Karthik Venkatakrishnan

Effects of the antifungal agents on oxidative drug metabolism: Clinical relevance

This article reviews the metabolic pharmacokinetic drug-drug interactions with the systemic antifungal agents: the azoles ketoconazole, miconazole, itraconazole and fluconazole, the allylamine terbinafine and the sulfonamide sulfamethoxazole. The majority of these interactions are metabolic and are caused by inhibition of cytochrome P450 (CYP)-mediated hepatic and/or small intestinal metabolism of coadministered drugs.Human liver microsomal studies in vitro, clinical case reports and controlled pharmacokinetic interaction studies in patients or healthy volunteers are reviewed. A brief overview of the CYP system and the contrasting effects of the antifungal agents on the different human drug-metabolising CYP isoforms is followed by discussion of the role of P-glycoprotein in presystemic extraction and the modulation of its function by the antifungal agents. Methods used for in vitro drug interaction studies and in vitro—in vivo scaling are then discussed, with specific emphasis on the azole antifungals.Ketoconazole and itraconazole are potent inhibitors of the major drugmetabolising CYP isoform in humans, CYP3A4. Coadministration of these drugs with CYP3A substrates such as cyclosporin, tacrolimus, alprazolam, triazolam, midazolam, nifedipine, felodipine, simvastatin, lovastatin, vincristine, terfenadine or astemizole can result in clinically significant drug interactions, some of which can be life-threatening. The interactions of ketoconazole with cyclosporin and tacrolimus have been applied for therapeutic purposes to allow a lower dosage and cost of the immunosuppressant and a reduced risk of fungal infections. The potency of fluconazole as a CYP3A4 inhibitor is much lower. Thus, clinical interactions of CYP3A substrates with this azole derivative are of lesser magnitude, and are generally observed only with fluconazole dosages of ≥200 mg/day.Fluconazole, miconazole and sulfamethoxazole are potent inhibitors of CYP2C9. Coadministration of phenytoin, warfarin, sulfamethoxazole and losartan with fluconazole results in clinically significant drug interactions. Fluconazole is a potent inhibitor of CYP2C19 in vitro, although the clinical significance of this has not been investigated. No clinically significant drug interactions have been predicted or documented between the azoles and drugs that are primarily metabolised by CYP 1A2, 2D6 or 2E1.Terbinafine is a potent inhibitor of CYP2D6 and may cause clinically significant interactions with coadministered substrates of this isoform, such as nortriptyline, desipramine, perphenazine, metoprolol, encainide and propafenone. On the basis of the existing in vitro and in vivo data, drug interactions of terbinafine with substrates of other CYP isoforms are unlikely.

Mechanism-Based Inactivation of Human Cytochrome P450 Enzymes and the Prediction of Drug-Drug Interactions

The ability to use vitro inactivation kinetic parameters in scaling to in vivo drug-drug interactions (DDIs) for mechanism-based inactivators of human cytochrome P450 (P450) enzymes was examined using eight human P450-selective marker activities in pooled human liver microsomes. These data were combined with other parameters (systemic Cmax, estimated hepatic inlet Cmax, fraction unbound, in vivo P450 enzyme degradation rate constants estimated from clinical pharmacokinetic data, and fraction of the affected drug cleared by the inhibited enzyme) to predict increases in exposure to drugs, and the predictions were compared with in vivo DDIs gathered from clinical studies reported in the scientific literature. In general, the use of unbound systemic Cmax as the inactivator concentration in vivo yielded the most accurate predictions of DDI with a mean -fold error of 1.64. Abbreviated in vitro approaches to identifying mechanism-based inactivators were developed. Testing potential inactivators at a single concentration (IC25) in a 30-min preincubation with human liver microsomes in the absence and presence of NADPH followed by assessment of P450 marker activities readily identified those compounds known to be mechanism-based inactivators and represents an approach that can be used with greater throughput. Measurement of decreases in IC50 occurring with a 30-min preincubation with liver microsomes and NADPH was also useful in identifying mechanism-based inactivators, and the IC50 measured after such a preincubation was highly correlated with the kinact/KI ratio measured after a full characterization of inactivation. Overall, these findings support the conclusion that P450 in vitro inactivation data are valuable in predicting clinical DDIs that can occur via this mechanism.

Human drug metabolism and the cytochromes P450 : Application and relevance of in vitro models

The cytochromes P450 (CYPs) constitute a superfamily of hemoprotein enzymes that are responsible for the biotransformation of numerous xenobiotics, including therapeutic agents. Studies of the biochemical and enzymatic properties of these enzymes and their molecular genetics and regulation of gene expression and activity have greatly enhanced our understanding of several aspects of clinical pharmacology such as pharmacokinetic variability, drug toxicity, and drug interactions. This review evaluates the major human hepatic drug‐metabolizing CYP enzymes and their clinically relevant substrates, inhibitors, and inducers. Also discussed are the molecular bases and clinical implications of genetic polymorphisms that affect the CYPs. Much of the information on the specificity of substrates and inhibitors of the CYP enzymes is derived from in vitro studies using human liver microsomes and heterologously expressed CYP enzymes. These methods are discussed, and guidelines are provided for designing enzyme kinetic and reaction phenotyping studies using multiple approaches. The strengths, weaknesses, and discrepancies among the different approaches are considered using representative examples. The mathematical models used in predicting the pharmacokinetic clearance of a drug from in vitro estimates of intrinsic clearance and the principles of quantitative in vitro‐in vivo scaling of metabolic drug interactions are also discussed.

Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes.

BackgroundOxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation. MethodsPropofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes. ResultsPropofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (Km < 8 &mgr;m), whereas low-activity livers displayed low-affinity kinetics (Km > 80 &mgr;m). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (Km = 10 ± 2 &mgr;m; mean ± SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (Km = 41 ± 8 &mgr;m), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 ± 22% inhibition; mean ± SD) compared with CYP2C isoforms (16 ± 7% inhibition) to hepatic microsomal activity. ConclusionsCytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.

Five Distinct Human Cytochromes Mediate Amitriptyline N-Demethylation In Vitro: Dominance of CYP 2C19 and 3A4

The human cytochromes P450 (CYPs) mediating amitriptyline N‐demethylation have been identified using a combination of enzyme kinetic and chemical inhibition studies. Amitriptyline was N‐demethylated to nortriptyline by microsomes from cDNA transfected human lymphoblastoid cells expressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no detectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, CYP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affinities. Based on these kinetic parameters and estimated relative abundance of the different CYPs in human liver, CYP 2C19 was identified as the major amitriptyline N‐demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitriptyline N‐demethylase at 100 μmol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 μmol/L. The CYP 1A2 inhibitor α‐naphthoflavone and the CYP 2C9 inhibitor sulfaphenazole produced much less inhibition of amitriptyline N‐demethylation at both substrate concentrations. Quinidine produced no detectable inhibition. The kinetics of amitriptyline N‐demethylation by human liver microsomes were consistent with a two enzyme model, with the high affinity component exhibiting Michaelis Menten kinetics and the low affinity component exhibiting Hill enzyme kinetics. No difference was apparent in the kinetics of amitriptyline N‐demethylation in two liver samples with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic parameters in vitro.

Drug metabolism and drug interactions: application and clinical value of in vitro models.

In vitro models of drug metabolism are being increasingly applied in the drug discovery and development process as tools for predicting human pharmacokinetics and for the prediction of drug-drug interaction risks associated with new chemical entities. The use of in vitro predictive approaches offers several advantages including minimization of compound attrition during development, with associated cost and time savings, as well as minimization of human risk due to the rational design of clinical drug-drug interaction studies. This article reviews the principles underlying the various mathematical models used to scale in vitro drug metabolism data to predict in vivo clearance and the magnitude of drug-drug interactions resulting from reversible as well as mechanism-based metabolic inhibition. Examples illustrating the predictive utility of specific in vitro approaches are critically reviewed. Commonly encountered uncertainties and sources of bias and error in the in vitro determination of intrinsic clearance and metabolic inhibitory potency, including nonspecific microsomal binding, solvent effects on enzyme activities, and uncertainties in estimating enzyme-available inhibitor concentrations are reviewed. In addition, the impact and clinical relevance of complexities such as dosing route-dependent effects, atypical multi-site kinetics of drug-metabolizing enzymes, non-cytochrome P450 determinants of metabolic clearance, and concurrent inhibition and induction, on the applicability and predictive accuracy of current in vitro models are discussed.

O- and N-demethylation of venlafaxine in vitro by human liver microsomes and by microsomes from cDNA-transfected cells: effect of metabolic inhibitors and SSRI antidepressants.

The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, Km = 41 μM, and Ks 22901 μM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 μM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 μM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, −2C19, and −2D6. CYP2D6 was dominant with the lowest Km, 23.2 μM, and highest intrinsic clearance (Vmax/Km ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and Km = 2504 μM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, −2C19, and −3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 μM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and −3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF.

In vitro cytochrome P450 inhibition data and the prediction of drug‐drug interactions: Qualitative relationships, quantitative predictions, and the rank‐order approach

q s Drug-drug interactions (DDIs) represent a serious roblem in clinical practice. However, with knowledge ained over the past 15 years on the human drugetabolizing enzymes, a better understanding of the nderlying mechanisms behind many of the pharmacoinetic DDIs has been obtained. Previously, studies of DIs for new drugs were carried out empirically or ere gained through random clinical observations. In he past combinations of drugs chosen for investigation f DDIs were selected on the basis of the potential for ntroduction of toxicity of a drug with a narrow theraeutic index (eg, digoxin, theophylline, and warfarin) r if there was frequent coprescription with another gent for a given condition. However, with an increased nderstanding of drug-metabolizing enzymes and their oles in the metabolism of specific drugs, it is possible o apply a more mechanistic approach to assessing DIs. In particular, the possibility of extrapolation of esults of clinical DDI studies with 1 drug known to be leared by a particular drug-metabolizing enzyme to ther drugs that are cleared by that same enzyme is ttractive.

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro : role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3‐methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA‐transfected human lymphoblastoid cells.

Phase I Pharmacokinetic/Pharmacodynamic Study of MLN8237, an Investigational, Oral, Selective Aurora A Kinase Inhibitor, in Patients with Advanced Solid Tumors

Purpose: Aurora A kinase (AAK) is a key regulator of mitosis and a target for anticancer drug development. This phase I study investigated the safety, pharmacokinetics, and pharmacodynamics of MLN8237 (alisertib), an investigational, oral, selective AAK inhibitor, in 59 adults with advanced solid tumors. Experimental Design: Patients received MLN8237 once daily or twice daily for 7, 14, or 21 consecutive days, followed by 14 days recovery, in 21-, 28-, or 35-day cycles. Dose-limiting toxicities (DLT) and the maximum-tolerated dose (MTD) for the 7- and 21-day schedules were determined. Pharmacokinetic parameters were derived from plasma concentration–time profiles. AAK inhibition in skin and tumor biopsies was evaluated and antitumor activity assessed. Results: Neutropenia and stomatitis were the most common DLTs. The MTD for the 7- and 21-day schedules was 50 mg twice daily and 50 mg once daily, respectively. MLN8237 absorption was fast (median time to maximum concentration, 2 hours). Mean terminal half-life was approximately 19 hours. At steady state, pharmacodynamic effects were shown by accumulation of mitotic and apoptotic cells in skin, and exposure-related increases in numbers of mitotic cells with characteristic spindle and chromosomal abnormalities in tumor specimens, supporting AAK inhibition by MLN8237. Stable disease was observed and was durable with repeat treatment cycles, administered over 6 months, in 6 patients, without notable cumulative toxicity. Conclusions: The recommended phase II dose of MLN8237 is 50 mg twice daily on the 7-day schedule, which is being evaluated further in a variety of malignancies, including in a phase III trial in peripheral T-cell lymphoma. Clin Cancer Res; 18(17); 4764–74. ©2012 AACR.