Deshi Shi

Anti-Influenza Prodrug Oseltamivir Is Activated by Carboxylesterase Human Carboxylesterase 1, and the Activation Is Inhibited by Antiplatelet Agent Clopidogrel

Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next influenza pandemic. This anti-influenza viral agent is an ester prodrug, and the antiviral activity is achieved by its hydrolytic metabolite: oseltamivir carboxylate. In this study, we report that the hydrolytic activation is catalyzed by carboxylesterase human carboxylesterase (HCE) 1. Liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the level of HCE1. Recombinant HCE1 but not HCE2 hydrolyzed this prodrug and produced similar kinetic parameters as the liver microsomes. Several HCE1 natural variants differed from the wild-type enzyme on the hydrolysis of oseltamivir. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as much as 90% when the equal concentration was assayed. Given the fact that hydrolysis of oseltamivir is required for its therapeutic activity, concurrent use of both drugs would inhibit the activation of oseltamivir, thus making this antiviral agent therapeutically inactive. This is epidemiologically of significance because people who receive oseltamivir and clopidogrel simultaneously may maintain susceptibility to influenza infection or a source of spreading influenza virus if already infected.

Antiplatelet Agents Aspirin and Clopidogrel Are Hydrolyzed by Distinct Carboxylesterases, and Clopidogrel Is Transesterificated in the Presence of Ethyl Alcohol

Aspirin (acetylsalicylic acid) and clopidogrel are two major antithrombogenic agents that are widely used for the treatment and prevention of cerebro- and cardiovascular conditions such as stroke. Combined use produces enhanced therapeutic effect. Aspirin and clopidogrel both are esters, and hydrolysis leads to decreased or inactivated therapeutic activity. The aim of the study was to determine whether aspirin and clopidogrel are hydrolyzed by the same enzyme(s), thus reciprocally prolonging the antithrombogenic activity. To test this possibility, microsomes from the liver and intestine were assayed for the hydrolysis of aspirin and clopidogrel. In contrary to the hypothesis, aspirin and clopidogrel were hydrolyzed in a tissue-differential manner. Liver microsomes hydrolyzed both drugs, whereas intestinal microsomes hydrolyzed aspirin only. Consistent with the tissue distribution of two carboxylesterases human carboxylesterase (HCE) 1 and HCE2, recombinant HCE1 hydrolyzed clopidogrel, whereas recombinant HCE2 hydrolyzed aspirin. In addition, hydrolysis of clopidogrel among liver samples was correlated well with the level of HCE1, and hydrolysis of aspirin with HCE2. Certain natural variants differed from the wild-type enzymes on the hydrolysis of aspirin or clopidogrel. In the presence of ethyl alcohol, clopidogrel is converted to ethyl clopidogrel. Carboxylesterases are important pharmacological determinants for drugs containing ester linkages and exhibit a large interindividual variation. The isoform-specific hydrolysis of aspirin and clopidogrel suggests that these two antithrombogenic agents may have pharmacokinetic interactions with different sets of ester drugs, and the altered hydrolysis by polymorphic mutants provides a molecular explanation to the interindividual variation.

Interleukin-6 Alters the Cellular Responsiveness to Clopidogrel, Irinotecan, and Oseltamivir by Suppressing the Expression of Carboxylesterases HCE1 and HCE2

Carboxylesterases constitute a class of enzymes that play important roles in the hydrolytic metabolism of drugs and other xenobiotics. Patients with liver conditions such as cirrhosis show increased secretion of proinflammatory cytokines [e.g., interleukin-6 (IL-6)] and decreased capacity of hydrolysis. In this study, we provide a molecular explanation linking cytokine secretion directly to the decreased capacity of hydrolytic biotransformation. In both primary hepatocytes and HepG2 cells, treatment with IL-6 decreased the expression of human carboxyl-esterases HCE1 and HCE2 by as much as 60%. The decreased expression occurred at both mRNA and protein levels, and it was confirmed by enzymatic assay. In cotransfection experiments, both HCE1 and HCE2 promoters were significantly repressed, and the repression was comparable with the decrease in HCE1 and HCE2 mRNA, suggesting that transrepression is responsible for the suppressed expression. In addition, pretreatment with IL-6 altered the cellular responsiveness in an opposite manner of overexpression of HCE1 and HCE2 toward various ester therapeutic agents (e.g., clopidogrel). Transfection of HCE1, for example, decreased the cytotoxicity induced by antithrombogenic agent clopidogrel, whereas pretreatment with IL-6 increased the cytotoxicity. Such a reversal was observed with other ester drugs, including anticancer agent irinotecan and anti-influenza agent oseltamivir. The altered cellular responsiveness was observed when drugs were assayed at sub- and low-micromolar concentrations, suggesting that suppressed expression of carboxylesterases by IL-6 has profound pharmacological consequences, particularly with those that are hydrolyzed in an isoform-specific manner.

Pregnane X receptor is required for interleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes

Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and metabolizes more than 60% of prescribed drugs in human body. Patients with liver conditions such as cirrhosis show increased secretion of cytokines (e.g., interleukin-6) and decreased capacity of oxidation of many drugs. In this study, we provided molecular evidence that cytokine secretion directly contributed to the decreased capacity of oxidative biotransformation in human liver. After human hepatocytes were treated with IL-6, the expression of CYP3A4 decreased at both mRNA and protein levels, so did the CYP3A4 enzymatic activity. Meanwhile, the repression of CYP3A4 by IL-6 occurred after the decrease of pregnane X receptor (PXR) in human hepatocytes. The PXR-overexpressed cells (transfected with human PXR) increased the CYP3A4 mRNA level, and the repression of CYP3A4 by IL-6 was greater in the PXR-overexpressed cells than in the control cells. Further, PXR knockdown (transfected with siPXR construct) decreased the CYP3A4 mRNA level with less repression by IL-6 than in the control cells transfected with corresponding vector. Collectively, our study suggests that PXR is necessary for IL-6-mediated repression of the CYP3A4 expression in human hepatocytes.

Surge in Expression of Carboxylesterase 1 During the Post-neonatal Stage Enables a Rapid Gain of the Capacity to Activate the Anti-influenza Prodrug Oseltamivir

BACKGROUNDnOseltamivir, a widely used anti-influenza drug, is hydrolytically activated by carboxylesterase 1 (CES1). The expression of this carboxylesterase is developmentally regulated. This study was performed to determine when after birth infants acquire competence of activating this prodrug.nnnMETHODSnLiver tissue samples were collected and divided into 5 age groups: group 1 (1-31 d old), group 2 (35-70 d old), group 3 (89-119 d old), group 4 (123-198 d old), and group 5 (>18 years of age). These samples were analyzed for oseltamivir hydrolysis and CES1 expression.nnnRESULTSnLiver samples in group 1 expressed the lowest level of CES1 with the lowest hydrolytic activity toward oseltamivir. A 4-7-fold increase between groups 1 and 2 (1-31 vs 35-70 d of age) was detected in the hydrolysis and expression analyses, respectively. Liver samples in the other 3 pediatric groups (35-198 d of age) exhibited similar expression and hydrolysis levels. Overall, liver samples in group 1 had CES1 expression and hydrolysis levels that were 10% of those of adults, whereas liver samples in the other 3 pediatric groups had levels that were ∼50% of adult levels.nnnCONCLUSIONSnThe post-neonatal surge in CES1 expression ensures the hydrolytic capacity to be gained rapidly after birth in infants, but the larger variability during this period suggests that caution should be exercised on the extrapolated dosing regimens of ester drugs from other age groups.

Dexamethasone transcriptionally increases the expression of the pregnane X receptor and synergistically enhances pyrethroid esfenvalerate in the induction of cytochrome P450 3A23

The pregnane X receptor (PXR) is recognized as a key regulator for the induction of a large number of genes in drug metabolism and transport. The transactivation of PXR is enhanced by the glucocorticoid dexamethasone and the enhancement is linked to the induction of PXR in humans and rats. The present study was undertaken to determine the mechanism for the induction and ascertain the synergistic effect on the expression of CYP3A23, a rat PXR target. In primary hepatocytes, significant induction of PXR was detected as early as 2h after the treatment and the maximal induction occurred at 1 microM dexamethasone. Similar induction kinetics was observed in the hepatoma line H4-II-E-C3. The induction was abolished by actinomycin D and dexamethasone efficaciously stimulated the rat PXR promoter. In addition, dexamethasone synergized esfenvalerate (an insecticide and a PXR activator) in inducing CYP3A23 and stimulating the CYP3A23 promoter. The full promoter of CYP3A23 (-1445/+74) was activated in a similar pattern as the changes in PXR mRNA in response to dexamethasone, esfenvalerate and co-treatment. In contrast, different responding patterns were detected on the stimulation of the CYP3A23 proximal promoter. Synergistic stimulation was also observed on the CYP3A4-DP-Luc reporter, the human counterpart of CYP3A23. These findings establish that transactivation is responsible for the induction of rat PXR and the induction presents potential interactions with insecticides in a species-conserved manner. The different responding patterns among CYP3A23 reporters point to an involvement of multiple transcriptional events in the regulation of CYP3A23 expression by dexamethasone, esfenvalerate and both.

Scoparone potentiates transactivation of the bile salt export pump gene and this effect is enhanced by cytochrome P450 metabolism but abolished by a PKC inhibitor

BACKGROUND AND PURPOSE Hyperbilirubinaemia and cholestasis are two major forms of liver abnormality. The Chinese herb Yin Chin has been used for thousands of years to treat liver dysfunctions. In mice, this herb and its principal ingredient scoparone were found to accelerate the clearance of bilirubin accompanied by the induction of uridine diphosphate‐5′‐glucuronosyltransferase‐1A1 (UGT1A1), a bilirubin processing enzyme. The aim of this study was to determine whether scoparone induces the expression of human UGT1A1. In addition, the expression of the bile salt export pump (BSEP), a transporter of bile acids, was determined.

Dexamethasone suppresses the expression of multiple rat carboxylesterases through transcriptional repression: Evidence for an involvement of the glucocorticoid receptor☆

Carboxylesterases play important roles in the metabolism of xenobiotics and detoxication of insecticides. Without exception, all mammalian species studied express multiple forms of carboxylesterases. Several rat carboxylesterases are well-characterized including hydrolase A, B and S, and the expression of these enzymes is significantly suppressed by glucocorticoid dexamethasone. In this study, we used multiple experimental systems and presented a molecular mechanism for the suppression. Rats receiving one or more daily injections of dexamethasone consistently expressed lower HA, HB and HS. The suppression occurred at the levels of mRNA, protein and hydrolytic activity. In hepatoma cell line H4-II-E-C3, nanomolar dexamethasone caused significant decreases in HA, HB and HS mRNA, and the decreases were abolished by antiglucocorticoid RU486. Additionally, dexamethasone at nanomolar concentrations repressed the promoters of carboxylesterases, and the repression was reduced by glucocorticoid receptor-beta, a dominant negative regulator of the glucocorticoid receptor (GR). In contrast, co-transfection of the pregnane X receptor (PXR) increased the reporter activities, but the increase occurred only at micromolar concentrations of dexamethasone. These findings establish that both GR and PXR are involved in the regulated expression of rat carboxylesterases by dexamethasone but their involvement depends on the concentrations.

Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs.

Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses.

Hypolipidemic agent Z-guggulsterone: metabolism interplays with induction of carboxylesterase and bile salt export pump.

Z-Guggulsterone is a major ingredient in the Indian traditional hypolipidemic remedy guggul. A study in mice has established that its hypolipidemic effect involves the farnesoid X receptor (FXR), presumably by acting as an antagonist of this receptor. It is generally assumed that the antagonism leads to induction of cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme converting free cholesterol to bile acids. In this study, we tested whether Z-guggulsterone indeed induces human CYP7A1. In addition, the expression of cholesteryl ester hydrolase CES1 and bile salt export pump (BSEP) was monitored. Contrary to the general assumption, Z-guggulsterone did not induce CYP7A1. Instead, this phytosterol significantly induced CES1 and BSEP through transactivation. Z-Guggulsterone underwent metabolism by CYP3A4, and the metabolites greatly increased the induction potency on BSEP but not on CES1. BSEP induction favors cholesterol elimination, whereas CES1 involves both elimination and retention (probably when excessively induced). Interestingly, clinical trials reported the hypolipidemic response rates from 18% to 80% and showed that higher dosages actually increased VLDL cholesterol. Our findings predict that better hypolipidemic outcomes likely occur in individuals who have a relatively higher capacity of metabolizing Z-guggulsterone with moderate CES1 induction, a scenario possibly achieved by lowering the dosing regimens.