Low molecular weight acids and OATP1B mediated hepatic clearance: In vitro and in vivo evaluation using novel hypoxia-inducible factor prolyl hydroxylase inhibitors (Dustats).

Organic anion transporting polypeptide (OATP1B) plays a key role in the hepatic clearance of a majority of high molecular weight (MW) acids and zwitterions. Here, we evaluated the role of OATP1B-mediated uptake in the clearance of novel hypoxia-inducible factor prolyl hydroxylase inhibitors ("Dustats"), which are typically low MW (300-400 daltons) aliphatic carboxylic acids. Five acid dustats, namely daprodustat, desidustat, enarodustat, roxadustat and vadadustat, showed specific transport by OATP1B1/1B3 in transporter-transfected HEK293 cells. Neutral compound, molidustat, was not a substrate to OATP1B1/1B3. None of the dustats showed transport by other hepatic uptake transporters, including NTCP, OAT2 and OAT7. In the primary human hepatocytes, uptake of all acids was significantly reduced by rifampin (OATP1B inhibitor); with an estimated fraction transported by OATP1B (ft ,OATP1B) of up to >80% (daprodustat). Molidustat uptake was minimally inhibited by rifampin; and low permeability acids (desidustat and enarodustat) also showed biliary efflux in sandwich culture human hepatocytes. In vivo, intravenous pharmacokinetics of all 5 acids was significantly altered by a single-dose rifampin (30 mg/kg) in Cynomolgus monkey. Hepatic clearance (non-renal) was about 4-fold (vadadustat) to >11-fod (daprodustat and roxadustat) higher in control group compared to rifampin-treated subjects. In vivo ft ,OATP1B was estimated to be ~70-90%. In the case of molidustat, rifampin had a minimal effect on overall clearance. Rifampin also considerably reduced volume of distribution of daprodustat and roxadustat. Overall, OATP1B significantly contribute to the hepatic clearance and pharmacokinetics of several dustats, which are low MW carboxylic acids. OATP1B activity should therefore by evaluated in this property space. Significance Statement Our in vitro and in vivo results suggest that OATP1B-mediated hepatic uptake play a significant role in the pharmacokinetics of low MW acidic dustats, which are being developed or approved for the treatment of anemia in chronic kidney disease. Significant active uptake mechanisms are not apparent for the neutral compound, molidustat. Characterization of uptake mechanisms is therefore important in predicting human pharmacokinetics and evaluating drug-drug interactions for low MW acids.

The Comparison of Machine Learning and Mechanistic In Vitro-In Vivo Extrapolation Models for the Prediction of Human Intrinsic Clearance.

Accurate prediction of human pharmacokinetics (PK) remains one of the key objectives of drug metabolism and PK (DMPK) scientists in drug discovery projects. This is typically performed by using in vitro-in vivo extrapolation (IVIVE) based on mechanistic PK models. In recent years, machine learning (ML), with its ability to harness patterns from previous outcomes to predict future events, has gained increased popularity in application to absorption, distribution, metabolism, and excretion (ADME) sciences. This study compares the performance of various ML and mechanistic models for the prediction of human IV clearance for a large (645) set of diverse compounds with literature human IV PK data, as well as measured relevant in vitro end points. ML models were built using multiple approaches for the descriptors: (1) calculated physical properties and structural descriptors based on chemical structure alone (classical QSAR/QSPR); (2) in vitro measured inputs only with no structure-based descriptors (ML IVIVE); and (3) in silico ML IVIVE using in silico model predictions for the in vitro inputs. For the mechanistic models, well-stirred and parallel-tube liver models were considered with and without the use of empirical scaling factors and with and without renal clearance. The best ML model for the prediction of in vivo human intrinsic clearance (CLint) was an in vitro ML IVIVE model using only six in vitro inputs with an average absolute fold error (AAFE) of 2.5. The best mechanistic model used the parallel-tube liver model, with empirical scaling factors resulting in an AAFE of 2.8. The corresponding mechanistic model with full in silico inputs achieved an AAFE of 3.3. These relative performances of the models were confirmed with the prediction of 16 Pfizer drug candidates that were not part of the original data set. Results show that ML IVIVE models are comparable to or superior to their best mechanistic counterparts. We also show that ML IVIVE models can be used to derive insights into factors for the improvement of mechanistic PK prediction.

Transporter-Enzyme Interplay in the Pharmacokinetics of PF-06835919, A First-in-class Ketohexokinase Inhibitor for Metabolic Disorders and Non-alcoholic Fatty Liver Disease.

Excess dietary fructose consumption promotes metabolic dysfunction thereby increasing the risk of obesity, type 2 diabetes, non-alcoholic steatohepatitis (NASH), and related comorbidities. PF-06835919, a first-in-class ketohexokinase (KHK) inhibitor, showed reversal of such metabolic disorders in preclinical models and clinical studies, and is under clinical development for the potential treatment of NASH. In this study, we evaluated the transport and metabolic pathways of PF-06835919 disposition and assessed pharmacokinetics in preclinical models. PF-06835919 showed active uptake in cultured primary human hepatocytes, and substrate activity to organic anion transporter (OAT)2 and organic anion transporting-polypeptide (OATP)1B1 in transfected cells. "SLC-phenotyping" studies in human hepatocytes suggested contribution of passive uptake, OAT2- and OATP1B-mediated transport to the overall uptake to be about 15%, 60% and 25%, respectively. PF-06835919 showed low intrinsic metabolic clearance in vitro, and was found to be metabolized via both oxidative pathways (58%) and acyl glucuronidation (42%) by CYP3A, CYP2C8, CYP2C9 and UGT2B7. Following intravenous dosing, PF-06835919 showed low clearance (0.4-1.3 mL/min/kg) and volume of distribution (0.17-0.38 L/kg) in rat, dog and monkey. Human oral pharmacokinetics are predicted within 20% error when considering transporter-enzyme interplay in a PBPK model. Finally, unbound liver-to-plasma ratio (Kpuu) measured in vitro using rat, NHP and human hepatocytes was found to be approximately 4, 25 and 10, respectively. Similarly, liver Kpuu in rat and monkey following intravenous dosing of PF-06835919 was found to be 2.5 and 15, respectively, and notably higher than the muscle and brain Kpuu, consistent with the active uptake mechanisms observed in vitro. Significance Statement This work characterizes the transport/metabolic pathways in the hepatic disposition of PF-06835919, a first-in-class KHK inhibitor for the treatment of metabolic disorders and NASH. Phenotyping studies using transfected systems, human hepatocytes and liver microsomes signifies the role of OAT2 and OATP1B1 in the hepatic uptake and multiple enzymes in the metabolism of PF-06835919. Data presented suggest hepatic transporter-enzyme interplay in determining its systemic concentrations and potential enrichment in liver, a target site for KHK inhibition.

In Vitro - in Vivo Extrapolation of Hepatic Clearance in Preclinical Species.

Accurate prediction of human clearance is of critical importance in drug discovery. In this study, in vitro - in vivo extrapolation (IVIVE) of hepatic clearance was established using large sets of compounds for four preclinical species (mouse, rat, dog, and non-human primate) to enable better understanding of clearance mechanisms and human translation. In vitro intrinsic clearances were obtained using pooled liver microsomes (LMs) or hepatocytes (HEPs) and scaled to hepatic clearance using the parallel-tube and well-stirred models. Subsequently, IVIVE scaling factors (SFs) were derived to best predict in vivo clearance. The SFs for extended clearance classification system (ECCS) class 2/4 compounds, involving metabolic clearance, were generally small (≤ 2.6) using both LMs and HEPs with parallel-tube model, with the exception of the rodents (~ 2.4-4.6), suggesting in vitro reagents represent in vivo reasonably well. SFs for ECCS class 1A and 1B are generally higher than class 2/4 across the species, likely due to the contribution of transporter-mediated clearance that is under-represented with in vitro reagents. The parallel-tube model offered lower variability in clearance predictions over the well-stirred model. For compounds that likely demonstrate passive permeability-limited clearance in vitro, rat LM predicted in vivo clearance more accurately than HEP. This comprehensive analysis demonstrated reliable IVIVE can be achieved using LMs and HEPs. Evaluation of clearance IVIVE in preclinical species helps to better understand clearance mechanisms, establish more reliable IVIVE in human, and enhance our confidence in human clearance and PK prediction, while considering species differences in drug metabolizing enzymes and transporters.

Organic Anion-Transporting Polypeptide 1B1/1B3-Mediated Hepatic Uptake Determines the Pharmacokinetics of Large Lipophilic Acids: In Vitro-In Vivo Evaluation in Cynomolgus Monkey.

It is generally presumed that uptake transport mechanisms are of limited significance in hepatic clearance for lipophilic or high passive-permeability drugs. In this study, we evaluated the mechanistic role of the hepato-selective organic anion-transporting polypeptides (OATPs) 1B1/1B3 in the pharmacokinetics of compounds representing large lipophilic acid space. Intravenous pharmacokinetics of 16 compounds with molecular mass ∼400-730 Da, logP ∼3.5-8, and acid pKa <6 were obtained in cynomolgus monkey after dosing without and with a single-dose rifampicin-OATP1B1/1B3 probe inhibitor. Rifampicin (30 mg/kg oral) significantly (P < 0.05) reduced monkey clearance and/or steady-state volume of distribution (VDss) for 15 of 16 acids evaluated. Additionally, clearance of danoprevir was reduced by about 35%, although statistical significance was not reached. A significant linear relationship was noted between the clearance ratio (i.e., ratio of control to treatment groups) and VDss ratio, suggesting hepatic uptake contributes to the systemic clearance and distribution simultaneously. In vitro transport studies using primary monkey and human hepatocytes showed uptake inhibition by rifampicin (100 µM) for compounds with logP ≤6.5 but not for the very lipophilic acids (logP > 6.5), which generally showed high nonspecific binding in hepatocyte incubations. In vitro uptake clearance and fraction transported by OATP1B1/1B3 (ft,OATP1B) were found to be similar in monkey and human hepatocytes. Finally, for compounds with logP ≤6.5, good agreement was noted between in vitro ft,OATP1B and clearance ratio (as well as VDss ratio) in cynomolgus monkey. In conclusion, this study provides mechanistic evidence for the pivotal role of OATP1B-mediated hepatic uptake in the pharmacokinetics across a wide, large lipophilic acid space. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into the pharmacokinetics of a broad range of large lipophilic acids. Organic anion-transporting polypeptides 1B1/1B3-mediated hepatic uptake is of key importance in the pharmacokinetics and drug-drug interactions of almost all drugs and new molecular entities in this space. Diligent in vitro and in vivo transport characterization is needed to avoid the false negatives often noted because of general limitations in the in vitro assays while handling compounds with such physicochemical attributes.

Effect of Human Plasma on Hepatic Uptake of Organic Anion-Transporting Polypeptide 1B Substrates: Studies Using Transfected Cells and Primary Human Hepatocytes.

Current challenges with the in vitro-in vivo extrapolation (IVIVE) of hepatic uptake clearance involving organic anion-transporting polypeptide (OATP) 1B1/1B3 hinder drug design strategies. Here we evaluated the effect of 100% human plasma on the uptake clearance using transfected human embryonic kidney (HEK) 293 cells and primary human hepatocytes and assessed IVIVE. Apparent unbound uptake clearance (PSinf,u) increased significantly (P < 0.05) in the presence of plasma (vs. buffer incubations) for about 50% of compounds in both OATP1B1-transfected and wild-type HEK cells. Thus, plasma showed a minimal effect on the uptake ratios. With cultured human hepatocytes, plasma significantly (P < 0.05) increased PSinf,u for 11 of 19 OATP1B substrates (median change of 2.1-fold). Cell accumulation in HEK cells and hepatocytes was also increased for tolbutamide, which is not an OATP substrate. Plasma-to-buffer ratio of PSinf,u obtained in hepatocytes showed a good correlation with unbound fraction in plasma, and the relationship was best described by a "facilitated-dissociation" model. IVIVE was evaluated for the 19 OATP1B substrates using hepatocyte data in the presence of buffer and plasma. PSinf,u from buffer incubations markedly underpredicted hepatic intrinsic clearance (calculated via well stirred and parallel tube models) with an estimated bias of 0.10-0.13. Predictions improved when using PSinf,u from plasma incubations; however, considerable systemic underprediction was still apparent (0.19-0.26 bias). Plasma data with a global scaling factor of 3.8-5.3 showed good prediction accuracy (95% predictions within 3-fold; average fold error = 1.7, bias = 1). In summary, this study offers insight into the effect of plasma on the uptake clearance and its scope in improving IVIVE. SIGNIFICANCE STATEMENT: Our study using diverse anionic compounds shows that human plasma facilitates organic anion-transporting polypeptide 1B-mediated as well as passive uptake clearance, particularly for the highly bound compounds. Leveraging data from transfected human embryonic kidney 293 cells and primary human hepatocytes, we further evaluated mechanisms involved in the observed plasma-facilitated uptake transport. Enhanced hepatic uptake rate in the presence of plasma could be of relevance, as such mechanisms likely prevail in vivo and emphasize the need to maintain physiologically relevant assay conditions to achieve improved translation of transport data.

Static and Dynamic Projections of Drug-Drug Interactions Caused by Cytochrome P450 3A Time-Dependent Inhibitors Measured in Human Liver Microsomes and Hepatocytes.

Cytochrome P450 3A (CYP3A) is a frequent target for time-dependent inhibition (TDI) that can give rise to drug-drug interactions (DDI). Yet many drugs that exhibit in vitro TDI for CYP3A do not result in DDI. There were 23 drugs with published clinical DDI evaluated for CYP3A TDI in human liver microsomes (HLM) and hepatocytes (HHEP), and these data were used in static and dynamic models for projecting DDI caused by inactivation of CYP3A in both liver and intestine. TDI parameters measured in HHEP, particularly the maximal rate of enzyme inactivation, were generally lower than those measured in HLM. In static models, the use of estimated average unbound organ exit concentrations offered the most accurate projections of DDI with geometric mean fold errors of 2.0 and 1.7 for HLM and HHEP, respectively. Use of maximum organ entry concentrations yielded marked overestimates of DDI. When evaluated in a binary fashion (i.e., projection of DDI of 1.25-fold or greater), data from HLM offered the greatest sensitivity (100%) and specificity (67%) and yielded no missed DDI when average unbound organ exit concentrations were used. In dynamic physiologically based pharmacokinetic modeling, accurate projections of DDI were obtained with geometric mean fold errors of 1.7 and 1.6 for HLM and HHEP, respectively. Sensitivity and specificity were 100% and 67% when using TDI data generated in HLM and Simcyp modeling. Overall, DDI caused by CYP3A-mediated TDI can be reliably projected using dynamic or static models. For static models, average organ unbound exit concentrations should be used as input values otherwise DDI will be markedly overestimated. SIGNIFICANCE STATEMENT: CYP3A time-dependent inhibitors (TDI) are important in the design and development of new drugs. The prevalence of CYP3A TDI is high among newly synthesized drug candidates, and understanding the potential need for running clinical drug-drug interaction (DDI) studies is essential during drug development. Ability to reliably predict DDI caused by CYP3A TDI has been difficult to achieve. We report a thorough evaluation of CYP3A TDI and demonstrate that DDI can be predicted when using appropriate models and input parameters generated in human liver microsomes or hepatocytes.

Quantitative Contribution of Six Major Transporters to the Hepatic Uptake of Drugs: "SLC-Phenotyping" Using Primary Human Hepatocytes.

Hepatic uptake transporters [solute carriers (SLCs)], including organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, sodium-dependent taurocholate cotransporting polypeptide (NTCP), and organic anion (OAT2) and organic cation (OCT1) transporters, play a key role in determining the systemic and liver exposure of chemically diverse drugs. Here, we established a phenotyping approach to quantify the contribution of the six SLCs, and passive diffusion, to the overall uptake using plated human hepatocytes (PHHs). First, selective inhibitor conditions were identified by screening about 20 inhibitors across the six SLCs using single-transfected human embryonic kidney 293 cells. Data implied rifamycin SV (20 µM) inhibits three OATPs, while rifampicin (5 µM) inhibits OATP1B1/1B3 only. Further, hepatitis B virus myristoylated-preS1 peptide (0.1 µM), quinidine (100 µM), and ketoprofen (100-300 µM) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported (ft ) by the individual SLCs was characterized for 20 substrates with PHH. Generally, extended clearance classification system class 1A/3A (e.g., warfarin) and 1B/3B compounds (e.g., statins) showed predominant OAT2 and OATP1B1/1B3 contribution, respectively. OCT1-mediated uptake was prominent for class 2/4 compounds (e.g., metformin). Third, in vitro ft values were corrected using quantitative proteomics data to obtain "scaled ft " Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/1B3 ft was assessed, leveraging statin clinical drug-drug interaction data with rifampicin as the perpetrator. Finally, we outlined a novel stepwise strategy to implement phenotypic characterization of SLC-mediated hepatic uptake for new molecular entities and drugs in a drug discovery and development setting.

A Novel Unified Approach to Predict Human Hepatic Clearance for Both Enzyme- and Transporter-Mediated Mechanisms Using Suspended Human Hepatocytes.

The accurate prediction of human pharmacokinetics is critically important in modern drug discovery since it drives both pharmacological and toxicological effects. Although significant progress has been made in predicting drug disposition by hepatic drug-metabolizing enzymes, predicting transporter-mediated clearance is still highly uncertain. Furthermore, different approaches are often used to predict clearance with and without transporter involvement, hence the major clearance pathway for a compound must first be determined to know which approach to use. As a result of these challenges, a novel unified method has been developed using cryopreserved suspended human hepatocytes to predict human hepatic clearance for both enzyme- and transporter-mediated mechanisms. This method hypothesizes that, once in vitro metabolic stability is scaled by partition coefficients between hepatocytes and buffer with 4% bovine serum albumin, in vivo clearance can be better predicted. With this method, good in vitro-in vivo correlation of human hepatic clearance has been obtained for a set of 32 structurally diverse compounds, including such transporters as organic anion-transporting polypeptide substrates. The clearance predictions for most compounds are within 3-fold of observed values. This is the first time that multiple compounds result in good in vitro-in vivo extrapolation using an entirely "bottom-up" approach without any empirical scaling factor when transporter-mediated clearance is involved. Potential exceptions are compounds with significant biliary and/or extra-hepatic clearance. The method offers an alternative approach to more accurately predict human hepatic clearance when multiple complex mechanisms are involved.

Organic Anion Transporter 2-Mediated Hepatic Uptake Contributes to the Clearance of High-Permeability-Low-Molecular-Weight Acid and Zwitterion Drugs: Evaluation Using 25 Drugs.

High-permeability-low-molecular-weight acids/zwitterions [i.e., extended clearance classification system class 1A (ECCS 1A) drugs] are considered to be cleared by metabolism with a minimal role of membrane transporters in their hepatic clearance. However, a marked disconnect in the in vitro-in vivo (IVIV) translation of hepatic clearance is often noted for these drugs. Metabolic rates measured using human liver microsomes and primary hepatocytes tend to underpredict. Here, we evaluated the role of organic anion transporter 2 (OAT2)-mediated hepatic uptake in the clearance of ECCS 1A drugs. For a set of 25 ECCS 1A drugs, in vitro transport activity was assessed using transporter-transfected cells and primary human hepatocytes. All but two drugs showed substrate affinity to OAT2, whereas four (bromfenac, entacapone, fluorescein, and nateglinide) also showed OATP1B1 activity in transfected cells. Most of these drugs (21 of 25) showed active uptake by plated human hepatocytes, with rifamycin SV (pan-transporter inhibitor) reducing the uptake by about 25%-95%. Metabolic turnover was estimated for 19 drugs after a few showed no measurable substrate depletion in liver microsomal incubations. IVIV extrapolation using in vitro data was evaluated to project human hepatic clearance of OAT2-alone substrates considering 1) uptake transport only, 2) metabolism only, and 3) transporter-enzyme interplay (extended clearance model). The transporter-enzyme interplay approach achieved improved prediction accuracy (average fold error = 1.9 and bias = 0.93) compared with the other two approaches. In conclusion, this study provides functional evidence for the role of OAT2-mediated hepatic uptake in determining the pharmacokinetics of several clinically important ECCS 1A drugs.

A Study on Pharmacokinetics of Bosentan with Systems Modeling, Part 2: Prospectively Predicting Systemic and Liver Exposure in Healthy Subjects.

Predicting human pharmacokinetics of novel compounds is a critical step in drug discovery and clinical study design but continues to be a challenging task for hepatic transporter substrates, particularly in predicting their liver exposures. In this study, using bosentan as an example, we prospectively predicted systemic exposure and the (pseudo) steady-state unbound liver-to-unbound plasma ratio (Kpuu) in healthy subjects using 1) a mechanistic approach solely based on in vitro hepatocyte assays and 2) an approach based on hepatic process rates from monkey in vivo data but Michaelis-Menten constants from in vitro data. Both methods reasonably match the observed human systemic time course data, but the second method leads to better prediction accuracy. In addition, the second method can predict a human Kpuu value that is close to the value deduced using clinical data. We also generated rat and monkey liver Kpuu values in terminal studies. However, these directly measured animal values are different from the deduced human value.

Comparison of Species and Cell-Type Differences in Fraction Unbound of Liver Tissues, Hepatocytes, and Cell Lines.

Fraction unbound (fu) of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. fu,liver and fu,cell are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for fu,liver and fu,cell of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as fu,cell of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat fu,liver Therefore, we recommend using rat fu,liver as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of fu for liver tissues, hepatocytes, and other cell types. In addition, fu under hepatocyte stability incubation conditions should not be confused with fu,cell, as one is a diluted fu and the other is an undiluted fu Cell density also plays a critical role in the accurate measurement of fu,cell.

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.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Quantitative Prediction of Human Renal Clearance and Drug-Drug Interactions of Organic Anion Transporter Substrates Using In Vitro Transport Data: A Relative Activity Factor Approach.

Organic anion transporters (OATs) are important in the renal secretion, and thus, the clearance, of many drugs; and their functional change can result in pharmacokinetic variability. In this study, we applied transport rates measured in vitro using OAT-transfected human embryonic kidney cells to predict human renal secretory and total renal clearance of 31 diverse drugs. Selective substrates to OAT1 (tenofovir), OAT2 (acyclovir and ganciclovir), and OAT3 (benzylpenicillin, oseltamivir acid) were used to obtain relative activity factors (RAFs) for these individual transporters by relating in vitro transport clearance (after physiologic scaling) to in vivo secretory clearance. Using the estimated RAFs (0.64, 7.3, and 4.1, respectively, for OAT1, OAT2, and OAT3, respectively) and the in vitro active clearances, renal secretory clearance and total renal clearance were predicted with average fold errors (AFEs) of 1.89 and 1.40, respectively. The results show that OAT3-mediated transport play a predominant role in renal secretion for 22 of the 31 drugs evaluated. This mechanistic static approach was further applied to quantitatively predict renal drug-drug interactions (AFE ∼1.6) of the substrate drugs with probenecid, a clinical probe OAT inhibitor. In conclusion, the proposed in vitro-in vivo extrapolation approach is the first comprehensive attempt toward mechanistic modeling of renal secretory clearance based on routinely employed in vitro cell models.

Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery.

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be different in vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similar enzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer be present if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

Study of the ketohexokinase inhibitor PF-06835919 as a clinical cytochrome P450 3A inducer: Integrated use of oral midazolam and liquid biopsy.

PF-06835919, a ketohexokinase inhibitor, presented as an inducer of cytochrome P450 3A4 (CYP3A4) in vitro (human primary hepatocytes), and static mechanistic modeling exercises predicted significant induction in vivo (oral midazolam area under the plasma concentration-time curve [AUC] ratio [AUCR] = 0.23-0.79). Therefore, a drug-drug interaction study was conducted to evaluate the effect of multiple doses of PF-06835919 (300 mg once daily × 10 days; N = 10 healthy participants) on the pharmacokinetics of a single oral midazolam 7.5 mg dose. The adjusted geometric means for midazolam AUC and its maximal plasma concentration were similar following co-administration with PF-06835919 (vs. midazolam administration alone), with ratios of the adjusted geometric means (90% confidence interval [CI]) of 97.6% (90% CI: 79.9%-119%) and 98.9% (90% CI: 76.4%-128%), respectively, suggesting there was minimal effect of PF-06835919 on midazolam pharmacokinetics. Lack of CYP3A4 induction was confirmed after the preparation of subject plasma-derived small extracellular vesicles (sEVs) and conducting proteomic and activity (midazolam 1'-hydroxylase) analysis. Consistent with the midazolam AUCR observed, the CYP3A4 protein expression fold-induction (geometric mean, 90% CI) was low in liver (0.9, 90% CI: 0.7-1.2) and non-liver (0.9, 90% CI: 0.7-1.2) sEVs (predicted AUCR = 1.0, 90% CI: 0.9-1.2). Likewise, minimal induction of CYP3A4 activity (geometric mean, 90% CI) in both liver (1.1, 90% CI: 0.9-1.3) and non-liver (0.9, 90% CI: 0.5-1.5) sEVs was evident (predicted AUCR = 0.9, 90% CI: 0.6-1.4). The results showcase the integrated use of an oral CYP3A probe (midazolam) and plasma-derived sEVs to assess a drug candidate as inducer.

Relationship of binding-site occupancy, transthyretin stabilisation and disease modification in patients with tafamidis-treated transthyretin amyloid cardiomyopathy.

BACKGROUND: Tafamidis inhibits progression of transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) by binding TTR tetramer and inhibiting dissociation to monomers capable of denaturation and deposition in cardiac tissue. While the phase 3 ATTR-ACT trial demonstrated the efficacy of tafamidis, the degree to which the approved dose captures the full potential of the mechanism has yet to be assessed. METHODS: We developed a model of dynamic TTR concentrations in plasma to relate TTR occupancy by tafamidis to TTR stabilisation. We then developed population pharmacokinetic-pharmacodynamic models to characterise the relationship between stabilisation and measures of disease progression. RESULTS: Modelling individual patient data of tafamidis exposure and increased plasma TTR confirmed that single-site binding provides complete tetramer stabilisation in vivo. The approved dose was estimated to reduce unbound TTR tetramer by 92%, and was associated with 53%, 56% and 49% decreases in the rate of change in NT-proBNP, KCCQ-OS, and six-minute walk test disease progression measures, respectively. Simulating complete TTR stabilisation predicted slightly greater reductions of 58%, 61% and 54%, respectively. CONCLUSIONS: These findings support the value of TTR stabilisation as a clinically beneficial treatment option in ATTR-CM and the ability of tafamidis to realise nearly the full therapeutic benefit of this mechanism. CLINICALTRIALS.GOV IDENTIFIER: NCT01994889.

In Vitro-In Vivo Extrapolation and Scaling Factors for Clearance of Human and Preclinical Species with Liver Microsomes and Hepatocytes.

In vitro-in vivo extrapolation ((IVIVE) and empirical scaling factors (SF) of human intrinsic clearance (CLint) were developed using one of the largest dataset of 455 compounds with data from human liver microsomes (HLM) and human hepatocytes (HHEP). For extended clearance classification system (ECCS) class 2/4 compounds, linear SFs (SFlin) are approximately 1, suggesting enzyme activities in HLM and HHEP are similar to those in vivo under physiological conditions. For ECCS class 1A/1B compounds, a unified set of SFs was developed for CLint. These SFs contain both SFlin and an exponential SF (SFß) of fraction unbound in plasma (fu,p). The unified SFs for class 1A/1B eliminate the need to identify the transporters involved prior to clearance prediction. The underlying mechanisms of these SFs are not entirely clear at this point, but they serve practical purposes to reduce biases and increase prediction accuracy. Similar SFs have also been developed for preclinical species. For HLM-HHEP disconnect (HLM > HHEP) ECCS class 2/4 compounds that are mainly metabolized by cytochrome P450s/FMO, HLM significantly overpredicted in vivo CLint, while HHEP slightly underpredicted and geometric mean of HLM and HHEP slightly overpredicted in vivo CLint. This observation is different than in rats, where rat liver microsomal CLint correlates well with in vivo CLint for compounds demonstrating permeability-limited metabolism. The good CLint IVIVE developed using HLM and HHEP helps build confidence for prospective predictions of human clearance and supports the continued utilization of these assays to guide structure-activity relationships to improve metabolic stability.