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Background: Aficamten, a novel cardiac myosin inhibitor, reversibly reduces cardiac hypercontractility in obstructive hypertrophic cardiomyopathy. We present a prespecified analysis of the pharmacokinetics, pharmacodynamics, and safety of aficamten in SEQUOIA-HCM (Safety, Efficacy, and Quantitative Understanding of Obstruction Impact of Aficamten in HCM)., Methods and Results: A total of 282 patients with obstructive hypertrophic cardiomyopathy were randomized 1:1 to daily aficamten (5-20 mg) or placebo between February 1, 2022, and May 15, 2023. Aficamten dosing targeted the lowest effective dose for achieving site-interpreted Valsalva left ventricular outflow tract gradient <30 mm Hg with left ventricular ejection fraction (LVEF) ≥50%. End points were evaluated during titration (day 1 to week 8), maintenance (weeks 8-24), and washout (weeks 24-28), and included major adverse cardiac events, new-onset atrial fibrillation, implantable cardioverter-defibrillator discharges, LVEF <50%, and treatment-emergent adverse events. At week 8, 3.6%, 12.9%, 35%, and 48.6% of patients achieved 5-, 10-, 15-, and 20-mg doses, respectively. Baseline characteristics were similar across groups. Aficamten concentration increased by dose and remained stable during maintenance. During the treatment period, LVEF decreased by -0.9% (95% CI, -1.3 to -0.6) per 100 ng/mL aficamten exposure. Seven (4.9%) patients taking aficamten underwent per-protocol dose reduction for site-interpreted LVEF <50%. There were no treatment interruptions or heart failure worsening for LVEF <50%. No major adverse cardiovascular events were associated with aficamten, and treatment-emergent adverse events were similar between treatment groups, including atrial fibrillation., Conclusions: A site-based dosing algorithm targeting the lowest effective aficamten dose reduced left ventricular outflow tract gradient with a favorable safety profile throughout SEQUOIA-HCM., Registration: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT05186818.

Firsocostat (FIR: previously GS-0976), a highly sensitive OATP substrate, reduces hepatic de novo lipogenesis (DNL) by inhibiting acetyl-CoA carboxylases (ACC). Measuring the pharmacodynamic (PD) efficacy of FIR on DNL provides a unique opportunity to determine optimal dosing strategies for liver-targeted OATP substrates in settings of altered OATP function. A randomized, four-way crossover drug-drug interaction study was conducted. Hepatic DNL, a marker for ACC activity, was measured in 28 healthy volunteers after reference, single dose FIR 10 mg, FIR 10 mg plus the OATP inhibitor rifampin (RIF) 300 mg i.v., or RIF 300 mg i.v. (control for DNL effect of RIF), each separated by a 7-day washout. Samples were collected for pharmacokinetic (PK) and PD assessments through 24 hours after each treatment. Hepatic DNL and its inhibition by FIR were assessed. Twenty-four subjects completed the study. All adverse events were mild. RIF alone increased hepatic DNL area under the effect curve from time of administration up to the time of the last quantifiable concentration (AUEC last ; 35.7%). Despite a 5.2-fold increase in FIR plasma exposure (area under the concentration-time curve from zero to infinity (AUC inf )) when administered with RIF, FIR alone, and FIR + RIF had the same hepatic PD effect, 37.1% and 34.9% reduction in DNL AUEC last , respectively, compared with their respective controls. These findings indicate that large decreases in OATP activity do not alter hepatic intracellular exposure (as inferred by no change in PD) for drugs that are primarily eliminated hepatically and permeability rate-limited, such as FIR. These results support PK theory that has been difficult to test and provide practical guidance on administration of liver-targeted drugs in settings of reduced OATP function., (© 2020 The Authors. Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

Remdesivir (RDV, Veklury ® ) is a once-daily, nucleoside ribonucleic acid polymerase inhibitor of severe acute respiratory syndrome coronavirus 2 replication. Remdesivir has been granted approvals in several countries for use in adults and children hospitalized with severe coronavirus disease 2019 (COVID-19). Inside the cell, remdesivir undergoes metabolic activation to form the intracellular active triphosphate metabolite, GS-443902 (detected in peripheral blood mononuclear cells), and ultimately, the renally eliminated plasma metabolite GS-441524. This review discusses the pre-clinical pharmacology of RDV, clinical pharmacokinetics, pharmacodynamics/concentration-QT analysis, rationale for dose selection for treatment of patients with COVID-19, and drug-drug interaction potential based on available in vitro and clinical data in healthy volunteers. Following single-dose intravenous administration over 2 h of an RDV solution formulation across the dose range of 3-225 mg in healthy participants, RDV and its metabolites (GS-704277and GS-441524) exhibit linear pharmacokinetics. Following multiple doses of RDV 150 mg once daily for 7 or 14 days, major metabolite GS-441524 accumulates approximately 1.9-fold in plasma. Based on pharmacokinetic bridging from animal data and available human data in healthy volunteers, the RDV clinical dose regimen of a 200-mg loading dose on day 1 followed by 100-mg maintenance doses for 4 or 9 days was selected for further evaluation of pharmacokinetics and safety. Results showed high intracellular concentrations of GS-443902 suggestive of efficient conversion from RDV into the triphosphate form, and further supporting this clinical dosing regimen for the treatment of COVID-19. Mathematical drug-drug interaction liability predictions, based on in vitro and phase I data, suggest RDV has low potential for drug-drug interactions, as the impact of inducers or inhibitors on RDV disposition is minimized by the parenteral route of administration and extensive extraction. Using physiologically based pharmacokinetic modeling, RDV is not predicted to be a clinically significant inhibitor of drug-metabolizing enzymes or transporters in patients infected with COVID-19 at therapeutic RDV doses.

Severe coronavirus disease 2019 (COVID-19) disease, including multisystem inflammatory syndrome, has been reported in children. This report summarizes development of a remdesivir physiologically-based pharmacokinetic (PBPK) model that accurately describes observed adult remdesivir and metabolites exposure and predicts pediatric remdesivir and metabolites exposure. The adult PBPK model was applied to predict pediatric remdesivir and metabolites steady-state exposures using the Pediatric Population Model in SimCYP and incorporated the relevant physiologic and mechanistic information. Model development was based on adult phase I exposure data in healthy volunteers who were administered a 200-mg loading dose of remdesivir intravenous (IV) over 0.5 hours on Day 1, then 100-mg daily maintenance doses of IV over 0.5 hours starting on Day 2 and continuing through Days 5 or 10. Simulations indicated that use of the adult therapeutic remdesivir dosage regimen (200-mg loading dose on Day 1 then 100-mg daily maintenance dose starting on Day 2) in pediatric patients ≥ 40 kg and a weight-based remdesivir dosage regimen (5-mg/kg loading dose on Day 1 then 2.5-mg/kg daily maintenance dose starting on Day 2) in pediatric patients weighing 2.5 to < 40 kg is predicted to maintain therapeutic exposures of remdesivir and its metabolites. The comprehensive PBPK model described in this report supported remdesivir dosing in planned pediatric clinical studies and dosing in the emergency use authorization and pediatric compassionate use programs that were initiated to support remdesivir as a treatment option during the pandemic., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

There is an increasing interest in transporter induction (i.e., decreased systemic drug exposure due to increased efflux-limited absorption or transporter-mediated clearance) as a mechanism of drug-drug interactions (DDIs), although evidence of clinical relevance is still evolving. Intestinal P-glycoprotein (P-gp) and hepatic organic anion transporting polypeptides 1B (OATP1B) can be important determinants of drug absorption and disposition, as well as targets for DDIs. Current data indicate that intestinal P-gp protein levels can be induced up to threefold to fourfold in humans primarily with pregnane X receptor (PXR) activators, and that this induction can decrease the systemic exposure of drugs with P-gp efflux-limited absorption (e.g., ≤ 67% decrease in the exposure of total dabigatran following rifampin multiple oral dosing). Evaluation of the clinical relevance of P-gp induction as a DDI mechanism must consider the induction potential of the perpetrator drug for P-gp and attenuation of exposure of the victim drug in the context of its therapeutic window. Practical drug development recommendations are provided herein. Reports are contradictory on OATP1B induction by PXR activators in human hepatocytes and liver biopsies. Some clinical investigations demonstrated that rifampin pretreatment decreased exposure of OATP1B substrates, while other studies found no differences, and the potential involvement of other mechanisms in these observed DDIs cannot be definitively ruled out. Thus, further studies are needed to understand hepatic OATP1B induction and potential involvement of other mechanisms contributing to reduced exposure of OATP1B substrates. This review critically summarizes the state-of-the-art on intestinal P-gp and hepatic OATP1B induction, and highlights implications for drug development., (© 2020 The Authors Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

Background: Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies., Methods: Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC 50 studies) or following a time course where inactivation kinetics were measured., Results: Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency k inact /K i was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a k inact /K i value of 2.4 ± 0.6 × 10 4  M -1  s -1 for BTK with selectivity against important off-targets., Conclusions: For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety., General Significance: This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases., Competing Interests: Declaration of Competing Interest All authors are current or former employees and shareholders of Gilead Sciences, Inc., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Selgantolimod is a novel oral, selective Toll-like receptor 8 (TLR8) agonist in development for the treatment of chronic hepatitis B (CHB). TLR8 is an endosomal innate immune receptor and a target for treatment of viral infections. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selgantolimod in healthy volunteers., Methods: Of 71 subjects enrolled, 59 received a single dose of selgantolimod (0.5, 1.5, 3 or 5 mg) or placebo, and 12 were evaluated for food effect. Safety, PK and PD activity by induction of cytokines, chemokines and acute phase proteins were assessed. PK/PD analyses were conducted., Results: Single doses of 0.5-5 mg were generally safe. No serious adverse events (AEs) or AEs leading to discontinuation were reported, and most were Grade 1 in severity. Selgantolimod displayed rapid absorption and dose-proportional PK and PD activity. Food had minimal effect on PK but resulted in diminished PD activity. In PK/PD analyses, near-saturation of induction for most evaluated biomarkers occurred at the 5-mg dose., Conclusions: Single doses of up to 5 mg selgantolimod were safe and induced dose-dependent PD responses. These data support evaluation of selgantolimod in combination with other agents in future clinical studies of CHB. Australian New Zealand Clinical Trials Registration: ACTRN12616001646437.

Drug transporter and cytochrome P450 expression is regulated by shared nuclear receptors and, hence, an inducer should induce both, although the magnitude may differ. The objective of this study was to establish relative induction relationships between CYP3A and drug transporters (P-glycoprotein (P-gp), organic anion transporting polypeptide (OATP), and breast cancer resistance protein (BCRP)) or other P450s (CYP2C9 and CYP1A2) using ascending doses of the prototypical pregnane xenobiotic receptor (PXR) agonist, rifampin, to elicit weak, moderate, and strong PXR agonism. Healthy subjects received dabigatran etexilate, pravastatin, rosuvastatin, and a midazolam/tolbutamide/caffeine cocktail before and after rifampin 2, 10, 75, or 600 mg q.d. Unlike CYP3A, only moderate induction of P-gp, OATP, and CYP2C9 was observed and dose-dependent induction of P-gp, OATP, and CYP2C9 was always one drug-drug interaction category lower than observed for CYP3A, even when correcting for probe drug sensitivity. Data from this study establish proof-of-concept that P450 induction data can be leveraged to inform on the effect on transporters., (© 2018 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Rifampin demonstrated dose-dependent relative induction between cytochrome P (CYP)3A and P-glycoprotein (P-gp), organic anion transporting polypeptides (OATPs), or CYP2C9; P-gp, OATP, and CYP2C9 induction was one drug-drug interaction (DDI) category lower than that observed for CYP3A across a wide range of pregnane X receptor (PXR) agonism. The objective of this study was to determine if these relationships could be utilized to predict transporter induction by other CYP3A inducers (rifabutin and carbamazepine) and of another P-gp substrate, sofosbuvir. Healthy subjects received sofosbuvir and a six-probe drug cassette before and after 300 mg q.d. rifabutin or 300 mg b.i.d. carbamazepine. Induction of P-gp, CYP2C9, and decreased sofosbuvir exposure were successfully predicted by observed CYP3A induction. Carbamazepine induction of OATP was underpredicted, likely due to reported additional non-PXR agonism. The results demonstrate that the effect of a PXR agonist on CYP3A can be leveraged to inform on induction liability for other primarily PXR-regulated P450s/transporters, allowing for prioritization of targeted DDI assessments during new drug development., (© 2018 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Aim: This open-label study investigated the effect of belatacept on cytokine levels and on the pharmacokinetics of caffeine, losartan, omeprazole, dextromethorphan and midazolam, as CYP probe substrates after oral administration of the Inje cocktail in healthy volunteers., Methods: Twenty-two evaluable subjects received the Inje cocktail on Days 1, 4, 7 and 11 and belatacept infusion on Day 4., Results: Since belatacept caused no major alterations to cytokine levels, there were no major effects on CYP-substrate pharmacokinetics, except for a slight (16-30%) increase in omeprazole exposure, which was probably due to omeprazole-mediated, time-dependent CYP inhibition. Belatacept did not cause major alterations in the pharmacokinetics, as measured by the geometric mean ratios and associated 90% confidence interval for area under the plasma concentration -time curve from time zero to infinity on Day 7 comparing administration with and without belatacept for caffeine (1.002 [0.914, 1.098]), dextromethorphan (1.031 [0.885, 1.200]), losartan (1.016 [0.938, 1.101)], midazolam (0.968 [0.892, 1.049]) or their respective metabolites., Conclusions: Therefore, no dose adjustments of CYP substrates are indicated with belatacept coadministration., (© 2016 The British Pharmacological Society.)

Aim: We sought to discover endogenous urinary biomarkers of human CYP2D6 activity., Patients & Methods: Healthy pediatric subjects (n = 189) were phenotyped using dextromethorphan and randomized for candidate biomarker selection and validation. Global urinary metabolomics was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Candidate biomarkers were tested in adults receiving fluoxetine, a CYP2D6 inhibitor., Results: A biomarker, M1 (m/z 444.3102) was correlated with CYP2D6 activity in both the pediatric training and validation sets. Poor metabolizers had undetectable levels of M1, whereas it was present in subjects with other phenotypes. In adult subjects, a 9.56-fold decrease in M1 abundance was observed during CYP2D6 inhibition., Conclusion: Identification and validation of M1 may provide a noninvasive means of CYP2D6 phenotyping.

Fluoxetine and its circulating metabolite norfluoxetine comprise a complex multiple-inhibitor system that causes reversible or time-dependent inhibition of the cytochrome P450 (CYP) family members CYP2D6, CYP3A4, and CYP2C19 in vitro. Although significant inhibition of all three enzymes in vivo was predicted, the areas under the concentration-time curve (AUCs) for midazolam and lovastatin were unaffected by 2-week dosing of fluoxetine, whereas the AUCs of dextromethorphan and omeprazole were increased by 27- and 7.1-fold, respectively. This observed discrepancy between in vitro risk assessment and in vivo drug-drug interaction (DDI) profile was rationalized by time-varying dynamic pharmacokinetic models that incorporated circulating concentrations of fluoxetine and norfluoxetine enantiomers, mutual inhibitor-inhibitor interactions, and CYP3A4 induction. The dynamic models predicted all DDIs with less than twofold error. This study demonstrates that complex DDIs that involve multiple mechanisms, pathways, and inhibitors with their metabolites can be predicted and rationalized via characterization of all the inhibitory species in vitro.

Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147 M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that coformulation of this transmission-blocking agent with asexual stage antimalarials such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission.

Recent guidance on drug-drug interaction (DDI) testing recommends evaluation of circulating metabolites. However, there is little consensus on how to quantitatively predict and/or assess the risk of in vivo DDIs by multiple time-dependent inhibitors (TDIs) including metabolites from in vitro data. Fluoxetine was chosen as the model drug to evaluate the role of TDI metabolites in DDI prediction because it is a TDI of both CYP3A4 and CYP2C19 with a circulating N-dealkylated inhibitory metabolite, norfluoxetine. In pooled human liver microsomes, both enantiomers of fluoxetine and norfluoxetine were TDIs of CYP2C19, (S)-norfluoxetine was the most potent inhibitor with time-dependent inhibition affinity constant (KI) of 7 μM, and apparent maximum time-dependent inhibition rate (k(inact,app)) of 0.059 min(-1). Only (S)-fluoxetine and (R)-norfluoxetine were TDIs of CYP3A4, with (R)-norfluoxetine being the most potent (K(I) = 8 μM, and k(inact,app) = 0.011 min(-1)). Based on in-vitro-to-in-vivo predictions, (S)-norfluoxetine plays the most important role in in vivo CYP2C19 DDIs, whereas (R)-norfluoxetine is most important in CYP3A4 DDIs. Comparison of two multiple TDI prediction models demonstrated significant differences between them in in-vitro-to-in-vitro predictions but not in in-vitro-to-in-vivo predictions. Inclusion of all four inhibitors predicted an in vivo decrease in CYP2C19 (95%) and CYP3A4 (60-62%) activity. The results of this study suggest that adequate worst-case risk assessment for in vivo DDIs by multiple TDI systems can be achieved by incorporating time-dependent inhibition by both parent and metabolite via simple addition of the in vivo time-dependent inhibition rate/cytochrome P450 degradation rate constant (λ/k(deg)) values, but quantitative DDI predictions will require a more thorough understanding of TDI mechanisms.

The aim of this study was to evaluate the contribution of metabolites to drug-drug interactions (DDI) using the inhibition of CYP2C19 and CYP3A4 by omeprazole and its metabolites as a model. Of the metabolites identified in vivo, 5-hydroxyomeprazole, 5'-O-desmethylomeprazole, omeprazole sulfone, and carboxyomeprazole had a metabolite to parent area under the plasma concentration-time curve (AUC(m)/AUC(p)) ratio ≥ 0.25 when either total or unbound concentrations were measured after a single 20-mg dose of omeprazole in a cocktail. All of the metabolites inhibited CYP2C19 and CYP3A4 reversibly. In addition omeprazole, omeprazole sulfone, and 5'-O-desmethylomeprazole were time dependent inhibitors (TDI) of CYP2C19, whereas omeprazole and 5'-O-desmethylomeprazole were found to be TDIs of CYP3A4. The in vitro inhibition constants and in vivo plasma concentrations were used to evaluate whether characterization of the metabolites affected DDI risk assessment. Identifying omeprazole as a TDI of both CYP2C19 and CYP3A4 was the most important factor in DDI risk assessment. Consideration of reversible inhibition by omeprazole and its metabolites would not identify DDI risk with CYP3A4, and with CYP2C19, reversible inhibition values would only identify DDI risk if the metabolites were included in the assessment. On the basis of inactivation data, CYP2C19 and CYP3A4 inhibition by omeprazole would be sufficient to identify risk, but metabolites were predicted to contribute 30-63% to the in vivo hepatic interactions. Therefore, consideration of metabolites may be important in quantitative predictions of in vivo DDIs. The results of this study show that, although metabolites contribute to in vivo DDIs, their relative abundance in circulation or logP values do not predict their contribution to in vivo DDI risk.

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Drugs that are mainly cleared by a single enzyme are considered more sensitive to drug-drug interactions (DDIs) than drugs cleared by multiple pathways. However, whether this is true when a drug cleared by multiple pathways is coadministered with an inhibitor of multiple P450 enzymes (multi-P450 inhibition) is not known. Mathematically, simultaneous equipotent inhibition of two elimination pathways that each contribute half of the drug clearance is equal to equipotent inhibition of a single pathway that clears the drug. However, simultaneous strong or moderate inhibition of two pathways by a single inhibitor is perceived as an unlikely scenario. The aim of this study was (i) to identify P450 inhibitors currently in clinical use that can inhibit more than one clearance pathway of an object drug in vivo and (ii) to evaluate the magnitude and predictability of DDIs caused by these multi-P450 inhibitors. Multi-P450 inhibitors were identified using the Metabolism and Transport Drug Interaction Database. A total of 38 multi-P450 inhibitors, defined as inhibitors that increased the AUC or decreased the clearance of probes of two or more P450s, were identified. Seventeen (45%) multi-P450 inhibitors were strong inhibitors of at least one P450, and an additional 12 (32%) were moderate inhibitors of one or more P450s. Only one inhibitor (fluvoxamine) was a strong inhibitor of more than one enzyme. Fifteen of the multi-P450 inhibitors also inhibit drug transporters in vivo, but such data are lacking on many of the inhibitors. Inhibition of multiple P450 enzymes by a single inhibitor resulted in significant (>2-fold) clinical DDIs with drugs that are cleared by multiple pathways such as imipramine and diazepam, while strong P450 inhibitors resulted in only weak DDIs with these object drugs. The magnitude of the DDIs between multi-P450 inhibitors and diazepam, imipramine, and omeprazole could be predicted using in vitro data with similar accuracy as probe substrate studies with the same inhibitors. The results of this study suggest that inhibition of multiple clearance pathways in vivo is clinically relevant, and the risk of DDIs with object drugs may be best evaluated in studies using multi-P450 inhibitors.

Introduction: Predictions of drug-drug interactions (DDIs) are commonly performed for single inhibitors, but interactions involving multiple inhibitors also frequently occur. Predictions of such interactions involving stereoisomer pairs, parent/metabolite combinations and simultaneously administered multiple inhibitors are increasing in importance. This review provides the framework for predicting inhibitory DDIs of multiple inhibitors with any combination of reversible inhibition mechanism., Areas Covered: The review provides an overview of the reliability of the in vitro determined reversible inhibition mechanism. Furthermore, the article provides a method to predict DDIs for multiple reversible inhibitors that allows substituting the inhibition constant (K(i)) with an inhibitor affinity (IC(50)) value determined at S << K(M)., Expert Opinion: A better understanding and the prediction methods of DDIs, resulting from multiple inhibitors, are important. The inhibition mechanism of a reversible inhibitor is often equivocal across studies and unreliable. Determination of the K(i) requires the assignment of reversible inhibition mechanism but in vitro-to-in vivo prediction of DDI risk can be achieved for multiple inhibitors from estimates of the inhibitor affinity (IC(50)) only, regardless of the inhibition mechanism.

Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3'-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC(50) 16-26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3'-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC(50) >15 μM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety.

All-trans-retinoic acid (atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower K(m) (19nM) and V(max) (0.8 pmol/min/pmol) than CYP26A1 (K(m)=50 nM and V(max)=10 pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2-10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Metabolites can have pharmacological or toxicological effects, inhibit metabolic enzymes, and be used as probes of drug-drug interactions or specific cytochrome P450 (P450) phenotypes. Thus, better understanding and prediction methods are needed to characterize metabolite exposures in vivo. This study aimed to test whether in vitro data could be used to predict and rationalize in vivo metabolite exposures using two model drugs and P450 probes: dextromethorphan and omeprazole with their primary metabolites dextrorphan, 5-hydroxyomeprazole (5OH-omeprazole), and omeprazole sulfone. Relative metabolite exposures were predicted using metabolite formation and elimination clearances. For dextrorphan, the formation clearances of dextrorphan glucuronide and 3-hydroxymorphinan from dextrorphan in human liver microsomes were used to predict metabolite (dextrorphan) clearance. For 5OH-omeprazole and omeprazole sulfone, the depletion rates of the metabolites in human hepatocytes were used to predict metabolite clearance. Dextrorphan/dextromethorphan in vivo metabolite/parent area under the plasma concentration versus time curve ratio (AUC(m)/AUC(p)) was overpredicted by 2.1-fold, whereas 5OH-omeprazole/omeprazole and omeprazole sulfone/omeprazole were predicted within 0.75- and 1.1-fold, respectively. The effect of inhibition or induction of the metabolite's formation and elimination on the AUC(m)/AUC(p) ratio was simulated. The simulations showed that unless metabolite clearance pathways are characterized, interpretation of the metabolic ratios is exceedingly difficult. This study shows that relative in vivo metabolite exposure can be predicted from in vitro data and characterization of secondary metabolism of probe metabolites is critical for interpretation of phenotypic data.

Background: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat., Results: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry., Conclusions: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

Importance of the Field: Due to growing concerns over toxic or active metabolites, significant efforts have been focused on qualitative identification of potential in vivo metabolites from in vitro data. However, limited tools are available to quantitatively predict their human exposures., Areas Covered in This Review: Theory of clearance predictions and metabolite kinetics is reviewed together with supporting experimental data. In vitro and in vivo data of known circulating metabolites and their parent drugs were collected and the predictions of in vivo exposures of the metabolites were evaluated., What the Reader Will Gain: The theory and data reviewed will be useful in early identification of human metabolites that will circulate at significant levels in vivo and help in designing in vivo studies that focus on characterization of metabolites. It will also assist in rationalization of metabolite-to-parent ratios used as markers of specific enzyme activity., Take Home Message: The relative importance of a metabolite in comparison to the parent compound as well as other metabolites in vivo can only be predicted using the metabolite's in vitro formation and elimination clearances, and the in vivo disposition of a metabolite can only be rationalized when the elimination pathways of that metabolite are known.

Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K(m) 9.4+/-3.3nM and V(max) 11.3+/-4.3pmolesmin(-1)pmoleP450(-1)) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.

The objectives of this research were to identify quantitative trait loci (QTL) for Stewart's wilt resistance from a mapping population derived from a sweet corn hybrid that is highly resistant to Pantoea stewartii and to determine if marker-based selection for those QTL could substantially improve Stewart's wilt resistance in a population derived from a cross of resistant lines and a highly susceptible sweet corn inbred. Three significant QTL for Stewart's wilt resistance on chromosomes 2 (bin 2.03), 5 (bin 5.03), and 6 (bin 6.06/6.07) explained 31% of the genetic variance in a population of 110 F(3:4) families derived from the sweet corn hybrid Bonus. The three QTL appeared to be additive in their effects on Stewart's wilt ratings. Based on means of families that were either homozygous or heterozygous for marker alleles associated with the resistance QTL, the QTL on chromosomes 2 and 6 appeared to have dominant or partially dominant gene action, while the QTL on chromosome 5 appeared to be recessive. A population of 422 BC(2)S(2) families was derived from crosses of a sweet corn inbred highly susceptible to Stewart's wilt, Green Giant Code 88 (GG88), and plants from two F(3:4) families (12465 and 12467) from the Bonus mapping population that were homozygous for marker alleles associated with Stewart's wilt resistance at the three QTL. Mean Stewart's wilt ratings for BC(2)S(2) families were significantly (P < 0.05) lower for families that were homozygous for the bnlg1902 marker allele (bin 5.03) from resistant lines 12465 or 12467 than for families that were heterozygous at this marker locus or homozygous for the bnlg1902 marker allele from GG88. Resistance associated with this QTL was expressed only if F(3:5) or BC(2)S(2) families were homozygous for marker alleles associated with the resistant inbred parent (P(1)). Marker alleles identified in the F(3:5) mapping population that were in proximity to the resistance QTL on chromosomes 2 and 6 were not polymorphic in crosses of GG88 with 12465 and 12467. Selection for other polymorphic marker loci adjacent to these two regions did not improve Stewart's wilt resistance of BC(2)S(2) families.

Purpose: To identify factors that predict survival in patients with variceal bleeding who have undergone transjugular intrahepatic portosystemic shunt (TIPS) placement., Patients and Methods: TIPS was performed in 64 of 65 patients. Indications were bleeding esophagogastric varices in 64 patients and hemorrhoidal bleeding in one. Child-Pugh classifications were A in two patients, B in 32, and C in 31. Acute bleeding was controlled before TIPS in 26 patients in stable condition but not in 39 patients whose condition was unstable., Results: Twelve patients died within 30 days of TIPS, and another 14 died thereafter. The cumulative survival rate was 67% at 6 months and 56% at 1 year. Cumulative 30-day survival was 96% for stable and 69% for unstable patients, a significant difference (P = .0135). Thirty-day survival was 91% for patients in Child-Pugh classes A and B combined and 71% for patients in class C (P = .042)., Conclusion: Efforts to control acute bleeding and improve a patient's metabolic status before TIPS are likely to improve 30-day survival.

Balloon-expandable, intraluminal stenting of the iliac arteries with the Palmaz stent was the subject of a multicenter study for 4 years. A total of 486 patients underwent 587 procedures. Four hundred and five patients had unilateral and 81 had bilateral iliac stent placements. Follow-up ranged from 1 to 48 months (mean 13.3 +/- 11 months). Sustained clinical benefit of the treated patients was obtained in 90.9% at 1 year, 84.1% at 2 years, and 68.6% at 43 months. Angiographic patency rate was 92%. Diabetes mellitus and poor runoff had significant negative influence on the clinical outcome. The 10% incidence of procedural complications was not altered by operator experience.

Plasma renin (PRA) and aldosterone activities were measured in 29 obese children and in 48 controls of the same age. Blood samples were taken at 8 AM after a night's sleep and after 3 days of a diet with normal sodium intake. The PRA in the obese children was lowered: m = 1.00 ng/ml/h (controls = 1.86 ng/ml/h; p = 0.01). The drop in plasma aldosterone activity was not significant: m = 134.1 pg/ml (controls: m = 167.5 pg/ml). Plasma aldosterone activity was positively correlated with PRA in controls (p less than 0.01): this correlation was not found in obese children. In obese children no dissociation could be found, in the resting state, between plasma aldosterone activity and PRA, contrary to obese adults under stimulation. In fact, 1) the aldosterone/PRA ratio did not differ significantly between obese children and controls, 2) the average value of plasma aldosterone activity that one could predict in the obese children from their average PRA level and the linear regression calculated from the controls exactly corresponded to the measured value. The elevated blood pressure found in the obese children could explain the drop in the PRA which would thus remain adapted to the blood pressure level.

The induction of nephrotoxic nephritis in rats with rabbit antibodies preparation results in proteinuria, hypoproteinemia and hyperlipidemia with little glomerular lesions. A study of some hydrolases in cortex and medulla on one hand and glomerular and tubules on the other, showed changes in the activities of following enzymes. 1) A 20-30 % decrease in Na+, K+ dependent ATP-ase in whole kidney. 2) A 20 % decrease in beta-galactosidase activity in glomerular and medulla. 3) A 20 % increase of arylsulphatase A activity in tubules. These results are discussed in the light of the present knowledge of sulphatide metabolism in kidney.

A congenital carotid--jugular aneurysm was responsible for severe heart failure in a two day old baby. The child recovered after surgery. The signs suggesting an arteriovenous fistula (a continuous murmur and thrill, hyperdynamic circulation) may be absent, as in this case, when the child is in severe cardiac failure. The signs should be sought when the circulation improves.

The authors presented a case of the Williams and Beuren's syndrome, special by the existence of aorta coarctation, high blood pressure, nephrotic syndrome and renal dysplasia. The Williams-Beuren's syndrome is characterised by the association of facial anomalies, mental retardation and supra-valvular aortic stenosis. The case presented in this study demonstrates: -- the symptomatic diversity of the Williams and Beuren's syndrome; -- and the relationship of this syndrome and severe idiopathic hypercalcemia of the infant. The etiopathogenesis is also discussed.