Simplifying the Extended Clearance Concept Classification System (EC3S) to Guide Clearance Prediction in Drug Discovery.

PURPOSE: The Extended Clearance Concept Classification System was established as a development-stage tool to provide a framework for identifying fundamental mechanism(s) governing drug disposition in humans. In the present study, the applicability of the EC3S in drug discovery has been investigated. In its current format, the EC3S relies on low-throughput hepatocyte uptake data, which are not frequently generated in a discovery setting. METHODS: A relationship between hepatocyte uptake clearance and MDCK permeability was first established along with intrinsic clearance from human liver microsomes. The performance of this approach was examined by categorizing 64 drugs into EC3S classes and comparing the predicted major elimination pathway(s) to that observed in humans. As an extension of the work, the ability of the simplified EC3S to predict human systemic clearance based on intrinsic clearance generated using in-vitro metabolic systems was evaluated. RESULTS: The assessment enabled the use of MDCK permeability and unscaled unbound intrinsic clearance to generate cut-off criteria to categorize compounds into four EC3S classes: Class 12ab, 2cd, 34ab, and 34cd, with major elimination mechanism(s) assigned to each class. The predictivity analysis suggested that systemic clearance could generally be predicted within threefold for EC3S class 12ab and 34ab compounds. For classes 2cd and 34cd, systemic clearance was poorly predicted using in-vitro systems explored in this study. CONCLUSION: Collectively, our simplified classification approach is expected to facilitate the identification of mechanism(s) involved in drug elimination, faster resolution of in-vitro to in-vivo disconnects, and better design of mechanistic pharmacokinetic studies in drug discovery.

Unbound Brain-to-Plasma Partition Coefficient, Kp,uu,brain-a Game Changing Parameter for CNS Drug Discovery and Development.

PURPOSE: More than 15 years have passed since the first description of the unbound brain-to-plasma partition coefficient (Kp,uu,brain) by Prof. Margareta Hammarlund-Udenaes, which was enabled by advancements in experimental methodologies including cerebral microdialysis. Since then, growing knowledge and data continue to support the notion that the unbound (free) concentration of a drug at the site of action, such as the brain, is the driving force for pharmacological responses. Towards this end, Kp,uu,brain is the key parameter to obtain unbound brain concentrations from unbound plasma concentrations. METHODS: To understand the importance and impact of the Kp,uu,brain concept in contemporary drug discovery and development, a survey has been conducted amongst major pharmaceutical companies based in Europe and the USA. Here, we present the results from this survey which consisted of 47 questions addressing: 1) Background information of the companies, 2) Implementation, 3) Application areas, 4) Methodology, 5) Impact and 6) Future perspectives. RESULTS AND CONCLUSIONS: From the responses, it is clear that the majority of the companies (93%) has established a common understanding across disciplines of the concept and utility of Kp,uu,brain as compared to other parameters related to brain exposure. Adoption of the Kp,uu,brain concept has been mainly driven by individual scientists advocating its application in the various companies rather than by a top-down approach. Remarkably, 79% of all responders describe the portfolio impact of Kp,uu,brain implementation in their companies as 'game-changing'. Although most companies (74%) consider the current toolbox for Kp,uu,brain assessment and its validation satisfactory for drug discovery and early development, areas of improvement and future research to better understand human brain pharmacokinetics/pharmacodynamics translation have been identified.

Passive Influx and Ion Trapping Are More Relevant to the Cellular Accumulation of Highly Permeable Low-Molecular-Weight Acidic Drugs than Is Organic Anion Transporter 2.

Recently published work suggests that highly permeable low-molecular-weight (LMW) acidic drugs are transported by organic anion transporter 2 (OAT2). However, an asymmetric distribution of ionizable drugs in subcellular organelles where pH gradients are significant may occur in the presence of an inhibitor relative to its absence (e.g., lysosomal trapping). In the present study, OAT2-mediated transport of highly permeable LMW anions could not be demonstrated using OAT2 transfected cells, despite robust transport of the OAT2 substrate penciclovir. Moreover, a rifamycin SV (RifSV)-dependent reduction in the accumulation of highly permeable LMW anions previously observed in hepatocytes could be qualitatively reproduced using HepG2 cells and also in Madin-Darby canine kidney (MDCK) cells, which lack expression of OAT2. Neither HepG2 nor MDCK cells demonstrated meaningful penciclovir transport, nor was the cellular accumulation of the highly permeable LMW anions sensitive to competitive inhibition by the neutral OAT2 substrate penciclovir. Both cell lines, however, demonstrated sensitivity to the mitochondrial uncoupler p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) in a manner similar to RifSV. Furthermore, the transepithelial MDCK permeability of the highly permeable LMW anions was measured in the absence and presence of RifSV and FCCP at concentrations that reduced the cellular accumulation of anions. Neither inhibitor, nor the OAT2 inhibitor ketoprofen, reduced the transepithelial flux of the anions as would be anticipated for transported substrate inhibition. The findings presented here are aligned with cellular accumulation of highly permeable LMW anions being significantly determined by ion trapping sensitive to mitochondrial uncoupling, rather than the result of OAT2-mediated transport. SIGNIFICANCE STATEMENT: The manuscript illustrates that passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low-molecular-weight acidic drugs than is the previously proposed mechanism of OAT2-mediated transport. The outcome illustrated here highlights a rare, and perhaps previously not reported, observation of anionic drug trapping in a compartment sensitive to mitochondrial uncoupling (e.g., the mitochondrial matrix) that may be confused for transporter-mediated uptake.

The impact of assay recovery on the apparent permeability, a function of lysosomal trapping.

In vitro permeability assessment tools, like PAMPA, Caco-2, and MDCK, are frequently used to assess permeability and provide input in to various classification systems. Frequently, the measured recovery values in permeability assays are poor. Poor recovery may be a result of lysosomal trapping of compound. It was hypothesized that a relationship existed between diminished assay recovery of compound due to lysosomal trapping and underestimation of the Papp value.To examine this hypothesis, a series of experiments were conducted measuring cellular accumulation, percent recovery, and permeability in the absence or presence of an inhibitor of the V-type H+-ATPase, bafilomycin A1, to determine if a quantifiable relationship between lysosomal trapping, recovery, and permeability existed.Displacing compounds from lysosomes using bafilomycin A1 resulted in an improved compound recovery in the assay and a corresponding elevated permeability, where for each 10% loss in recovery, a Papp underestimate of ∼2.2 × 10-6 cm/s was observed. The findings highlight the potential for compound misclassification in various classification systems when assay recovery is not considered. Consideration of lysosomal trapping in the context of permeability assays may yield permeability values more reflective of the intrinsic permeability and the appropriate permeability classification.

Organic anion transporting polypeptide 2B1 (OATP2B1), an expanded substrate profile, does it align with OATP2B1's hypothesized function?

An expanded view of the substrate landscape of organic anion transporting polypeptide (OATP) 2B1 was pursued with the goal of understanding if the identification of novel in vitro substrates could shed additional light on the impact of OATP2B1 on intestinal absorption and brain penetration.To examine this hypothesis, a series of experiments measured the cellular accumulation of a diverse array of compounds. Representative angiotensin II receptor blockers (ARBs) and other compounds of interest were subsequently investigated for inhibition, time dependence, and kinetics.The study identified ARBs as a class of OATP2B1 substrates and found balsalazide, olsalzine, and gavestinel to be novel substrates of OATP2B1 too. Some compounds previously reported to be OATP2B1 substrates in the literature, aliskiren, erlotinib, montelukast, fexofenadine, and taurocholate could not be confirmed as substrates.Literature describing in vivo outcomes for OATP2B1 substrates, coproporphyrin III, ARBs, balsalazide, olsalzine, and gavestinel highlight the absence of a substantial impact of OATP2B1 on the oral absorption and/or brain penetration of OATP2B1 substrates. Suggestions of including OATP2B1 assessment as part of the drug approval process are likely premature and further mechanistic work with more robust OATP2B1 substrates, which may include some of those described here, is desirable.

Using Membrane Partitioning Simulations To Predict Permeability of Forty-Nine Drug-Like Molecules.

A simple descriptor calculated from molecular dynamics simulations of the membrane partitioning event is found to correlate well with experimental measurements of passive membrane permeation from the high-throughput MDCK-LE assay using a data set of 49 drug-like molecules. This descriptor approximates the energy cost of translocation across the hydrophobic membrane core (flip-flop), which for many molecules limits permeability. Performance is found to be superior in comparison to calculated properties such as clogP, clogD, or polar surface area. Furthermore, the atomistic simulations provide a structural understanding of the partitioned drug-membrane complex, facilitating medicinal chemistry optimization of membrane permeability.

Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III.

1. Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are polyspecific transporters that mediate the transport of organic acids into hepatocytes. Inactivating mutations of both OATP1B1 and OATP1B3 alleles lead to Rotor syndrome, a disease characterized by coproporphyrinuria, an elevated urinary excretion of coproporphyrins I and III. It was hypothesized that transport of coproporphyrins I and III was mediated by OATP1B1 and OATP1B3. 2. This hypothesis was tested using cells transfected with OATP1B1 and OATP1B3. OATP1B-mediated transport of coproporphyrin was time-dependent and concentration-dependent. OATP1B1-mediated transport of coproporphyrins I and III (Km = 0.13 and 0.22 µM, respectively), as did OATP1B3 (Km = 3.25 and 4.61 µM, respectively). The OATP1B-mediated transport of each coproporphyrin was inhibited by rifampicin. 3. The specificity of coproporphyrin transport was also investigated where OATP2B1 demonstrated meaningful transport of coproporphyrin III (Km = 0.31 µM), while OCT1, OCT2, OAT1, OAT3 and NTCP were negative for coproporphyrin transport. 4. The identification of coproporphyrins as OATP substrates in vitro more clearly defines the role of OATPs in the hepatic disposition and renal excretion of coproporphyrins I and III and provides compelling evidence for future in vivo exploration of coproporphyrins as biomarkers of OATP activity.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Application of receiver operating characteristic analysis to refine the prediction of potential digoxin drug interactions.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.

Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily.

Interactions of bilastine, a new oral H1 antihistamine, with human transporter systems.

Membrane transporters play a significant role in facilitating transmembrane drug movement. For new pharmacological agents, it is important to evaluate potential interactions (e.g., substrate specificity and/or inhibition) with human transporters that may affect their pharmacokinetics, efficacy, or toxicity. Bilastine is a new nonsedating H1 antihistamine indicated for the treatment of allergic rhinoconjunctivitis and urticaria. The in vitro inhibitory effects of bilastine were assessed on 12 human transporters: four efflux [multidrug resistance protein 1 (MDR1) or P-glycoprotein, breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2), and bile salt export pump) and eight uptake transporters (sodium taurocholate cotransporting polypeptide, organic cation transporter (OCT)1, organic anion transporter (OAT)1, OAT3, OCT2, OATP2B1, OATP1B1, and OATP1B3). Only mild inhibition was found for MDR1-, OCT1-, and OATP2B1-mediated transport of probe substrates at the highest bilastine concentration assayed (300 µM; half-maximal inhibitory concentration: ≥300 µM). Bilastine transport by MDR1, BCRP, OAT1, OAT3, and OCT2 was also investigated in vitro. Only MDR1 active transport of bilastine was relevant, whereas it did not appear to be a substrate of OCT2, OAT1, or OAT3, nor was it transported substantially by BCRP. Drug-drug interactions resulting from bilastine inhibition of drug transporters that would be generally regarded as clinically relevant are unlikely. Additionally, bilastine did not appear to be a substrate of human BCRP, OAT1, OAT3, or OCT2 and thus is not a potential victim of inhibitors of these transporters. On the other hand, based on in vitro evaluation, clinically relevant interactions with MDR1 inhibitors are anticipated.

Paradoxical Increase of Permeability and Lipophilicity with the Increasing Topological Polar Surface Area within a Series of PRMT5 Inhibitors.

An imidazolone → triazolone replacement addressed the limited passive permeability of a series of protein arginine methyl transferase 5 (PRMT5) inhibitors. This increase in passive permeability was unexpected given the increase in the hydrogen bond acceptor (HBA) count and topological polar surface area (TPSA), two descriptors that are typically inversely correlated with permeability. Quantum mechanics (QM) calculations revealed that this unusual effect was due to an electronically driven disconnect between TPSA and 3D-PSA, which manifests in a reduction in overall HBA strength as indicated by the HBA moment descriptor from COSMO-RS (conductor-like screening model for real solvation). HBA moment was subsequently deployed as a design parameter leading to the discovery of inhibitors with not only improved passive permeability but also reduced P-glycoprotein (P-gp) transport. Our case study suggests that hidden polarity as quantified by TPSA-3DPSA can be rationally designed through QM calculations.

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3.

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug-drug interactions. Numerous methods exist for identifying potential drug-drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (K(m)=2.9 and 1.8 microM, V(max)=0.20 and 0.33 pmol/min/cm(2), respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug-drug interactions with OATP1B1 and OATP1B3.

The activities of drug inactive ingredients on biological targets.

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

Potent nonimmunosuppressive cyclophilin inhibitors with improved pharmaceutical properties and decreased transporter inhibition.

Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.

Influence of molecular structure on substrate binding to the human organic cation transporter, hOCT1.

Organic cation transporters play a critical role in the elimination of therapeutic compounds in the liver and the kidney. We used computational quantitative structure activity approaches to predict molecular features that influence interaction with the human ortholog of the organic cation transporter (hOCT1). [(3)H]tetraethylammonium uptake in HeLa cells stably expressing hOCT1 was inhibited to varying extents by a diverse set of 30 molecules. A subset of 22 of these was used to produce, using Catalyst, a pharmacophore that consisted of three hydrophobic features and a positive ionizable feature. The correlation coefficient of observed versus predicted IC(50) was 0.86 for this training set, which was superior to calculated logP alone (r = 0.73) as a predictor of hOCT1 inhibition. A descriptor-based quantitative structure-activity relationship study using Cerius(2) resulted in an equation relating five molecular descriptors to log IC(50) with a correlation coefficient of 0.95. Furthermore, a group of phenylpyridinium and quinolinium compounds were used to investigate the spatial limitations of the hOCT1 binding site. The affinity for hOCT was higher for 4-phenylpyridiniums > 3-phenylpyridiniums > quinolinium, indicating that substrate affinity was influenced by the distribution of hydrophobic mass. In addition, supraplanar hydrophobic mass was found to increase the affinity for binding hOCT1. These results indicate how a combination of computational and in vitro approaches may yield insight into the binding affinity of transporters and may be applicable to predicting these properties for new therapeutics.