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1. Meperidine is an opioid analgesic that undergoes N-demethylation to form the neurotoxic metabolite normeperidine. Previous studies indicate that meperidine N-demethylation is catalyzed by cytochrome P450 2B6 (CYP2B6), CYP3A4, and CYP2C19. 2. The purpose of this study was to examine the relative P450 contributions to meperidine N-demethylation and to evaluate the effect of CYP2C19 polymorphism on normeperidine generation. Experiments were performed using recombinant P450 enzymes, selective chemical inhibitors, enzyme kinetic assays, and correlation analysis with individual CYP2C19-genotyped human liver microsomes. 3. The catalytic efficiency (kcat/Km) for meperidine N-demethylation was similar between recombinant CYP2B6 and CYP2C19, but markedly lower by CYP3A4. 4. In CYP2C19-genotyped human liver microsomes, normeperidine formation was significantly correlated with CYP2C19 activity (S-mephenytoin 4´-hydroxylation). 5. CYP2C19 inhibitor (+)-N-3-benzylnirvanol and CYP3A inhibitor ketoconazole significantly reduced microsomal normeperidine generation by an individual donor with high CYP2C19 activity, whereas donors with lower CYP2C19 activity were sensitive to inhibition by ketoconazole but not benzylnirvanol. 6. These findings demonstrate that the relative CYP3A4, CYP2B6, and CYP2C19 involvement in meperidine N-demethylation depends on the enzyme activities in individual human liver microsomal samples. CYP2C19 is likely an important contributor to normeperidine generation in individuals with high CYP2C19 activity, but additional factors influence inter-individual metabolite accumulation. [ABSTRACT FROM AUTHOR]

Topoisomerasesare essential enzymes that are involved in DNA metabolism.Topoisomerase II generates transient DNA strand breaks that are stabilizedby anticancer drugs, such as doxorubicin, causing an accumulationof DNA damage. However, doxorubicin causes cardiac toxicity and, likeetoposide and other topoisomerase II-targeted agents, can induce DNAdamage, resulting in secondary cancers. The cannabinoid quinone HU-331has been identified as a potential anticancer drug that demonstratesmore potency in cancer cells with less off-target toxicity than thatof doxorubicin. Reports indicate that HU-331 does not promote celldeath via apoptosis, cell cycle arrest, caspase activation, or DNAstrand breaks. However, the precise mechanism of action is poorlyunderstood. We employed biochemical assays to study the mechanismof action of HU-331 against purified topoisomerase IIα. Theseassays examined DNA binding, cleavage, ligation, relaxation, and ATPaseactivities of topoisomerase IIα. Our results demonstrate thatHU-331 inhibits topoisomerase IIα-mediated DNA relaxation atmicromolar levels. We find that HU-331 does not induce DNA strandbreaks in vitro. When added prior to the DNA substrate,HU-331 blocks DNA cleavage and relaxation activities of topoisomeraseIIα in a redox-sensitive manner. The action of HU-331 can beblocked, but not reversed, by the presence of dithiothreitol. Ourresults also show that HU-331 inhibits the ATPase activity of topoisomeraseIIα using a noncompetitive mechanism. Preliminary binding studiesalso indicate that HU-331 decreases the ability of topoisomerase IIαto bind DNA. In summary, HU-331 inhibits relaxation activity withoutpoisoning DNA cleavage. This action is sensitive to reducing agentsand appears to involve noncompetitive inhibition of the ATPase activityand possibly inhibition of DNA binding. These studies provide a promisingfoundation for the exploration of HU-331 as a catalytic inhibitorof topoisomerase IIα. [ABSTRACT FROM AUTHOR]

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2-3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIa-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIa in the absence of reducing agents. Under these conditions, etoposide quinone was 5-fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIa when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis. [ABSTRACT FROM AUTHOR]

Gamma-hydroxybutyrate (GHB) is used to treat narcolepsy but is also abused. GHB has many actions in common with the GABAB receptor agonist baclofen. To further study the role of GABAB receptors in the effects of GHB. The experiments examined the ability of the GABAB receptor antagonist CGP35348 to attenuate GHB-induced catalepsy in comparison with its ability to attenuate the cataleptic effects of GABAB receptor agonists. In C57BL/6J mice, GHB, the GHB precursor gamma-butyrolactone (GBL), and the GABAB receptor agonists baclofen and SKF97541 all produced catalepsy but differed in potency (i.e., SKF97541>baclofen>GBL>GHB) and in onset of action. The cataleptic effects of drug combinations were assessed at the time of peak effect of each compound, i.e., 60 min after CGP35348 and 60, 30, 30, and 15 min after baclofen, SKF97541, GHB, and GBL, respectively. At 100 mg/kg, CGP35348 shifted the dose–response curves of baclofen and SKF97541 to the right but not those of GHB and GBL; at 320 mg/kg, CGP35348 shifted the curves of all four compounds to the right. The finding that CGP35348 was about threefold less potent to antagonize GHB and GBL than baclofen and SKF97541 is further evidence that the mechanisms mediating the effects of GHB and GABAB agonists are not identical. Differential involvement of GABAB receptor subtypes, or differential interactions with GABAB receptors, may possibly explain why GHB is effective for treating narcolepsy and is abused whereas baclofen is not. [ABSTRACT FROM AUTHOR]

Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.