In situ evaluation of the impact of metformin or verapamil coadministration with vildagliptin on its regional absorption from the rabbit's intestine.

This research aims to identify regional differences in vildagliptin absorption across the intestinal membrane. Furthermore, it was to investigate the effect of verapamil or metformin on vildagliptin absorptive clearance. The study utilized an in situ rabbit intestinal perfusion technique to determine vildagliptin oral absorption from duodenum, jejunum, ileum, and ascending colon. This was conducted both with and without perfusion of metformin or verapamil. The findings revealed that the vildagliptin absorptive clearance per unit length varied by site and was in the order as follows: ileum < jejunum < duodenum < ascending colon, implying that P-gp is significant in the reduction of vildagliptin absorption. Also, the arrangement cannot reverse intestinal P-gp, but the observations suggest that P-gp is significant in reducing vildagliptin absorption. Verapamil co-perfusion significantly increased the vildagliptin absorptive clearance by 2.4 and 3.2 fold through the jejunum and ileum, respectively. Metformin co-administration showed a non-significant decrease in vildagliptin absorptive clearance through all tested segments. Vildagliptin absorption was site-dependent and may be related to the intestinal P-glycoprotein content. This may aid in understanding the important elements that influence vildagliptin absorption, besides drug-drug interactions that can occur in type 2 diabetic patients taking vildagliptin in conjunction with other drugs that can modify the P-glycoprotein level.

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

Navigating Transporter Sciences in Pharmacokinetics Characterization Using the Extended Clearance Classification System.

Membrane transporters play an important role in the absorption, distribution, clearance, and elimination of drugs. Supported by the pharmacokinetics data in human, several transporters including organic anion transporting polypeptide (OATP)1B1, OATP1B3, organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, multidrug and toxin extrusion (MATE) proteins, P-glycoprotein and breast cancer resistance protein are suggested to be of clinical relevance. An early understanding of the transporter role in drug disposition and clearance allows reliable prediction/evaluation of pharmacokinetics and changes due to drug-drug interactions (DDIs) or genetic polymorphisms. We recently proposed an extended clearance classification system (ECCS) based on simple drug properties (i.e., ionization, permeability, and molecular weight) to predict the predominant clearance mechanism. According to this framework, systemic clearance of class 1B and 3B drugs is likely determined by the OATP-mediated hepatic uptake. Class 3A and 4 drugs, and certain class 3B drugs, are predominantly cleared by renal, wherein, OAT1, OAT3, OCT2, and MATE proteins could contribute to their active renal secretion. Intestinal efflux and uptake transporters largely influence the oral pharmacokinetics of class 3A, 3B, and 4 drugs. We discuss the paradigm of applying the ECCS framework in mapping the role of clinically relevant drug transporters in early discovery and development; thereby implementing the right strategy to allow optimization of drug exposure and evaluation of clinical risk due to DDIs and pharmacogenomics.

A Study on Pharmacokinetics of Bosentan with Systems Modeling, Part 1: Translating Systemic Plasma Concentration to Liver Exposure in Healthy Subjects.

Understanding liver exposure of hepatic transporter substrates in clinical studies is often critical, as it typically governs pharmacodynamics, drug-drug interactions, and toxicity for certain drugs. However, this is a challenging task since there is currently no easy method to directly measure drug concentration in the human liver. Using bosentan as an example, we demonstrate a new approach to estimate liver exposure based on observed systemic pharmacokinetics from clinical studies using physiologically based pharmacokinetic modeling. The prediction was verified to be both accurate and precise using sensitivity analysis. For bosentan, the predicted pseudo steady-state unbound liver-to-unbound systemic plasma concentration ratio was 34.9 (95% confidence interval: 4.2, 50). Drug-drug interaction (i.e., CYP3A and CYP2B6 induction) and inhibition of hepatic transporters (i.e., bile salt export pump, multidrug resistance-associated proteins, and sodium-taurocholate cotransporting polypeptide) were predicted based on the estimated unbound liver tissue or plasma concentrations. With further validation and refinement, we conclude that this approach may serve to predict human liver exposure and complement other methods involving tissue biopsy and imaging.

Involvement of Drug Transporters in Organ Toxicity: The Fundamental Basis of Drug Discovery and Development.

Membrane transporters play a pivotal role in many organs to maintain their normal physiological functions and contribute significantly to drug absorption, distribution, and elimination. Knowledge gained from gene modified animal models or human genetic disorders has demonstrated that interruption of the transporter activity can lead to debilitating diseases or organ toxicities. Herein we describe transporter associated diseases and organ toxicities resulting from transporter gene deficiency or functional inhibition in the liver, kidney, gastrointestinal tract (GIT), and central nervous system (CNS). While proposing additional transporters as targets for drug-induced organ toxicity, strategies and future perspectives are discussed for transporter risk assessment in drug discovery and development.

Projecting ADME Behavior and Drug-Drug Interactions in Early Discovery and Development: Application of the Extended Clearance Classification System.

PURPOSE: To assess the utility of Extended Clearance Classification System (ECCS) in understanding absorption, distribution, metabolism, and elimination (ADME) attributes and enabling victim drug-drug interaction (DDI) predictions. METHODS: A database of 368 drugs with relevant ADME parameters, main metabolizing enzymes, uptake transporters, efflux transporters, and highest change in exposure (%AUC) in presence of inhibitors was developed using published literature. Drugs were characterized according to ECCS using ionization, molecular weight and estimated permeability. RESULTS: Analyses suggested that ECCS class 1A drugs are well absorbed and systemic clearance is determined by metabolism mediated by CYP2C, esterases, and UGTs. For class 1B drugs, oral absorption is high and the predominant clearance mechanism is hepatic uptake mediated by OATP transporters. High permeability neutral/basic drugs (class 2) showed high oral absorption, with metabolism mediated generally by CYP3A, CYP2D6 and UGTs as the predominant clearance mechanism. Class 3A/4 drugs showed moderate absorption with dominant renal clearance involving OAT/OCT2 transporters. Class 3B drugs showed low to moderate absorption with hepatic uptake (OATPs) and/or renal clearance as primary clearance mechanisms. The highest DDI risk is typically seen with class 2/1B/3B compounds manifested by inhibition of either CYP metabolism or active hepatic uptake. Class 2 showed a wider range in AUC change likely due to a variety of enzymes involved. DDI risk for class 3A/4 is small and associated with inhibition of renal transporters. CONCLUSIONS: ECCS provides a framework to project ADME profiles and further enables prediction of victim DDI liabilities in drug discovery and development.

Predicting Clearance Mechanism in Drug Discovery: Extended Clearance Classification System (ECCS).

Early prediction of clearance mechanisms allows for the rapid progression of drug discovery and development programs, and facilitates risk assessment of the pharmacokinetic variability associated with drug interactions and pharmacogenomics. Here we propose a scientific framework--Extended Clearance Classification System (ECCS)--which can be used to predict the predominant clearance mechanism (rate-determining process) based on physicochemical properties and passive membrane permeability. Compounds are classified as: Class 1A--metabolism as primary systemic clearance mechanism (high permeability acids/zwitterions with molecular weight (MW) ≤400 Da), Class 1B--transporter-mediated hepatic uptake as primary systemic clearance mechanism (high permeability acids/zwitterions with MW >400 Da), Class 2--metabolism as primary clearance mechanism (high permeability bases/neutrals), Class 3A--renal clearance (low permeability acids/zwitterions with MW ≤400 Da), Class 3B--transporter mediated hepatic uptake or renal clearance (low permeability acids/zwitterions with MW >400 Da), and Class 4--renal clearance (low permeability bases/neutrals). The performance of the ECCS framework was validated using 307 compounds with single clearance mechanism contributing to ≥70% of systemic clearance. The apparent permeability across clonal cell line of Madin - Darby canine kidney cells, selected for low endogenous efflux transporter expression, with a cut-off of 5 × 10(-6) cm/s was used for permeability classification, and the ionization (at pH7) was assigned based on calculated pKa. The proposed scheme correctly predicted the rate-determining clearance mechanism to be either metabolism, hepatic uptake or renal for ~92% of total compounds. We discuss the general characteristics of each ECCS class, as well as compare and contrast the framework with the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS). Collectively, the ECCS framework is valuable in early prediction of clearance mechanism and can aid in choosing the right preclinical tool kit and strategy for optimizing drug exposure and evaluating clinical risk of pharmacokinetic variability caused by drug interactions and pharmacogenomics.

Development and validation of an UPLC-MS/MS assay for quantitative analysis of the ghrelin receptor inverse agonist PF-5190457 in human or rat plasma and rat brain.

PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. Our aim was to develop and validate a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain using liquid chromatography-tandem mass spectrometry. The analyte and stable isotope internal standard were extracted from 50 µL plasma or rat brain homogenate by protein precipitation using 0.1% formic acid in acetonitrile. Chromatography was carried out on an Acquity UPLC BEH C18 (2.1 mm × 50 mm) column with 1.7 µm particle size and 130 Å pore size. The flow rate was 0.5 mL/min and total chromatographic run time was 2.2 min. The mobile phase consisted of a gradient mixture of water: acetonitrile 95:5% (v/v) containing 0.1% formic acid (solvent A) and 100% acetonitrile containing 0.1% formic acid (solvent B). Multiple reaction monitoring was carried out in positive electro-spray ionization mode using m/z 513.35 → 209.30 for PF-5190457 and m/z 518.47 → 214.43 for the internal standard. The recovery ranged from 102 to 118% with coefficient of variation (CV) less than 6% for all matrices. The calibration curves for all matrices were linear over the studied concentration range (R(2) ≥ 0.998, n = 3). The lower limit of quantification was 1 ng/mL in rat or human plasma and 0.75 ng/g in rat brain. Intra- and inter-run mean percent accuracies were between 85 and 115% and percent imprecision was ≤15%. The assays were successfully utilized to measure the concentration of PF-5190457 in pre-clinical and clinical pharmacology studies of the compound.

Quantitative prediction of repaglinide-rifampicin complex drug interactions using dynamic and static mechanistic models: delineating differential CYP3A4 induction and OATP1B1 inhibition potential of rifampicin.

Repaglinide is mainly metabolized by cytochrome P450 enzymes CYP2C8 and CYP3A4, and it is also a substrate to a hepatic uptake transporter, organic anion transporting polypeptide (OATP)1B1. The purpose of this study is to predict the dosing time-dependent pharmacokinetic interactions of repaglinide with rifampicin, using mechanistic models. In vitro hepatic transport of repaglinide, characterized using sandwich-cultured human hepatocytes, and intrinsic metabolic parameters were used to build a dynamic whole-body physiologically-based pharmacokinetic (PBPK) model. The PBPK model adequately described repaglinide plasma concentration-time profiles and successfully predicted area under the plasma concentration-time curve ratios of repaglinide (within ± 25% error), dosed (staggered 0-24 hours) after rifampicin treatment when primarily considering induction of CYP3A4 and reversible inhibition of OATP1B1 by rifampicin. Further, a static mechanistic "extended net-effect" model incorporating transport and metabolic disposition parameters of repaglinide and interaction potency of rifampicin was devised. Predictions based on the static model are similar to those observed in the clinic (average error ∼19%) and to those based on the PBPK model. Both the models suggested that the combined effect of increased gut extraction and decreased hepatic uptake caused minimal repaglinide systemic exposure change when repaglinide is dosed simultaneously or 1 hour after the rifampicin dose. On the other hand, isolated induction effect as a result of temporal separation of the two drugs translated to an approximate 5-fold reduction in repaglinide systemic exposure. In conclusion, both dynamic and static mechanistic models are instrumental in delineating the quantitative contribution of transport and metabolism in the dosing time-dependent repaglinide-rifampicin interactions.

Quantitative prediction of renal transporter-mediated clinical drug-drug interactions.

Kidney plays a critical role in the elimination of xenobiotics. Drug-drug interactions (DDIs) via inhibition of renal organic anion (OAT) and organic cation (OCT) transporters have been observed in the clinic. This study examined the quantitative predictability of renal transporter-mediated clinical DDIs based on basic and mechanistic models. In vitro transport and clinical pharmacokinetics parameters were used to quantitatively predict DDIs of victim drugs when coadministrated with OAT or OCT inhibitors, probenecid and cimetidine, respectively. The predicted changes in renal clearance (CLr) and area under the plasma concentration-time curve (AUC) were comparable to that observed in clinical studies. With probenecid, basic modeling predicted 61% cases within 25% and 94% cases within 50% of the observed CLr changes in clinic. With cimetidine, basic modeling predicted 61% cases within 25% and 92% cases within 50% of the observed CLr changes in clinic. Additionally, the mechanistic model predicted 54% cases within 25% and 92% cases within 50% of the observed AUC changes with probenecid. Notably, the magnitude of AUC changes attributable to the renal DDIs is generally less than 2-fold, unlike the DDIs associated with inhibition of CYPs and/or hepatic uptake transporters. The models were further used to evaluate the renal DDIs of Pfizer clinical candidates/drugs, and the overall predictability demonstrates their utility in the drug discovery and development settings.

Mechanistic modeling to predict the transporter- and enzyme-mediated drug-drug interactions of repaglinide.

PURPOSE: Quantitative prediction of complex drug-drug interactions (DDIs) is challenging. Repaglinide is mainly metabolized by cytochrome-P-450 (CYP)2C8 and CYP3A4, and is also a substrate of organic anion transporting polypeptide (OATP)1B1. The purpose is to develop a physiologically based pharmacokinetic (PBPK) model to predict the pharmacokinetics and DDIs of repaglinide. METHODS: In vitro hepatic transport of repaglinide, gemfibrozil and gemfibrozil 1-O-ß-glucuronide was characterized using sandwich-culture human hepatocytes. A PBPK model, implemented in Simcyp (Sheffield, UK), was developed utilizing in vitro transport and metabolic clearance data. RESULTS: In vitro studies suggested significant active hepatic uptake of repaglinide. Mechanistic model adequately described repaglinide pharmacokinetics, and successfully predicted DDIs with several OATP1B1 and CYP3A4 inhibitors (<10% error). Furthermore, repaglinide-gemfibrozil interaction at therapeutic dose was closely predicted using in vitro fraction metabolism for CYP2C8 (0.71), when primarily considering reversible inhibition of OATP1B1 and mechanism-based inactivation of CYP2C8 by gemfibrozil and gemfibrozil 1-O-ß-glucuronide. CONCLUSIONS: This study demonstrated that hepatic uptake is rate-determining in the systemic clearance of repaglinide. The model quantitatively predicted several repaglinide DDIs, including the complex interactions with gemfibrozil. Both OATP1B1 and CYP2C8 inhibition contribute significantly to repaglinide-gemfibrozil interaction, and need to be considered for quantitative rationalization of DDIs with either drug.

Physicochemical property space of hepatobiliary transport and computational models for predicting rat biliary excretion.

Biliary excretion (BE) is a major elimination pathway, and its prediction is particularly important for optimization of systemic and/or target-site exposure of new molecular entities. The objective is to characterize the physicochemical space associated with hepatobiliary transport and rat BE and to develop in silico models. BE of 123 in-house compounds was obtained using the bile-duct cannulated rat model. Human and rat hepatic uptake transporters (hOATP1B1, hOATP1B3, hOATP2B1, and rOatp1b2) substrates (n = 183) were identified using transfected cells. Furthermore, the datasets were extended by adding BE of 163 compounds and 97 organic anion transporting polypeptide (OATP) substrates from the literature. Approximately 60% of compounds showing percentage of BE (%BE) ≥ 10 are anions, with mean BE of anions (36%) more than 3-fold higher than that of nonacids (11%). Compounds with %BE ≥ 10 are found to have high molecular mass, large polar surface area, more rotatable bonds, and high H-bond count, whereas the lipophilicity and passive membrane permeability are lower compared with compounds with %BE < 10. According to statistical analysis and principal component analysis, hOATPs and rOatp1b2 substrates showed physicochemical characteristics that were similar to those of the %BE ≥ 10 dataset. We further build categorical in silico models to predict rat BE, and the models (gradient boosting machine and scoring function) developed showed 80% predictability in identifying the rat BE bins (%BE ≥ 10 or < 10). In conclusion, the significant overlap of the property space of OATP substrates and rat BE suggests a predominant role of sinusoidal uptake transporters in biliary elimination. Categorical in silico models to predict rat BE were developed, and successful predictions were achieved.

In vitro evaluation of hepatic transporter-mediated clinical drug-drug interactions: hepatocyte model optimization and retrospective investigation.

To assess the feasibility of using sandwich-cultured human hepatocytes (SCHHs) as a model to characterize transport kinetics for in vivo pharmacokinetic prediction, the expression of organic anion-transporting polypeptide (OATP) proteins in SCHHs, along with biliary efflux transporters, was confirmed quantitatively by liquid chromatography-tandem mass spectrometry. Rifamycin SV (Rif SV), which was shown to completely block the function of OATP transporters, was selected as an inhibitor to assess the initial rates of active uptake. The optimized SCHH model was applied in a retrospective investigation of compounds with known clinically significant OATP-mediated uptake and was applied further to explore drug-drug interactions (DDIs). Greater than 50% inhibition of active uptake by Rif SV was found to be associated with clinically significant OATP-mediated DDIs. We propose that the in vitro active uptake value therefore could serve as a cutoff for class 3 and 4 compounds of the Biopharmaceutics Drug Disposition Classification System, which could be integrated into the International Transporter Consortium decision tree recommendations to trigger clinical evaluations for potential DDI risks. Furthermore, the kinetics of in vitro hepatobiliary transport obtained from SCHHs, along with protein expression scaling factors, offer an opportunity to predict complex in vivo processes using mathematical models, such as physiologically based pharmacokinetics models.

Mechanistic pharmacokinetic modeling for the prediction of transporter-mediated disposition in humans from sandwich culture human hepatocyte data.

With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic anion-transporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under- and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.

Differential modulation of cytochrome P450 activity and the effect of 1-aminobenzotriazole on hepatic transport in sandwich-cultured human hepatocytes.

Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.

Characterization of organic anion transporting polypeptide (OATP) expression and its functional contribution to the uptake of substrates in human hepatocytes.

Since the substrate specificities of OATP1B1, 1B3, and 2B1 are broad and overlapping, the contribution of each isoform to the overall hepatic uptake is of concern when assessing transporter-mediated drug-drug interactions (DDIs) or genetic polymorphism impact in the clinic. Herein, we quantitatively measured OATP proteins in cryopreserved hepatocytes, sandwich-cultured human hepatocytes (SCHH), and the liver, and examined the relationship with functional uptake of OATP substrates in an effort to identify the OATP isoform(s) contributing to the hepatic uptake of pitavastatin. The modulation of OATP expression in SCHH was found to be lot-dependent. However, OATP protein measurements averaged from 5 lots of SCHH were comparable to that of suspended hepatocytes. All three OATP transporters in suspended hepatocytes and SCHH were significantly lower than those in the liver. In SCHH, the uptake of CCK-8 and pravastatin was found to be associated with the expression of OATP1B3 and OATP1B1, respectively. In suspended hepatocytes, OATP1B1 appeared to show a positive trend with respect to the uptake of pitavastatin, which suggests a selective contribution of OATP1B1 to pitavastatin transport and thus an OATP quantitative protein expression-activity relationship. While the passive diffusion of rosuvastatin in SCHH was consistent across hepatocyte lots, the passive diffusion of pitavastatin varied over a broad range (>4-fold) in suspended hepatocytes and was inversely correlated with transporter-mediated uptake, presumably due to cell membrane alterations imparted by cryopreservation. Collectively, SCHH maintains OATP protein expression and membrane integrity and, if feasible when considering research goals, would be considered a superior tool for the characterization of in vitro transport parameters without the complication of membrane leakage.

pH-Dependent solubility and permeability criteria for provisional biopharmaceutics classification (BCS and BDDCS) in early drug discovery.

The Biopharmaceutics Classification System (BCS) is a scientific framework that provides a basis for predicting the oral absorption of drugs. These concepts have been extended in the Biopharmaceutics Drug Disposition Classification System (BDDCS) to explain the potential mechanism of drug clearance and understand the effects of uptake and efflux transporters on absorption, distribution, metabolism, and elimination. The objective of present work is to establish criteria for provisional biopharmaceutics classification using pH-dependent passive permeability and aqueous solubility data generated from high throughput screening methodologies in drug discovery settings. The apparent permeability across monolayers of clonal cell line of Madin-Darby canine kidney cells, selected for low endogenous efflux transporter expression, was measured for a set of 105 drugs, with known BCS and BDDCS class. The permeability at apical pH 6.5 for acidic drugs and at pH 7.4 for nonacidic drugs showed a good correlation with the fraction absorbed in human (Fa). Receiver operating characteristic (ROC) curve analysis was utilized to define the permeability class boundary. At permeability ≥ 5 × 10(-6) cm/s, the accuracy of predicting Fa of ≥ 0.90 was 87%. Also, this cutoff showed more than 80% sensitivity and specificity in predicting the literature permeability classes (BCS), and the metabolism classes (BDDCS). The equilibrium solubility of a subset of 49 drugs was measured in pH 1.2 medium, pH 6.5 phosphate buffer, and in FaSSIF medium (pH 6.5). Although dose was not considered, good concordance of the measured solubility with BCS and BDDCS solubility class was achieved, when solubility at pH 1.2 was used for acidic compounds and FaSSIF solubility was used for basic, neutral, and zwitterionic compounds. Using a cutoff of 200 µg/mL, the data set suggested a 93% sensitivity and 86% specificity in predicting both the BCS and BDDCS solubility classes. In conclusion, this study identified pH-dependent permeability and solubility criteria that can be used to assign provisional biopharmaceutics class at early stage of the drug discovery process. Additionally, such a classification system will enable discovery scientists to assess the potential limiting factors to oral absorption, as well as help predict the drug disposition mechanisms and potential drug-drug interactions.

Full efficacy with no CNS side-effects: unachievable panacea or reality? DMPK considerations in design of drugs with limited brain penetration.

Optimising drug properties can be an important strategy to limit penetration into the CNS and offers advantages in reducing the risk of undesirable neurological effects When considering the design of these drugs it is important to consider the relative influx and efflux rates at the relevant biological membranes The highest degree of restriction at the brain is probably achievable by utilising active transport to exclude compounds from the brain Affinity for the efflux transporters Pgp and BCRP has been achieved in two in-house chemistry programmes by increasing polar surface area, which resulted in highly orally bioavailable low CNS penetrant compounds in preclinical species.

Anxiolytic-like activity of pregabalin in the Vogel conflict test in α2δ-1 (R217A) and α2δ-2 (R279A) mouse mutants.

The α(2)δ auxiliary subunits (α(2)δ-1 and α(2)δ-2) of voltage-sensitive calcium channels are thought to be the site of action of pregabalin (Lyrica), a drug that has been shown to be anxiolytic in clinical trials for generalized anxiety disorder. Pregabalin and the chemically related drug gabapentin have similar binding and pharmacology profiles, demonstrating high-affinity, in vitro binding to both α(2)δ-1 and α(2)δ-2 subunits. Two independent point mutant mouse strains were generated in which either the α(2)δ-1 subunit (arginine-to-alanine mutation at amino acid 217; R217A) or the α(2)δ-2 subunit (arginine-to-alanine mutation at amino acid 279; R279A) were rendered insensitive to gabapentin or pregabalin binding. These strains were used to characterize the activity of pregabalin in the Vogel conflict test, a measure of anxiolytic-like activity. Pregabalin showed robust anticonflict activity in wild-type littermates from each strain at a dose of 10 mg/kg but was inactive in the α(2)δ-1 (R217A) mutants up to a dose of 320 mg/kg. In contrast, pregabalin was active in the α(2)δ-2 (R279A) point mutants at 10 and 32 mg/kg. The positive control phenobarbital was active in mice carrying either mutation. These data suggest that the anxiolytic-like effects of pregabalin are mediated by binding of the drug to the α(2)δ-1 subunit.

pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1.

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 µM, 0.53 ± 0.2 µM and 2.2 ± 0.4 µM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.