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.

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.

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.

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.

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.

Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry.

A liquid chromatography/mass spectrometry (LC/MS) method using an atmospheric pressure chemical ionisation source was used to measure the metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivative (1) in human liver microsomes. After 15 min incubation with human liver microsomes, compound 1 exhibited metabolic turnover of 44%. Data-dependent tandem mass spectrometry (MS/MS) scanning was used to generate product ion spectra from the protonated ions of the compound and its metabolites. An unusual metabolite at m/z 407 corresponding to the [M-24+H]+ ion was identified for compound 1. Interestingly, the formation of the [M-24+H]+ ion was not observed in the analogues wherein the fused thieno double bond was substituted (2) and the thieno group replaced by a fused benzo derivative (3). Compounds 2 and 3 exhibited metabolic turnovers of 24 and 30%, yielding oxidative metabolites corresponding to [M+16] and [M+32]+, respectively. Based on these facts the mechanism for [M-24]+ formation in compound 1 through an initial epoxide formation on the double bond of the fused thieno ring followed by hydrolytic ring opening and deacylation is envisaged.

The use of liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to explore the in vitro metabolism of cyanoalkyl piperidine derivatives.

A LC/MS method using atmospheric pressure chemical ionization, positive ion mode and full scan to measure the in vitro metabolic stability of cyanoalkyl functionalized compounds with the human liver microsomes was employed. Percentage metabolism examined for the five cyanoalkyl piperidines revealed the optimal chain length and positioning of these functions to produce the most metabolically stable compound. The 4-cyanomethyl piperidine derivative was the most stable compound with 15% metabolism after 15 min incubation with human liver microsomes. In general, the major metabolites formed from the cyanoalkyl piperidine derivatives were due to oxidation of the cyanoalkyl chain or the piperidine fragment, resulting in a M+16 ion. However, the 2-cyanomethyl piperidine derivative exhibited an interesting biotransformation pathway with unusual metabolite peaks corresponding to M+5, M-11 and M+21 ions. Data-dependent MS/MS scanning was used to generate daughter ion spectra from the parent compound and its metabolite peaks. Based on the fragmentation analysis, a carboxylic acid, aldehyde and oxidative metabolite of the carboxylic acid structure have been proposed for M+5, M-11 and M+21 ions, respectively.

Simultaneous measurement of drug metabolic stability and identification of metabolites using ion-trap mass spectrometry.

The use of in vitro drug metabolism data in the understanding of in vivo pharmacokinetic, safety and toxicity data has become a large area of scientific interest. This has stemmed from a trend in the pharmaceutical industry to use in vitro data generated from human tissue as a criterion to select compounds for further investigation. As well as measuring metabolic stability in vitro using human liver microsomal preparations, the identification of possible metabolite(s) formed may play a vital role in Hit-to-Lead and Lead optimisation processes. The data-dependent scan function mode with the ion-trap instrumentation provides the ability to measure the metabolic stability and identification of possible metabolites of a compound. A gradient liquid chromatographic method with a run time of 6 min/injection was developed for this purpose. The approach of simultaneous metabolic stability measurements and rapid identification of metabolites of drugs with high (verapamil), medium (propranolol and cisapride) and low (flunarazine) metabolic stabilities using ion-trap mass spectrometry is described. The metabolites identified after 15 min incubation for verapamil, propranolol and cisapride are in good agreement with those reported as the major metabolites in human in vivo studies.