Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy.

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

Discovery and Pharmacological Characterization of JNJ-42756493 (Erdafitinib), a Functionally Selective Small-Molecule FGFR Family Inhibitor.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.

Mechanistic models enable the rational use of in vitro drug-target binding kinetics for better drug effects in patients.

INTRODUCTION: Drug-target binding kinetics are major determinants of the time course of drug action for several drugs, as clearly described for the irreversible binders omeprazole and aspirin. This supports the increasing interest to incorporate newly developed high-throughput assays for drug-target binding kinetics in drug discovery. A meaningful application of in vitro drug-target binding kinetics in drug discovery requires insight into the relation between in vivo drug effect and in vitro measured drug-target binding kinetics. AREAS COVERED: In this review, the authors discuss both the relation between in vitro and in vivo measured binding kinetics and the relation between in vivo binding kinetics, target occupancy and effect profiles. EXPERT OPINION: More scientific evidence is required for the rational selection and development of drug-candidates on the basis of in vitro estimates of drug-target binding kinetics. To elucidate the value of in vitro binding kinetics measurements, it is necessary to obtain information on system-specific properties which influence the kinetics of target occupancy and drug effect. Mathematical integration of this information enables the identification of drug-specific properties which lead to optimal target occupancy and drug effect in patients.

Fragment-based discovery of type I inhibitors of maternal embryonic leucine zipper kinase.

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 µM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.

Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase.

A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK.

Novel antibiotics targeting respiratory ATP synthesis in Gram-positive pathogenic bacteria.

Emergence of drug-resistant bacteria represents a high, unmet medical need, and discovery of new antibacterials acting on new bacterial targets is strongly needed. ATP synthase has been validated as an antibacterial target in Mycobacterium tuberculosis, where its activity can be specifically blocked by the diarylquinoline TMC207. However, potency of TMC207 is restricted to mycobacteria with little or no effect on the growth of other Gram-positive or Gram-negative bacteria. Here, we identify diarylquinolines with activity against key Gram-positive pathogens, significantly extending the antibacterial spectrum of the diarylquinoline class of drugs. These compounds inhibited growth of Staphylococcus aureus in planktonic state as well as in metabolically resting bacteria grown in a biofilm culture. Furthermore, time-kill experiments showed that the selected hits are rapidly bactericidal. Drug-resistant mutations were mapped to the ATP synthase enzyme, and biochemical analysis as well as drug-target interaction studies reveal ATP synthase as a target for these compounds. Moreover, knockdown of the ATP synthase expression strongly suppressed growth of S. aureus, revealing a crucial role of this target in bacterial growth and metabolism. Our data represent a proof of principle for using the diarylquinoline class of antibacterials in key Gram-positive pathogens. Our results suggest that broadening the antibacterial spectrum for this chemical class is possible without drifting off from the target. Development of the diarylquinolines class may represent a promising strategy for combating Gram-positive pathogens.

Pharmacokinetics and pharmacodynamics of TMC207 and its N-desmethyl metabolite in a murine model of tuberculosis.

TMC207 is a first-in-class diarylquinoline with a new mode of action against mycobacteria targeting the ATP synthase. It is metabolized to an active derivative, N-desmethyl TMC207, and both compounds are eliminated with long terminal half-lives (50 to 60 h in mice) reflecting slow release from tissues such as lung and spleen. In vitro, TMC207 is 5-fold more potent against Mycobacterium tuberculosis than N-desmethyl TMC207, and the effects of the two compounds are additive. The pharmacokinetic and pharmacodynamic (PK-PD) response was investigated in the murine model of tuberculosis (TB) infection following oral administration of different doses of TMC207 or N-desmethyl TMC207 at 5 days per week for 4 weeks starting the day after intravenous infection with M. tuberculosis and following administration of different doses of TMC207 at various dosing frequencies for 6 weeks starting 2 weeks after infection. Upon administration of N-desmethyl TMC207, maximum plasma concentration (C(max)), area under the plasma concentration-time curve from time zero to 168 h postdose (AUC(168h)), and minimum plasma concentration (C(min)) were approximately dose proportional between 8 and 64 mg/kg, and the lung CFU counts were strongly correlated with these pharmacokinetic parameters using an inhibitory sigmoid maximum effect (E(max)) model. Administration of the highest dose (64 mg/kg) produced a 4.0-log(10) reduction of the bacillary load at an average exposure (average concentration [C(avg)] or AUC(168h) divided by 168) of 2.7 µg/ml. Upon administration of the highest dose of TMC207 (50 mg/kg) 5 days per week for 4 weeks, the total reduction of the bacillary load was 4.7 log(10). TMC207 was estimated to contribute to a 1.8-log(10) reduction and its corresponding exposure (C(avg)) was 0.5 µg/ml. Optimal bactericidal activity with N-desmethyl TMC207 was reached at a high exposure compared to that achieved in humans, suggesting a minor contribution of the metabolite to the overall bactericidal activity in TB-infected patients treated with TMC207. Following administration of TMC207 at a total weekly dose of 15, 30, or 60 mg/kg fractionated for either 5 days per week, twice weekly, or once weekly, the bactericidal activity was correlated to the total weekly dose and was not influenced by the frequency of administration. Exposures (AUC(168h)) to TMC207 and N-desmethyl TMC207 mirrored this dose response, indicating that the bactericidal activity of TMC207 is concentration dependent and that AUC is the main PK-PD driver on which dose optimization should be based for dosing frequencies up to once weekly. The PK-PD profile supports intermittent administration of TMC207, in agreement with its slow release from tissues.

Prediction of human oral plasma concentration-time profiles using preclinical data: comparative evaluation of prediction approaches in early pharmaceutical discovery.

BACKGROUND AND OBJECTIVES: Empirically based methods remain one of our tools in human pharmacokinetic predictions. The Dedrick approach and the steady-state plasma drug concentration (C(ss))-mean residence time (MRT) approach are based on the assumption that concentration-time profiles are similar among species, including man, and that curves derived from a variety of animal species can be superimposed after mathematical transformation. In the Dedrick approach the transformation is based on the slope and intercept of the allometric relationship. The C(ss)-MRT approach is based on the implementation of measured animal and predicted human MRT and dose/volume of distribution at steady state (V(ss)). The aims of the present study were to compare the predictive performance of concentration-time profiles obtained by these approaches, to evaluate the prediction of individual pharmacokinetic parameters by these approaches and to further refine these approaches incorporating the experience from our previous work. METHODS: A retrospective analysis using 35 proprietary compounds developed at Johnson & Johnson Pharmaceutical Research and Development was conducted to compare the accuracies of the Dedrick and C(ss)-MRT approaches for predicting oral concentration-time profiles and pharmacokinetic parameters in man. In the first step, input for the transformation was based on simple allometry. Then we assessed whether both methods could be fine-tuned by systematically incorporating correction factors (maximum life span potential, brain weight and plasma protein binding), depending on the interspecies relationship. In addition, for the C(ss)-MRT approach, we used formulas based on multivariate regression analysis as input for the transformation. RESULTS: Inclusion of correction factors significantly improved the profile predictability for the Dedrick and C(ss)-MRT approaches. This was mainly linked to an improved prediction of terminal elimination half-life (t(½)), MRT and the ratio between the maximum plasma concentration and the concentration at the last observed time point (C(max)/C(last)). No significant differences were observed between the Dedrick approach with correction factors, the C(ss)-MRT approach with correction factors and the C(ss)-MRT approach, based on the regression equations. CONCLUSIONS: Based on the dataset evaluated in this study, we demonstrated that human plasma concentration-time profiles and pharmacokinetic parameters could be predicted with the Dedrick and C(ss)-MRT approaches and that if correction factors were implemented, the predictions improved significantly. With the requirement of only a limited preclinical in vivo pharmacokinetic dataset, these empirical methods could offer potential in the early stages of drug discovery.

Towards a better prediction of peak concentration, volume of distribution and half-life after oral drug administration in man, using allometry.

BACKGROUND: It is imperative that new drugs demonstrate adequate pharmacokinetic properties, allowing an optimal safety margin and convenient dosing regimens in clinical practice, which then lead to better patient compliance. Such pharmacokinetic properties include suitable peak (maximum) plasma drug concentration (C(max)), area under the plasma concentration-time curve (AUC) and a suitable half-life (t(½)). The C(max) and t(½) following oral drug administration are functions of the oral clearance (CL/F) and apparent volume of distribution during the terminal phase by the oral route (V(z)/F), each of which may be predicted and combined to estimate C(max) and t(½). Allometric scaling is a widely used methodology in the pharmaceutical industry to predict human pharmacokinetic parameters such as clearance and volume of distribution. In our previous published work, we have evaluated the use of allometry for prediction of CL/F and AUC. In this paper we describe the evaluation of different allometric scaling approaches for the prediction of C(max), V(z)/F and t(½) after oral drug administration in man. METHODS: Twenty-nine compounds developed at Janssen Research and Development (a division of Janssen Pharmaceutica NV), covering a wide range of physicochemical and pharmacokinetic properties, were selected. The C(max) following oral dosing of a compound was predicted using (i) simple allometry alone; (ii) simple allometry along with correction factors such as plasma protein binding (PPB), maximum life-span potential or brain weight (reverse rule of exponents, unbound C(max) approach); and (iii) an indirect approach using allometrically predicted CL/F and V(z)/F and absorption rate constant (k(a)). The k(a) was estimated from (i) in vivo pharmacokinetic experiments in preclinical species; and (ii) predicted effective permeability in man (P(eff)), using a Caco-2 permeability assay. The V(z)/F was predicted using allometric scaling with or without PPB correction. The t(½) was estimated from the allometrically predicted parameters CL/F and V(z)/F. Predictions were deemed adequate when errors were within a 2-fold range. RESULTS: C(max) and t(½) could be predicted within a 2-fold error range for 59% and 66% of the tested compounds, respectively, using allometrically predicted CL/F and V(z)/F. The best predictions for C(max) were obtained when k(a) values were calculated from the Caco-2 permeability assay. The V(z)/F was predicted within a 2-fold error range for 72% of compounds when PPB correction was applied as the correction factor for scaling. CONCLUSIONS: We conclude that (i) C(max) and t(½) are best predicted by indirect scaling approaches (using allometrically predicted CL/F and V(z)/F and accounting for k(a) derived from permeability assay); and (ii) the PPB is an important correction factor for the prediction of V(z)/F by using allometric scaling. Furthermore, additional work is warranted to understand the mechanisms governing the processes underlying determination of C(max) so that the empirical approaches can be fine-tuned further.

Essentiality of FASII pathway for Staphylococcus aureus.

Recently, Brinster et al. suggested that type II fatty-acid biosynthesis (FASII) is not a suitable antibacterial target for Gram-positive pathogens because they use fatty acids directly from host serum rather than de novo synthesis. Their findings, if confirmed, are relevant for further scientific and financial investments in the development of new drugs targeting FASII. We present here in vitro and in vivo data demonstrating that their observations do not hold for Staphylococcus aureus, a major Gram-positive pathogen causing several human infections. The observed differences among Gram-positive pathogens in FASII reflects heterogeneity either in fatty-acid synthesis or in the capacity for fatty-acid uptake from the environment.

Identification of a series of substituted 2-piperazinyl-5-pyrimidylhydroxamic acids as potent histone deacetylase inhibitors.

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Cardio-vascular safety beyond hERG: in silico modelling of a guinea pig right atrium assay.

As chemists can easily produce large numbers of new potential drug candidates, there is growing demand for high capacity models that can help in driving the chemistry towards efficacious and safe candidates before progressing towards more complex models. Traditionally, the cardiovascular (CV) safety domain plays an important role in this process, as many preclinical CV biomarkers seem to have high prognostic value for the clinical outcome. Throughout the industry, traditional ion channel binding data are generated to drive the early selection process. Although this assay can generate data at high capacity, it has the disadvantage of producing high numbers of false negatives. Therefore, our company applies the isolated guinea pig right atrium (GPRA) assay early-on in discovery. This functional multi-channel/multi-receptor model seems much more predictive in identifying potential CV liabilities. Unfortunately however, its capacity is limited, and there is no room for full automation. We assessed the correlation between ion channel binding and the GPRA's Rate of Contraction (RC), Contractile Force (CF), and effective refractory frequency (ERF) measures assay using over six thousand different data points. Furthermore, the existing experimental knowledge base was used to develop a set of in silico classification models attempting to mimic the GPRA inhibitory activity. The Naïve Bayesian classifier was used to built several models, using the ion channel binding data or in silico computed properties and structural fingerprints as descriptors. The models were validated on an independent and diverse test set of 200 reference compounds. Performances were assessed on the bases of their overall accuracy, sensitivity and specificity in detecting both active and inactive molecules. Our data show that all in silico models are highly predictive of actual GPRA data, at a level equivalent or superior to the ion channel binding assays. Furthermore, the models were interpreted in terms of the descriptors used to highlight the undesirable areas in the explored chemical space, specifically regions of low polarity, high lipophilicity and high molecular weight. In conclusion, we developed a predictive in silico model of a complex physiological assay based on a large and high quality set of experimental data. This model allows high throughput in silico safety screening based on chemical structure within a given chemical space.

JNJ-26481585, a novel "second-generation" oral histone deacetylase inhibitor, shows broad-spectrum preclinical antitumoral activity.

PURPOSE: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a "second-generation" class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy. EXPERIMENTAL DESIGN: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors. RESULTS: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC(50), 0.16 nmol/L). CONCLUSIONS: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising "second-generation" HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies.

Pharmacological profile of JNJ-27141491 [(S)-3-[3,4-difluorophenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester], as a noncompetitive and orally active antagonist of the human chemokine receptor CCR2.

The interaction between CC chemokine receptor 2 (CCR2) with monocyte chemoattractant proteins, such as MCP-1, regulates the activation and recruitment of inflammatory leukocytes. In this study, we characterized (S)-3-[3,4-difluoro-phenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester (JNJ-27141491) as a noncompetitive and orally active functional antagonist of human (h)CCR2. JNJ-27141491 strongly suppressed hCCR2-mediated in vitro functions, such as MCP-1-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate binding; MCP-1, -3, and -4-induced Ca(2+) mobilization; and leukocyte chemotaxis toward MCP-1 (IC(50) = 7-97 nM), whereas it had little or no effect on the function of other chemokine receptors tested. The inhibition of CCR2 function was both insurmountable and reversible, consistent with a noncompetitive mode of action. JNJ-27141491 blocked the binding of (125)I-MCP-1 to human monocytes (IC(50) = 0.4 microM), but it failed to affect MCP-1 binding to mouse, rat, and dog cells (IC(50) > 10 microM). Therefore, transgenic mice, in which the mouse (m)CCR2 gene was replaced by the human counterpart, were generated for in vivo testing. In these mice, oral administration of JNJ-27141491 dose-dependently [5-40 mg/kg q.d. (once daily) or b.i.d.] inhibited monocyte and neutrophil recruitment to the alveolar space 48 h after intratracheal mMCP-1/lipopolysaccharide instillation. Furthermore, treatment with JNJ-27141491 (20 mg/kg q.d.) significantly delayed the onset and temporarily reduced neurological signs in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Taken together, these results identify JNJ-27141491 as a noncompetitive, functional antagonist of hCCR2, capable of exerting oral anti-inflammatory activity in transgenic hCCR2-expressing mice.

Discovery of potent, orally bioavailable small-molecule inhibitors of the human CCR2 receptor.

We recently reported the discovery of a series of 2-thioimidazoles as CCR2 antagonists. The most potent molecules of this series, the 4,5-diesters, were rapidly hydrolyzed to the inactive acids and were found to be metabolically unstable. Herein we describe the synthesis of a number of analogues with heterocyclic bioisosteric replacements of the ester group(s). Small 5-membered heterocyclic substituents at the 4-position gave highly potent CCR2 antagonists. Hydrolysis of the 5-ester is diminished, thus imparting these compounds with sufficient stability and systemic exposure after oral administration to warrant further study of the in vivo pharmacology of these functional CCR2 inhibitors.

Predicting oral clearance in humans: how close can we get with allometry?

BACKGROUND: Oral clearance (CL/F) is an important pharmacokinetic parameter and plays an important role in the selection of a safe and tolerable dose for first-in-human studies. Throughout the pharmaceutical industry, many drugs are administered via the oral route; however, there are only a handful of published scaling studies for the prediction of oral pharmacokinetic parameters. METHODS: We evaluated the predictive performances of four different allometric approaches -- simple allometry (SA), the rule of exponents, the unbound CL/F approach, and the unbound fraction corrected intercept method (FCIM) -- for the prediction of human CL/F and the oral area under the plasma concentration-time curve (AUC). Twenty-four compounds developed at Johnson and Johnson Pharmaceutical Research and Development, covering a wide range of physicochemical and pharmacokinetic properties, were selected. The CL/F was predicted using these approaches, and the oral AUC was then estimated using the predicted CL/F. RESULTS: The results of this study indicated that the most successful predictions of CL/F and the oral AUC were obtained using the unbound CL/F approach in combination with the maximum lifespan potential or the brain weight as correction factors based on the rule of exponents. We also observed that the unbound CL/F approach gave better predictions when the exponent of SA was between 0.5 and 1.2. However, the FCIM seemed to be the method of choice when the exponent of SA was <0.50 or >1.2. CONCLUSIONS: Overall, we were able to predict CL/F and the oral AUC within 2-fold of the observed value for 79% and 83% of the compounds, respectively, by selecting the allometric approaches based on the exponents of SA.

Prediction of human pharmacokinetics using physiologically based modeling: a retrospective analysis of 26 clinically tested drugs.

The aim of this study was to evaluate different physiologically based modeling strategies for the prediction of human pharmacokinetics. Plasma profiles after intravenous and oral dosing were simulated for 26 clinically tested drugs. Two mechanism-based predictions of human tissue-to-plasma partitioning (P(tp)) from physicochemical input (method Vd1) were evaluated for their ability to describe human volume of distribution at steady state (V(ss)). This method was compared with a strategy that combined predicted and experimentally determined in vivo rat P(tp) data (method Vd2). Best V(ss) predictions were obtained using method Vd2, providing that rat P(tp) input was corrected for interspecies differences in plasma protein binding (84% within 2-fold). V(ss) predictions from physicochemical input alone were poor (32% within 2-fold). Total body clearance (CL) was predicted as the sum of scaled rat renal clearance and hepatic clearance projected from in vitro metabolism data. Best CL predictions were obtained by disregarding both blood and microsomal or hepatocyte binding (method CL2, 74% within 2-fold), whereas strong bias was seen using both blood and microsomal or hepatocyte binding (method CL1, 53% within 2-fold). The physiologically based pharmacokinetics (PBPK) model, which combined methods Vd2 and CL2 yielded the most accurate predictions of in vivo terminal half-life (69% within 2-fold). The Gastroplus advanced compartmental absorption and transit model was used to construct an absorption-disposition model and provided accurate predictions of area under the plasma concentration-time profile, oral apparent volume of distribution, and maximum plasma concentration after oral dosing, with 74%, 70%, and 65% within 2-fold, respectively. This evaluation demonstrates that PBPK models can lead to reasonable predictions of human pharmacokinetics.

The prediction of drug metabolism, tissue distribution, and bioavailability of 50 structurally diverse compounds in rat using mechanism-based absorption, distribution, and metabolism prediction tools.

The aim of this study was to assess a physiologically based modeling approach for predicting drug metabolism, tissue distribution, and bioavailability in rat for a structurally diverse set of neutral and moderate-to-strong basic compounds (n = 50). Hepatic blood clearance (CL(h)) was projected using microsomal data and shown to be well predicted, irrespective of the type of hepatic extraction model (80% within 2-fold). Best predictions of CL(h) were obtained disregarding both plasma and microsomal protein binding, whereas strong bias was seen using either blood binding only or both plasma and microsomal protein binding. Two mechanistic tissue composition-based equations were evaluated for predicting volume of distribution (V(dss)) and tissue-to-plasma partitioning (P(tp)). A first approach, which accounted for ionic interactions with acidic phospholipids, resulted in accurate predictions of V(dss) (80% within 2-fold). In contrast, a second approach, which disregarded ionic interactions, was a poor predictor of V(dss) (60% within 2-fold). The first approach also yielded accurate predictions of P(tp) in muscle, heart, and kidney (80% within 3-fold), whereas in lung, liver, and brain, predictions ranged from 47% to 62% within 3-fold. Using the second approach, P(tp) prediction accuracy in muscle, heart, and kidney was on average 70% within 3-fold, and ranged from 24% to 54% in all other tissues. Combining all methods for predicting V(dss) and CL(h) resulted in accurate predictions of the in vivo half-life (70% within 2-fold). Oral bioavailability was well predicted using CL(h) data and Gastroplus Software (80% within 2-fold). These results illustrate that physiologically based prediction tools can provide accurate predictions of rat pharmacokinetics.

Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4.

Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the separation and quantitation of reaction components. In this study, the feasibility of using CE in an in vitro metabolism study with CYP was tested. Verapamil was chosen as the substrate for CYP 3A4 isozyme (Supersome). A chiral capillary electrophoretic method was developed and validated for the simultaneous determination of R,S-verapamil (VER) and their major metabolites, R,S-norverapamil (NOR). A method for CYP 3A4 activity assay was proposed with VER as a probe. At the same time, the enantioselective metabolism of VER was studied. Michaelis-Menten constants of R- and S-VER were determined. S-VER was metabolised faster and more extensively than R-VER, with K(m)=167+/-23 microM, V(max)=3,418+/-234 pmol/min/mg for S-VER, and K(m)=168+/-35 microM, V(max)=2,502+/-275 pmol/min/mg for R-VER.

HERG mutation predicts short QT based on channel kinetics but causes long QT by heterotetrameric trafficking deficiency.

OBJECTIVE: Mutations in the KCNH2 (hERG, human ether-à-go-go related gene) gene may cause a reduction of the delayed rectifier current I(Kr), thereby leading to the long QT syndrome (LQTS). The reduced I(Kr) delays the repolarisation of cardiac cells and renders patients vulnerable to ventricular arrhythmias and sudden death. We identified a novel mutation in a LQTS family and investigated its functional consequences using molecular and microscopic techniques. METHODS AND RESULTS: Genetic screening in the LQTS family revealed a heterozygous frameshift mutation p.Pro872fs located in the C-terminus of the KCNH2 gene. The mutation leads to a premature truncation of the C-terminus of the hERG protein. p.Pro872fs channels lack 282 amino acids at the C-terminus and possess an extra 4-amino acid tail. Both the kinetic and biochemical properties of the p.Pro872fs and p.Pro872fs/WT channels were studied in HEK293 cells and resulted in a novel proof of concept for heterozygous LQTS mutations: homotetrameric p.Pro872fs channels displayed near-normal expression, trafficking, and channel kinetics. Unexpectedly, upon co-expression of p.Pro872fs and WT channels, the repolarising power (the proportion of hERG current contributing to the action potential as the percentage of the total current available) was substantially higher during action potential clamp experiments as compared to WT channels alone. This would lead to a shorter rather than a prolonged QT interval. However, at the same time, heterotetramerisation of p.Pro872fs and WT channels also caused a dominant negative effect on trafficking by an increase in ER retention of these heterotetrameric channels, which surpassed the former gain in repolarising power. CONCLUSION: The LQTS phenotype in the studied family is caused by a mutation with novel properties. We demonstrate that a KCNH2 mutation that clinically leads to long QT syndrome causes at the cellular level both a "gain" and a "loss" of HERG channel function due to a kinetic increase in repolarising power and a decrease in trafficking efficiency of heteromultimeric channels.