ABSTRACT:The potency of methylenedioxymethamphetamine (MDMA) as a mechanism-based inhibitor of CYP2D6 has been defined using microsomes prepared from yeast expressing the enzyme and from three human livers. The inhibitory effect was increased by preincubation through formation of a metabolic intermediate complex. MDMA (commonly known as "Ecstasy") is a synthetic amphetamine derivative that has become a popular recreational drug, particularly in the "rave" culture (Cole and Sumnall, 2003). Case reports of exaggerated responses and death associated with its use suggest that some individuals are at an increased risk of toxicity (Tucker et al., 1994). Hyperthermia, tachycardia, convulsions, rhabdomyolysis, and acute liver and renal failure following MDMA use in nightclubs are well documented, and longer-term use is linked to irreversible changes in serotoninergic function (Ramamoorthy et al., 2002). Although administration of MDMA and its metabolite methylenedioxyamphetamine to rats produces serotonergic nerve terminal degeneration, these compounds are not neurotoxic when injected directly into the brain. This suggests a requirement for their peripheral metabolism to neurotoxic metabolites (Easton et al., 2003) which, presumably, are then actively transported into the brain. These metabolites are believed to be downstream products formed after the opening of the methylendioxyphenyl ring, a process which is mainly mediated by CYP2D6 as a high-affinity enzyme (Tucker et al., 1994;Kreth et al., 2000), with low-affinity contributions from CYP1A2, CYP2B6, and CYP3A4 (Kreth et al., 2000).CYP2D6 is a polymorphic enzyme and a functional form is absent in 5 to 9% of Caucasians as a result of autosomal recessive inheritance of gene mutations (Zanger et al., 2004). Thus, it has been suggested that genetically deficient metabolism of MDMA may help to explain why some users of "Ecstasy" appear to be more sensitive to its acute effects (Tucker et al., 1994;Lin et al., 1997). However, recent studies have found no obvious link between inherited CYP2D6 deficiency and MDMA intoxication (O'Donohoe et al., 1998;Schwab et al., 1999;Gilhooly and Daly, 2002). Wu et al. (1997) reported that MDMA is a potent competitive inhibitor of CYP2D6 in human liver microsomes. In addition, they showed that the inhibitory effect of MDMA was increased following preincubation with human liver microsomes in the presence of NADPH. Later studies (Delaforge et al., 1999) confirmed that MDMA forms a metabolic intermediate complex (MIC) with human CYP2D6 expressed in yeast microsomes. However, a quantitative definition of the potency of MDMA as a mechanismbased inhibitor of CYP2D6 is lacking. Thus, the kinetics of the inhibitory effects of MDMA on CYP2D6 in human liver microsomes from extensive metabolizer (EM) subjects and microsomes prepared from yeast expressing the human enzyme were investigated in this study. Materials and MethodsDrugs and Chemicals. Racemic MDMA was provided by Professor R. Forrest (Department of Forensic Pathology, Royal Hallamshire Hos...

The aim of this study was to model the in vivo kinetic consequences of mechanism-based inhibition (MBI) of CYP2D6 by 3,4 methylenedioxymethamphetamine (MDMA, ecstasy). A model with physiologically-based components of drug metabolism was developed, taking account of change in the hepatic content of active CYP2D6 due to MBI by MDMA. Based on the in vitro information, plasma concentration time profiles of MDMA after various doses were computed and compared with reported observations. The analysis suggested that a typical recreational MDMA dose could inactivate most hepatic CYP2D6 within an hour, and the return to a basal level of CYP2D6 could take at least 10 days. Thus, the genetic polymorphism of CYP2D6 and coadministration of CYP2D6 inhibitors may have less impact on MDMA pharmacokinetics and the risk of acute toxicity than previously thought. This is consistent with clinical observations that indicate no obvious link between inherited CYP2D6 deficiency and acute MDMA intoxication.

MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely k(inact) (the maximal inhibition rate) and KI (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of k(inact) and KI using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 microM) gave mean (+/-SE) values of k(inact) (min-1) of 0.20+/-0.06, 0.21+/-0.05, 0.31+/-0.06, 0.37+/-0.11, 0.51+/-0.10 and 0.58+/-0.08, respectively, and KI (microM) values (after correction for non-specific microsomal binding) of 2.22+/-1.90, 2.80+/-1.34, 5.78+/-2.07, 6.36+/-2.93, 3.99+/-1.57 and 4.86+/-1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p <0.05). Varying DEX concentration (10-100 microM) was not associated with significant changes in k(inact) and KI values when a five-fold dilution was used (with the exception of a lower KI at 10 microM DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50-100 mg), since the potency of inhibition was high. The different values of the kinetic parameters were predicted to have a marginal influence on the time for recovery of enzyme activity following re-synthesis of CYP2D6.

Mono-targeting by imatinib as a main antitumor agent does not always accomplish complete cancer suppression. 2,5-dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor, celecoxib, that lacks COX-2 inhibitory function. In this study, we aimed to show the apoptotic effects of imatinib in combination with DMC in human HT-29 colorectal cancer (CRC) cells. HT-29 CRC cells were treated with IC50 dose of imatinib (6.60 μM), DMC (23.45 μM), and their combination (half dose of IC50) for 24 h. The caspase-3 activity was estimated with colorimetric kit. The caspase-3 gene expression was evaluated by real-time PCR method. There was a significant up-regulation in caspase-3 enzyme activity and caspase-3 expression by imatinib and its half dose combination with DMC as compared to control. As a summary, the results of this study strongly suggest that half dose combination of imatinib with DMC induced apoptosis as potent as full dose imatinib in human HT-29 CRC cells, while minimizing undesired side effects related to imatinib mono-therapy. This study also pointed towards possible caspase-dependent actions of imatinib and DMC.

No abstract