Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures.

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

CTP Synthase 1 Is a Novel Therapeutic Target in Lymphoma.

Lymphoma is the most common hematological malignancy and is among the 10 most prevalent cancers worldwide. Although survival has been improved by modern immunochemotherapeutic regimens, there remains a significant need for novel targeted agents to treat both B-cell and T-cell malignancies. Cytidine triphosphate synthase 1 (CTPS1), which catalyzes the rate-limiting step in pyrimidine synthesis, plays an essential and nonredundant role in B-cell and T-cell proliferation but is complemented by the homologous CTPS2 isoform outside the hemopoietic system. This report describes the identification and characterization of CTPS1 as a novel target in B- and T-cell cancers. A series of small molecules have been developed which show potent and highly selective inhibition of CTPS1. Site-directed mutagenesis studies identified the adenosine triphosphate pocket of CTPS1 as the binding site for this small molecule series. In preclinical studies, a potent and highly selective small molecule inhibitor of CTPS1 blocked the in vitro proliferation of human neoplastic cells, showing the highest potency against lymphoid neoplasms. Importantly, pharmacological CTPS1 inhibition induced cell death by apoptosis in the majority of lymphoid cell lines tested, thus demonstrating a cytotoxic mechanism of action. Selective CTPS1 inhibition also inhibited the growth of neoplastic human B- and T- cells in vivo. These findings identify CTPS1 as a novel therapeutic target in lymphoid malignancy. A compound from this series is in phase 1/2 clinical studies for the treatment of relapsed/refractory B- and T-cell lymphoma (NCT05463263).

Identification of novel inhibitors of rat Mrp3.

Multidrug resistance-associated protein (MRP; ABCC gene family) mediated efflux transport plays an important role in the systemic and tissue exposure profiles of many drugs and their metabolites, and also of endogenous compounds like bile acids and bilirubin conjugates. However, potent and isoform-selective inhibitors of the MRP subfamily are currently lacking. Therefore, the purpose of the present work was to identify novel rat Mrp3 inhibitors. Using 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFDA) as a model-(pro)substrate for Mrp3 in an oil-spin assay with primary rat hepatocytes, the extent of inhibition of CDF efflux was determined for 1584 compounds, yielding 59 hits (excluding the reference inhibitor) that were identified as new Mrp3 inhibitors. A naive Bayesian prediction model was constructed in Pipeline Pilot to elucidate physicochemical and structural features of compounds causing Mrp3 inhibition. The final Bayesian model generated common physicochemical properties of Mrp3 inhibitors. For instance, more than half of the hits contain a phenolic structure. The identified compounds have an AlogP between 2 and 4.5, between 5 to 8 hydrogen bond acceptor atoms, a molecular weight between 260 and 400, and 2 or more aromatic rings. Compared to the depleted dataset (i.e. 90% remaining compounds), the Mrp3 hit rate in the enriched set was 7.5-fold higher (i.e. 17.2% versus 2.3%). Several hits from this first screening approach were confirmed in an additional study using Mrp3 transfected inside-out membrane vesicles. In conclusion, several new and potent inhibitors of Mrp3 mediated efflux were identified in an optimized in vitro rat hepatocyte assay and confirmed using Mrp3 transfected inside-out membrane vesicles. A final naive Bayesian model was developed in an iterative way to reveal common physicochemical and structural features for Mrp3 inhibitors. The final Bayesian model will enable in silico screening of larger libraries and in vitro identification of more potent Mrp3 inhibitors.

LC-MS/MS Analysis of Bile Acids in In Vitro Samples.

Over the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the quantification of bile acids (BA) and their conjugates in different matrices, such as plasma, blood, urine, and cell lysates. Numerous reports have indeed been published describing methods for quantitative determination of bile acids in plasma samples obtained during in vivo studies. However, information on bioanalytical methods suitable for determination of bile acids in in vitro samples remained scarce. Therefore, we presently report a simple and accurate LC-MS/MS method for the quantification of BA in cells (e.g., cultured human hepatocytes) and corresponding cell culture medium, obtained during in vitro experiments.

Population Pharmacokinetic Modeling of Canagliflozin in Healthy Volunteers and Patients with Type 2 Diabetes Mellitus.

BACKGROUND AND OBJECTIVES: Canagliflozin is an orally active, reversible, selective sodium-glucose co-transporter-2 inhibitor. A population pharmacokinetic (popPK) model of canagliflozin, including relevant covariates as sources of inter-individual variability, was developed to describe phase I, II, and III data in healthy volunteers and in patients with type 2 diabetes mellitus (T2DM). METHODS: The final analysis included 9061 pharmacokinetic (PK) samples from 1616 volunteers enrolled in nine phase I, two phase II, and three phase III studies and was performed using NONMEM(®) 7.1. Inter-individual variability was evaluated using an exponential model and the residual error model was additive in the log domain. The first-order conditional estimation method with interaction was applied and the model was parameterized in terms of rate constants. Covariate effects were explored graphically on empirical Bayes estimates of PK parameters, as shrinkage was low. Clinical relevance of statistically significant covariates was evaluated. The predictive properties of the model were illustrated by prediction-corrected visual predictive checks. RESULTS: A two-compartment PK model with lag-time and sequential zero- and first-order absorption and first-order elimination best described the observed data. Sex, age, and weight on apparent volume of distribution of the central compartment, body mass index on first-order absorption rate constant, and body mass index and over-encapsulation on lag-time, and estimated glomerular filtration rate (eGFR, by MDRD equation), dose, and genetic polymorphism (carriers of UGT1A9*3 allele) on elimination rate constant were identified as statistically significant covariates. The prediction-corrected visual predictive checks revealed acceptable predictive performance of the model. CONCLUSION: The popPK model adequately described canagliflozin PK in healthy volunteers and in patients with T2DM. Because of the small magnitude of statistically significant covariates, they were not considered clinically relevant. However, dosage adjustments are recommended for T2DM patients with renal impairment (eGFR ≥60 mL/min/1.73 m(2): 100 or 300 mg/day; eGFR of 45 to <60 mL/min/1.73 m(2): 100 mg/day).

Pharmacokinetic and pharmacodynamic characterisation of JNJ-40411813, a positive allosteric modulator of mGluR2, in two randomised, double-blind phase-I studies.

Metabotropic glutamate receptor-2 positive allosteric modulator, JNJ-40411813 (ADX71149), was characterised for clinical effects in healthy volunteers in two phase-1 studies. In study 1, healthy men received 50-, 100-, 150- or 225 mg and women received 100 mg JNJ-40411813 (n=6, each cohort) or placebo (n=2, each cohort) twice daily for seven days; smoking men (n=30) received placebo twice daily on days 1-7, 100 mg JNJ-40411813 (n=20) or placebo (n=10) on days 8-14. In study 2, healthy men received intravenous 0.005 mg/kg S(+) ketamine over 60 min at 3 (n=24; cohort 1), 12 h (n=8; cohort 3), and 24 h (n=8; cohort 2) after a single oral dose of 500 mg JNJ-40411813 or placebo. The pharmacokinetics and effects of JNJ-40411813 on cognition and subjective awareness were evaluated. Plasma JNJ-40411813 exposure was dose-dependent, t max ranged from 3-4 h and t 1/2 19.4-34.2 h across the dose levels. JNJ-40411813 significantly (p=0.02) reduced continuity of attention score (150 mg dose) and ameliorated smoking withdrawal-induced changes in power of attention and quality of episodic memory versus placebo. A modest reduction in alertness was observed at 150-225 mg doses, JNJ-40411813 (500 mg) reduced S(+) ketamine-induced negative symptoms by approximately 43% and 30% in cohorts 1 and 3, respectively. JNJ-40411813 was generally well-tolerated.

Population pharmacokinetics of JNJ-37822681, a selective fast-dissociating dopamine D2-receptor antagonist, in healthy subjects and subjects with schizophrenia and dose selection based on simulated D2-receptor occupancy.

BACKGROUND AND OBJECTIVES: JNJ-37822681 is a selective, fast-dissociating dopamine D2-receptor antagonist currently in development as a candidate antipsychotic. The aim of the analyses was to develop a population pharmacokinetic model to describe the pharmacokinetics of JNJ-37822681 in healthy subjects and patients with schizophrenia and to identify covariates of interest. The model was then used to simulate D2-receptor occupancy in support of dose selection for subsequent studies. METHODS: Data were obtained from 378 subjects enrolled in three phase I and two phase II studies. Nonlinear mixed effects modeling of pooled data was conducted using NONMEM(®) to estimate population pharmacokinetic parameters and the effect of covariates on these parameters. The model was evaluated on a subset of data that was not used for model building and was subsequently used to simulate steady-state exposure for each subject in the phase IIb study. Striatal D2-receptor occupancy was predicted using simulated exposure combined with pharmacodynamic parameters from a sigmoid maximum effect model established from previous [(11)C]raclopride positron emission tomography studies. RESULTS: A two-compartment disposition model with zero-order input in a depot compartment followed by first-order absorption into and first-order elimination from the central compartment combined with a transit compartment provided the best fit to the data. Significant covariates were sex and bioavailability on apparent clearance and food intake on the absorption rate constant. Clearance was 11 % higher in females compared with males. The model passed external evaluation. The estimated pharmacokinetic parameters for the phase IIb study were similar to those observed in the phase IIa study. D2-receptor occupancy was predicted to be in the 65-80 % range at 10 mg twice daily and partially or fully reaching the 80 % threshold at doses of 20 and 30 mg twice daily. CONCLUSION: The population pharmacokinetic model of JNJ-37822681 successfully described the pharmacokinetics of JNJ-37822681 and allowed the reliable determination of individual exposure parameters in a phase IIb study. It was concluded that 5 or 7.5 mg twice daily would likely be minimal- or no-effect doses, whereas 10 mg twice daily was expected to provide an optimal balance of efficacy and tolerability.

Dose-response relationship after single oral dose administrations of morphine and oxycodone using laser-evoked potentials on UVB- and capsaicin-irritated skin in healthy male subjects.

The aim of the study was to evaluate the analgesic/antihyperalgesic efficacy and to establish the dose-response relationship of morphine immediate release (IR) and oxycodone IR in a human experimental algesimetric model. Calculated effect ratios for peak-to-peak (PtP) amplitudes of laser-evoked potentials (LEPs) and visual analog scales (VAS) postlaser pain on UVB-irradiated skin (main target variables) were 1.68 and 1.18 respectively for oxycodone 10mg/morphine 20mg, 3.00 and 1.63 respectively for oxycodone 15 mg/morphine 30 mg, and 1.12 and 1.25 respectively for oxycodone 20mg/morphine 40 mg. The effect on the laser-PtP amplitude of morphine at the highest dose (40 mg) and of oxycodone at all doses (10, 15, 20mg) was considered to be clinically relevant based on a difference from placebo of ≥ 2.5 µV. For both compounds, a statistically significant linear trend was observed between dose groups in at least 1 of the 2 main target variables (adjusted P value for both end points <.001 at all doses). Hyperalgesia developed over time vs baseline due to acute exposure to UVB irradiation and to topical/occlusive 1% capsaicin solution. For both compounds, the principal onset of analgesic/antihyperalgesic drug effects was around 0.5 hours with an average peak at about 1 to 2 hours and the effect lasting for more than 3 hours (morphine 20 and 30 mg) or 6 hours (morphine 40 mg and oxycodone all doses). In conclusion, the study demonstrated a solid outcome of a mixed objective/subjective human experimental algesimetric model to approach dose-response relationships and analgesic/antihyperalgesic effects of 2 opioids.