Homologous Recombination Deficiency: Concepts, Definitions, and Assays.

BACKGROUND: Homologous recombination deficiency (HRD) is a phenotype that is characterized by the inability of a cell to effectively repair DNA double-strand breaks using the homologous recombination repair (HRR) pathway. Loss-of-function genes involved in this pathway can sensitize tumors to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy, which target the destruction of cancer cells by working in concert with HRD through synthetic lethality. However, to identify patients with these tumors, it is vital to understand how to best measure homologous repair (HR) status and to characterize the level of alignment in these measurements across different diagnostic platforms. A key current challenge is that there is no standardized method to define, measure, and report HR status using diagnostics in the clinical setting. METHODS: Friends of Cancer Research convened a consortium of project partners from key healthcare sectors to address concerns about the lack of consistency in the way HRD is defined and methods for measuring HR status. RESULTS: This publication provides findings from the group's discussions that identified opportunities to align the definition of HRD and the parameters that contribute to the determination of HR status. The consortium proposed recommendations and best practices to benefit the broader cancer community. CONCLUSION: Overall, this publication provides additional perspectives for scientist, physician, laboratory, and patient communities to contextualize the definition of HRD and various platforms that are used to measure HRD in tumors.

Angiogenic and immunomodulatory biomarkers in axitinib-treated patients with advanced renal cell carcinoma.

Aim: Immunomodulatory mechanisms contributing to angiogenic inhibition in renal tumors are not well characterized. We report associations between efficacy and tumor-associated immune cells and mRNA/miRNA expression in patients from AXIS. Materials & methods: Immunohistochemistry (n = 52) and mRNA/miRNA expression analyses (n = 72) were performed on tumor samples. Results: In axitinib-treated patients, higher CXCR4 and TLR3 expression, respectively, was associated with longer progression-free survival (hazard ratio; 95% CI: 0.3; 0.1-0.8 and 0.4; 0.2-0.9) and showed interaction with treatment (p = 0.029 and p < 0.001); lower CCR7 expression was associated with objective response (odds ratio: 0.1; 95% CI: 0.01-1.0) and longer overall survival (hazard ratio: 3.9; 95% CI: 1.4-10.3). Conclusion: CCR7, CXCR4 and TLR3 expression levels may be prognostic/predictive of clinical benefit with axitinib. Clinical trial identifier: ClinicalTrials.gov NCT00678392.

A case for the use of receiver operating characteristic analysis of potential clinical efficacy biomarkers in advanced renal cell carcinoma.

AIM: Assess patient-level utility of suggested pretreatment biomarkers of sunitinib in advanced renal cell carcinoma. PATIENTS & METHODS: Kaplan-Meier analysis of data from a randomized, Phase II study (n = 292) suggested baseline predictive value for circulating soluble Ang-2 and MMP-2 and HIF-1α percentage of tumor expression. Using this dataset, the sensitivity, specificity and area under the curve (AUC) were calculated, using receiver operating characteristic (ROC) curves. RESULTS: Based on a ROC (sensitivity vs 1 - specificity) threshold AUC value of >0.8, neither Ang-2 (0.67) nor MMP-2 (0.65), nor HIF-1α percentage of tumor expression (0.65), performed appropriately from a patient-selection standpoint. CONCLUSION: To properly assess potential biomarkers, sensitivity and specificity characteristics should be obtained by ROC analysis.

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 patient's 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.

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

PURPOSE: Axitinib, 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. METHODS: A 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. RESULTS: Up 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. CONCLUSIONS: No 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.

Application of the fluorescent probe 1-anilinonaphthalene-8-sulfonate to the measurement of the nonspecific binding of drugs to human liver microsomes.

The fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) in the presence of human liver microsomes (HLMs) is altered by drugs that bind nonspecifically to the lipid bilayer. The present study characterized the relationship between the nonspecific binding (NSB) of drugs to HLMs as measured by equilibrium dialysis and the magnitude of the change in baseline ANS fluorescence. Fraction unbound in incubations of HLMs (f(u(mic))) was determined for 16 drugs (12 bases, 3 acids, and 1 neutral) with log P values in the range 0.1 to 6.7 at three concentrations (100, 200, and 500 µM). Changes in ANS fluorescence induced by each of the drugs in the presence of HLMs were measured by spectrofluorometry. Values of f(u(mic)) determined by equilibrium dialysis ranged from 0.08 to 1.0. Although NSB of the basic drugs tended to increase with increasing log P, exceptions occurred. Basic drugs generally caused an increase in ANS fluorescence, whereas the acidic and neutral drugs resulted in a decrease in ANS fluorescence. There were highly significant (p < 0.001) linear relationships between the modulus (absolute value) of the increment/decrement in ANS fluorescence and both f(u(mic)) (r = 0.90 to 0.96) and log(1 - f(u(mic))/f(u(mic))) (r = 0.85 to 0.92) at the three drug concentrations. Agreement between measured f(u(mic)) and that predicted by ANS fluorescence was very good (<10% variance) for a validation set of six compounds. The ANS fluorescence method provides an accurate measure of the NSB of drugs to HLMs. Physicochemical determinants other than log P and charge type influence the NSB of drugs to HLMs.

Optimal PD-L1-high cutoff for association with overall survival in patients with urothelial cancer treated with durvalumab monotherapy.

BACKGROUND: Studies have indicated that programmed death ligand 1 (PD-L1) expression may have utility as a predictive biomarker in patients with advanced/metastatic urothelial carcinoma (UC). Different immunohistochemical (IHC) assays are in development to assess PD-L1 expression on tumor cells (TCs) and tumor-infiltrating immune cells (ICs). METHODS: In this post hoc analysis of the single-arm, phase 1/2 Study 1108 (NCT01693562), PD-L1 expression was evaluated from tumor samples obtained prior to second-line treatment with durvalumab in patients with advanced/metastatic UC using the VENTANA (SP263) IHC Assay. The primary objective was to determine whether the TC ≥ 25%/IC ≥ 25% algorithm (i.e., cutoff of ≥ 25% TC or ≥ 25% IC with PD-L1 staining at any intensity above background) was optimal for predicting response to durvalumab. PD-L1 expression data were available from 188 patients. RESULTS: After a median follow-up of 15.8 and 14.6 months, higher PD-L1 expression was associated with longer overall survival (OS) and progression-free survival (PFS), respectively, with significant separation in survival curves for PD-L1-high and-low expressing patients for the TC ≥ 25%/IC ≥ 25% cutoff (median OS: 19.8 vs 4.8 months; hazard ratio: 0.46; 90% confidence interval: 0.33, 0.639). OS was also prolonged for PD-L1-high compared with-low patients when samples were categorized using TC/IC combined positive score ≥ 10 and IC≥ 5% cutoffs. In multivariate analysis, IC but not TC PD-L1 expression was significantly associated with OS, PFS, and objective response rate (P < 0.001 for each), although interaction analysis showed similar directionality of benefit for ICs and TCs. CONCLUSIONS: These findings support the utility of a combined TC/IC algorithm for predicting response to durvalumab in patients with UC, with the TC≥ 25%/IC≥ 25% cutoff optimal when used with the VENTANA (SP263) IHC Assay.

Development of a PD-L1 Complementary Diagnostic Immunohistochemistry Assay (SP142) for Atezolizumab.

Cancer immunotherapies, such as atezolizumab, are proving to be a valuable therapeutic strategy across indications, including non-small cell lung cancer (NSCLC) and urothelial cancer (UC). Here, we describe a diagnostic assay that measures programmed-death ligand 1 (PD-L1) expression, via immunohistochemistry, to identify patients who will derive the most benefit from treatment with atezolizumab, a humanized monoclonal anti-PD-L1 antibody. We describe the performance of the VENTANA PD-L1 (SP142) Assay in terms of specificity, sensitivity, and the ability to stain both tumor cells (TC) and tumor-infiltrating immune cells (IC), in NSCLC and UC tissues. The reader precision, repeatability and intermediate precision, interlaboratory reproducibility, and the effectiveness of pathologist training on the assessment of PD-L1 staining on both TC and IC were evaluated. We detail the analytical validation of the VENTANA PD-L1 (SP142) Assay for PD-L1 expression in NSCLC and UC tissues and show that the assay reliably evaluated staining on both TC and IC across multiple expression levels/clinical cut-offs. The reader precision showed high overall agreement when compared with consensus scores. In addition, pathologists met the predefined training criteria (≥85.0% overall percent agreement) for the assessment of PD-L1 expression in NSCLC and UC tissues with an average overall percent agreement ≥95.0%. The assay evaluates PD-L1 staining on both cell types and is robust and precise. In addition, it can help to identify those patients who may benefit the most from treatment with atezolizumab, although treatment benefit has been demonstrated in an all-comer NSCLC and UC patient population.

Novel polymorphic human UDP-glucuronosyltransferase 2A3: cloning, functional characterization of enzyme variants, comparative tissue expression, and gene induction.

UDP-glucuronosyltransferases (UGTs) are critical to the detoxification of numerous drugs, environmental pollutants, and endogenous molecules. However, as yet not all of the human UGTs have been cloned and characterized. cDNA clones from the UGT2A3 gene (located on chromosome 4q13) were isolated using pooled human liver RNA. Approximately 10% of clones contained a c.1489A>G nucleotide substitution, yielding proteins with a residue 497 alanine (UGT2A3.2) instead of a threonine (UGT2A3.1). The allele frequency of this polymorphism (rs13128286) was 0.13 in a European-American population as determined by direct DNA sequencing. Of 81 structurally diverse glucuronidation substrates tested, UGT2A3 expressed by a baculovirus system selectively glucuronidated bile acids, particularly hyodeoxycholic acid at the 6-hydroxy position. Apparent K(m) values of UGT2A3.1 and UGT2A3.2 for hyodeoxycholic acid 6-glucuronidation were 69 +/- 7 and 44 +/- 12 microM, respectively. Of 29 different extrahepatic tissues evaluated by real-time polymerase chain reaction, UGT2A3 mRNA was most highly expressed in small intestine (160% of liver), colon (78% of liver), and adipose tissue (91% of liver). An in silico scan of the proximal UGT2A3 promoter/5'-regulatory region identified transcription factor consensus elements consistent with tissue-selective expression in liver (HNF1) and intestine (CXD2), as well as induction by rifampicin (pregnane X receptor). In LS180 human intestinal cells, rifampicin increased UGT2A3 mRNA by more than 4.5-fold compared with vehicle, whereas levels were not significantly affected by the arylhydrocarbon receptor ligand beta-naphthoflavone. This is the first report establishing UGT2A3 as a functional enzyme, and it represents significant progress toward the goal of having a complete set of recombinant human UGTs for comparative functional analyses.

Kinetic modeling of the interactions between 4-methylumbelliferone, 1-naphthol, and zidovudine glucuronidation by udp-glucuronosyltransferase 2B7 (UGT2B7) provides evidence for multiple substrate binding and effector sites.

Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.

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.

Regional Differences in Efficacy, Safety, and Biomarkers for Second-Line Axitinib in Patients with Advanced Hepatocellular Carcinoma: From a Randomized Phase II Study.

BACKGROUND: An unmet need exists for treatment of patients with advanced hepatocellular carcinoma (HCC) who progress on or are intolerant to sorafenib. A global randomized phase II trial (ClinicalTrial.gov No. NCT01210495) of axitinib, a vascular endothelial growth factor receptor 1-3 inhibitor, in combination with best supportive care (BSC) did not prolong overall survival (OS) over placebo/BSC, but showed improved progression-free survival in some patients. Subgroup analyses were conducted to identify potential predictive/prognostic factors. METHODS: The data from this phase II study were analyzed for the efficacy and safety of axitinib/BSC in patients from Asia versus non-Asia versus Asian subgroups (Japan, Korea, or mainland China/Hong Kong/Taiwan) and predictive/prognostic values of baseline microRNAs and serum soluble proteins, using the Cox proportional hazards model. RESULTS: Of 202 patients, 78 were from non-Asia and 124 from Asia (37 Japanese, 36 Korean, and 51 Chinese). No significant differences in OS were found between axitinib/BSC and placebo/BSC in non-Asians, Asians, or Asian subgroups. However, in an exploratory analysis, axitinib/BSC showed favorable OS in Asians, especially Japanese, when patients intolerant to prior antiangiogenic therapy were excluded from the data set. Axitinib/BSC was well tolerated by non-Asians and Asians alike. The presence of 4 circulating microRNAs, including miR-5684 and miR-1224-5p, or a level lower than or equal to the median protein level of stromal cell-derived factor 1 at baseline was significantly associated with longer OS in axitinib/BSC-treated Asians or non-Asians. CONCLUSIONS: Axitinib/BSC did not prolong survival over placebo/BSC in non-Asians, Asians, or Asian subgroups, but favorable OS with axitinib/BSC was observed in a subset of Japanese patients. A patient population that excludes sorafenib-intolerant patients might potentially be more suitable for clinical trials of new agents in advanced HCC. Since these results are very preliminary, further investigation is warranted. The potential predictive/prognostic value of several baseline microRNAs and soluble proteins identified in this study would require validation in prospective studies on a large cohort of patients.

Germline Genetic Biomarkers of Sunitinib Efficacy in Advanced Renal Cell Carcinoma: Results From the RENAL EFFECT Trial.

BACKGROUND: Sunitinib, the vascular endothelial growth factor pathway inhibitor, is an established standard-of-care for advanced renal cell carcinoma (RCC). This study aimed to assess correlations between candidate germline single nucleotide polymorphisms (SNPs) and sunitinib efficacy in patients from the RENAL EFFECT trial (NCT00267748), a randomized phase II study in patients with metastatic RCC comparing the 4-weeks-on/2-weeks-off schedule and a continuous daily dosing schedule. PATIENTS AND METHODS: Informed consent for pharmacogenetics research was obtained from 202 out of 289 treated patients in the trial. Associations between 9 SNP variants (CXCL8, LOXL2, CCDC26, SH3GL2, CLLU1, IL2RA, AURKB, and 2 SNPs on Chromosomes 7 and 12) and progression-free survival (PFS), objective response rate, and overall survival were assessed using Kaplan-Meier analysis, Cox proportional hazard model, and the Fisher exact test. RESULTS: CXCL8 rs1126647 A/A versus A/T (P = .004) or T/T (P < .0001) and SH3GL2 rs10963287 C/C versus C/T (P = .005) or T/T (P = .018) were associated with improved overall survival in all patients. CLLU1 rs525810 A/A genotype versus A/G (P = .014) or G/G (P = .048) was associated with improved PFS in the continuous daily dosing arm. IL2RA rs7893467 T/G versus T/T was associated with improved PFS (P = .034) in the 4-weeks-on/2-weeks-off arm and objective response rate (P = .034) in all patients. No significant associations between improved efficacy and genotype were found for other SNPs. CONCLUSION: Germline variants in CLLU1, IL2RA, CXCL8, and SH3GL2 warrant further retrospective study in independent cohorts of patients with metastatic RCC treated with vascular endothelial growth factor-class inhibitors, to test their biological significance and potential clinical fitness as biomarkers to guide treatment.

PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project.

INTRODUCTION: The Blueprint Programmed Death Ligand 1 (PD-L1) Immunohistochemistry (IHC) Assay Comparison Project is an industrial-academic collaborative partnership to provide information on the analytical and clinical comparability of four PD-L1 IHC assays used in clinical trials. METHODS: A total of 39 NSCLC tumors were stained with four PD-L1 IHC assays (22C3, 28-8, SP142, and SP263), as used in the clinical trials. Three experts in interpreting their respective assays independently evaluated the percentages of tumor and immune cells staining positive at any intensity. Clinical diagnostic performance was assessed through comparisons of patient classification above and below a selected expression cutoff and by agreement using various combinations of assays and cutoffs. RESULTS: Analytical comparison demonstrated that the percentage of PD-L1-stained tumor cells was comparable when the 22C3, 28-8, and SP263 assays were used, whereas the SP142 assay exhibited fewer stained tumor cells overall. The variability of immune cell staining across the four assays appears to be higher than for tumor cell staining. Of the 38 cases, 19 (50.0%) were classified above and five (13%) were classified below the selected cutoffs of all assays. For 14 of the 38 cases (37%), a different PD-L1 classification would be made depending on which assay/scoring system was used. CONCLUSIONS: The Blueprint PD-L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD-L1 expression for three assays, interchanging assays and cutoffs would lead to "misclassification" of PD-L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy-related PD-L1 cutoffs.

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.

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.

In vitro ADME phenotyping in drug discovery: current challenges and future solutions.

Drug-metabolizing enzymes and drug transporters are key regulators of drug disposition and pharmacodynamics, which are closely linked to drug efficacy and safety. In this article, current challenges and future solutions to predicting their influence on pharmacokinetics and inter-organ distribution in humans, from data generated during the drug discovery decision-making process, are presented. In vitro phenotyping strategies for drug metabolizing enzymes (eg, CYP3A4, UGT1A1) and transporters (eg, OATP1B1) are offered, including perspectives on a selection of in vitro systems, novel in vitro phenotyping reagents and remaining technology gaps, challenges in extrapolating in vitro data to the in vivo situation, in silico models for the prediction of whether compounds are enzyme or transporter substrates, and the impact of pharmacogenomics.

A novel simultaneous modeling approach to estimate linearity of pharmacokinetic parameters, including saturation of intestinal efflux, in the rat.

INTRODUCTION: Various independent methods exist for the estimation of linearity of pharmacokinetic parameters in vivo. A novel simultaneous modeling approach has been developed in the rat that in combination allows estimation of the rate and extent of duodenal absorption, hepatic first pass extraction and saturation of potential dose-dependent duodenal bioavailability (saturation of intestinal efflux). METHODS: Simultaneous modeling of plasma concentrations of a Pfizer compound in the rat were conducted using NONMEM after (1) accelerated intravenous, intraduodenal, and intraportal infusions over 5 h and (2) 5-min intravenous infusions of 0.28 and 1.4 mg doses. RESULTS: The data was best described by a two-compartment linear pharmacokinetic model with good agreement between observed and model predicted plasma concentrations following the various routes of administration. Clearance was estimated to be linear up to plasma concentrations of 1200 ng/ml. The estimated rate constants (+/-asymptotic errors) for intraduodenal absorption (KA), movement of drug from plasma to tissue (K23) and movement of drug from tissue to plasma (K32) were 0.645+/-0.107, 18.0+/-2.98, and 2.02+/-0.209 h(-1), respectively. The rate constants for drug elimination from the central compartment (K20) after 5-min intravenous infusion or accelerated infusion were 3.24+/-0.6 or 6.26+/-1.64 h(-1). The estimated maximal extent of first pass extraction was 17%. The model (including increasing duodenal bioavailability as the amount in the duodenum increases, from a minimum of 5% to a maximum estimated intraportal bioavailability value-83%) indicated a saturable intestinal efflux process. DISCUSSION: This novel study design and the proposed method for data analysis provides a robust and efficient means for assessing the linearity of multiple pharmacokinetic processes while accounting for the multi-compartmental distribution characteristics of the test compound.

Genotype Correlations With Blood Pressure and Efficacy From a Randomized Phase III Trial of Second-Line Axitinib Versus Sorafenib in Metastatic Renal Cell Carcinoma.

BACKGROUND: In the phase III axitinib second-line (AXIS) trial, axitinib significantly prolonged progression-free survival (PFS) versus sorafenib in patients with previously treated metastatic renal cell carcinoma (mRCC). Analyses of associations between germline single-nucleotide polymorphisms (SNPs) and outcomes are reported. PATIENTS AND METHODS: DNA samples from blood were genotyped using TaqMan allelic discrimination. Logistic/Cox regression analyses were used to evaluate association of 15 SNPs in vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)1, VEGFR2, or hypoxia-inducible factor (HIF)-1α with outcomes for blood pressure (BP; Grade ≥ 3 hypertension, diastolic BP > 90 mm Hg, and increase ≥ 15 mm Hg from baseline) and efficacy (independent review committee-assessed objective response rate and PFS, and overall survival [OS]). Multivariate analyses assessed SNPs and baseline characteristics as potential predictors of PFS and OS. RESULTS: Genotype data were available for 305 (42.7%) of 714 patients; 159 received axitinib and 146 sorafenib. After Bonferroni adjustment, no SNP was associated with BP outcomes. In axitinib-treated patients, VEGF-A rs699947 (A/A vs. C/C) and rs833061 (C/C vs. T/T) were associated with longer OS (27.0 vs. 13.4 months; hazard ratio [HR], 0.39; Padjusted = .015). In sorafenib-treated patients, VEGFR2 rs2071559 (G/G vs. A/A) was associated with longer OS (26.8 vs. 13.8 months; HR, 0.41; Padjusted = .030). In multivariate analyses, no SNP predicted axitinib efficacy; VEGFR2 rs2071559 predicted PFS (P = .0053) and OS (P = .0027) for sorafenib. Sensitivity/specificity of VEGFR2 rs2071559 for OS was < 80%. CONCLUSION: No SNP predicted axitinib outcomes. Although VEGFR2 rs2071559 predicted sorafenib efficacy in patients with mRCC, sensitivity/specificity limitations preclude its use for selecting individual patients for sorafenib treatment.

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

OBJECTIVES: Our objectives were to evaluate the contribution of bergamottin to the grapefruit juice-felodipine interaction and to characterize bergamottin disposition. METHODS: In this study 250 mL grapefruit juice; 2-, 6-, or 12-mg capsules of bergamottin plus water; or water was administered with 5 mg extended-release felodipine to 11 volunteers in a partially randomized, 5-way crossover study. Plasma concentrations of felodipine, its primary metabolite (dehydrofelodipine), bergamottin, and 6',7'-dihydroxybergamottin were determined. RESULTS: Grapefruit juice (containing 1.7 mg bergamottin) increased peak plasma concentration (C max ) and area under the plasma concentration-time curve (AUC) of felodipine by 89% (P < .025) and 54% (P < .025), respectively, compared with water. With 2 mg bergamottin, felodipine C max increased by 33% (P < .05). The increase by bergamottin was markedly variable among individuals (range, -33% to 125%). With 6 mg bergamottin, felodipine C max was enhanced by 35% (P < .025), and with 12 mg bergamottin, felodipine C max increased by 40% (P < .05) and AUC increased by 37% (P < .05) compared with water. Bergamottin measured in plasma after administration of 6 and 12 mg produced C max values of 2.1 and 5.9 ng/mL, respectively, and times to reach C max of 0.8 and 1.1 hours, respectively. The bergamottin metabolite 6',7'-dihydroxybergamottin was detected in plasma of some subjects after bergamottin administration. CONCLUSIONS: Bergamottin enhanced the oral bioavailability of felodipine and may cause a clinically relevant drug interaction in susceptible individuals. Grapefruit juice-drug interactions likely also involve other furanocoumarins, possibly acting in combination by additive or synergistic mechanisms. Bergamottin has systemic availability and is metabolized in vivo to 6',7'-dihydroxybergamottin.