Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

Hypoxia aggravates non-alcoholic steatohepatitis in mice lacking hepatocellular PTEN

The metabolic disorders that predispose patients to NASH (non-alcoholic steatohepatitis) include insulin resistance and obesity. Repeated hypoxic events, such as occur in obstructive sleep apnoea syndrome, have been designated as a risk factor in the progression of liver disease in such patients, but the mechanism is unclear, in particular the role of hypoxia. Therefore we studied the influence of hypoxia on the development and progression of steatohepatitis in an experimental mouse model. Mice with a hepatocellular-specific deficiency in the Pten (phosphatase and tensin homologue deleted on chromosome 10) gene, a tumour suppressor, were exposed to a 10% O2 (hypoxic) or 21% O2 (control) atmosphere for 7 days. Haematocrit, AST (aspartate aminotransferase), glucose, triacylglycerols (triglycerides) and insulin tolerance were measured in blood. Histological lesions were quantified. Expression of genes involved in lipogenesis and mitochondrial beta-oxidation, as well as FOXO1 (forkhead box O1), hepcidin and CYP2E1 (cytochrome P450 2E1), were analysed by quantitative PCR. In the animals exposed to hypoxia, the haematocrit increased (60+/-3% compared with 50+/-2% in controls; P<0.01) and the ratio of liver weight/body weight increased (5.4+/-0.2% compared with 4.7+/-0.3% in the controls; P<0.01). Furthermore, in animals exposed to hypoxia, steatosis was more pronounced (P<0.01), and the NAS [NAFLD (non-alcoholic fatty liver disease) activity score] (8.3+/-2.4 compared with 2.3+/-10.7 in controls; P<0.01), serum AST, triacylglycerols and glucose were higher. Insulin sensitivity decreased in mice exposed to hypoxia relative to controls. The expression of the lipogenic genes SREBP-1c (sterol-regulatory-element-binding protein-1c), PPAR-gamma (peroxisome-proliferator-activated receptor-gamma), ACC1 (acetyl-CoA carboxylase 1) and ACC2 (acetyl-CoA carboxylase 2) increased significantly in mice exposed to hypoxia, whereas mitochondria beta-oxidation genes [PPAR-alpha (peroxisome-proliferator-activated receptor-alpha) and CPT-1 (carnitine palmitoyltransferase-1)] decreased significantly. In conclusion, the findings of the present study demonstrate that hypoxia alone aggravates and accelerates the progression of NASH by up-regulating the expression of lipogenic genes, by down-regulating genes involved in lipid metabolism and by decreasing insulin sensitivity.

Pharmacogenetics and forensic toxicology

Large inter-individual variability in drug response and toxicity, as well as in drug concentrations after application of the same dosage, can be of genetic, physiological, pathophysiological, or environmental origin. Absorption, distribution and metabolism of a drug and interactions with its target often are determined by genetic differences. Pharmacokinetic and pharmacodynamic variations can appear at the level of drug metabolizing enzymes (e.g., the cytochrome P450 system), drug transporters, drug targets or other biomarker genes. Pharmacogenetics or toxicogenetics can therefore be relevant in forensic toxicology. This review presents relevant aspects together with some examples from daily routines.

Personalized therapy in pain management: where do we stand?

Genomic variations influencing response to pharmacotherapy of pain are currently under investigation. Drug-metabolizing enzymes represent a major target of ongoing research in order to identify associations between an individual's drug response and genetic profile. Polymorphisms of the cytochrome P450 enzymes (CYP2D6) influence metabolism of codeine, tramadol, hydrocodone, oxycodone and tricyclic antidepressants. Blood concentrations of some NSAIDs depend on CYP2C9 and/or CYP2C8 activity. Genomic variants of these genes associate well with NSAIDs' side effect profile. Other candidate genes, such as those encoding (opioid) receptors, transporters and other molecules important for pharmacotherapy in pain management, are discussed; however, study results are often equivocal. Besides genetic variants, further variables, for example, age, disease, comorbidity, concomitant medication, organ function as well as patients' compliance, may have an impact on pharmacotherapy and need to be addressed when pain therapists prescribe medication. Although pharmacogenetics as a diagnostic tool has the potential to improve patient therapy, well-designed studies are needed to demonstrate superiority to conventional dosing regimes.

A randomised determination of the effect of fluvastatin and atorvastatin on top of dual antiplatelet treatment on platelet aggregation after implantation of coronary drug-eluting stents. The EFA-Trial

Drug-drug interaction between statins metabolised by cytochrome P450 3A4 and clopidogrel have been claimed to attenuate the inhibitory effect of clopidogrel. However, published data regarding this drug-drug interaction are controversial. We aimed to determine the effect of fluvastatin and atorvastatin on the inhibitory effect of dual antiplatelet therapy with acetylsalicylic acid (ASA) and clopidogrel. One hundred one patients with symptomatic stable coronary artery disease undergoing percutaneous coronary intervention and drug-eluting stent implantation were enrolled in this prospective randomised study. After an interval of two weeks under dual antiplatelet therapy with ASA and clopidogrel, without any lipid-lowering drug, 87 patients were randomised to receive a treatment with either fluvastatin 80 mg daily or atorvastatin 40 mg daily in addition to the dual antiplatelet therapy for one month. Platelet aggregation was assessed using light transmission aggregometry and whole blood impedance platelet aggregometry prior to randomisation and after one month of receiving assigned statin and dual antiplatelet treatment. Platelet function assessment after one month of statin and dual antiplatelet therapy did not show a significant change in platelet aggregation from 1st to 2nd assessment for either statin group. There was also no difference between atorvastatin and fluvastatin treatment arms. In conclusion, neither atorvastatin 40 mg daily nor fluvastatin 80 mg daily administered in combination with standard dual antiplatelet therapy following coronary drug-eluting stent implantation significantly interfere with the antiaggregatory effect of ASA and clopidogrel.

Garlic extract induces intestinal P-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans

Garlic extracts have been shown to decrease drug exposure for saquinavir, a P-glycoprotein and cytochrome P450 3A4 substrate. In order to explore the underlying mechanisms and to study the effects of garlic on pre-systemic drug elimination, healthy volunteers were administered garlic extract for 21 days. Prior to and at the end of this period, expression of duodenal P-glycoprotein and cytochrome P450 3A4 protein were assayed and normalized to villin, while hepatic cytochrome P450 3A4 function and simvastatin, pravastatin and saquinavir pharmacokinetics were also evaluated. Ingestion of garlic extract increased expression of duodenal P-glycoprotein to 131% (95% CI, 105-163%), without increasing the expression of cytochrome P450 3A4 which amounted to 87% (95% CI, 67-112%), relative to baseline in both cases. For the erythromycin breath test performed, the average result was 96% (95% CI, 83-112%). Ingestion of garlic extract had no effect on drug and metabolite AUCs following a single dose of simvastatin or pravastatin, although the average area under the plasma concentration curve (AUC) of saquinavir decreased to 85% (95% CI, 66-109%), and changes in intestinal P-glycoprotein expression negatively correlated with this change. In conclusion, garlic extract induces intestinal expression of P-glycoprotein independent of cytochrome P450 3A4 in human intestine and liver.

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

Prediction of codeine toxicity in infants and their mothers using a novel combination of maternal genetic markers

Substantial variation exists in response to standard doses of codeine ranging from poor analgesia to life-threatening central nervous system (CNS) depression. We aimed to discover the genetic markers predictive of codeine toxicity by evaluating the associations between polymorphisms in cytochrome P450 2D6 (CYP2D6), UDP-glucuronosyltransferase 2B7 (UGT2B7), P-glycoprotein (ABCB1), mu-opioid receptor (OPRM1), and catechol O-methyltransferase (COMT) genes, which are involved in the codeine pathway, and the symptoms of CNS depression in 111 breastfeeding mothers using codeine and their infants. A genetic model combining the maternal risk genotypes in CYP2D6 and ABCB1 was significantly associated with the adverse outcomes in infants (odds ratio (OR) 2.68; 95% confidence interval (CI) 1.61-4.48; P(trend) = 0.0002) and their mothers (OR 2.74; 95% CI 1.55-4.84; P(trend) = 0.0005). A novel combination of the genetic and clinical factors predicted 87% of the infant and maternal CNS depression cases with a sensitivity of 80% and a specificity of 87%. Genetic markers can be used to improve the outcome of codeine therapy and are also probably important for other opioids sharing common biotransformation pathways.

Drug interaction potential of resveratrol

Resveratrol is a naturally occurring polyphenol that is often used as a food supplement. Many positive health effects, including cardio protection, tumor suppression, and immune modulation, are associated with the intake of resveratrol. Resveratrol is well tolerated in healthy subjects without any comedication. However, supplemental doses of resveratrol in the range of 1 g/day or above by far exceed the natural intake through food. Whether resveratrol-drug interactions can be harmful in patients taking additional medications remains unknown. Recent in vivo studies and clinical trials indicate a possible drug-drug interaction potential using high-dosage formulations. In this review, the known in vitro and in vivo effects of resveratrol on various cytochrome P450 (CYP) isoenzymes are summarized. They are discussed in relation to clinically relevant plasma concentrations in humans. We conclude that resveratrol may lead to interactions with various CYPs, especially when taken in high doses. Aside from systemic CYP inhibition, intestinal interactions must also be considered. They can potentially lead to reduced first-pass metabolism, resulting in higher systemic exposure to certain coadministrated CYP substrates. Therefore, patients who ingest high doses of this food supplement combined with additional medications may be at risk of experiencing clinically relevant drug-drug interactions.

Hepatocellular toxicity of clopidogrel: Mechanisms and risk factors

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100μM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100µM) in HepG2/CYP3A4 supersome. Co-incubation with 1μM ketoconazole or 10mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.

Toxicity of thienopyridines on human neutrophil granulocytes and lymphocytes

Thienopyridines can cause neutropenia and agranulocytosis. The aim of the current investigations was to compare cytotoxicity of ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel for human neutrophil granulocytes with the toxicity for lymphocytes and to investigate underlying mechanisms. For granulocytes, clopidogrel, ticlopidine, clopidogrel carboxylate and prasugrel were concentration-dependently toxic starting at 10μM. Cytotoxicity could be prevented by the myeloperoxidase inhibitor rutin, but not by the cytochrome P450 inhibitor ketoconazole. All compounds were also toxic for lymphocytes, but cytotoxicity started at 100μM and could not be prevented by rutin or ketoconazole. Granulocytes metabolized ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel, and metabolization was inhibited by rutin, but not by ketoconazole. Metabolism of these compounds by lymphocytes was much slower and could not be inhibited by ketoconazole or rutin. In neutrophils, all compounds investigated decreased the electrical potential across the inner mitochondrial membrane, were associated with cellular accumulation of ROS, mitochondrial loss of cytochrome c and induction of apoptosis starting at 10μM. All of these effects could be inhibited by rutin, but not by ketoconazole. Similar findings were obtained in lymphocytes; but compared to neutrophils, the effects were detectable only at higher concentrations and were not inhibited by rutin. In conclusion, ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel are toxic for both granulocytes and lymphocytes. In granulocytes, cytotoxicity is more accentuated than in lymphocytes and depends on metabolization by myeloperoxidase. These findings suggest a mitochondrial mechanism for cytotoxicity for both myeloperoxidase-associated metabolites and, at higher concentrations, also for the parent compounds.

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.

Effect of atorvastatin and pravastatin on platelet inhibition by aspirin and clopidogrel treatment in patients with coronary stent thrombosis

We sought to determine a potential interaction between statins and antiplatelet therapy with aspirin and clopidogrel. Previous laboratory studies have shown a possible drug-drug interaction of statins metabolized by cytochrome P450 3A4 and clopidogrel (prodrug metabolized by cytochrome P450 3A4), resulting in an impaired inhibitory effect of clopidogrel on platelet aggregation. However, conclusive prospective data assessing this potentially relevant interaction are lacking. In 73 patients, 23 with previous coronary stent thrombosis (ST) (ST group) and 50 without coronary ST (control group), platelet aggregation was measured 3 times in monthly intervals using light transmission aggregometry (adenosine diphosphate [ADP] and arachidonic acid induction). Measurements were carried out with aspirin monotherapy (100 mg/day), dual antiplatelet therapy with aspirin plus clopidogrel (75 mg/day), and additional treatment of 20 mg/day of atorvastatin or 40 mg/day of pravastatin. ADP (5 and 20 micromol)-induced platelet aggregation was significantly decreased with clopidogrel (p <0.001) but remained stable under additional treatment with atorvastatin or pravastatin in the 2 groups. Patients with previous ST showed a higher ADP-induced aggregation level than control subjects. This difference was not influenced by clopidogrel or statin treatment. In conclusion, patients with previous ST show a higher aggregation level than control subjects independent of statin treatment. Atorvastatin and pravastatin do not interfere with the antiaggregatory effect of aspirin and clopidogrel. In conclusion, drug-drug interaction between dual antiplatelet therapy and atorvastatin or pravastatin seems not to be associated with ST.

Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.