Mechanism-based Inactivation Of CYP2D6 Research Articles

Mechanism-Based Inactivation of CYP2D6 by Phellopterin and Related Drug-Drug Interaction with Metoprolol.

Phellopterin (PLP) is a linear furanocoumarin widely found in citrus fruits and herbal medicines. The study aims to comprehensively investigate the mechanism of inhibition of CYP2D6 enzyme activity by PLP and its alteration of metoprolol pharmacokinetics. PLP was found to irreversibly inhibit CYP2D6 in time-, concentration-, and nicotinamide adenine dinucleotide phosphate-dependent manners. Coincubation with quinidine, which is a competitive inhibitor of CYP2D6, attenuated this time-dependent inhibition. Glutathione (GSH) and catalase/superoxide dismutase failed to reverse the PLP-induced CYP2D6 inactivation. GSH trapping experiments provided strong evidence that PLP metabolic activation produces epoxide or γ-ketoaldehyde intermediates. In addition, pretreatment with PLP resulted in significant increases in Cmax and area under curve of plasma metoprolol in rats.

Mechanism-based inactivation of cytochrome P450 2D6 by Notopterol

Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of KI and kinact were 10.8 μM and 0.62 min−1, respectively. The calculated kobs at 10 μM was 0.29 min−1, above the 0.02 min−1 risk level. After incubation with NOT at 10 μM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme.

Rolapitant Is a Reversible Inhibitor of CYP2D6.

Rolapitant [(Varubi), 5S,8S)-8-[[(1R)-1-[3,5 bis(trifluoromethyl phenyl]ethoxy]methyl]-8-phenyl-1,7-diazaspiro[4.5]decan-2-one] is a high-affinity NK1 receptor antagonist that was approved in September 2015 as a treatment for nausea and vomiting caused by chemotherapy. In vivo rolapitant moderately inhibits CYP2D6 for at least 7 days after one 180 mg dose. Due to the long inhibition time, we investigated rolapitant as a possible mechanism-based inactivator of CYP2D6. Rolapitant docked in the active site of CYP2D6 and displayed type I binding to CYP2D6 with a K s value of 1.2 ± 0.4 µM. However, in NADPH-, time-, and concentration-dependent assays of CYP2D6 activity, no evidence for mechanism-based inactivation and no metabolites of rolapitant were observed. Stopped-flow binding studies yielded a kon /koff (K d) value of 6.2 µM. The IC50 value for rolapitant inhibition of CYP2D6 activity was 24 µM, suggesting that inhibition is not due to tight binding of rolapitant to CYP2D6. By Lineweaver-Burk analysis, rolapitant behaved as a mixed, reversible inhibitor. The K i values of 20 and 34 µM were determined by Dixon analysis, with bufuralol and dextromethorphan as reporter substrates, respectively, and drug-drug interaction modeling did not predict the reported in vivo inhibition. The interaction of rolapitant with CYP2D6 was also examined in 1 microsecond molecular dynamics simulations. Rolapitant adopted multiple low-energy binding conformations near the active site, but at distances not consistent with metabolism. Given these findings, we do not see evidence that rolapitant is a mechanism-based inactivator. Moreover, the reversible inhibition of CYP2D6 by rolapitant may not fully account for the moderate inhibition described in vivo.

Mechanism-based inactivation of cytochrome P450 2D6 by chelidonine.

Chelidonine (CHE) is a major bioactive constituent of greater celandine, a plant used in traditional herbal medicines. CHE has widely been used as an analgesic in clinical settings. We evaluated the inhibitory effects of CHE on human cytochrome P450 enzymes. CHE produced time-, concentration-, and NADPH-dependent inhibition of CYP2D6, with K I and k inact values of 20.49 μM and 11.05 min -1 , respectively. Approximately 76% of CYP2D6 activity was suppressed after 9 minute incubation with CHE (50 μM). The loss of enzyme activity was not restored following dialysis. The estimated partition ratio of the inactivation was about 156. Quinidine, a competitive inhibitor of CYP2D6, attenuated the CHE-mediated enzyme inactivation, while glutathione and catalase/superoxide dismutase did not markedly ameliorate the inhibitory effect. Upon oxidation using potassium ferricyanide, the 15.1% activity of CYP2D6 was restored. These findings indicate that CHE acted as a mechanism-based inactivator of CYP2D6 and the observed effects may induce potential drug-drug interactions.

Nitidine Chloride Is a Mechanism-Based Inactivator of CYP2D6.

Nitidine chloride (NC) is a benzophenanthridine alkaloid isolated from the roots of Zanthoxylum nitidum (Roxb.) DC, a widely used traditional herbal medicine. Several reports have revealed NC's multiple pharmacologic properties. The inhibitory effects of NC on human cytochrome P450 enzymes were investigated in the present study. We found that NC caused time- and concentration-dependent inhibition of CYP2D6, and more than 50% of CYP2D6 activity was suppressed after a 15-minute incubation with NC at 100 μM in the primary incubation mixtures, with KI of 4.36 μM, kinact of 0.052 minute-1, and a partition ratio of approximately 290. Moreover, the loss of CYP2D6 activity required the presence of NADPH. Superoxide dismutase/catalase and glutathione showed minor protection against the NC-induced enzyme inhibition. Quinidine as a competitive inhibitor of CYP2D6 slowed down the inactivation by NC. Trapping experiments using N-acetylcysteine demonstrated that quinone and/or carbene intermediate(s) were/was generated in human liver microsomal incubations with NC. In addition, potassium ferricyanide prevented the enzyme from the inactivation mediated by NC, which provided evidence that inhibition of CYP2D6 resulted from heme destruction by the formation of a carbene-iron complex. CYP1A2 was found to be the major enzyme involved in the generation of NC quinone metabolites. In conclusion, NC is a mechanism-based inactivator of CYP2D6. The generation of a carbene intermediate might be mainly responsible for the enzyme inactivation.

Metoclopramide is metabolized by CYP2D6 and is a reversible inhibitor, but not inactivator, of CYP2D6

1. Metoclopramide is a widely used clinical drug in a variety of medical settings with rare acute dystonic events reported. The aim of this study was to assess a previous report of inactivation of CYP2D6 by metoclopramide, to determine the contribution of various CYPs to metoclopramide metabolism, and to identify the mono-oxygenated products of metoclopramide metabolism.2. Metoclopramide interacted with CYP2D6 with Type I binding and a Ks value of 9.56 ± 1.09 µM. CYP2D6 was the major metabolizer of metoclopramide and the two major products were N-deethylation of the diethyl amine and N-hydroxylation on the phenyl ring amine. CYPs 1A2, 2C9, 2C19, and 3A4 also metabolized metoclopramide.3. While reversible inhibition of CYP2D6 was noted, CYP2D6 inactivation by metoclopramide was not observed under conditions of varying concentration or varying time using SupersomesTM or pooled human liver microsomes.4. The major metabolites of metoclopramide were N-hydroxylation and N-deethylation formed most efficiently by CYP2D6 but also formed by all CYPs examined. Also, while metoclopramide is metabolized primarily by CYP2D6, it is not a mechanism-based inactivator of CYP2D6 in vitro.

The effect of CTP-347 on CYP2D6-mediated tamoxifen metabolism

11552 Background: The activity of tamoxifen is primarily mediated through its 4-hydroxy metabolites which are potent antagonists of the estrogen receptor in breast tissue. The 4-hydroxylation reaction is mediated through the cytochrome P450 isozyme CYP2D6. Unfortunately, high concentrations of endoxifen, the major 4-hydroxy metabolite, have also been implicated in the occurrence of hot flashes. Although paroxetine has been demonstrated to reduce the frequency of hot flashes in investigator trials of patients with breast cancer, co- administration would reduce the effectiveness of tamoxifen since paroxetine has been shown to be a mechanism-based inactivator of CYP2D6. CTP-347 is a new chemical entity in which key hydrogen atoms of paroxetine are replaced with deuterium atoms. CTP-347 is being developed for the treatment of post-menopausal and tamoxifen-induced hot flashes and will enter clinical trials in mid-2008. Methods: Serotonin and norepinephrine reuptake inhibition was measured in rat synaptosomes u...

THE EFFECT OF CIMETIDINE ON DEXTROMETHORPHAN O-DEMETHYLASE ACTIVITY OF HUMAN LIVER MICROSOMES AND RECOMBINANT CYP2D6

Clinically, cimetidine therapy impairs the clearance of various drugs metabolized by CYP2D6, such as desipramine and sparteine. Cimetidine is known to reversibly inhibit CYP2D6 in vitro; however, Ki values are greater than plasma concentrations observed in vivo. There is evidence suggesting that this drug may act as an inactivator of cytochrome P450 (P450) enzymes after metabolic activation. Therefore, the purpose of this study was to determine whether cimetidine acts as a mechanism-based inactivator of CYP2D6. Dextromethorphan O-demethylation was used as a probe of CYP2D6 activity. The Vmax and Km of this reaction were 0.82 +/- 0.06 nmol/min/nmol of P450 and 4.1 +/- 0.1 microM, respectively, in pooled human liver microsomes; and 15.9 +/- 0.8 nmol/min/nmol P450 and 1.4 +/- 0.6 microM, respectively, with recombinant CYP2D6. With human liver microsomes, cimetidine competitively inhibited CYP2D6 (Ki = 38 +/- 5 microM) and was a mixed inhibitor of recombinant CYP2D6 (Ki = 103 +/- 17 microM). Preincubation of human liver microsomes with cimetidine and NADPH did not increase the inhibitory potency of cimetidine; however, preincubation with recombinant CYP2D6 resulted in enzyme inactivation that could be attenuated by the CYP2D6 inhibitor quinidine. The KI and kinact were estimated to be 77 microM and 0.03 min-1, respectively, and the half-life of inactivation was 25 min. Therefore, cimetidine may represent a class of compounds capable of inactivating specific cytochromes P450 in vivo, but for which conditions may not be achievable in vitro using human liver microsomes.