J. Andrew Williams

DRUG-DRUG INTERACTIONS FOR UDP-GLUCURONOSYLTRANSFERASE SUBSTRATES: A PHARMACOKINETIC EXPLANATION FOR TYPICALLY OBSERVED LOW EXPOSURE (AUCI/AUC) RATIOS

Glucuronidation is a listed clearance mechanism for 1 in 10 of the top 200 prescribed drugs. The objective of this article is to encourage those studying ligand interactions with UDP-glucuronosyltransferases (UGTs) to adequately consider the potential consequences of in vitro UGT inhibition in humans. Spurred on by interest in developing potent and selective inhibitors for improved confidence around UGT reaction phenotyping, and the increased availability of recombinant forms of human UGTs, several recent studies have reported in vitro inhibition of UGT enzymes. In some cases, the observed potency of UGT inhibitors in vitro has been interpreted as having potential relevance in humans via pharmacokinetic drug-drug interactions. Although there are reported examples of clinically relevant drug-drug interactions for UGT substrates, exposure increases of the aglycone are rarely greater than 100% in the presence of an inhibitor relative to its absence (i.e., AUCi/AUC ≤2). This small magnitude in change is in contrast to drugs primarily cleared by cytochrome P450 enzymes, where exposures have been reported to increase as much as 35-fold on coadministration with an inhibitor (e.g., ketoconazole inhibition of CYP3A4-catalyzed terfenadine metabolism). In this article the evidence for purported clinical relevance of potent in vitro inhibition of UGT enzymes will be assessed, taking the following into account: in vitro data on the enzymology of glucuronide formation from aglycone, pharmacokinetic principles based on empirical data for inhibition of metabolism, and clinical data on the pharmacokinetic drug-drug interactions of drugs primarily cleared by glucuronidation.

In vitro and pharmacophore insights into CYP3A enzymes

The cytochrome P450 3A (CYP3A) enzymes have a major role in the metabolism of drugs in humans. Their wide substrate specificity and induction by a vast array of structurally diverse compounds presents the possibility of metabolic drug-drug interactions. Understanding the enzymes themselves is crucial. Over the past decade, this has occurred mostly with in vitro studies, although more recent approaches incorporate computational models to predict CYP inhibition and substrate potential. The three-dimensional displacement, or pharmacophore, of chemical features in space that are derived from inhibition data have produced pharmacophores for CYP3A4, CYP3A5 and CYP3A7, and provide new insights into ligand binding for each enzyme.

Fluorouracil, Leucovorin, and Irinotecan Plus Either Sunitinib or Placebo in Metastatic Colorectal Cancer: A Randomized, Phase III Trial

PURPOSE This double-blind, phase III study aimed to demonstrate that sunitinib plus FOLFIRI (fluorouracil, leucovorin, and irinotecan) was superior to placebo plus FOLFIRI in previously untreated metastatic colorectal cancer (mCRC). PATIENTS AND METHODS Patients were randomly assigned to receive FOLFIRI and either sunitinib (37.5 mg per day) or placebo (4 weeks on treatment, followed by 2 weeks off [schedule 4/2]) until disease progression. The primary end point was progression-free survival (PFS). Secondary end points included overall survival, safety, and patient-reported outcomes. The correlation between genotype and clinical outcomes was also analyzed. RESULTS In all, 768 patients were randomly assigned to sunitinib plus FOLFIRI (n = 386) or placebo plus FOLFIRI (n = 382). Following a second prespecified interim analysis, the study was stopped because of potential futility of sunitinib plus FOLFIRI. Final results are reported. The PFS hazard ratio was 1.095 (95% CI, 0.892 to 1.344; one-sided stratified log-rank P = .807), indicating a lack of superiority for sunitinib plus FOLFIRI. Median PFS for the sunitinib arm was 7.8 months (95% CI, 7.1 to 8.4 months) versus 8.4 months (95% CI, 7.6 to 9.2 months) for the placebo arm. Sunitinib plus FOLFIRI was associated with more grade ≥ 3 adverse events and laboratory abnormalities than placebo (especially diarrhea, stomatitis/oral syndromes, fatigue, hand-foot syndrome, neutropenia, thrombocytopenia, anemia, and febrile neutropenia). More deaths as a result of toxicity (12 v four) and significantly more dose delays, dose reductions, and treatment discontinuations occurred in the sunitinib arm. CONCLUSION Sunitinib 37.5 mg per day (schedule 4/2) plus FOLFIRI is not superior to FOLFIRI alone and has a poorer safety profile. This combination regimen is not recommended for previously untreated mCRC.

A 16-gene assay to predict recurrence after surgery in localised renal cell carcinoma: development and validation studies

BACKGROUND The likelihood of tumour recurrence after nephrectomy in localised clear cell renal cell carcinoma is well characterised by clinical and pathological parameters. However, these assessments can be improved and personalised by the addition of molecular characteristics of each patients tumour. We aimed to develop and validate a prognostic multigene signature to improve prediction of recurrence risk in clear cell renal cell carcinoma. METHODS In the development stage, we investigated the association between expression of 732 genes, measured by reverse-transcription PCR, and clinical outcome in 942 patients with stage I-III clear cell renal cell carcinoma who had undergone a nephrectomy at the Cleveland Clinic (OH, USA). 516 genes were associated with recurrence-free interval. 11 of these genes were selected by further statistical analyses, and were combined with five reference genes (ie, 16 genes in total), from which a recurrence score algorithm was developed. The recurrence score was then validated in an independent cohort of 626 patients from France with stage I-III clear cell renal cell carcinoma who had also undergone nephrectomy. The association between the recurrence score and the risk of recurrence and cancer-specific survival in the first 5 years after surgery was assessed using Cox proportional hazard regression, stratified by tumour stage (stage I vs stage II vs III). FINDINGS In our primary univariate analysis, the continuous recurrence score (median 37, IQR 31-45) was significantly associated with recurrence-free interval (hazard ratio 3·91 [95% CI 2·63-5·79] for a 25-unit increase in score, p<0·0001). In multivariable analyses, the recurrence score was significantly associated with the risk of tumour recurrence (hazard ratio per 25-unit increase in the score 3·37 [95% CI 2·23-5·08], p<0·0001) after stratification by stage and adjustment for tumour size, grade, or Leibovich score. The recurrence score was able to identify a clinically significant number of both high-risk stage I and low-risk stage II-III patients. A heterogeneity study on separate samples showed little to no intratumoural variability among the 16 genes. INTERPRETATION Our findings validate the recurrence score as a predictor of clinical outcome in patients with stage I-III clear cell renal cell carcinoma, providing a more accurate and individualised risk assessment beyond existing clinical and pathological parameters. FUNDING Genomic Health Inc and Pfizer Inc.

Bergamottin contribution to the grapefruit juice–felodipine interaction and disposition in humans

Our objectives were to evaluate the contribution of bergamottin to the grapefruit juice–felodipine interaction and to characterize bergamottin disposition.

PhRMA White Paper on ADME Pharmacogenomics

Pharmacogenomic (PGx) research on the absorption, distribution, metabolism, and excretion (ADME) properties of drugs has begun to have impact for both drug development and utilization. To provide a cross‐industry perspective on the utility of ADME PGx, the Pharmaceutical Research and Manufacturers of America (PhRMA) conducted a survey of major pharmaceutical companies on their PGx practices and applications during 2003–2005. This white paper summarizes and interprets the results of the survey, highlights the contributions and applications of PGx by industrial scientists as reflected by original research publications, and discusses changes in drug labels that improve drug utilization by inclusion of PGx information. In addition, the paper includes a brief review on the clinically relevant genetic variants of drug‐metabolizing enzymes and transporters most relevant to the pharmaceutical industry.

Reaction Phenotyping in Drug Discovery: Moving Forward with Confidence?

For the pharmaceutical industry, one of the challenges in evaluating the risk of future compound attrition at the discovery stage is the successful prediction of the major routes of clearance in humans. For compounds cleared by metabolism, such information will help to avoid the development of compounds that will exhibit large interpatient differences in pharmacokinetics via 1). routes of metabolism catalyzed by functionally polymorphic enzymes and/or 2). clinically significant metabolic drug-drug interactions, in the later stages of development. The degree of intersubject variability that is acceptable for a drug candidate is uncertain in the discovery stage where knowledge of other important factors is limited or unavailable (i.e. therapeutic index, pharmacodynamic variability, etc). Reaction phenotyping is the semi-quantitative in vitro estimation of the relative contributions of specific drug-metabolizing enzymes to the metabolism of a test compound. However, reaction phenotyping in the discovery stage of drug development is complicated by the absence of radiolabelled parent compound or metabolite bioanalytical standards relative to later stages of development. In this commentary, some of the approaches, based on published data, which can be taken to overcome these challenges are discussed. In addition, knowledge of the molecular structure (i.e. specific chemical substituents), physicochemical properties, and routes of clearance in animals can all help in making a successful prediction for the routes of clearance in humans. In combination, the objective of these studies should be to reduce to a minimum the risk of finding significant inter-patient differences in pharmacokinetics at a later stage in development due to significant metabolism by polymorphic enzymes or drug-drug interactions. Consequently, this data should be used to avoid costly late stage attrition.

N-acetyltransferases, sulfotransferases and heterocyclic amine activation in the breast

Heterocyclic amines are mammary carcinogens in rats and their N-hydroxy metabolites are substrates for subsequent metabolic activation by N-acetyltransferases (NAT) and sulfotransferases (SULT) in man. We investigated the expression of these enzymes in human breast tissue and the relationship between NAT genotype and NAT mRNA expression or enzyme activity. Immunohistochemical staining of sections of breast tissue identified expression of NAT1 and NAT2 protein in human mammary epithelial cells, but not in the stroma. We also measured the formation of DNA adducts of the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in calf thymus DNA after incubation of their promutagenic N-hydroxy metabolites with mammary cytosols prepared from reduction mammoplasty tissue. Experimental observations gained from use of enzyme cofactors and NAT and/or SULT inhibitors on cytosolic enzyme activity, recombinant NAT1 activity and heterocyclic amine-DNA adduct formation suggest that both NAT1 and SULT1A enzymes contribute significantly to the activation of N-hydroxylated heterocyclic amines in mammary tissue. NAT1 mRNA transcript levels were found to be two- to three-fold higher than mRNA transcripts of the NAT2 gene in reduction mammoplasty tissue and mammary epithelial cells. NAT1-specific p-aminobenzoic acid acetylation activity, but not NAT2-specific sulfamethazine acetylation activity, was detectable in mammary cytosols. There was no association apparent between NAT genotype and the levels of NAT mRNA or NAT enzyme activity, or between NAT1 genotype and IQ-DNA adduct formation mediated by mammary cytosols. Western blot analysis of mammary cytosolic protein showed detectable levels of SULT1A1 and SULT1A3.

Meta-analysis of contribution of genetic polymorphisms in drug-metabolizing enzymes or transporters to axitinib pharmacokinetics

PurposeAxitinib, an orally administered inhibitor of vascular endothelial growth factor 1, 2 and 3, is primarily metabolized by cytochrome P450 (CYP) 3A4/5 but is also a substrate for CYP1A2, CYP2C19, UDP-glucuronosyltransferase (UGT)1A1 and the drug transporters P-glycoprotein (encoded by the ABCB1 gene) and OATP1B1 (encoded by SLC01B1). The potential contribution of polymorphisms in genes encoding these enzymes and transporters to axitinib pharmacokinetic variability was assessed.MethodsA fixed effects meta-analysis was performed using data pooled from 11 healthy volunteer clinical pharmacology trials to investigate the potential association between axitinib exposure and major polymorphisms in these genes following a 5-mg dose of axitinib.ResultsUp to 15 variant alleles were evaluated and up to 315 healthy volunteers per polymorphism were assayed. None of the polymorphisms analysed was a statistically significant predictor of axitinib pharmacokinetic variability. Amongst genotypes and inferred phenotypes, CYP2C19 genotype and the ABCB1 (G2677T/A) polymorphism were the closest to statistical significance in influencing axitinib pharmacokinetic variability after multiple-testing adjustment. However, no enzyme or transporter genotype/inferred phenotype contributed >5% to the overall pharmacokinetic variability of axitinib.ConclusionsNo statistically significant associations between the specific polymorphisms analysed and axitinib plasma exposure were observed, suggesting that genotype- or inferred phenotype-based adjustment of axitinib dose in individual subjects is not warranted.

So Many Studies, Too Few Subjects: Establishing Functional Relevance of Genetic Polymorphisms on Pharmacokinetics

Based on current literature, greater clarity in defining the magnitude of polymorphism effects on pharmacokinetics can be achieved by addressing key components of study design, including adequate subject numbers per study group. Convincing evidence of functional relevance exists for polymorphisms in genes such as CYP2D6 and UGT1A1, whereas the published evidence for similar effects for CYP3A5, OATP1B1, and ABCB1 is still emerging or equivocal. Polymorphism‐associated differences in pharmacokinetic parameters were simulated to incorporate (1) the ratio of group mean parameter values for homozygous wild‐type subjects versus homozygous variants, (2) pharmacokinetic variability, and (3) sample size needed to achieve 80% power, assuming 69% coefficient of variation. Subject selection by genotype and choice of probe substrate are also considered. Simulation results and literature examples are incorporated to define key recommendations for future investigations. This will allow for more definitive statements in publications regarding genotype influence on pharmacokinetics.