The molecular basis of dapsone activation of CYP2C9-catalyzed nonsteroidal anti-inflammatory drug oxidation.

Positive heterotropic cooperativity, or "activation," results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of nonsteroidal anti-inflammatory drugs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4'-hydroxylation and S-naproxen O-demethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MDS additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4'-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of nonsteroidal anti-inflammatory drugs with a planar or near-planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.

Drug-drug interactions that alter the exposure of glucuronidated drugs: Scope, UDP-glucuronosyltransferase (UGT) enzyme selectivity, mechanisms (inhibition and induction), and clinical significance.

Drug-drug interactions (DDIs) arising from the perturbation of drug metabolising enzyme activities represent both a clinical problem and a potential economic loss for the pharmaceutical industry. DDIs involving glucuronidated drugs have historically attracted little attention and there is a perception that interactions are of minor clinical relevance. This review critically examines the scope and aetiology of DDIs that result in altered exposure of glucuronidated drugs. Interaction mechanisms, namely inhibition and induction of UDP-glucuronosyltransferase (UGT) enzymes and the potential interplay with drug transporters, are reviewed in detail, as is the clinical significance of known DDIs. Altered victim drug exposure arising from modulation of UGT enzyme activities is relatively common and, notably, the incidence and importance of UGT induction as a DDI mechanism is greater than generally believed. Numerous DDIs are clinically relevant, resulting in either loss of efficacy or an increased risk of adverse effects, necessitating dose individualisation. Several generalisations relating to the likelihood of DDIs can be drawn from the known substrate and inhibitor selectivities of UGT enzymes, highlighting the importance of comprehensive reaction phenotyping studies at an early stage of drug development. Further, rigorous assessment of the DDI liability of new chemical entities that undergo glucuronidation to a significant extent has been recommended recently by regulatory guidance. Although evidence-based approaches exist for the in vitro characterisation of UGT enzyme inhibition and induction, the availability of drugs considered appropriate for use as 'probe' substrates in clinical DDI studies is limited and this should be a research priority.

Understanding the Risk of Drug Interactions Between Ritonavir-Containing COVID-19 Therapies and Small-Molecule Kinase Inhibitors in Patients With Cancer.

PURPOSE: The introduction of COVID-19 therapies containing ritonavir has markedly expanded the scope of use for this medicine. As a strong cytochrome P450 3A4 inhibitor, the use of ritonavir is associated with a high drug interaction risk. There are currently no data to inform clinician regarding the likely magnitude and duration of interaction between ritonavir-containing COVID-19 therapies and small-molecule kinase inhibitors (KIs) in patients with cancer. METHODS: Physiologically based pharmacokinetic modeling was used to conduct virtual clinical trials with a parallel group study design in the presence and absence of ritonavir (100 mg twice daily for 5 days). The magnitude and time course of changes in KI exposure when coadministered with ritonavir was evaluated as the primary outcome. RESULTS: Dosing of ritonavir resulted in a > 2-fold increase in steady-state area under the plasma concentration-time curve and maximal concentration for six of the 10 KIs. When the KI was coadministered with ritonavir, dose reductions to between 10% and 75% of the original dose were required to achieve an area under the plasma concentration-time curve within 1.25-fold of the value in the absence of ritonavir. CONCLUSION: To our knowledge, this study provides the first data to assist clinicians' understanding of the drug interaction risk associated with administering ritonavir-containing COVID-19 therapies to patients with cancer who are currently being treated with KIs. These data may support clinicians to make more informed dosing decisions for patients with cancer undergoing treatment with KIs who require treatment with ritonavir-containing COVID-19 antiviral therapies.

Inhibitory effects of non-steroidal anti-inflammatory drugs on human liver microsomal morphine glucuronidation: Implications for drug-drug interaction liability.

The inhibitory effects of fifteen NSAIDs from six structurally distinct classes on human liver microsomal morphine glucuronidation were investigated. Ki values of selected NSAIDs were generated and employed to assess DDI liability in vivo. Potent inhibition was observed for mefenamic acid and tolfenamic acid; respective IC50 values for morphine 3- and 6-glucuronidation were 9.2 and 13.5 µM, and 5.3 and 8.3 µM. Diclofenac and celecoxib showed moderate inhibition with IC50 values of 78 and 52 µM, and 83 and 214 µM, respectively. Estimated IC50 values for the other NSAIDs screened were >100 µM. Mefenamic acid, diclofenac, and S-naproxen competitively inhibited morphine 3- and 6-glucuronidation, with the Ki values of 11 and 12 µM, 110 and 76 µM, and 319 and 650 µM, respectively. Using the static mechanistic IVIVE approach, an approximate 40% increase in the AUC of morphine was predicted when co-administered with mefenamic acid, whereas the increase was <10% with diclofenac and S-naproxen. PBPK modeling predicted <15% increases in the morphine AUC from diclofenac and S-naproxen inhibition in virtual healthy and cirrhotic subjects. The data suggest that potential clinically significant DDIs arising from NSAID inhibition of morphine glucuronidation is unlikely, with the possible exception of some fenamates.

Binding of SEP-363856 within TAAR1 and the 5HT1A receptor: implications for the design of novel antipsychotic drugs.

Current medications for schizophrenia typically modulate dopaminergic neurotransmission. While affecting positive symptoms, antipsychotic drugs have little clinical effect on negative symptoms and cognitive impairment. Moreover, newer 'atypical' antipsychotic drugs also have significant metabolic adverse-effects. The recent positive clinical trial of the novel drug candidate SEP-363856, which targets non-dopamine receptors (trace amine-associated receptor and the 5HT1A receptor), is a potentially promising development for the management of schizophrenia. In this perspective, we briefly overview the role of TAAR1 and the 5HT1A receptor in schizophrenia and explore the specific binding characteristics of SEP-363856 at these receptors. Molecular dynamics simulations (MDS) indicate that SEP-363856 interacts with a small, common set of conserved residues within the TAAR1 and 5HT1A ligand-binding domain. The primary interaction of SEP-363856 involves binding to the negatively charged aspartate residue (Asp1033.32, TAAR1; Asp1163.32, 5HT1A). In general, the binding of SEP-363856 within TAAR1 involves a greater number of aromatic contacts compared to 5HT1A. MDS provides important insights into the molecular basis of binding site interactions of SEP-363856 with TAAR1 and the 5HT1A receptor, which will be beneficial for understanding the pharmacological uniqueness of SEP-363856 and for the design of novel drug candidates for these newly targeted receptors in the treatment of schizophrenia and related disorders.

Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs).

Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation.

Effect of concomitant use of antihypertensives and immune check point inhibitors on cancer outcomes.

OBJECTIVES: Antihypertensives and cancer have a complex relationship. Among the antihypertensives, renin--angiotensin system inhibitors have strong immune modulatory activities that may affect immune check point inhibitors-related outcomes in cancer patients. We evaluated the association between concomitant use of renin--angiotensin system inhibitors and other antihypertensive agents with survival/toxicity outcomes from atezolizumab. METHODS: A post hoc analysis of individual patient data from seven clinical trials of lung, renal or urothelial cancers was performed. Users and nonusers of antihypertensive classes were compared for overall survival, progression-free survival and immune adverse events. Cox proportional hazards were calculated between the groups and reported as hazards ratio and 95% confidence interval (95% CI). RESULTS: Of the 3695 patients, 2539 were treated with atezolizumab and the rest with chemotherapy. Twenty-four percent of patients were on a renin--angiotensin system inhibitor at trial commencement. No statistically significant difference in overall survival (hazard ratio 0.92, 95% CI 0.79-1.07, P = 0.29), progression-free survival (hazard ratio 0.95, 95% CI 0.84-1.08, P = 0.42) or immune adverse events (odds ratio 0.94, 95% CI 0.76-1.15, P = 0.55) between renin--angiotensin system inhibitor users and nonusers were identified in the atezolizumab-treated cohort. Other classes of antihypertensives were also not associated with survival. CONCLUSION: Concomitant use of antihypertensives including RASi was not associated with survival and immune-related safety outcomes during atezolizumab therapy for solid cancers. Future studies should evaluate the association between antihypertensives and other ICI as well as ICI combination interventions in clinical trials and real-world settings.

Proton Pump Inhibitors and Survival in Patients With Colorectal Cancer Receiving Fluoropyrimidine-Based Chemotherapy.

BACKGROUND: Concomitant use of proton pump inhibitors (PPIs) may negatively affect the efficacy of anticancer drugs such as fluoropyrimidines in patients with colorectal cancer (CRC). The primary objective of this study was to assess whether there is an association between concomitant PPI use and survival outcomes in patients with CRC treated with a fluoropyrimidine-based chemotherapy. PATIENTS AND METHODS: A secondary analysis of 6 randomized controlled clinical trials in patients with advanced CRC was conducted using individual patient data through data-sharing platforms. The outcome measures were progression-free survival and overall survival in PPI users and nonusers. Subgroup analysis included the type of chemotherapy, capecitabine versus 5-FU, line of therapy, and addition of a vascular endothelial growth factor receptor inhibitor. Overall pooled hazard ratios (HRs) with 95% confidence intervals were calculated using a random effects model. RESULTS: A total of 5,594 patients with advanced CRC across 6 trials and 11 trial arms were included; 902 patients were receiving a PPI at trial entry and initiation of chemotherapy. PPI use was significantly associated with worse overall survival (pooled HR, 1.20; 95% CI, 1.03-1.40; P=.02; I2 for heterogeneity = 69%) and progression-free survival (overall pooled HR, 1.20; 95% CI, 1.05-1.37; P=.009; I2 = 65%) after adjusting for clinical covariates. Furthermore, the association between concomitant PPI use and survival outcomes was similar across most treatment subgroups. CONCLUSIONS: We speculate that alterations in the gut microbiome, altered immune milieu within the tumor, and interactions through transporters are potential mechanisms behind this association between PPI use and chemotherapy in patients with CRC, which warrant further study. Concomitant use of PPIs is associated with worse survival outcomes in patients with CRC treated with fluoropyrimidine-based chemotherapy. Clinicians should cautiously consider the concomitant use of PPIs in such patients.

Application of Model Informed Precision Dosing to Address the Impact of Pregnancy Stage and CYP2D6 Phenotype on Foetal Morphine Exposure.

Guidance regarding the effect of codeine and its metabolites on foetal development is limited by small studies and inconsistent findings. The primary objective was to use physiologically based pharmacokinetic modelling to investigate the impact of gestational stage and maternal CYP2D6 phenotype on foetal morphine exposure following codeine administration. Full body physiologically based pharmacokinetic models were developed and verified for codeine and morphine using Simcyp (version 19.1). The impact of gestational age and maternal CYP2D6 phenotype on foetal and maternal morphine and codeine exposure following oral codeine administration was modelled in a cohort of 250 pregnant females and foetuses at gestational weeks 0 (mothers only), 6, 12, 24 and 36. Consistent with the known effect on codeine metabolism, a clinically meaningful (> 1.65-fold) increase in foetal morphine AUC was observed in the CYP2D6 UM phenotype cohort compared to the CYP2D6 EM and PM phenotype cohorts. The mean (95% CI) foetal morphine AUC in the CYP2D6 UM cohort of 0.988 (0.902 to 1.073) ng/mL.h was 1.8-fold higher than the CYP2D6 EM cohort of 0.546 (0.492 to 0.600) ng/mL.h (p < 0.001). Despite a 2.8-fold increase in maternal CYP2D6 protein abundance between gestational weeks 6 and 36, the mean foetal morphine AUC in the CYP2D6 EM and UM phenotype cohorts reduced by 1.55- and 1.75-fold, respectively, over this period. Maternal CYP2D6 phenotype is a significant determinant of foetal morphine AUC. Simulations suggest that the greatest risk with respect to foetal morphine exposure is during the first trimester of pregnancy, particularly in CYP2D6 UM phenotype mothers.

Evidence-based strategies for the characterisation of human drug and chemical glucuronidation in vitro and UDP-glucuronosyltransferase reaction phenotyping.

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily contribute to the elimination of drugs from almost all therapeutic classes. Awareness of the importance of glucuronidation as a drug clearance mechanism along with increased knowledge of the enzymology of drug and chemical metabolism has stimulated interest in the development and application of approaches for the characterisation of human drug glucuronidation in vitro, in particular reaction phenotyping (the fractional contribution of the individual UGT enzymes responsible for the glucuronidation of a given drug), assessment of metabolic stability, and UGT enzyme inhibition by drugs and other xenobiotics. In turn, this has permitted the implementation of in vitro - in vivo extrapolation approaches for the prediction of drug metabolic clearance, intestinal availability, and drug-drug interaction liability, all of which are of considerable importance in pre-clinical drug development. Indeed, regulatory agencies (FDA and EMA) require UGT reaction phenotyping for new chemical entities if glucuronidation accounts for ≥25% of total metabolism. In vitro studies are most commonly performed with recombinant UGT enzymes and human liver microsomes (HLM) as the enzyme sources. Despite the widespread use of in vitro approaches for the characterisation of drug and chemical glucuronidation by HLM and recombinant enzymes, evidence-based guidelines relating to experimental approaches are lacking. Here we present evidence-based strategies for the characterisation of drug and chemical glucuronidation in vitro, and for UGT reaction phenotyping. We anticipate that the strategies will inform practice, encourage development of standardised experimental procedures where feasible, and guide ongoing research in the field.

Arginine-259 of UGT2B7 Confers UDP-Sugar Selectivity.

Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions.

Multiorgan Immune-Related Adverse Events During Treatment With Atezolizumab.

BACKGROUND: Immune-related adverse events (irAEs) are known to occur in patients with cancer who are treated with immune checkpoint inhibitors. However, limited literature exists on the incidence, time of onset, and risk factors for irAEs, particularly those affecting multiple organs, associated with anti-PD-L1 inhibitors. METHODS: A post hoc pooled analysis was conducted using individual patient data from atezolizumab monotherapy arms of 4 non-small cell lung cancer clinical trials. Incidence, clinical patterns, outcomes, and risk factors were investigated of selected organ-specific and multiorgan irAEs during treatment using the anti-PD-L1 inhibitor atezolizumab. RESULTS: From a total of 1,548 patients, 730 irAE episodes were reported in 424 patients (27%). Skin irAEs were the most common (42%), followed by laboratory abnormalities (27%) and endocrine (11.6%), neurologic (7.6%), and pulmonary (6.2%) irAEs. A total of 84 patients (5.4%) had multiorgan irAEs, 70 had 2, 13 had 3, and 1 had 4 different organs affected. "Skin plus" or "laboratory plus" were the most common irAE multiorgan clusters. Patients with multiorgan irAEs were more likely to be white and have a good performance status, a lower baseline neutrophil-lymphocyte ratio, and a good or intermediate lung immune prognostic index score. Multiorgan irAEs were also associated with improved overall survival (hazard ratio, 0.47; 95% CI, 0.28-0.78; P<.0001) but not with progression-free survival (hazard ratio, 0.92; 95% CI, 0.62-1.35; P=.74) compared with the cohort with no irAEs. CONCLUSIONS: Multiorgan irAEs occurred in 5.4% of patients treated with atezolizumab in non-small cell lung cancer trials. Future trials should consider routine reporting of data on multiorgan toxicities in addition to organ-specific toxicities.

Slow-, Tight-Binding Inhibition of CYP17A1 by Abiraterone Redefines Its Kinetic Selectivity and Dosing Regimen.

Substantial evidence underscores the clinical efficacy of inhibiting CYP17A1-mediated androgen biosynthesis by abiraterone for treatment of prostate oncology. Previous structural analysis and in vitro assays revealed inconsistencies surrounding the nature and potency of CYP17A1 inhibition by abiraterone. Here, we establish that abiraterone is a slow-, tight-binding inhibitor of CYP17A1, with initial weak binding preceding the subsequent slow isomerization to a high-affinity CYP17A1-abiraterone complex. The in vitro inhibition constant of the final high-affinity CYP17A1-abiraterone complex ( ( K i * = 0.39 nM )yielded a binding free energy of -12.8 kcal/mol that was quantitatively consistent with the in silico prediction of -14.5 kcal/mol. Prolonged suppression of dehydroepiandrosterone (DHEA) concentrations observed in VCaP cells after abiraterone washout corroborated its protracted CYP17A1 engagement. Molecular dynamics simulations illuminated potential structural determinants underlying the rapid reversible binding characterizing the two-step induced-fit model. Given the extended residence time (42 hours) of abiraterone within the CYP17A1 active site, in silico simulations demonstrated sustained target engagement even when most abiraterone has been eliminated systemically. Subsequent pharmacokinetic-pharmacodynamic (PK-PD) modeling linking time-dependent CYP17A1 occupancy to in vitro steroidogenic dynamics predicted comparable suppression of downstream DHEA-sulfate at both 1000- and 500-mg doses of abiraterone acetate. This enabled mechanistic rationalization of a clinically reported PK-PD disconnect, in which equipotent reduction of downstream plasma DHEA-sulfate levels was achieved despite a lower systemic exposure of abiraterone. Our novel findings provide the impetus for re-evaluating the current dosing paradigm of abiraterone with the aim of preserving PD efficacy while mitigating its dose-dependent adverse effects and financial burden. SIGNIFICANCE STATEMENT: With the advent of novel molecularly targeted anticancer modalities, it is becoming increasingly evident that optimal dose selection must necessarily be predicated on mechanistic characterization of the relationships between target exposure, drug-target interactions, and pharmacodynamic endpoints. Nevertheless, efficacy has always been perceived as being exclusively synonymous with affinity-based measurements of drug-target binding. This work demonstrates how elucidating the slow-, tight-binding inhibition of CYP17A1 by abiraterone via in vitro and in silico analyses was pivotal in establishing the role of kinetic selectivity in mediating time-dependent CYP17A1 engagement and eventually downstream efficacy outcomes.

Binding of clozapine to the GABAB receptor: clinical and structural insights.

Clozapine is the gold-standard agent for treatment resistant schizophrenia but its mechanism of action remains unclear. There is emerging evidence of the potential role of the GABAB receptor in the pathogenesis of schizophrenia. It has been hypothesised that clozapine can mediate its actions via the GABAB receptor. Baclofen is currently recognised as the prototype GABAB receptor agonist. There are some potential clinical similarities between clozapine and baclofen. Indeed, baclofen has been previously proposed for use as an antipsychotic agent. Our analysis of the X-ray crystal structure of GABAB receptor along with molecular docking calculations, suggests that clozapine could directly bind to the GABAB receptor similar to that of baclofen. This finding could lead to a better understanding of the pharmacological uniqueness of clozapine, potential development of a biomarker for treatment resistant schizophrenia and the development of more targeted treatments leading to personalisation of treatment.

Relationship between vemurafenib plasma concentrations and survival outcomes in patients with advanced melanoma.

Purpose To validate a plasma vemurafenib steady-state trough concentration (Css,min) threshold that predicts survival outcomes of patients with BrafV600 mutated melanoma. METHODS: A pooled analysis of individual patient data from two advanced melanoma trials involving vemurafenib ± cobimetinib therapy was performed. Day 23 was chosen as the landmark time when steady-state concentration reached. Optimal Css,min threshold was determined via assessment of discriminative performance and model fitting. Association between vemurafenib Css,min and survival was modelled using Cox proportional hazards regression. RESULTS: Vemurafenib plasma concentration data were available for 402 patients who were on stable dose for the first 3 weeks. When compared to a previously described plasma vemurafenib Css,min threshold of 42 mg/L, we identified that a cutoff concentration of 50 mg/L by day 23 was strongly associated with progression-free survival and overall survival. The association remained statistically significant after adjusting for important clinical confounding variables. Sub-group analysis showed that while the addition of cobimetinib resulted in a lower day 23 plasma vemurafenib Css,min, the threshold was still associated with overall survival and not in the monotherapy cohort. CONCLUSION: A plasma vemurafenib Css,min threshold of 50 mg/L is strongly associated with survival outcomes in patients with advanced melanoma. This new threshold needs to be validated prospectively in future studies.

Association Between Body Mass Index and Overall Survival With Immune Checkpoint Inhibitor Therapy for Advanced Non-Small Cell Lung Cancer.

Importance: High body mass index (BMI) is independently associated with overall survival benefit from immune checkpoint inhibitor therapy in patients with melanoma, yet whether BMI is associated with outcomes in patients with advanced non-small cell lung cancer treated with atezolizumab remains unknown. Objective: To examine whether BMI is associated with survival outcomes and adverse events in patients with non-small cell lung cancer (NSCLC) treated with atezolizumab. Design, Setting, and Participants: A pooled analysis of individual patient-level data from 4 international, multicenter clinical trials was performed. Two were single-arm phase 2 trials (BIRCH [data cutoff of May 28, 2015] and FIR [data cutoff of January 7, 2015]), and 2 were 2-arm randomized clinical trials (POPLAR [phase 2; data cutoff of May 8, 2015] and OAK [phase 3; data cutoff of July 7, 2016]). Patients with advanced NSCLC previously untreated or treated with at least 1 line of systemic therapy, with measurable disease and good organ function and without contraindications for chemotherapy or immune checkpoint inhibitor therapy, were included in these trials. Data analyses were performed from February 28, 2019, to September 30, 2019. Interventions: The control group was treated with docetaxel once every 3 weeks until disease progression or unacceptable toxic effects occurred in POPLAR and OAK. The experimental group was treated with atezolizumab once every 3 weeks until disease progression or unacceptable toxic effects occurred in all available trials. Main Outcomes and Measures: Association between BMI and overall survival (OS), progression-free survival (PFS), and treatment-related adverse events. Intention-to-treat analysis was conducted. Results: Adequate data were available for 2110 patients from a total pool of 2261 across 4 trials. Of those 2110, 1434 patients (median age, 64 years [range, 57-70 years]; 890 men [62%]) received atezolizumab and 676 patients (median age, 63 years[range, 57-69 years]; 419 men [62%]) received docetaxel. There was a linear association between increasing BMI and OS in patients treated with atezolizumab. Obesity (BMI ≥30 [calculated as weight in kilograms divided by height in meters squared]) was associated with significantly improved OS in patients treated with atezolizumab, but not in those who received docetaxel after adjusting for confounding variables. The association between BMI and OS/PFS was the strongest in the high PD-L1 expression subgroup. Overall survival for patients with the highest category of PD-L1 expression (≥50% of tumor cells or ≥10% of tumor-infiltrating immune cells; n = 436) had hazard ratios of 0.36 (95% CI, 0.21-0.62) for the group with obesity and 0.69 (95% CI, 0.48-0.98) for the group with overweight. Patients with the highest category of PD-L1 expression had PFS hazard ratios of 0.68 (95% CI, 0.49-0.94) for the group with obesity and 0.72 (95% CI, 0.56-0.92) for the group with overweight. Treatment-related adverse events were not associated with BMI. Conclusions and Relevance: High BMI appears to be independently associated with improved survival with atezolizumab in patients with NSCLC, raising the possibility that baseline BMI should be considered as a stratification factor in future immune checkpoint inhibitor therapy trials.

Inhibition of human UDP-glucuronosyltransferase (UGT) enzymes by kinase inhibitors: Effects of dabrafenib, ibrutinib, nintedanib, trametinib and BIBF 1202.

We have demonstrated previously that the kinase inhibitors (KIs) lapatinib, pazopanib, regorafenib and sorafenib are potent inhibitors of UGT1A1 and UGTs 1A7-1A10. The present study characterised the effects of four additional drugs in this class, dabrafenib, ibrutinib, nintedanib and trametinib, on human UGT enzyme activities in vitro. Dabrafenib, ibrutinib, nintedanib and trametinib were potent inhibitors of human liver microsomal UGT1A1; Ki,u values ranged from 1.1 to 7.5 µM. Similarly, these KIs inhibited UGT 1A7, 1A8, 1A9 and 1A10, albeit less potently than UGT1A1. Despite the potent inhibition of UGT1A1, in vitro - in vivo extrapolation excluded the likelihood that dabrafenib, ibrutinib, nintedanib and trametinib would precipitate drug-drug interactions (DDIs) due to the low unbound plasma concentrations of these drugs observed in patients. The structures of dabrafenib, ibrutinib, lapatinib, nintedanib, pazopanib, regorafenib, trametinib and 22 other KIs overlaid well on that of sorafenib, a potent inhibitor of UGT1A1 and UGTs 1A7-1A10. Taken together, kinetic and computational modelling data suggest that all currently marketed KIs are likely to be potent inhibitors of UGT1A1, and are also likely to inhibit UGTs 1A7-1A10 to some extent due to the structural and chemical features shared in common by these drugs. By contrast, BIBF 1202, the major metabolite of nintedanib, did not appreciably inhibit human UGTs, due to the presence of a terminal electronegative group which appears to disfavor enzyme inhibition. Given the potent inhibition of several UGT enzymes, especially UGT1A1, by KIs, characterisation of the DDI potential of newly developed agents in this class is warranted.

Identification of the caffeine to trimethyluric acid ratio as a dietary biomarker to characterise variability in cytochrome P450 3A activity.

PURPOSE: Cytochrome P450 (CYP) 3A plays an important role in the metabolism of many clinically used drugs and exhibits substantial between-subject variability (BSV) in activity. Current methods to assess variability in CYP3A activity have limitations and there remains a need for a minimally invasive clinically translatable strategy to define CYP3A activity. The purpose of this study was to evaluate the potential for a caffeine metabolic ratio to describe variability in CYP3A activity. METHODS: The metabolic ratio 1,3,7-trimethyluric acid (TMU) to caffeine was evaluated as a biomarker to describe variability in CYP3A activity in a cohort (n = 28) of healthy 21 to 35-year-old males. Midazolam, caffeine, and TMU concentrations were assessed at baseline and following dosing of rifampicin (300 mg daily) for 7 days. RESULTS: At baseline, correlation coefficients for the relationship between apparent oral midazolam clearance (CL/F) with caffeine/TMU ratio measured at 3, 4, and 6 h post dose were 0.82, 0.79, and 0.65, respectively. The strength of correlations was retained post rifampicin dosing; 0.72, 0.87, and 0.82 for the ratios at 3, 4, and 6 h, respectively. Weaker correlations were observed between the change in midazolam CL/F and change in caffeine/TMU ratio post/pre-rifampicin dosing. CONCLUSION: BSV in CYP3A activity was well described by caffeine/TMU ratios pre- and post-induction. The caffeine/TMU ratio may be a convenient tool to assess BSV in CYP3A activity, but assessment of caffeine/TMU ratio alone is unlikely to account for all sources of variability in CYP3A activity.

Computational Prediction of the Site(s) of Metabolism and Binding Modes of Protein Kinase Inhibitors Metabolized by CYP3A4.

Protein kinase inhibitors (KIs), which are mainly biotransformed by CYP3A4-catalyzed oxidation, represent a rapidly expanding class of drugs used primarily for the treatment of cancer. Ligand- and structure-based methods were applied here to investigate whether computational approaches may be used to predict the site(s) of metabolism (SOM) of KIs and to identify amino acids within the CYP3A4 active site involved in KI binding. A data set of the experimentally determined SOMs of 31 KIs known to undergo biotransformation by CYP3A4 was collated. The structure-based (molecular docking) approach employed three CYP3A4 X-ray crystal structures to account for structural plasticity of this enzyme. Docking pose and SOM predictivity were influenced by the X-ray crystal template used for docking and the scoring function used for ranking binding poses. The best prediction of SOM (77%) was achieved using the substrate (bromoergocryptine)-bound X-ray crystal template together with the potential of mean force score. Binding interactions of KIs with CYP3A4 active site residues were generally similar to those observed for other substrates of this enzyme. The ligand-based molecular superposition approach, using bromoergocryptine from the X-ray cocrystal structure as a template, poorly predicted (42%) the SOM of KIs, although predictivity improved to 71% when the docked conformation of sorafenib was used as the template. Among the web-based approaches examined, all web servers provided excellent predictivity, with one web server predicting the SOM of 87% of the data set molecules. Computational approaches may be used to predict the SOM of KIs, and presumably other classes of CYP3A4 substrates, but predictivity varies between methods.

Drug and Chemical Glucosidation by Control Supersomes and Membranes from Spodoptera frugiperda (Sf) 9 Cells: Implications for the Apparent Glucuronidation of Xenobiotics by UDP-glucuronosyltransferase 1A5.

Accumulating evidence indicates that several human UDP-glucuronosyltransferase (UGT) enzymes catalyze both glucuronidation and glucosidation reactions. Baculovirus-infected insect cells [Trichoplusia ni and Spodoptera frugiperda (Sf9)] are used widely for the expression of recombinant human UGT enzymes. Following the observation that control Supersomes (c-SUP) express a native enzyme capable of glucosidating morphine, we characterized the glucosidation of a series of aglycones with a hydroxyl (aliphatic or phenolic), carboxylic acid, or amine functional group by c-SUP and membranes from uninfected Sf9 cells. Although both enzyme sources glucosidated the phenolic substrates investigated, albeit with differing activities, differences were observed in the selectivities of the native UDP-glucosyltransferases toward aliphatic alcohols, carboxylic acids, and amines. For example, zidovudine was solely glucosidated by c-SUP. By contrast, c-SUP lacked activity toward the amines lamotrigine and trifluoperazine and did not form the acyl glucoside of mycophenolic acid, reactions all catalyzed by uninfected Sf9 membranes. Glucosidation intrinsic clearances were high for several substrates, notably 1-hydroxypyrene (∼1400-1900 µl/min⋅mg). The results underscore the importance of including control cell membranes in the investigation of drug and chemical glucosidation by UGT enzymes expressed in T. ni (High-Five) and Sf9 cells. In a coincident study, we observed that UGT1A5 expressed in Sf9, human embryonic kidney 293T, and COS7 cells lacked glucuronidation activity toward prototypic phenolic substrates. However, Sf9 cells expressing UGT1A5 glucosidated 1-hydroxypyrene with UDP-glucuronic acid as the cofactor, presumably due to the presence of UDP-glucose as an impurity. Artifactual glucosidation may explain, at least in part, a previous report of phenolic glucuronidation by UGT1A5.