, clearance per body (CL/F) in TSOD mice did not differ compared with Tsumura, Suzuki, non-obesity (TSNO) mice. In STZ mice, CL/F was greater than in control mice. Small intestinal cytochrome P450 (Cyp) 3a expression in TSOD mice was significantly lower than in TSNO mice. No significant difference existed in small intestinal Cyp3a expression between STZ mice and control mice. In insulin-treated mice, small intestinal Cyp3a expression was significantly lower than in control mice.

3. These results suggested that the differences in changes in small intestinal Cyp3a expression between T1DM and T2DM may be due to differences in plasma insulin concentrations. This may be a factor in the difference in the drug pharmacokinetics between T2DM and T1DM patients.

     

Alterations in gene expression in vitamin D‐deficiency: Down‐regulation of liver Cyp7a1 and renal Oat3 in mice

The vitamin D‐deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D‐deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α‐hydroxylase (Cyp7a1, rate‐limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up‐regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D‐deficiency. When vitamin D‐sufficient or D‐deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D‐deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D‐sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)₂D₃/cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D‐deficient mice, while 1,25(OH)₂D₃ treatment led to hypercalcemia. In conclusion, vitamin D‐deficiency resulted in down‐regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.     

Identification and characterization of metabolites of ASP015K,a novel oral Janus kinase inhibitor,in rats,chimeric mice with humanized liver,and humans

Abstract

1.?Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice).

2.?Rat biological samples collected after oral dosing of ¹⁴C-labelled ASP015K were examined using a liquid chromatography–radiometric detector and mass spectrometer (LC–RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC–MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance.

3.?Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.     

Roles of cytochrome P450 3A enzymes in the 2-hydroxylation of 1,4-cineole,a monoterpene cyclic ether,by rat and human liver microsomes

1. Oxidation of 1,4-cineole, a monoterpene cyclic ether, was studied in rat and human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes expressed in insect cells in which human P450 and NADPH-P450 reductase cDNAs have been introduced. On analysis with gas chromatography/mass spectrometry, 2- exo -hydroxy-1,4-cineole was identified as a principal oxidation product of 1,4-cineole catalysed by rat and human P450 enzymes. 2. CYP3A4 was a major enzyme involved in the 2-hydroxylation of 1,4-cineole by human liver microsomes, based on the following lines of evidence. First, 1,4-cineole 2-hydroxylation activities catalysed by human liver microsomes were inhibited by ketoconazole, a potent inhibitor of CYP3A activities, and an anti-CYP3A4 antibody. Second, there was a good correlation between CYP3A4 contents and 1,4-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples examined. Finally, of 10 recombinant human P450 enzymes examined, CYP3A4 had the highest activity for 1,4-cineole 2-hydroxylation. 3. Liver microsomal 1,4-cineole 2-hydroxylation activities were induced in rat by pregnenolone 16 α-carbonitrile and dexamethasone and extensively inhibited by ketoconazole, indicative of the possible roles of CYP3A enzymes in this reaction. 4. Kinetic analysis showed that V max / K m for 1,4-cineole 2-hydroxylation catalysed by liver microsomes was higher in a human sample HL-104 (4.6 μM -1?min -1) than those of rat treated with pregnenolone 16 α-carbonitrile (0.49 μM -1?min -1) and dexamethasone (0.36 μM -1?min -1). 5. 1,8-Cineole, a structurally related monoterpene previously shown to be catalysed by CYP3A enzymes, inhibited 1,4-cineole 2-hydroxylation catalysed by human liver microsomes, whereas 1,4-cineole did not inhibit 1,8-cineole 2-hydroxylation activities. Both compounds caused inhibition of testosterone 6 β -hydroxylation by human liver microsomes, the former compound being more inhibitory than the latter. 6. These results suggest that 1,4-cineole and 1,8-cineole, two plant essential oils present in Citrus medica L. var. acida and Eucalyptus polybractea, respectively, are converted to 2-hydroxylated products by CYP3A enzymes in rat and human liver microsomes. It is unknown at present whether the 2-hydroxylation products of these compounds are more active biologically than the parent compound.     

Use of MGLUR2 and MGLUR3 knockout mice to explore in vivo receptor specificity of the MGLUR2/3 selective antagonist LY341495

LY341495 is a metabotropic glutamate receptor (mGluR) antagonist showing selectivity to mGluR2/3 but having measurable antagonist efficacy across all mGluR subtypes at 10-1000 fold higher concentrations. In vivo in rodents it increases locomotor activity and wakefulness, enhances cognition and modulates emotions. It also induces widespread neuronal activation measured as c-Fos expression. To further investigate the receptor subtypes through which LY341495 might act in vivo we analyzed how its effects are altered in mGluR2-knockout (KO) and mGluR3-KO brains. In most regions, LY341495 (3 mg/kg, i.p., 2.5 h) -induced c-Fos expression was not altered in either KO brain. However, in mGluR3-KO mice, LY341495 was almost inactive in the central extended amygdala [central nucleus of the amygdala, lateral (CeL) and bed nucleus of the stria terminalis, laterodorsal (BSTLD)], suggesting that acute blockade of mGluR3 is activating these neurons in wildtype brain. In the ventrolateral nucleus of the thalamus (VL), LY341495 produced a significantly enhanced response in mGluR3-KO mice and attenuated response in mGluR2-KO mice. We also analyzed locomotion in familiar environment and found that locomotor activity was dose-dependently increased by LY341495 (1-30 mg/kg, i.p.) regardless of the genotype. In unfamiliar environment, both KO strains showed enhanced sensitivity to LY341495 in reducing locomotor habituation. Together our results indicate that certain effects of LY341495 may not be mediated by a blockade of either mGluR2 or mGluR3, but may involve other mGluR subtypes. Alternatively, functions of mGluR2 and mGluR3 may be redundant, resulting similar effects irrespective the receptor subtype being antagonized in vivo by LY341495.     

Unremarkable impact of Oatp inhibition on the liver concentration of fluvastatin,lovastatin and pitavastatin in wild-type and Oatp1a/1b knockout mouse

1. Oatp inhibitors have been shown to significantly increase the plasma exposure of statins. However, understanding alterations of liver concentration is also important. While modeling has simulated liver concentration changes, availability of experimental data is limited, especially when concerning drug–drug interactions (DDI). The objective of this work was to determine blood and liver concentrations of fluvastatin, lovastatin and pitavastatin, when blocking uptake transporters.

2. In wild-type mouse, rifampin pre-treatment decreased the unbound liver-to-plasma ratio (K_p,uu) of fluvastatin by 4.2-fold to 2.2, lovastatin by 4.9-fold to 0.81 and pitavastatin by 10-fold to 0.21. Changes in K_p,uu were driven by increases in systemic exposures as liver concentrations were not greatly altered.

3. In Oatp1a/1b knockout mouse (KO), rifampin exerted no additional effect on fluvastatin and lovastatin. Contrarily, rifampin further decreased pitavastatin K_p,uu by 3.4-fold, suggesting that the KO is inadequate to completely block liver uptake of pitavastatin as there are additional rifampin-sensitive uptake mechanism(s) not captured in the KO model.

4. This work provides experimental data showing that the plasma compartment is more sensitive to Oatp modulation than the liver compartment, even for rifampin-mediated DDI. Consistent with previous simulations, inhibiting or targeting Oatps may change K_p,uu, but exhibit only a minimal effect on absolute liver concentrations.     

Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience

Introduction

Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay.

Methods

Liver samples were obtained from ~ 1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored.

Results

Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250 mg/kg/day) to 160-fold (750 mg/kg/day), which declined slightly at 2000 mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750 mg/kg/day).

Discussion

These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay.     

Induction of liver microsomal cytochrome P-450 And associated monooxygenases by octachlorostyrene in inbred strains of mice: Lack of correlation with the murinE Ah locus

Single intraperitoneal injections of octachlorostyrene (OCS) and hexachlorobenzene in genetically polycyclic aromatic hydrocarbon ‘responsive’ C57/BL/6 (B6) mice led to a time- and dose-dependent increase in the levels of liver microsomal cytochromes P-450 and b₅ as well as in the activities of NADPH cytochrome P-450 (cytochrome c) reductase, ethylmorphine (EM) N-demethylase, 4-nitroanisole (PNA) O-demethylase and acetanilide 4-hydroxylase (AcA hydroxylase). No, or only a very moderate, increase in the activity of aryl hydrocarbon hydroxylase was seen after OCS and HCB, respectively. Pretreatments with phenobarbital (PB) or 3-methylcholanthrene (MC) both increased AcA hydroxylase activity to a similar degree, whereas pretreatment with polychlorinated biphenyls (Aroclor 1254) had an effect equal to the sum of PB and MC. Judged from sodium dodecylsulfate polyacrylamide gel electrophoresis studies, OCS and HCB predominantly increased a microsomal polypeptide of apparent mol. wt 52,000, similar to PB. A reduced response was seen after OCS or HCB treatment of aromatic hydrocarbon ‘non-responsive’ DBA/2 (D2) mice compared to B6 mice, both with respect to AcA hydroxylase as well as EM demethylase and PNA demethylase activities. OCS treatment of B6D2F1 mice resulted in a doubling of AcA hydroxylase activity, but in mice of the (B6D2)D2 backcross no distinct subgroupings of individual AcA hydroxylase activities were apparent. These results demonstrate that OCS is an inducer of the PB-type in mice and that induction of AcA hydroxylase by OCS is not regulated by the Ah locus.     

黄精成方治疗早期糖尿病肾病的荟萃分析

目的 对黄精成方治疗早期糖尿病肾病进行荟萃分析。方法 检索黄精成方治疗早期糖尿病肾病的随机对照研究（RCTs）,采用RevMan 5.3进行荟萃分析。结果 与对照组相比,黄精成方组治疗早期糖尿病肾病的有效率明显较高（P<0.000 01）,尿白蛋白排泄率下降也较明显（P<0.000 1）,但尿β2微球蛋白和血肌酐下降相当。结论 荟萃分析表明,黄精成方治疗早期糖尿病肾病疗效显著。但本次研究存在一定的局限性,仍需设计良好、多中心、大规模临床随机对照研究来验证。     

Effects of microsomal enzyme inducers in vivo and inhibitors in vitro on the covalent binding of benzo[a]pyrene metabolites to DNA catalyzed by liver microsomes from genetically responsive and nonresponsive mice.

The in vitro DNA binding of benzo[a]pyrene metabolites generated by mouse liver microsomes can be resolved into at least nine distinct peaks by elution of a Sephadex LH20 column with a water-methanol gradient. These peaks, representing metabolite-nucleoside complexes, are named A (most polar) through I (least polar). 3-Methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital, Aroclor 1254, pregnenolone-16α-carbonitrile, or ethanol was administered in vivo to genetically “responsive” C57BL/6N or “nonresponsive” DBA/2N mice, in an attempt to understand and identify increases or decreases in reactive BP intermediates that bind to DNA. Rises or falls in these peaks are also noted when liver microsomes from control or 3-methylcholanthrene-treated C57BL/6N or DBA/2N mice were incubated in vitro with [³H]benzo[a]pyrene and microsomal enzyme inhibitors such as α-naphthoflavone, metyrapone or cyclohexene oxide. All of our interpretations concerning the binding of metabolites to DNA are consistent with non-K-region oxygenation of benzo[a]pyrene being mediated predominantly by cytochrome(s) P₁-450 and K-region oxygenation of benzo[a]pyrene being catalysed predominantly by form(s) of P-450 other than P₁-450. All of the biological perturbations are consistent with the following assignments. The major reactive intermediate of benzo[a]pyrene contributing to each peak is suggested to be: peaks A and C, an unknown dihydrodiol oxide; peaks B, D, F and I, quinones oxygenated further (or quinone-derived free radicals); peak E, both cis- and tans-7,8-diol-9,10-epoxides; peak F′, the 7.8-oxide; peak G, the 4.5-oxide; and peak H, an unknown phenol oxide. The DNA nucleosides are not identified in this study. Of the ten peaks listed here, it is of interest that the major metabolite(s) contributing to eight of the peaks (all except peaks F' and G) involve(s) more than a single mono-oxygenation by forms of cytochrome P-450. All peaks, with the exception of peak G, appear to be predominantly associated with benzo[a]pyrene metabolism mediated by P₁-450 and, therefore, controlled by the Ah locus. The use of these microsomal enzyme inducers or inhibitors—combined with the underlying genetic predisposition of the individual, tissue, or cell culture system under study—demonstrates that the balance between P-450 and epoxide hydrase, and the ratio of each form of P-450 to the other forms of P-450, can influence markedly the quantity and quality of reactive intermediates of benzo[a]pyrene that bind to DNA.     

Review of the effects of 17α‐estradiol in humans: a less feminizing estrogen with neuroprotective potential

17α‐Estradiol is a less feminizing isomer of the potent hormonal estrogen, 17β‐estradiol. 17α‐Estradiol is an orally active small molecule with conflicting reports of efficacy in preclinical models of degenerative diseases. A number of studies suggest neuroprotective potential in human neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease. Several studies have established an antioxidant effect of 17α‐estradiol in humans. The sodium salt of 17α‐estradiol 3‐sulfate is a minor component (2.5–9.5%) of several widely marketed estrogen hormone replacement products, such as Premarin^®, that are approved by the U.S. Food and Drug Administration and have been prescribed and studied in women and men for more than 65 years. Most of the more than 100 published reports on the neurological effects of feminizing estrogens found positive responses in at least one measure relating to cognition or prevention and treatment of AD, notwithstanding the negative results in the Women's Health Initiative studies. Whether these limited, and often not statistically significant, findings are clinically meaningful remains unknown. In many in vitro and in vivo preclinical neuroprotection and related studies, 17α‐estradiol and 17β‐estradiol are active at similar concentrations and doses. However, 17α‐estradiol is less pleiotropic than 17β‐estradiol, and thus its potential toxicity might be lower. Given decades of mixed reports regarding the potential efficacy and safety of strongly feminizing hormones in neurodegenerative diseases, the weakly feminizing 17α‐estradiol might be a suitable candidate for clinical testing of the neuroprotective potential of this chemical class because it avoids, or significantly reduces, the adverse effects of potent hormonal compounds. Drug Dev Res 70:1–21, 2009. © 2009 Wiley‐Liss, Inc.     

In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: a comparison with human liver microsomes

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V(max) and intrinsic metabolic clearance (CL(int)) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL(int) and previously reported values of MMDX LD(50) for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation.     

Signal transduction pathways implicated in the decrease in CYP1A1, 1A2 and 3A6 activity produced by serum from rabbits and humans with an inflammatory reaction

Incubation of serum from rabbits with a turpentine-induced inflammatory reaction and from humans with an upper respiratory viral infection with hepatocytes from rabbits with a turpentine-induced inflammatory reaction for 4h reduces total cytochrome P450 content and activity of cytochrome P450 isoforms CYP1A1/1A2 and 3A6 without affecting the expression of these proteins. To document the signal transduction pathways implicated in the decrease in CYP1A1/1A2 and 3A6 activity, hepatocytes from rabbits with a turpentine-induced inflammatory reaction were incubated with serum from rabbits with a turpentine-induced inflammatory reaction, serum from individuals with a viral infection and interleukin-6 for 4h in presence of inhibitors of protein kinases. The sera-induced decrease in CYP1A1/1A2 and 3A6 activity was partially prevented by the inhibition of Janus-associated protein tyrosine kinase, double-stranded RNA-dependent protein kinase, protein kinase C, and p42/44 mitogen-activated protein kinase. The serum from rabbits with a turpentine-induced inflammatory reaction increased the phosphorylation of Erk1/2, effect prevented by PD98059 but not by bis-indolylmaleimide, a specific inhibitor of protein kinase C. The results demonstrated that the decrease in total cytochrome P450 content and in CYP1A1/1A2 and 3A6 activity by sera and interleukin-6 involves the activation of protein tyrosine kinases, p42/44 mitogen-activated protein kinase and protein kinase C. Indirect evidence supported that nitric oxide is implicated in the decrease in activity of these enzymes.