Metabolism-dependent Inhibition Research Articles

Metabolism-dependent inhibition of CYP2E1 by isoniazid, a mediator of idiosyncratic liver injury

Liver injury is a well-known adverse effect of the anti-tuberculosis drug isoniazid (INH); however, animal models that accurately replicate this effect as seen in humans have not been constructed, and the mechanism of its pathogenesis remains unclear. Recently, an immune-mediated mechanism have been proposed based on clinical studies, suggesting the involvement of cytochrome P450-mediated formation of reactive metabolites and covalent adducts in severe cases. In the present study, we investigated the role of CYP2E1 in this mechanism. Liver microsomes from humans, rats, and mice were preincubated with INH and NADPH; thereafter, residual CYP2E1 activity was measured. The inhibition of CYP2E1 by INH was potentiated by preincubation, indicating time-dependent inhibition. There were no major species-based differences in inhibition among humans, rats, and mice. Further to our findings on the inhibition kinetics, resistance of the inhibition to glutathione and catalase indicated that the reactive metabolites of INH covalently bonded to CYP2E1 in a suicidal manner. A similar time-dependent inhibition was also observed for the known metabolites acetylhydrazine and hydrazine; however, the conditions that inhibited the hydrolysis or activated the acetylation of INH did not affect inhibition by INH, suggesting that the reactive metabolites contributing to the inhibition were generated via alternative pathways. This indicates that CYP2E1 alone generates reactive INH metabolites and that haptenized CYP2E1 may be involved in immune-mediated liver injury.

Comparison of the CYP3A selective inhibitors CYP3cide, clobetasol, and azamulin for their potential to distinguish CYP3A7 activity in the presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ~50% of the total CYP content in fetal and neonatal livers and is the predominant CYP involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-S (sulfate). The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low, but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We utilized three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and CYP inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that CP was not an MDI of CYP3A5, but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7 and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. Significance Statement These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect probe selection may have on CYP3A P450 inhibition studies.

Evaluation of Strategies for the Assessment of Drug-Drug Interactions Involving Cytochrome P450 Enzymes.

Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated. Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC50-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding. Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred. This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.

Metoprolol-pridopidine drug-drug interaction and food effect assessments of pridopidine, a new drug for treatment of Huntington's disease

AimsPridopidine is an oral drug in clinical development for treatment of patients with Huntington's disease. This study examined the interactions of pridopidine with in vitro cytochrome P450 activity and characterized the effects of pridopidine on CYP2D6 activity in healthy volunteers using metoprolol as a probe substrate. The effect of food on pridopidine exposure was assessed.MethodsThe ability of pridopidine to inhibit and/or induce in vitro activity of drug metabolizing enzymes was examined in human liver microsomes and fresh hepatocytes. CYP2D6 inhibition potency and reversibility was assessed using dextromethorphan. For the clinical assessment, 22 healthy subjects were given metoprolol 100 mg alone and concomitantly with steady‐state pridopidine 45 mg twice daily. Food effect on a single 90 mg dose of pridopidine was evaluated in a crossover manner. Safety assessments and pharmacokinetic sampling occurred throughout the study.ResultsPridopidine was found to be a metabolism dependent inhibitor of CYP2D6, the main enzyme catalysing its own metabolism. Flavin‐containing monooxygenase heat inactivation of liver microsomes did not affect pridopidine metabolism‐dependent inhibition of CYP2D6 and its inhibition of CYP2D6 was not reversible with addition of FeCN3. Exposure to metoprolol was markedly increased when coadministered with pridopidine; the ratio of the geometric means (90% confidence interval) for maximum observed plasma concentration, and area under the plasma concentration–time curve from time 0 to the time of the last quantifiable concentration and extrapolated to infinity were 3.5 (2.9, 4.22), 6.64 (5.27, 8.38) and 6.55 (5.18, 8.28), respectively. Systemic exposure to pridopidine was unaffected by food conditions.ConclusionsAs pridopidine is a metabolism‐dependent inhibitor of CYP2D6, systemic levels of drugs metabolized by CYP2D6 may increase with chronic coadministration of pridopidine. Pridopidine can be administered without regard to food.

POTENTIAL FOR DRUG INTERACTION OF THE NOVEL COMT INHIBITOR OPICAPONE

ObjectivesThe effect of opicapone, a third generation COMT inhibitor, on metabolic enzymes and drug transporters was evaluated in vitro to anticipate potential drug‐drug interactions.Methodsin vitro studies using human liver microsomes and cell‐lines overexpressing a panel of selected drug‐transporters.ResultsWhen tested against P450 cytochromes (CYP), opicapone was found to be reversible inhibitor of CYP2C8 (IC50 = 3.6 μg/ml and IC50 = 6.7 μg/ml, respectively) and CYP2C9 (IC50 = 9.6 μg/ml, and IC50 = 20.7 μg/ml, respectively). No metabolism dependent inhibition by opicapone was observed for all tested CYPs. Opicapone was a substrate for the efflux transporters breast cancer resistance protein (BCRP) and P‐glycoprotein (P‐gp) and for the organic anion transporting polypeptide (OATP1B3). Opicapone showed moderate inhibition of bile salt export pump (BSEP), and organic anionic transporters (OAT1 and OAT3), but inhibited OATP1B1 and OATP1B3 at lower concentrations (IC50 values of 0.45 and 1.4 μg/ml, respectively).ConclusionsWhen the 99.9% binding of opicapone to human serum proteins is considered, the 1.55 μg/mL Cmax in humans receiving 50 mg/day of opicapone equates to 0.002 μg/mL of unbound material. This is 225‐fold lower than the lowest IC50 against transporters (OATP1B1, OATP1B3) and metabolic enzymes (CYP2C8 and CYP2C9) measured in the present study. As such, the observed in vitro effect upon these biological targets is highly unlikely to have any clinical impact.

Retrospective use of PBPK modelling to understand a clinical drug–drug interaction between dextromethorphan and GSK1034702

1. In a clinical trial, a strong drug–drug interaction (DDI) was observed between dextromethorphan (DM, the object or victim drug) and GSK1034702 (the precipitant or perpetrator drug), following single and repeat doses. This study determined the inhibition parameters of GSK1034702 in vitro and applied PBPK modelling approaches to simulate the clinical observations and provide mechanistic hypotheses to understand the DDI.2. In vitro assays were conducted to determine the inhibition parameters of human CYP2D6 by GSK1034702. PBPK models were populated with the in vitro parameters and DDI simulations conducted and compared to the observed data from a clinical study with DM and GSK1034702.3. GSK1034702 was a potent direct and metabolism-dependent inhibitor of human CYP2D6, with inhibition parameters of: IC50 = 1.6 μM, Kinact = 3.7 h−1 and KI = 0.8 μM. Incorporating these data into PBPK models predicted a DDI after repeat, but not single, 5 mg doses of GSK1034702.4. The DDI observed with repeat administration of GSK1034702 (5 mg) can be attributed to metabolism-dependent inhibition of CYP2D6. Further, in vitro data were generated and several potential mechanisms proposed to explain the interaction observed following a single dose of GSK1034702.

Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole.

Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (K(I) = 18.0 µM; k(inact) = 0.056 minute(-1)), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes).

The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments.

In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which Ki = IC₅₀/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). Values of Ki and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to Km (for IC₅₀ determinations) and spanned Km (for Ki determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC₅₀/2, correlated strongly with experimentally observed Ki determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2.

Development of a Rapid and Cost Effective Assay for the Screening of Reversible Cytochrome P450 Inhibition in Parallel with Cyp3a4 Metabolism-Dependent Inhibition Using Recombinant Proteins

In the present study we have developed a high quality, rapid and cost effective CYP450 inhibition assay that does have the ability to detect both reversible and CYP3A4 metabolism-dependent inhibition (MDI), using recombinantly expressed P450 isoforms and fluorogenic P450 substrates. CYP3A4 isoform is screened with diethoxyflourescein (DEF) as probe substrate. The IC50 values can then be calculated for test compounds against the CYP3A4 isoform, based on the rate of metabolism of the probe substrate, measured for 10 minutes. In addition, the CYP3A4 metabolism-dependent inhibitory potential of test compounds is determined by extending for 30 minutes the determination of the rate of metabolism of diethoxyflourescein and calculating IC50 values every 5 minutes of the incubation period. An estimate of the CYP3A4 metabolism-dependent inhibitory potential of the test compounds can be determined comparing IC50 values, measured following 10 and 30 minutes incubation. The incubation was performed using the selective CYP inhibitors miconazole, for direct P450 inhibition, and troleandromycin, for metabolism-dependent inhibition, as positive controls. The entire screening process was fully-automated in 96-well plate format with the use of Hamilton liquid-handling robot technology coupled with two fluorimeters (Tecan) and a custom laboratory-information management. This assay is currently applied to screen compounds early in the lead optimization process and identify those compounds that cause reversible and/or metabolism-based CYP450 inhibition and therefore progress those molecules or chemical series with the lowest DDI potential possible. The high number of data generated through this assay can also be used to build an informative database and improve predictive models.

In vitro inhibition of CYP2C9-mediated warfarin 7-hydroxylation by iguratimod: possible mechanism of iguratimod-warfarin interaction.

Iguratimod is a novel disease-modifying antirheumatic drug. A blue letter (safety advisory) for drug interaction between iguratimod and warfarin was issued by the Ministry of Health, Labour and Welfare of Japan in May 2013. Iguratimod may affect warfarin metabolism catalyzed by CYP. However, it is not clear whether iguratimod inhibits warfarin oxidation. This study was performed to investigate the effects of iguratimod on warfarin 7-hydroxylation with human liver microsomes (HLMs) and recombinant CYP enzymes. Iguratimod concentration-dependently inhibited R,S-warfarin 7-hydroxylase activity of HLMs with an IC50 value of 15.2 µM. The inhibitory effect was examined with S-warfarin and R-warfarin to determine which enantiomer was more potently inhibited by iguratimod. Iguratimod potently inhibited the S-warfarin 7-hydroxylase activity of HLMs with an IC50 value of 14.1 µM, but showed only slight inhibition of R-warfarin 7-hydroxylation. Furthermore, iguratimod inhibited the S-warfarin 7-hydroxylase activity of recombinant CYP2C9.1 (rCYP2C9.1) and rCYP2C9.3 in a concentration-dependent manner with IC50 values of 10.8 and 20.1 µM, respectively. Kinetic analysis of the inhibition of S-warfarin 7-hydroxylation by iguratimod indicated competitive-type inhibition for HLMs and rCYP2C9.1 but mixed-type inhibition for rCYP2C9.3. The Ki values for HLMs, rCYP2C9.1, and rCYP2C9.3 were 6.74, 4.23, and 14.2 µM, respectively. Iguratimod did not exert metabolism-dependent inhibition of S-warfarin 7-hydroxylation. These results indicated that iguratimod is a potent direct inhibitor of CYP2C9-mediated warfarin 7-hydroxylation and that its inhibitory effect on CYP2C9.1 was more sensitive than that on CYP2C9.3.

Evaluation of in vitro inhibition and induction of cytochrome P450 activities by hydrolyzed ginkgolides

Ethnopharmacological relavanceThe extracts of Ginkgo biloba leaves are effective in treating cerebral infarction, of which ginkgolides have been demonstrated to be the active ingredients. The purpose of this study was to determine whether hydrolyzed ginkgolides would cause potential drug–drug interactions (DDI) during its clinical use via inhibition or induction of the major human cytochrome P450s (CYPs). Materials and methodsThe inhibition (direct and metabolism-dependent inhibiton on CYP activities) and induction (mRNA expression level and activity of CYPs) by the hydrolyzed ginkgolides were evaluated in human liver microsomes and cryopreserved human hepatocytes, respectively. ResultsWithin 0.1 to 10μg/mL, the hydrolyzed ginkgolides showed negligible direct inhibition against CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4m (midazolam as substrate) and 3A4t (testosterone as substrate), with IC50 values determined to be >10μg/mL (concentrations expressed as the sum of equivalent concentrations of ginkgolide A, B and K). For the metabolism-dependent inhibition studies, the preincubation of 30min did not substantially alter the IC50 values when compared with the corresponding values in the direct inhibition studies. The activities and mRNA expression levels for CYP1A2 and 2B6 within each drug-treated group (0.1, 1 and 10μg/mL) were not affected after the 48-h incubation. For CYP3A4, the activity and mRNA expression level were not altered when incubated with 0.1 and 1μg/mL of hydrolyzed ginkgolides. When incubated with hydrolyzed ginkgolides at 10μg/mL, the relative activity and relative mRNA expression level of CYP3A4 remarkably increased to 4.59±3.67 and 17.2±9.16-fold of the corresponding vehicle control values, respectively. ConclusionsThe hydrolyzed ginkgolides is not likely to cause DDI via inhibition of the major human CYPs. However, the CYP3A4 induction might be clinically relevant.

Time- and NADPH-dependent inhibition of cytochrome P450 3A4 by the cyclopentapeptide cilengitide: significance of the guanidine group and accompanying spectral changes.

Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15-19% direct inhibition, 10-23% time-dependent inhibition (30-minute preincubation), and 54-60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min(-1) mM(-1) and was 0.04 ± 0.01 min(-1) mM(-1) with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 μM and 6 μM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.

The metabolic drug-drug interaction profile of Dabrafenib: in vitro investigations and quantitative extrapolation of the P450-mediated DDI risk.

Dabrafenib is a potent ATP-competitive inhibitor for the V600 mutant b-rapidly accelerated fibrosarcoma (b-raf) kinase currently approved in the United States for the treatment of metastatic melanoma. Studies were conducted in human liver microsomes, recombinant human cytochrome P450 (P450) enzymes, and human hepatocytes to investigate the potential of dabrafenib and its major circulating metabolites to perpetrate pharmacokinetic drug-drug interactions (DDIs) as well as have their own pharmacokinetics affected (victim) by coadministered drugs. Dabrafenib metabolism was mediated by CYP2C8 (56% to 67%) and CYP3A4 (24%); in addition, it has demonstrated inhibition of CYP2C8, 2C9, 2C19, 3A4 (atorvastatin), and (nifedipine), with calculated IC50 values of 8.2, 7.2, 22.4, 16, and 32 μM. It also demonstrated metabolism-dependent inhibition of CYP3A4 with a maximal inactivation rate constant of 0.040 minute(-1) and a concentration required to achieve half-maximal inactivation for CYP3A4 of 38 μM. Hydroxy-dabrafenib inhibited CYP1A2, 2C9, and 3A4 (midazolam) with calculated IC50 values of 83, 29, and 44 μM, and carboxy-dabrafenib did not inhibit any of the P450 enzymes tested. Desmethyl-dabrafenib inhibited CYP2B6, 2C8, 2C9, 2C19, and 3A4 (midazolam, atorvastatin, and nifedipine) with calculated IC50 values of 78, 47, 6.3, 36, 17, 20, and 28 μM, respectively. At 30 μM dabrafenib showed increases in CYP2B6 and CYP3A4 mRNA expression indicative of induction. The potential clinical relevance of these findings was explored by using mechanistic static mathematical models to estimate the magnitude of change (area under the curve change) as a result of P450-mediated DDI interactions. This risk-assessment approach indicated that dabrafenib is unlikely to perpetrate any in vivo DDIs by inhibition mechanisms, but is a likely inducer of CYP3A4 and a victim of CYP3A4 and CYP2C8 inhibitors. Furthermore, inclusion of the in vitro drug interaction data for dabrafenib metabolites did not impact the overall clinical risk assessment.

Characterization of the structural determinants required for potent mechanism-based inhibition of human cytochrome P450 1A1 by cannabidiol

We previously demonstrated that cannabidiol (CBD) was a potent mechanism-based inhibitor of human cytochrome P450 1A1 (CYP1A1). However, the moiety of CBD that contributes to the potent mechanism-based inhibition of human CYP1A1 remains unknown. Thus, the effects of compounds structurally related to CBD on CYP1A1 activity were examined with recombinant human CYP1A1 in order to characterize the structural requirements for potent inactivation by CBD. When preincubated in the presence of NADPH for 20min, olivetol, which corresponds to the pentylresorcinol moiety of CBD, enhanced the inhibition of the 7-ethoxyresorufin O-deethylase activity of CYP1A1. In contrast, d-limonene, which corresponds to the terpene moiety of CBD, failed to inhibit CYP1A1 activity in a metabolism-dependent manner. Pentylbenzene, which lacks two free phenolic hydroxyl groups, also did not enhance CYP1A1 inhibition. On the other hand, preincubation of the CBD-2′-monomethyl ether (CBDM) and CBD-2′,6′-dimethyl ether (CBDD) enhanced the inhibition of CYP1A1 activity. Inhibition by cannabidivarin (CBDV), which possessed a propyl side chain, was strongly potentiated by its preincubation. Orcinol, which has a methyl group, augmented CYP1A1 inhibition, whereas its derivative without an alkyl side chain, resorcinol, did not exhibit any metabolism-dependent inhibition. The preincubation of CBD-hydroxyquinone did not markedly enhance CYP1A1 inhibition. We further confirmed that olivetol, CBDM, CBDD, CBDV, and orcinol, as well as CBD (kinact=0.215min−1), inactivated CYP1A1 activity; their kinact values were 0.154, 0.0638, 0.0643, 0.226, and 0.0353min−1, respectively. These results suggest that the methylresorcinol structure in CBD may have structurally important roles in the inactivation of CYP1A1.

The effect of mirabegron, a potent and selective β3-adrenoceptor agonist, on the pharmacokinetics of CYP2D6 substrates desipramine and metoprolol

Mirabegron is a potent and selective β3-adrenoceptor agonist developed for the treatment of overactive bladder. In vitro studies demonstrated that mirabegron partly acts as a (quasi-) irreversible, metabolism-dependent inhibitor of CYP2D6. The effect of steady-state mirabegron on single doses of the sensitive CYP2D6 substrates metoprolol (100mg) and desipramine (50mg) was assessed in two open-label, one-sequence crossover studies in healthy subjects (CYP2D6 extensive metabolizers). Mirabegron 160mg/day increased metoprolol maximum plasma concentration (C max) 1.90-fold (90% confidence interval [CI] 1.54; 2.33) and total exposure (AUC0-∞) 3.29-fold (90% CI 2.70; 4.00) in 12 males (study 1). Mean metoprolol half-life increased from 2.96 to 4.11h. α-Hydroxymetoprolol C max and AUC to last measurable concentration decreased 2.6-fold and 2.2-fold, respectively. In study 2, mirabegron 100mg/day increased desipramine C max 1.79-fold (90% CI 1.69; 1.90) and AUC0-∞ 3.41-fold (90% CI 3.07; 3.80) in 14 males and 14 females. Mean desipramine half-life increased from 19.5 to 35.8h. C max of 2-hydroxydesipramine decreased ~twofold, while AUC increased ~1.3-fold. Desipramine was administered again 2weeks after the last mirabegron dose. Desipramine C max and AUC0-∞ were still ~1.13-fold increased; the 90% CIs fell within the 0.80-1.25 interval. All treatments were well tolerated. In conclusion, mirabegron is a moderate CYP2D6 inhibitor (ratio and 90% CI <5.0).

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Objectives:An extended release formulation of dalfampridine (4-aminopyridine; 4-AP), a potassium channel blocker is available in the USA to improve walking in patients with multiple sclerosis. This study investigated the human metabolites of 4-AP and the cytochrome P450 (CYP450) pathways responsible for 4-AP metabolism.Methods:Metabolites were identified, using thin layer chromatography, high performance liquid chromatography, and gas chromatography/mass spectroscopy, in plasma and urine samples obtained during an excretion balance study of four subjects who were administered a single oral 15-mg dose of 14C-4-AP. Samples were compared with authentic standards of 4-AP, 2-hydroxy-4AP, 3-hydroxy-4AP, and 4-AP-N-oxide. Reaction phenotyping was performed in vitro using human liver microsomes and recombinant CYP450 enzymes with incubation in the presence of direct and time-dependent inhibitors to determine the CYP450 pathways involved in metabolite formation.Results:While most (∼70%) of the radioactivity detected in plasma at each time point corresponded to unchanged 4-AP, two major metabolites were recovered. One metabolite co-localized with the authentic reference standard of 3-hydroxy-4-AP, and the other metabolite was identified as the sulfate conjugate of 3-hydroxy-4-AP. Two minor components were observed, one accounting for 2% of radioactivity and the other below the level of quantitation. Reaction phenotyping showed moderate correlations for conversion of 4-AP to 3-hydroxy-4AP with both CYP2E1 (r = 0.596; p < 0.001) and CYP2C8 (r = 0.608; p < 0.001). Use of a CYP2E1 metabolism-dependent inhibitor inhibited formation of 3-hydroxy-4-AP with and without pre-incubation (higher inhibition with pre-incubation), further supporting the likelihood of CYP2E1 as a metabolic pathway. The main limitation of this study was the inability to identify the CYP enzymes responsible for the 3-hydroxylation of 4-AP, although this conversion represents only a minor metabolic pathway.Conclusion:There is limited metabolism of 4-AP in humans. The two major metabolites were 3-hydroxy-4-AP and 3-hydroxy-4-AP sulfate, likely through CYP2E1 pathways; the possibility of other CYP enzymes playing a minor role in 4-AP metabolism could not be established unequivocally. Overall, these data suggest that there is a low risk for drug–drug interactions via an impact on 4-AP metabolism through cytochrome pathways.

Metabolism-Dependent Inhibition of CYP3A4 by Lapatinib: Evidence for Formation of a Metabolic Intermediate Complex with a Nitroso/Oxime Metabolite Formed via a Nitrone Intermediate

Metabolism-dependent inhibition (MDI) of cytochrome P450 (P450) enzymes has the potential to cause clinically relevant drug-drug interactions. In the case of several alkylamine drugs, MDI of P450 involves formation of a metabolite that binds quasi-irreversibly to the ferrous heme iron to form a metabolic intermediate (MI) complex. The specific metabolites coordinately bound to ferrous iron and the pathways leading to MI complex formation are the subject of debate. We describe an approach combining heme iron oxidation with potassium ferricyanide and metabolite profiling to probe the mechanism of MI complex-based CYP3A4 inactivation by the secondary alkylamine drug lapatinib. Ten metabolites formed from lapatinib by CYP3A4-mediated heteroatom dealkylation, C-hydroxylation, N-oxygenation with or without further oxidation, or a combination thereof, were detected by accurate mass spectrometry. The abundance of one metabolite, the N-dealkylated nitroso/oxime lapatinib metabolite (M9), correlated directly with the prevalence or the disruption of the MI complex with CYP3A4. Nitroso/oxime metabolite formation from secondary alkylamines has been proposed to occur through two possible pathways: (1) sequential N-dealkylation, N-hydroxylation, and dehydrogenation (primary hydroxylamine pathway) or (2) N-hydroxylation with dehydrogenation to yield a nitrone followed by N-dealkylation (secondary hydroxylamine pathway). All intermediates for the secondary hydroxylamine pathway were detected but the primary N-hydroxylamine intermediate of the primary hydroxylamine pathway was not. Our findings support the mechanism of lapatinib CYP3A4 inactivation as MI complex formation with the nitroso metabolite formed through the secondary hydroxylamine and nitrone pathway, rather than by N-dealkylation to the primary amine followed by N-hydroxylation and dehydrogenation as is usually assumed.

Central glucagon-like peptide 1 receptor-induced anorexia requires glucose metabolism-mediated suppression of AMPK and is impaired by central fructose

Glucagon-like peptide-1 (GLP-1) suppresses food intake via activation of a central (i.e., brain) GLP-1 receptor (GLP-1R). Central AMP-activated protein kinase (AMPK) is a nutrient-sensitive regulator of food intake that is inhibited by anorectic signals. The anorectic effect elicited by hindbrain GLP-1R activation is attenuated by the AMPK stimulator AICAR. This suggests that central GLP-1R activation suppresses food intake via inhibition of central AMPK. The present studies examined the mechanism(s) by which central GLP-1R activation inhibits AMPK. Supporting previous findings, AICAR attenuated the anorectic effect elicited by intracerebroventricular (icv) administration of the GLP-1R agonist exendin-4 (Ex-4). We demonstrate that Ex-4 stimulates glycolysis and suppresses AMPK phosphorylation in a glucose-dependent manner in hypothalamic GT1-7 cells. This suggests that inhibition of AMPK and food intake by Ex-4 requires central glucose metabolism. Supporting this, the glycolytic inhibitor 2-deoxyglucose (2-DG) attenuated the anorectic effect of Ex-4. However, icv glucose did not enhance the suppression of food intake by Ex-4. AICAR had no effect on Ex-4-mediated reduction in locomotor activity. We also tested whether other carbohydrates affect the anorectic response to Ex-4. Intracerebroventricular pretreatment with the sucrose metabolite fructose, an AMPK activator, attenuated the anorectic effect of Ex-4. This potentially explains the increased food intake observed in sucrose-fed mice. In summary, we propose a model whereby activation of the central GLP-1R reduces food intake via glucose metabolism-dependent inhibition of central AMPK. We also suggest that fructose stimulates food intake by impairing central GLP-1R action. This has significant implications given the correlation between sugar consumption and obesity.

Preclinical Strategy to Reduce Clinical Hepatotoxicity Using in Vitro Bioactivation Data for &gt;200 Compounds

Drug-induced liver injury is the most common cause of market withdrawal of pharmaceuticals, and thus, there is considerable need for better prediction models for DILI early in drug discovery. We present a study involving 223 marketed drugs (51% associated with clinical hepatotoxicity; 49% non-hepatotoxic) to assess the concordance of in vitro bioactivation data with clinical hepatotoxicity and have used these data to develop a decision tree to help reduce late-stage candidate attrition. Data to assess P450 metabolism-dependent inhibition (MDI) for all common drug-metabolizing P450 enzymes were generated for 179 of these compounds, GSH adduct data generated for 190 compounds, covalent binding data obtained for 53 compounds, and clinical dose data obtained for all compounds. Individual data for all 223 compounds are presented here and interrogated to determine what level of an alert to consider termination of a compound. The analysis showed that 76% of drugs with a daily dose of <100 mg were non-hepatotoxic (p < 0.0001). Drugs with a daily dose of ≥100 mg or with GSH adduct formation, marked P450 MDI, or covalent binding ≥200 pmol eq/mg protein tended to be hepatotoxic (∼ 65% in each case). Combining dose with each bioactivation assay increased this association significantly (80-100%, p < 0.0001). These analyses were then used to develop the decision tree and the tree tested using 196 of the compounds with sufficient data (49% hepatotoxic; 51% non-hepatotoxic). The results of these outcome analyses demonstrated the utility of the tree in selectively terminating hepatotoxic compounds early; 45% of the hepatotoxic compounds evaluated using the tree were recommended for termination before candidate selection, whereas only 10% of the non-hepatotoxic compounds were recommended for termination. An independent set of 10 GSK compounds with known clinical hepatotoxicity status were also assessed using the tree, with similar results.

In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron, a potent and selective β3-adrenoceptor agonist

The potential for mirabegron, a β3-adrenoceptor agonist for the treatment of overactive bladder, to cause drug–drug interactions via inhibition or induction of cytochrome P450 (CYP) enzymes was investigated in vitro.Mirabegron was shown to be a time-dependent inhibitor of CYP2D6 in the presence of NADPH as the IC50 value in human liver microsomes decreased from 13 to 4.3 μM after 30-min pre-incubation. Further evaluation indicated that mirabegron may act partly as an irreversible or quasi-irreversible metabolism-dependent inhibitor of CYP2D6. Therefore, the potential of mirabegron to inhibit the metabolism of CYP2D6 substrates in vivo cannot be excluded. Mirabegron was predicted not to cause clinically significant metabolic drug–drug interactions via inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 because the IC50 values for these enzymes both with and without pre-incubation were >100 μM (370 times maximum human plasma concentration [Cmax]).Whereas positive controls (100 µM omeprazole and 10 µM rifampin) caused the anticipated CYP induction, the highest concentration of mirabegron (10 µM; 37 times plasma Cmax) had minimal effect on CYP1A2 and CYP3A4/5 activity, and CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that mirabegron is not an inducer of these enzymes.