Short Communication EFFECTIVE DOSING REGIMEN OF 1-AMINOBENZOTRIAZOLE FOR INHIBITION OF ANTIPYRINE CLEARANCE IN RATS, DOGS, AND MONKEYS

This article is available online at http://dmd.aspetjournals.org ABSTRACT: 1-Aminobenzotriazole (ABT) has been extensively used as a nonspecific inhibitor of cytochromes P450 (P450s) in animals for mechanistic studies, and antipyrine (AP) has been used as a probe for hepatic oxidative metabolic capacity determination in vivo. The method of use of ABT has been variable from lab to lab due largely to unknown pharmacokinetics of ABT itself and incomplete information on various P450s inhibited. The oral pharmacokinetic profiles of ABT were generated in rats, dogs, and monkeys in the dose range of 5 to 200 mg/kg. The results showed that after single oral doses of 50 mg/kg in rats, and 20 mg/kg in dogs and monkeys, the plasma concentrations were high and were sustained for over 24 h. In vitro, inhibition of various expressed P450s upon 30-min preincubation with ABT (0-500 M) showed that CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were inhibited in a dose-dependent manner. The intravenous pharmacokinetics of AP also was affected in a dose-dependent manner in all species, treated 2 h earlier with ABT. Thus, the plasma clearance of AP was inhibited by 88% in rats pretreated with 50 mg/kg ABT and 96% in dogs and 83% in monkeys pretreated with 20 mg/kg ABT. Based on these data in rats, dogs, and monkeys, and the established safety profile of ABT in rats dosed up to 100 mg/kg, a pretreatment at 2 h with a single oral dose of ABT at 100 mg/kg in rats (providing 93% inhibition) and 20 mg/kg in dogs and monkeys effectively inhibited the clearance of the probe compound. ABT 1 has been extensively used as a nonspecific inhibitor of cytochromes P450 in animals for mechanistic studies Materials and Methods ABT and antipyrine (AP) were obtained from Sigma-Aldrich (St. Louis, MO). Single oral dose pharmacokinetics of ABT, in 0.5% aqueous methylcellulose, were studied in fasted male Sprague-Dawley rats at 10, 50, and 200 mg/kg (n Ն 3) and in Beagle dogs and Cynomolus monkeys at 5, 20, and 100 mg/kg (n ϭ 2 each). All animals were obtained from Charles River Laboratories (Wilmington, MA). The plasma samples were collected over a period of 72 h, and the concentrations of ABT as well as of antipyrine (from experiments described below) were determined simultaneously by precipitation of protein from 0.1 ml of plasma by 0.2 ml of acetonitrile, followed by liquid chromatography/tandem mass spectrometry on a SCIEX API 4000 (PerkinElmerSciex Instruments, Boston, MA) using a Monolithic RP-18 column (4.6 ϫ 100 mm, 2 ). The mobile phase involved a mixture of acetonitrile and water containing 0.1% formic acid flowing at 2.5 ml/min. In vitro inhibition of rP450 activities was measured in a 96-well plate format involving 30-min preincubation of individual human P450 supersomes (40 nM) with ABT (0 -500 M) in the presence of 1 mM NADPH and 0.05 M potassium phosphate buffer, at 37°C. The incubation mixtures were then diluted 10 times with NADPH and marker substrates at 100 M: 7-benzyloxy-4-trifluoromethylcoumarin (CYP1A2, 3A4), 4-(Aminomethyl)-7-methoxycoumarin (CYP2D6), 7-methoxy-4-trifluoromethylcoumarin (CYP2C9), or 7-ethoxy-4-trifluoromethylcoumarin (CYP2B6 and 2C19). The metabolism was determined by fluorometric assays as described by GENTEST (www.gentest.com). The substrates, rP450s and fluorescent metabolite standards, were obtained from BD Gentest Corporation (Woburn, MA). The metabolism was determined by fluorometric assays as described by GENTEST. Two hours prior to administration of AP (20 mg/kg, i.v.), ABT was administered orally to rats at 50 and 100 mg/kg (n ϭ 5 each), and dogs and monkeys at 20 (n ϭ 2) and 100 mg/kg (n ϭ 1), for the interaction study. The control groups involved dosing of animals (n ϭ 2) with 20 mg/kg AP alone and with a low dose of ABT alone (n ϭ 1). Plasma samples were collected up to 24 h and processed and analyzed as above by the liquid chromatography/tandem mass spectrometry method for simultaneous quantitation of both the analytes. Results and Discussion ABT is a nonspecific and effective inhibitor of P450s. It is a metabolism-based inactivator of cytochromes P450 by the mechanism of N-alkylation of heme moiety (Ortiz de Montellano and Mathews, DMD 30:1059DMD 30: -1062DMD 30: , 2002 Printed in U.S.A. 0090-9556/02/3010-1059-1062$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 10 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 738/1009411 1059 1981; AP is a nonspecific substrate of cytochromes P450 The plasma concentration-time profiles of ABT following single oral doses in fasted male Sprague-Dawley rats at 10, 50, and 200 mg/kg (n Ն 3), and beagle dogs and cynomolgus monkeys at 5, 20, and 100 mg/kg (n ϭ 2 each), are shown in The results of the in vitro inhibition of rP450 activities following a 30-min preincubation of individual human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 supersomes with ABT (0 -500 M) showed that all P450s were inhibited dose dependently by ABT pretreatment. ABT has previously been shown to inhibit CYP2E1 as well FIG. 1. Plasma concentration-time profiles of ABT after single oral doses in rats, dogs, and monkeys. FIG. 2. In vitro inhibition of marker activities for human rP450 isoforms following 30-min preincubation with ABT. The extent of metabolism was measured by fluorescence of metabolite generated. BALANI ET AL. Based on the observed, high plasma concentrations in animals and the potential for inhibition of P450s to a significant extent after 30-min ABT pretreatment, ABT doses of 50 and 100 mg/kg for rats, and 20 and 100 mg/kg for dogs and monkeys, were selected for coadministration with AP. The control studies involved dosing of animals with 20 mg/kg AP alone and with a low dose of ABT alone. The pretreatment of animals 2 h prior to the intravenous AP administration was considered to provide substantial inhibitory effect on the P450s, based on reports of profound loss of cytochrome P450 contents of liver and kidneys in rats 2 h post the ABT dose Acknowledgments. We thank Drs. Check Quon and Gerald Miwa for their helpful discussions

Zero-Emission Vessels 2030: How do we get there?

Fossil fuels provide society in general, as well as shipping, with a high-density and low-cost energy source that is comparatively easy to store, handle and transport. We have had decades to optimise the design, maintenance and operation of the shipping system to suit the fossil ‘paradigm’. But the world is changing. It is, therefore, unsurprising that when looking for a non-fossil, zero-emission and sustainable energy source, as we must urgently now do, it’s difficult to see an obvious ‘silver bullet’

Metformin versus placebo in obese pregnant women without diabetes mellitus

Background: Obesity is associated with increased risk of adverse pregnancy outcomes. Lifestyle intervention studies have not improved outcome. Metformin improves insulin sensitivity and leads to less weight gain. Methods: Our double-blind placebo-controlled trial randomized non-diabetic pregnant women with a body mass index >35 kg/m2 to metformin or placebo from 12-18 weeks’ gestation until delivery. Primary outcome was median neonatal birth weight z-score reduction by 0.3 standard deviations (equivalent to a 50% reduction in incidence of large-for-gestational-age neonates from 20% to 10%). Secondary outcomes included maternal gestational weight gain and incidence of gestational diabetes, and preeclampsia, as well as adverse neonatal outcomes. Women were randomized, by computer generated random numbers, to either daily metformin 3.0 grams (n=225) or to placebo (n=225). Analysis was by intention to treat. Results: Fifty women withdrew consent, leaving 202 in the metformin group and 198 in the placebo group. There was no significant difference in median neonatal birth weight z-score (metformin: 0.05, IQR -0.71 to 0.92; placebo: 0.17, IQR -0.62 to 0.89; p=0.655). In the metformin group, compared to placebo, median maternal gestational weight gain was lower (4.6 kg, IQR 1.3-7.2 vs. 6.3 kg, IQR 2.9-9.2, p<0.0001) and incidence of preeclampsia was lower (3.0% vs 11.3%; odds ratio 0.24, 95% CI 0.10-0.61; p=0.001), incidence of side effects was higher; there were no significant differences in gestational diabetes, large for gestational age neonates and adverse neonatal outcomes. Conclusions: In non-diabetic women with BMI >35 kg/m2, antenatal administration of metformin reduces maternal weight gain but not neonatal birth weight. (ClinicalTrials.gov number, NCT01273584

Rapid interrogation of the physical and chemical characteristics of salbutamol sulphate aerosol from a pressurised metered-dose inhaler (pMDI)

Individual micron-sized solid particles from a Salamols pharmaceutical inhaler are stably captured in air using an optical trap for the first time. Raman spectroscopy of the levitated particles allows online interrogation of composition and deliquescent phase change within a high humidity environment that mimics the particle’s travel from inhaler to lun

The BCoS cognitive profile screen: Utility and predictive value for stroke

Objective: We examined the utility of the Birmingham Cognitive Screen (BCoS) in discriminating cognitive profiles and recovery of function across stroke survivors. BCoS was designed for stroke-specific problems across 5 cognitive domains: (a) controlled and spatial attention, (b) language, (c) memory, (d) number processing, and (e) praxis. Method: On the basis of specific inclusion criteria, this cross-section observational study analyzed cognitive profiles of 657 subacute stroke patients, 331 of them reassessed at 9 months. Impairments on 32 measures were evaluated by comparison with 100 matched healthy controls. Measures of affect, apathy, and activities of daily living were also taken. Between-subjects group comparisons of mean performance scores and impairment rates and within-subject examination of impairment rates over time were conducted. Logistic regressions and general linear modeling were used for multivariate analysis of domain-level effects on outcomes. Results: Individuals with repeated stroke experienced significantly less cognitive recovery at 9 months than those with a first stroke despite similar initial level of cognitive performance. Individuals with left hemisphere lesions performed more poorly than those with right hemisphere lesions, but both groups showed similar extent of recovery at 9 months. BCoS also revealed lesion-side-specific deficits and common areas of persistent problems. Functional outcome at 9 months correlated with domain-level deficits in controlled attention, spatial attention, and praxis over and above initial dependency and concurrent levels of affect and apathy. Conclusion: The study demonstrates how BCoS can identify differential cognitive profiles across patient groups. This can potentially help predict outcomes and inform rehabilitation. (PsycINFO Database Record (c) 2014 APA, all rights reserved)

Quantitative Prediction and Clinical Observation of a CYP3A Inhibitor-Based Drug-Drug Interactions with MLN3897, a Potent C-C Chemokine Receptor-1 Antagonist

ABSTRACT A novel in vitro model was recently developed in our laboratories for the prediction of magnitude of clinical pharmacokinetic drugdrug interactions (DDIs), based on reversible hepatic cytochrome P450 (P450) inhibition. This approach, using inhibition data from human hepatocytes incubated in human plasma, and quantitative P450 phenotyping data from hepatic microsomal incubations, successfully predicted DDIs for 15 marketed drugs with ketoconazole, a strong competitive inhibitor of CYP3A4/5, generally used to demonstrate a &quot;worst-case scenario&quot; for CYP3A inhibition. In addition, this approach was successfully extended to DDI predictions with the moderate competitive CYP3A inhibitor fluconazole for nine marketed drugs. In the current report, the general applicability of the model has been demonstrated by prospectively predicting the degree of inhibition and then conducting DDI studies in the clinic for an investigational CCR1 antagonist MLN3897, which is cleared predominantly by CYP3A. The clinical studies involved treatment of healthy volunteers (n ϭ 17-20), in a crossover design, with ketoconazole (200 mg b.i.d.) or fluconazole (400 mg once a day), while receiving MLN3897. Administration of MLN3897 and ketoconazole led to an average 8.28-fold increase in area under the curve of plasma concentration-time plot (AUC) of MLN3897 at steady state, compared with the 8.33-fold increase predicted from the in vitro data. Similarly for fluconazole, an average increase of 3.93-fold in AUC was observed for MLN3897 in comparison with a predicted value of 3.26-fold. Thus, our model reliably predicted the exposure changes for MLN3897 in interaction studies with competitive CYP3A inhibitors in humans, further strengthening the utility of our in vitro model. Prediction of clinical DDIs using in vitro studies is one of the major challenges in the pharmaceutical industry. The main utility of such DDI predictions is to help foresee any safety issues anticipated from higher exposures and thus help design clinical trials with better safety. In some instances, clinical DDI studies can be avoided if no significant pharmacokinetic interaction is predicted. Traditionally, DDI predictions have been based on the ratio of the inhibitor concentration [I] and the enzyme inhibition constant K i ([I]/K i ratio

Remdesivir Versus Standard-of-Care for Severe Coronavirus Disease 2019 Infection: An Analysis of 28-Day Mortality

BACKGROUND: Remdesivir is FDA approved for the treatment of hospitalized patients with COVID-19 and has been shown to shorten time to recovery and improve clinical outcomes in randomized trials. METHODS: This was the final day 28 comparative analysis of data from a phase 3, randomized, open-label study comparing 2 remdesivir regimens (5 vs 10 days, combined for this analysis [remdesivir cohort]) and a real-world retrospective longitudinal cohort study of patients receiving standard-of-care treatment (non-remdesivir cohort). Eligible patients, aged ≥18 years, had confirmed SARSCoV-2, oxygen saturation ≤94% on room air or required supplemental oxygen, with pulmonary infiltrates. Propensity score matching (up to 1:10 ratio) was used to ensure comparable populations. We assessed day 14 clinical recovery (determined using a 7-point ordinal scale) and day 28 all-cause mortality (coprimary endpoints). RESULTS: Altogether, 368 (remdesivir) and 1399 (non-remdesivir) patients were included in the matched analysis. The day 14 clinical recovery rate was significantly higher among the remdesivir versus the non-remdesivir cohort (65.2% vs 57.1%; OR 1.49, 95% CI 1.16–1.90; P = .002). The day 28 mortality rate was significantly lower in the remdesivir cohort versus the non-remdesivir cohort (12.0% vs 16.2%; OR 0.67, 95% CI 0.47–0.95; P = .03). CONCLUSIONS: Remdesivir was associated with significantly higher rates of day 14 clinical recovery, and lower day 28 mortality, compared with standard-of-care treatment in hospitalized patients with COVID-19. Collectively, these data support the use of remdesivir to improve clinical recovery and decrease mortality from SARS-CoV-2 infection

Plasticity of the Mycobacterium tuberculosis respiratory chain and its impact on tuberculosis drug development.

The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb's respiratory chain, and highlight the challenges associated with targeting the pathogen's respiratory enzymes for tuberculosis drug development

Role of Biotransformation Studies in Minimizing Metabolism-Related Liabilities in Drug Discovery

Metabolism-related liabilities continue to be a major cause of attrition for drug candidates in clinical development. Such problems may arise from the bioactivation of the parent compound to a reactive metabolite capable of modifying biological materials covalently or engaging in redox-cycling reactions leading to the formation of other toxicants. Alternatively, they may result from the formation of a major metabolite with systemic exposure and adverse pharmacological activity. To avert such problems, biotransformation studies are becoming increasingly important in guiding the refinement of a lead series during drug discovery and in characterizing lead candidates prior to clinical evaluation. This article provides an overview of the methods that are used to uncover metabolism-related liabilities in a pre-clinical setting and offers suggestions for reducing such liabilities via the modification of structural features that are used commonly in drug-like molecules