Using the Dynamic Well-Stirred Model to Extrapolate Hepatic Clearance of Organic Anion-Transporting Polypeptide Transporter Substrates without Assuming Albumin-Mediated Hepatic Drug Uptake.

Extrapolating in vivo hepatic clearance from in vitro uptake data is a known challenge, especially for organic anion-transporting polypeptide transporter (OATP) substrates, and the well-stirred model (WSM) commonly yields systematic underpredictions for those anionic drugs, hypothetically due to "albumin-mediated hepatic drug uptake". In the present study, we demonstrate that the WSM incorporating the dynamic free fraction (f D), a measure of drug protein binding affinity, performs reasonably well in predicting hepatic clearance of OATP substrates. For a selection of anionic drugs, including atorvastatin, fluvastatin, pravastatin, rosuvastatin, pitavastatin, cerivastatin, and repaglinide, this dynamic well-stirred model (dWSM) correctly predicts hepatic plasma clearance within 2-fold error for six out of seven OATP substrates examined. The geometric mean of clearance ratios between the predicted and the observed values falls in the range of 1.21-1.38. As expected, the WSM with unbound fraction (f u) systematically underpredicts hepatic clearance with greater than 2-fold error for five out of seven drugs, and the geometric mean of clearance ratios between the predicted and the observed values is in the range of 0.20-0.29. The results suggest that, despite its simplicity, the dWSM operates well for transporter-mediated uptake clearance, and that clearance under-prediction of OATP substrates may not necessarily be associated with the chemical class of the anionic drugs, nor is it a result of albumin-mediated hepatic drug uptake as currently hypothesized. Instead, the superior prediction power of the dWSM confirms the utility of the dynamic free fraction in clearance prediction and the importance of drug plasma binding kinetics in hepatic uptake clearance. SIGNIFICANCE STATEMENT: The traditional well-stirred model (WSM) consistently underpredicts organin anion-transporting polypeptide transporter (OATP)-mediated hepatic uptake clearance, hypothetically due to the albumin-mediated hepatic drug uptake. In this manuscript, we apply the dynamic WSM to extrapolate hepatic clearance of the OATP substrates, and our results show significant improvements in clearance prediction without assuming albumin-mediated hepatic drug uptake.

An Integrated Hepatocyte Stability Assay for Simultaneous Metabolic Stability Assessment and Metabolite Profiling.

The determination of metabolic stability is critical for drug discovery programs, allowing for the optimization of chemical entities and compound prioritization. As such, it is common to perform high-volume in vitro metabolic stability experiments early in the lead optimization process to understand metabolic liabilities. Additional metabolite identification experiments are subsequently performed for a more comprehensive understanding of the metabolic clearance routes to aid medicinal chemists in the structural design of compounds. Collectively, these experiments require extensive sample preparation and a substantial amount of time and resources. To overcome the challenges, a high-throughput integrated assay for simultaneous hepatocyte metabolic stability assessment and metabolite profiling was developed. This assay platform consists of four parts: 1) an automated liquid-handling system for sample preparation and incubation, 2) a liquid chromatography and high-resolution mass spectrometry-based system to simultaneously monitor the parent compound depletion and metabolite formation, 3) an automated data analysis and report system for hepatic clearance assessment; and 4) streamlined autobatch processing for software-based metabolite profiling. The assay platform was evaluated using eight control compounds with various metabolic rates and biotransformation routes in hepatocytes across three species. Multiple sample preparation and data analysis steps were evaluated and validated for accuracy, repeatability, and metabolite coverage. The combined utility of an automated liquid-handling instrument, a high-resolution mass spectrometer, and multiple streamlined data processing software improves the process of these highly demanding screening assays and allows for simultaneous determination of metabolic stability and metabolite profiles for more efficient lead optimization during early drug discovery. SIGNIFICANCE STATEMENT: Metabolic stability assessment and metabolite profiling are pivotal in drug discovery to fully comprehend metabolic liabilities for chemical entity optimization and lead selection. Process of these assays can be repetitive and resource demanding. Here, we developed an integrated hepatocyte stability assay that combines automation, high-resolution mass spectrometers, and batch-processing software to improve and combine the workflow of these assays. The integrated approach allows simultaneous metabolic stability assessment and metabolite profiling, significantly accelerating screening and lead optimization in a resource-effective manner.

Introducing the Dynamic Well-Stirred Model for Predicting Hepatic Clearance and Extraction Ratio.

The well-stirred model (WSM) incorporating the fraction of unbound drug (fu) to account for the effect of plasma binding on intrinsic clearance has been widely used for predicting hepatic clearance under the assumption that drug protein binding reaches equilibrium instantaneously. Our theoretical analysis reveals that the effect of protein binding on intrinsic clearance is better accounted for with the dynamic free fraction (fD), a measure of drug protein binding affinity, which leads to a putative dynamic well-stirred model (dWSM) without the instantaneous equilibrium assumption. Using recombinant CYP3A4 as the in vitro clearance system, we demonstrate that the binding effect of albumin on the intrinsic clearance of both highly bound midazolam and highly free verapamil is fully corrected by their corresponding fD values, respectively. On the other hand, fu only corrects the binding effect of albumin on the intrinsic clearance of verapamil, and yields severe over-correction of the intrinsic clearance of midazolam. The results suggest that the traditional WSM is suitable for highly free drugs like verapamil but not necessarily for highly bound drugs such as midazolam due to the violation of the instantaneous equilibrium assumption or under-estimating the true free drug concentration. In comparison, the dWSM incorporating fD holds true as long as drug elimination follows steady-state kinetics, and hence, it is more broadly applicable to drugs with different protein binding characteristics. Here we demonstrate with 36 diverse drugs, that the dWSM significantly improves the accuracy of predicting human hepatic clearance and liver extraction ratio from in vitro microsomal clearance data, highlighting the importance of drug plasma protein binding kinetics in addressing the under-prediction of hepatic clearance by the WSM.

Using Counter Equilibrium Dialysis (CED) to Increase Confidence in the Measurement of Free Fraction for Challenging Compounds.

The confidence in fraction unbound (ƒu) using equilibrium dialysis (ED) is often questioned (e.g., highly bound, labile compounds) due to uncertainty in whether true equilibrium is achieved. Different methods have been developed to increase confidence in ƒu measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒu measurement can still suffer due to non-specific binding and inter-run variations introduced during equilibrium and analysis. To address this concern, we introduce an orthogonal approach called counter equilibrium dialysis (CED) in which non-labeled and isotope-labeled compounds are dosed counter-directionally in rapid equilibrium dialysis (RED). ƒu values of both non-labeled and labeled compounds are measured simultaneously in the same run. These tactics not only minimize non-specific binding and inter-run variability but also enable the confirmation of true equilibrium. If equilibrium is reached in both dialysis directions, the ƒu for the non-labeled compound and the labeled compound will converge. The refined methodology was extensively tested with various compounds of diverse physicochemical properties and plasma binding characteristics. Our results demonstrated that, by using the CED method, ƒu values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds.

New Methodology for Determining Plasma Protein Binding Kinetics Using an Enzyme Reporter Assay Coupling with High-Resolution Mass Spectrometry.

Determination of drug binding kinetics in plasma is important yet extremely challenging. Accordingly, we introduce "dynamic free fraction" as a new binding parameter describing drug-protein binding kinetics. We demonstrate theoretically and experimentally that the dynamic free fraction can be determined by coupling the drug binding assay with a reporter enzyme in combination with high-resolution mass spectrometry measuring the relative initial steady-state rates of enzymatic reactions in the absence and presence of matrix proteins. This novel and simple methodology circumvents a long-standing challenge inherent in existing methods for determining binding kinetics constants, such as kon and koff, and enables assessment of the impact of protein binding kinetics on pharmaceutical properties of drugs. As demonstrated with nine model drugs, the predicted liver extraction ratio, a measure of efficiency of drug removal by the liver, correlates significantly better to the observed extraction ratio when using the dynamic free fraction (fD) in place of the unbound fraction (fu) of the drug in plasma. Similarly, the in vivo hepatic clearance of these drugs, a measure of liver drug elimination, is highly comparable to the clearance values calculated with the dynamic free fraction (fD), which is markedly better than those calculated with the unbound fraction (fu). In contrast to the prevailing view, these results indicate that protein binding kinetics is an important pharmacokinetic property of a drug. As plasma protein binding is one of the most important drug properties, this new methodology may represent a breakthrough and could have a real impact on the field.

High-throughput protein binding assay using cross-sample pooling in combination with high-resolution mass spectrometry.

RATIONALE: The fraction of unbound drugs (ƒu ) is a useful pharmacokinetic parameter in understanding drug disposition (Absorption, Distribution, Metabolism, Excretion), pharmacological activity and toxicity. Therefore, protein binding assays are frequently performed in drug development, creating a high demand for biological, experimental and analytical resources. Our work aims to increase binding assay throughput and comprehensiveness, while reducing biological and experimental consumption without compromising data quality by introducing cross-pooling and cassetting procedures, followed by a rapid and informative high-resolution mass spectrometry (HRMS) analysis. METHODS: Individual drugs were spiked into a test matrix and incubated in a rapid equilibrium dialysis device. After incubation, a cross-pooling procedure was performed, in which the samples of one drug were equalized with the complementary matrix provided from a different drug. The same drugs were also assayed with a conventional method, in which samples were equalized with the newly prepared complementary matrix. Cross-pooled samples were further cassetted to increase throughput. The samples were analyzed by high-performance liquid chromatography coupled with an Orbitrap HRMS, and the fu values were calculated and compared between the cross-pooling and conventional sampling procedures. RESULTS: Highly comparable human plasma fu values of 27 drugs representing different chemical classes and wide-ranging fu values were obtained by conventional and cross-pooling procedures, The tight correlation was further validated in other species (rat, mouse) and matrices (microsomes, brain). In addition, the cassetted samples showed highly consistent fu values compared to their noncassetted counterparts. Moreover, HRMS analysis not only showed highly consistent and repeatable quantification results compared to the "gold standard" triple quadrupole (QqQ) analysis, but also demonstrated outstanding advantage over QqQ in enabling a high-throughput, informative and versatile analysis. CONCLUSIONS: This work demonstrates that the cross-pooling procedure with further sample cassetting using HRMS is experimentally and analytically feasible to allow a higher throughput (increased by up to 8-fold), resource-effective (reducing matrix consumption by 50%, minimizing time spent on method development and platemap design), analytically dependable (accurate quantification), and versatile (metabolite elucidation and low recovery troubleshooting) analysis.

Design and Measurement of Drug Tissue Concentration Asymmetry and Tissue Exposure-Effect (Tissue PK-PD) Evaluation.

The "free drug hypothesis" assumes that, in the absence of transporters, the steady state free plasma concentrations equal to that at the site of action that elicit pharmacologic effects. While it is important to utilize the free drug hypothesis, exceptions exist that the free plasma exposures, either at Cmax, Ctrough, and Caverage, or at other time points, cannot represent the corresponding free tissue concentrations. This "drug concentration asymmetry" in both total and free form can influence drug disposition and pharmacological effects. In this review, we first discuss options to assess total and free drug concentrations in tissues. Then various drug design strategies to achieve concentration asymmetry are presented. Last, the utilities of tissue concentrations in understanding exposure-effect relationships and translational projections to humans are discussed for several therapeutic areas and modalities. A thorough understanding in plasma and tissue exposures correlation with pharmacologic effects can provide insightful guidance to aid drug discovery.

Application of empirical scalars to enable early prediction of human hepatic clearance using IVIVE in drug discovery: an evaluation of 173 drugs.

The utilization of in vitro data to predict drug pharmacokinetics (PK) in vivo has been a consistent practice in early drug discovery for decades. However, its success is hampered by mispredictions attributed to uncharacterized biological phenomena/experimental artifacts. Predicted drug clearance (CL) from experimental data (i.e. hepatocyte intrinsic clearance: CLint, fraction unbound in plasma: fu,p) is often systematically underpredicted using the well-stirred model (WSM). The objective of this study was to evaluate using empirical scalars in the WSM to correct for CL mispredictions. Drugs (N=28) were used to generate numerical scalars on CLint (α), and fu,p (ß) to minimize the error (AAFE) for CL predictions. These scalars were validated using an additional dataset (N=28 drugs) and applied to a non-redundant AstraZeneca (AZ) dataset available in the literature (N=117 drugs) for a total of 173 compounds. CL predictions using the WSM were improved for most compounds using an α value of 3.66 (~64%<2-fold) compared to no scaling (~46%<2-fold). Similarly, using a ß value of 0.55 or combination of α and ß scalars (values of 1.74 and 0.66, respectively) resulted in a similar improvement in predictions (~64%<2-fold and ~65%<2-fold, respectively). For highly bound compounds (fu,p{less than or equal to}0.01), AAFE was substantially reduced across all scaling methods. Using the ß scalar alone or a combination of α and ß appeared optimal; and produce larger magnitude corrections for highly-bound compounds. Some drugs are still disproportionally mispredicted, however the improvements in prediction error and simplicity of applying these scalars suggests its utility for early-stage CL predictions. Significance Statement In early drug discovery, prediction of human clearance using in vitro experimental data plays an essential role in triaging compounds prior to in vivo studies. These predictions have been systematically underestimated. Here we introduce empirical scalars calibrated on the extent of plasma protein binding that appear to improve clearance prediction across multiple datasets. This approach can be used in early phases of drug discovery prior to the availability of pre-clinical data for early quantitative predictions of human clearance.

Re-Examining the Impact of Minimal Scans in Liquid Chromatography-Mass Spectrometry Analysis.

Liquid chromatography-mass spectrometry (LC-MS) is one of the most widely used analytical tools. High analysis volumes and sample complexity often demand more informative LC-MS acquisition schemes to improve efficiency and throughput without compromising data quality, and such a demand has been always hindered by the prerequisite that a minimum of 13-20 MS scans (data points) across an analyte peak are required for accurate quantitation. The current study systematically re-evaluated and compared the impact of different scan numbers on quantitation analysis using both triple quadrupoles mass spectrometry (TQMS) and high-resolution mass spectrometry (HRMS). Contrary to the 13-20 minimal scan prerequisite, the data obtained from a group of eight commercial drugs in the absence and presence of biological matrices suggest that 6 scans per analyte peak are sufficient to achieve highly comparable quantitation results compared to that obtained using 10 and 20 scans, respectively. The fewer minimal scan prerequisite is presumably attributed to an improved LC system and advanced column technology, better MS detector, and more intelligent peak detection and integration algorithms leading to a more symmetric peak shape and smaller peak standard deviation. As a result, more informative acquisition schemes can be broadly set up for higher throughput and more data-rich LC-MS/MS analysis as demonstrated in a hepatocyte clearance assay in which fewer MS scans executed on HRMS led to broader metabolite coverage without compromising data quality in hepatic clearance assessment. The demonstrated acquisition scheme would substantially increase the throughput, robustness, and richness of the nonregulatory analysis, which can be broadly applied in diverse fields including pharmaceutical, environmental, forensic, toxicological, and biotechnological.

Comparative assessment for rat strain differences in metabolic profiles of 14 drugs in Wistar Han and Sprague Dawley hepatocytes.

Knowledge of inter-strain and inter-gender differences in drug metabolism studies is important for animal selection in pharmacokinetic and toxicological studies. The effects of rat strain and gender in in vitro metabolism were investigated in Sprague Dawley (SD) and Wister Han (WH) rats based on the hepatocyte metabolic profiles of 14 small molecule drugs. Similarities were found between the hepatocyte metabolic clearances of SD and WH strains, suggesting that only one strain can be confidently used for the evaluation of hepatic clearance. Neither strain of rat was preferable over the other to cover human metabolites. Higher similarities in metabolic pathways were found between the same gender than the same strain. Differences in metabolite identities, metabolite formation rates and potential biotransformation pathways were observed between SD and WH rat strains. Eleven metabolites from six drugs were "disproportionally" formed between SD and WH rats. The use of a specific rat strain model and gender for ADME and toxicity testing should, therefore, be carefully considered as metabolic profiles may differ, even though metabolic clearance was similar between SD and WH rats.

Inter-individual and inter-regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donors.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12-inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non-P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway-selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12-inch segments of the proximal small intestine shows that the segments immediately after the first 12-inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.

Strategy for Determining the Free Fraction of Labile Covalent Modulators in Plasma Using Equilibrium Dialysis.

Determination of free drug fraction (fu) in plasma can be challenging for labile covalent modulators due to the off-target reactivity of chemical warheads to matrix proteins. The resulting poor drug recovery yields low confidence in fu. Two approaches using diluted plasma and low temperature (4 & 20 °C) for equilibrium dialysis (ED) have been investigated using covalent modulators including osimertinib, ibrutinib, rociletinib, afatinib, neratinib and acalabrutinib. Our data indicate that stability of covalent modulators in plasma varies in different species, and drug depletion may lead to overestimation of fu if true equilibrium is not reached. Additionally, although ED at low temperature improves the recovery of covalent modulators, the impact of low temperature may lead to underestimate of fu. Overall, ED using diluted plasma is a preferred method because of its faster equilibrium, improved recovery and free of temperature effect on fu. If low temperature ED must be used for extremely labile compounds, precaution must be taken to ensure no temperature dependence of fu in plasma. Nevertheless, an orthogonal ED approach is recommended for labile covalent modulators to confirm the true equilibrium and impact of temperature on fu. Additionally, this strategy can be used for determining fu of other liable compounds.

NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD: Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition.

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.

Improving Confidence in the Determination of Free Fraction for Highly Bound Drugs Using Bidirectional Equilibrium Dialysis.

Equilibrium dialysis has been widely used for the measurement of the fraction of unbound drug (fu) in plasma, but it suffers from the accuracy and reliability for low fu values. To address this concern, an orthogonal approach, called the bidirectional equilibrium dialysis, is described to simultaneously measure a pair of fu values for each drug based on equilibration in 2 opposite dialysis directions: from plasma to buffer (fu,p/b) and from buffer to plasma (fu,b/p). Hypothetically, if true equilibrium is attained in both dialysis directions, the measured fu,b/p and fu,p/b values for a given drug should converge, and thus, the ratio of fu,b/p to fu,p/b becomes unity (1.0). Thus, the ratio can be used as a tangible readout for data reliability. This methodology has been extensively tested in the present study using various drugs with distinct plasma binding characteristics. Our results clearly showed that low fu values (<0.01) could be reliably determined and verified using either the standard or dilution bidirectional equilibrium dialysis method for some known highly bound drugs; for extensively bound drugs with high logD7.4, such as montelukast, bedaquiline, and venetoclax, only a range of fu can be reported with confidence because of uncertainty in the true equilibrium.

Development of a mass spectrometry-based tryptophan 2, 3-dioxygenase assay using liver cytosol from multiple species.

A novel and rapid method to determine the potency of inhibitors for tryptophan 2, 3-dioxygenase (TDO2) activities in human and preclinical species was successfully developed and validated utilizing LC-MS/MS. Previously reported TDO2 activity assays are resource intensive, requiring cloning and overexpression of TDO2. Here, we demonstrated that liver cytosol contained sufficient active TDO2 for evaluating the potency of TDO2 inhibitors across multiple species. TDO2 expression in human cytosol was estimated by LC-MS/MS to be 41 pmoL/mg cytosolic protein, with similar levels in dogs and monkeys, whereas mice and rats had 9.6 and 5.0-fold greater expression, respectively. Reaction conditions for TDO2-mediated conversion of l-tryptophan to kynurenine were optimized. Marked differences in kinetic parameters and inhibition potency were observed in TDO2 across species, with different Km values in dog (0.055 mM), monkey (0.070 mM), human (0.19 mM), mouse (0.32 mM) and rat (0.36 mM). Subsequently, IC50 values were determined for a series of TDO2 inhibitors in liver cytosol of five species, and good agreement with the literature values was observed for human enzyme. Taken together, these data indicate that TDO2 inhibition can be rapidly determined in readily available hepatic cytosol to assess potential species differences in potency.

Utility of Pooled Cryopreserved Human Enterocytes as an In vitro Model for Assessing Intestinal Clearance and Drug-Drug Interactions.

BACKGROUND: A recent advancement in isolation and cryopreservation has resulted in commercially available primary human enterocytes that express various drug metabolizing enzymes (DMEs) and transporters. The main objective of this study was to further evaluate the utility of pooled cryopreserved enterocytes, specifically MetMax™ cryopreserved human enterocytes (In vitro ADMET Laboratories), as an in vitro model for assessing intestinal clearance in comparison to hepatocytes. METHODS: It was found that, for CYP3A4/5 substrates such as midazolam, amprenavir and loperamide, in vitro metabolic clearance is generally lower in enterocytes compared to that of hepatocytes, which is consistent with the relative abundance of the enzyme between the intestine and liver. In contrast, raloxifene, a surrogate UGT activity substrate, showed 3-fold greater turnover in enterocytes than hepatocytes, which is likely attributed to the differential expression of individual UGTs in human liver and intestine. For procaine, a known CES2 substrate, the measured apparent clearance was higher in hepatocytes, but formation of 4-aminobenzoic acid, a CE2-specific metabolite, was more pronounced in enterocytes, suggesting that CE2 is more active in enterocytes. Salbutamol, a SULT1A3 substrate, showed little turnover in both enterocytes and hepatocytes, and more abundant sulfate conjugate was detected in enterocytes, indicating higher SULT activity in enterocytes than hepatocytes. As expected, ketoconazole inhibited CYP3A4/5-mediated metabolite formation in enterocytes for midazolam, amprenavir and loperamide, suggesting that cryopreserved enterocytes may be useful in determining intestinal CYP3A inhibition parameters. Interestingly, elacridar, a P-gp inhibitor, suppressed metabolite formation in enterocytes for loperamide, a substrate of CYP3A4 and P-gp, suggesting that enterocytes in suspension do not have active P-gp efflux functions, and the suppression of metabolism in enterocytes is probably caused by inhibition of CYP3A4/5 by elacridar. RESULTS: Our results suggest that pooled cryopreserved human enterocytes, specifically the MetMax™ cryopreserved human enterocytes, represent a valuable in vitro model for assessing first-pass clearance and potential drug interactions in human intestine.

Industry Perspective on Contemporary Protein-Binding Methodologies: Considerations for Regulatory Drug-Drug Interaction and Related Guidelines on Highly Bound Drugs.

Regulatory agencies have recently issued drug-drug interaction guidelines, which require determination of plasma protein binding (PPB). To err on the conservative side, the agencies recommend that a 0.01 lower limit of fraction unbound (fu) be used for highly bound compounds (>99%), irrespective of the actual measured values. While this may avoid false negatives, the recommendation would likely result in a high rate of false positive predictions, resulting in unnecessary clinical studies and more stringent inclusion/exclusion criteria, which may add cost and time in delivery of new medicines to patients. In this perspective, we provide a review of current approaches to measure PPB, and important determinants in enabling the accuracy and precision in these measurements. The ability to measure fu is further illustrated by a cross-company data comparison of PPB for warfarin and itraconazole, demonstrating good concordance of the measured fu values. The data indicate that fu values of ≤0.01 may be determined accurately across laboratories when appropriate methods are used. These data, along with numerous other examples presented in the literature, support the use of experimentally measured fu values for drug-drug interaction predictions, rather than using the arbitrary cutoff value of 0.01 as recommended in current regulatory guidelines.

Ester Prodrugs of IHVR-19029 with Enhanced Oral Exposure and Prevention of Gastrointestinal Glucosidase Interaction.

IHVR-19029 (6) is a lead endoplasmic reticulum α-glucosidases I and II inhibitor, which efficiently protected mice from lethal Ebola and Marburg virus infections via injection route, but suffered from low bioavailability and off-target interactions with gut glucosidases when administered orally. In an effort to improve efficacious exposure levels and avoid side effects, we designed and synthesized ester prodrugs. Not only were the prodrugs stable in simulated gastric and intestinal fluids and were inactive against glucosidases but they also exhibited antiviral activities against dengue virus infection in a cell based assay. Further in vitro evaluation showed that the bioconversion of the prodrugs is species dependent: in mice, the prodrugs were converted to 6 in the plasma and liver; while in human, the conversion occurred mainly in liver. An in vivo pharmacokinetic study in mice demonstrated that the tetrabutyrate prodrug 8 achieved the most improved overall exposure of 6 upon both oral and intravenous administration.

Highly predictive and interpretable models for PAMPA permeability.

Cell membrane permeability is an important determinant for oral absorption and bioavailability of a drug molecule. An in silico model predicting drug permeability is described, which is built based on a large permeability dataset of 7488 compound entries or 5435 structurally unique molecules measured by the same lab using parallel artificial membrane permeability assay (PAMPA). On the basis of customized molecular descriptors, the support vector regression (SVR) model trained with 4071 compounds with quantitative data is able to predict the remaining 1364 compounds with the qualitative data with an area under the curve of receiver operating characteristic (AUC-ROC) of 0.90. The support vector classification (SVC) model trained with half of the whole dataset comprised of both the quantitative and the qualitative data produced accurate predictions to the remaining data with the AUC-ROC of 0.88. The results suggest that the developed SVR model is highly predictive and provides medicinal chemists a useful in silico tool to facilitate design and synthesis of novel compounds with optimal drug-like properties, and thus accelerate the lead optimization in drug discovery.

Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis.

PURPOSE: To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). METHODS: Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. RESULTS: In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). CONCLUSIONS: In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.