Larry M. Tremaine

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.

Pharmacokinetics of Sertraline and its N -Demethyl Metabolite in Elderly and Young Male and Female Volunteers

SummaryA nonblinded study was conducted to compare the pharmacokinetic properties of the selective serotonin reuptake inhibitor sertraline in 22 young (aged 18 to 45 years) and 22 elderly (> 65 years) volunteers, of whom half were male and half were female. In this study, sertraline was administered at a dosage of 200mg once daily (the maximum recommended daily dosage) for 21 days after upward dosage titration from 50 mg/day over a 9-day period. Thus, this study was designed to measure the effect of age and gender on the pharmacokinetic properties of sertraline at the maximum dosage recommended for clinical use.The terminal elimination half-life (t½β) of sertraline was similar in young females, elderly males and elderly females (mean t½β ranged from 32.1 to 36.7 hours in these groups) but shorter (22.4 hours) in the young males. The mean maximum plasma sertraline concentration (Cmax) and the mean steady-state area under the plasma concentration-time curve from time zero to 24 hours postdose (AUC24) were also similar between the young females, elderly males and elderly females, but were approximately 25% lower in the young males. The time to Cmax was unaffected by age or gender and ranged from 6.4 to 6.9 hours. N-Demethylsertraline is the principal metabolite of sertraline and does not contribute significantly to its serotonergic actions. The mean values for N-demethylsertraline trough plasma concentrations, AUC24 and Cmax were comparable in elderly males and females and young females but lower in young males. The ratios of mean AUC24 and Cmax for N-demethylsertraline to the AUC24 and Cmax for sertraline were similar between the 4 groups.

PREDICTION OF DRUG-DRUG INTERACTIONS FROM IN VITRO INDUCTION DATA Application of the Relative Induction Score Approach Using Cryopreserved Human Hepatocytes

Cytochrome P450 induction-mediated drug-drug interaction (DDI) is one of the major concerns in clinical practice and for the pharmaceutical industry. Previously, a novel approach [the relative induction score (RIS)] was developed using the Fa2N-4 immortalized human hepatocyte line and proposed as a tool for predicting magnitude of clinical DDIs caused by induction of CYP3A. The approach is based on combining in vitro induction parameters (EC50 and Emax) with the efficacious free plasma concentrations to calculate a relative induction score, which is correlated to the magnitude of clinical DDI for midazolam or ethinyl estradiol. To expand the applicability of the RIS model, we have measured induction caused by ten drugs in two different lots of human cryopreserved hepatocytes and correlated the data to clinical DDIs using the RIS. The results demonstrated that, as with Fa2N-4 hepatocytes, sigmoidal relationships can be derived between RIS and magnitude of induction of midazolam and ethinyl estradiol clearance in cryopreserved human hepatocytes. This study demonstrates the general applicability of the relative induction score approach using the human cryopreserved hepatocyte model to predict clinical DDI.

A Novel Relay Method for Determining Low-Clearance Values

A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml · min−1 · kg−1. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.

Deuterium Isotope Effects on Drug Pharmacokinetics. I. System- Dependent Effects of Specific Deuteration with Aldehyde Oxidase Cleared Drugs

The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzymes reaction mechanism, species differences in the role played by other enzymes in a drugs metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clearance mechanism. Differences in pharmacokinetic outcomes in guinea pig and rat, with the same metabolic clearance mechanism, show how species differences in the systemic clearance mechanism can affect the in vivo outcome. Overall, to gain from the application of deuteration as a strategy to alter drug pharmacokinetics, these studies demonstrate the importance of understanding the systemic clearance mechanism and knowing the identity of the metabolic enzymes involved, the extent to which they contribute to metabolic clearance, and the extent to which metabolism contributes to the systemic clearance.

P-glycoprotein has differential effects on the disposition of statin acid and lactone forms in mdr1a/b knockout and wild-type mice.

In the present study we examined the disposition of atorvastatin, lovastatin, and simvastatin in acid and lactone forms and pravastatin in acid form in multidrug-resistant gene (mdr1a/b) knockout (KO), and wild-type (WT) mice. Each statin was administered s.c. to mdr1a/b KO and WT mice at 3.0 mg/kg (n ≥ 3 mice/time point). Blood, brain, and liver samples were harvested at 0, 0.5, 1.5, and 3 h postdose. Plasma and tissue concentrations of the acid and lactone (only the acid form was determined for pravastatin) were determined using a liquid chromatography-mass spectrometry method. Both lactone and acid were observed in plasma when lactones were administered, but only acids were detected when the acid forms were administered. The plasma and liver concentrations of acid or lactone were similar between the KO and WT mice. Two- to 23-fold higher concentrations were observed in liver than in plasma, suggesting potential uptake transporters involved. A significantly higher (p < 0.05) brain penetration in the KO compared with the WT mice was observed for lovastatin acid (but the brain/plasma ratio was low for both KO and WT mice) and lactone and simvastatin lactone but not for atorvastatin or pravastatin. The present results suggest that mouse P-glycoprotein does not affect the lactone-acid interconversion or liver-plasma distribution. Furthermore, P-glycoprotein plays a limited role in restricting the brain penetration of the acid forms of atorvastatin, pravastatin, simvastatin, lovastatin, and atorvastatin lactone but may limit the brain availability of the lactone forms of simvastatin and lovastatin.

Impact of drug transporter pharmacogenomics on pharmacokinetic and pharmacodynamic variability – considerations for drug development

Introduction: Drug transporter proteins are expressed on the cell membrane, regulating substrate exposure in systemic circulation and/or peripheral tissues. Genetic polymorphism of drug transporter genes encoding these proteins could alter the functional activity and/or protein expression, having effects on absorption, distribution, metabolism and excretion (ADME), efficacy and adverse effects. Areas covered: The authors provide the reader with an overview of the pharmacogenetics (PGx) of 12 membrane transporters. The clinical literature is summarized as to the quantitative significance on pharmacokinetics (PK) and implications on pharmacodynamics (PD) and adverse effects, due to transporter influence on intracellular drug concentrations. Expert opinion: Unlike polymorphisms for cytochrome P450s (CYPs) resulting in large magnitude of PK variation, genetic mutations for membrane transporters are typically less than threefold alteration in systemic PK for drugs with a few exceptions. However, substantially greater changes in intracellular drug levels may result. We are aware of 1880 exome variants in 12 of the best-studied transporters to date, and nearly 40% of these change the amino acid. However, the functional consequences of most of these variants remain to be determined, and have only been empirically evaluated for a handful. To the extent that genetic polymorphisms impact ADME, it is a variable that will contribute to ethnic differences due to substantial frequency differences for the known variants.

Automated gas chromatographic-electron-capture assay for the selective serotonin uptake blocker sertraline.

This report describes a modified assay that permitted the automated analysis of a few thousand human plasma samples. The major changes from the previous assay included a derivatization step with trifluoroacetic anhydride to enhance electron-capture sensitivity and improve peak shape, improvements in sample clean-up to permit quantitation of 1.0 ng/ml by electron-capture detection and alterations in GC conditions to permit direct on-line injection for automated sample analysis

Tissue Distribution and Elimination of [14C]Apixaban in Rats

Apixaban, a potent and highly selective factor Xa inhibitor, is currently under development for treatment of arterial and venous thrombotic diseases. The distribution, metabolism, and elimination of [14C]apixaban were investigated in male, female, pregnant, and lactating rats after single oral doses. Tissue distribution of radioactivity in rats was measured using quantitative whole-body autoradiography. After a single oral administration, radioactivity distributed quickly in rats with Cmax at 1 h for most tissues. The elimination t1/2 of radioactivity in blood was 1.7 to 4.2 h. The blood area under the plasma concentration-time curve of radioactivity was similar between male and female rats and was slightly higher in pregnant rats and lower in lactating rats. The radioactivity concentration in tissues involved in elimination was greater than that in blood with the highest concentration in the gastrointestinal tract, liver, and urinary bladder/contents and lowest level in brains. In pregnant rats, the whole-body autoradiogram showed that low levels of radioactivity were present in fetal blood, liver, and kidney and were much lower than the radioactivity in the respective maternal organs. The fecal route was the major pathway (74% of dose), and the urinary route was the minor pathway (14%) for apixaban elimination. After single oral doses of [14C]apixaban to lactating rats, apixaban exhibited extensive lacteal excretion with apixaban as the major component. In summary, tissue distribution of apixaban in rats was extensive but with limited transfer to fetal and brain tissues and extensive secretion into rat milk with the parent drug as the major component. Milk excretion could account for 10% of apixaban dose, which was comparable to urinary elimination in rats. Tissue distribution and drug excretion of apixaban are consistent with those for a moderately permeable drug that is a substrate for P-glycoprotein and breast cancer resistance protein efflux transporters.

Application of Liquid Chromatography-Accelerator Mass Spectrometry (LC-AMS) to Evaluate the Metabolic Profiles of a Drug Candidate in Human Urine and Plasma

Metabolite profiling of 100- and 1,000-fold diluted urine and plasma samples from a conventional radiolabeled human ADME study is described using a highly sensitive LC-AMS technique. The concentration of radioactivity and the metabolic profiles in urine and plasma determined using this technique were similar to those employing standard off-line (i.e. LSC) or in-line (i.e. beta-RAM or LC-ARC dynamic-flow) radioactivity monitoring techniques. The results indicate that at a simulated ca. 100 nCi clinical dose, plasma and urine concentrations of (14)C, as well as their metabolic profiles, may be determined routinely by LC-AMS. This approach opens the possibility of using LC-AMS for both the high-throughput quantitation of biological samples and the generation of high-resolution chromatographic profiles of complex mixtures at a lower cost than current AMS analyses that require the conversion of sample carbon to graphite, a laborious and time consuming process.