Mechanistic account of distinct change in organic anion transporting polypeptide 1B (OATP1B) substrate pharmacokinetics during OATP1B-mediated drug-drug interactions using physiologically-based pharmacokinetic modeling.

The role of transporters in drug clearance is widely acknowledged, directly and indirectly by facilitating tissue/enzyme exposure. Through the latter, transporters also affect volume of distribution. Drug-drug interactions (DDIs) involving organic anion transporting polypeptides (OATPs) 1B1/1B3 and SLCO1B1 pharmacogenetics lead to altered pharmacokinetics of OATP1B substrates; however, several factors may confound direct interpretation of pharmacokinetic parameters from these clinical studies using noncompartmental analysis (NCA). A review of clinical data herein indicates a single dose of OATP1B inhibitor rifampin almost never leads to increased substrate half-life but often a decrease, and that most clinical OATP1B substrates are CYP3A4 substrates and/or undergo enterohepatic cycling (EHC). Using hypothetically simple OATP1B substrate physiologically-based pharmacokinetic (PBPK) models, simulated effect of rifampin differed from specific OATP1B inhibition, due to short rifampin half-life causing dissipation of OATP1B inhibition over time combined with CYP3A4 induction. Calculated using simulated tissue data, volume of distribution indeed decreased with OATP1B inhibition and was expectedly limited to the contribution of liver volume. However, an apparent and counterintuitive effect of rifampin on volume greater than that on clearance resulted for CYP3A4 substrates, using NCA. Effect of OATP1B inhibition and rifampin on OATP1B substrate models incorporating EHC +/- renal clearance was distinct compared to simpler models. Using PBPK models incorporating reversible lactone metabolism for clinical OATP1B substrates atorvastatin and pitavastatin, DDIs reporting decreased half-life with rifampin were reproduced. These simulations provide explanation for the distinct change in OATP1B substrate pharmacokinetics observed in clinical studies, including changes in volume of distribution and additional mechanisms. Significance Statement Transporters are involved in both drug clearance and volume of distribution and distinct changes in OATP1B substrate pharmacokinetics are observed with OATP1B inhibitor rifampin. Using hypothetical and validated PBPK models and simulations we address the limitations of single-dose rifampin and complicated clinical OATP1B substrate disposition in evaluating the pharmacokinetic parameters of OATP1B substrates during rifampin DDIs. These models account for the change in volume of distribution and identify additional mechanisms underlying apparent pharmacokinetic changes in OATP1B DDIs.

Utilization of OATP1B Biomarker Coproporphyrin-I to Guide Drug-Drug Interaction Risk Assessment: Evaluation by the Pharmaceutical Industry.

Drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptides 1B1/1B3 (OATP1B) can be substantial, however, challenges remain for predicting interaction risk. Emerging evidence suggests that endogenous biomarkers, particularly coproporphyrin-I (CP-I), can be used to assess in vivo OATP1B activity. The present work under the International Consortium for Innovation and Quality in Pharmaceutical Development was aimed primarily at assessing CP-I as a biomarker for informing OATP1B DDI risk. Literature and unpublished CP-I data along with pertinent in vitro and clinical DDI information were collected to identify DDIs primarily involving OATP1B inhibition and assess the relationship between OATP1B substrate drug and CP-I exposure changes. Static models to predict changes in exposure of CP-I, as a selective OATP1B substrate, were also evaluated. Significant correlations were observed between CP-I area under the curve ratio (AUCR) or maximum concentration ratio (Cmax R) and AUCR of substrate drugs. In general, the CP-I Cmax R was equal to or greater than the CP-I AUCR. CP-I Cmax R < 1.25 was associated with absence of OATP1B-mediated DDIs (AUCR < 1.25) with no false negative predictions. CP-I Cmax R < 2 was associated with weak OATP1B-mediated DDIs (AUCR < 2). A correlation was identified between CP-I exposure changes and OATP1B1 static DDI predictions. Recommendations for collecting and interpreting CP-I data are discussed, including a decision tree for guiding DDI risk assessment. In conclusion, measurement of CP-I is recommended to inform OATP1B inhibition potential. The current analysis identified changes in CP-I exposure that may be used to prioritize, delay, or replace clinical DDI studies.

Physiologically based pharmacokinetic modeling of tadalafil to inform pediatric dose selection in children with pulmonary arterial hypertension.

Tadalafil, a phosphodiesterase 5 inhibitor, is being investigated as a treatment for pulmonary arterial hypertension (PAH) in children aged 6 months to less than 18 years. Tadalafil pharmacokinetic (PK) data in children less than 2 years old are unavailable, therefore a physiologically based pharmacokinetic (PBPK) model was developed to enable estimation of tadalafil doses in children less than 2 years old. The model was verified in adults and extended for use in children by modifying CYP3A-mediated intrinsic clearance to include CYP3A7. To account for co-dosing of the commonly prescribed moderate CYP3A4 inducer bosentan, predicted exposures were increased by a factor of 1.54 based on changes in exposure in adults with PAH. This factor was predictable using a bosentan PBPK model. The tadalafil model was verified in children aged greater than or equal to 2 years by comparing predicted and observed exposures. Tadalafil doses for children less than 2 years old were calculated as target area under the concentration curve from zero to 24 h (AUC0-24 )/predicted AUC0-24 , with target AUC0-24 of 10,000 ng*h/ml based on adult 40 mg single dose exposures determined in patients without bosentan background treatment. These doses were 2 mg, 3 mg, 4 mg, and 6 mg, respectively, for children aged birth to less than 1 month, 1 month to less than 6 months, 6 months to less than 1 year, and 1 to less than 2 years. Due to uncertainties in CYP maturation, a nonmechanistic steady-state volume scalar, and lack of PK data in children less than 2 years old, accumulation of tadalafil to steady-state in children less than 2 years was not verifiable. Safety of proposed doses is supported by postmarketing research and investigator-led trials.

Comparison of Hepatic Transporter Tissue Expression in Rodents and Interspecies Hepatic OCT1 Activity.

Hepatic clearance may be uptake rate limited by organic anion transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1). While comparison of OATP activity has been investigated across species, little has been reported for OCT1. Additionally, while data on interspecies transporter expression in the liver exist, quantitative comparison of these transporters in multiple tissues is lacking. In the current research, the pharmacokinetics of OCT1 substrates (sumatriptan and metformin) were assessed in Oct knockout rats for comparison with previous Oct1/2-/- mice data and OCT1 pharmacogenetics in humans. Effect of OCT1 inhibitors verapamil and erlotinib on OCT1 substrate liver partitioning was also evaluated in rats. Expression of 18 transporters, including Oatps and Octs, in 9 tissues from mice and rats was quantitated using nanoLC/MS-MS, along with uptake transporters in hepatocytes from 5 species. Interspecies differences in OCT1 activity were further evaluated via uptake of OCT1 substrates in hepatocytes with corresponding in vivo liver partitioning in rodents and monkey. In Oct1-/- rats, sumatriptan hepatic clearance and liver partitioning decreased; however, metformin pharmacokinetics were unaffected. OCT1 inhibitor coadministration decreased sumatriptan liver partitioning. In rodents, Oatp expression was highest in the liver, although comparable expression of Oatps in other tissues was determined. Expression of Octs was highest in the kidney, with liver Oct1 expression comparably lower than Oatps. Liver partitioning of OCT1 substrates was lower in rodents than in monkey, in agreement with the highest OCT1 expression and uptake of OCT1 substrates in monkey hepatocytes. Species-dependent OCT1 activity requires consideration when translating preclinical data to the clinic.

Expansion of Knowledge on OCT1 Variant Activity In Vitro and In Vivo Using Oct1/2-/- Mice.

The role of organic cation transporter 1 (OCT1) in humans is gaining attention as data emerges regarding its role in physiology, drug exposure, and drug response. OCT1 variants with decreased in vitro function correlate well with altered exposure of multiple OCT1 substrates in variant carriers. In the current research, we investigate mechanisms behind activity of OCT1 variants in vitro by generating cell lines expressing known OCT1 variants and quantifying membrane OCT1 protein expression with corresponding OCT1 activity and kinetics. Oct knockout mice have provided additional insight into the role of Oct1 in the liver and have reproduced effects of altered OCT1 activity observed in the clinic. To assess the complex effect of Oct1 depletion on pharmacokinetics of prodrug proguanil and its active moiety cycloguanil, both of which are OCT1 substrates, Oct1/2-/- mice were used. Decreased membrane expression of OCT1 was demonstrated for all variant cell lines, although activity was substrate-dependent, as reported previously. Lack of change in activity for OCT1*2 resulted in increased intrinsic activity per pmol of OCT1 protein, particularly for sumatriptan but also for proguanil and cycloguanil. Similar to that reported in humans with decreased OCT1 function, systemic exposure of proguanil was minimally affected in Oct1/2-/- mice. However, proguanil liver partitioning and exposure decreased. Cycloguanil exposure decreased following proguanil administration in Oct1/2-/- mice, as did the systemic metabolite:parent ratio. When administered directly, systemic exposure of cycloguanil decreased slightly; however liver partitioning and exposure were decreased in Oct1/2-/- mice. Unexpectedly, following proguanil administration, the metabolite ratio in the liver changed only minimally, and liver partitioning of cycloguanil was affected in Oct1/2-/- mice to a lesser extent following proguanil administration than direct administration of cycloguanil. In conclusion, these in vitro and in vivo data offer additional complexity in understanding mechanisms of OCT1 variant activity as well as the effects of these variants in vivo. From cell lines, it is apparent that intrinsic activity is not directly related to OCT1 membrane expression. Additionally, in situations with a more complicated role of OCT1 in drug pharmacokinetics there is difficulty translating in vivo impact simply from intrinsic activity from cellular data.

γ-Hydroxybutyric Acid: Pharmacokinetics, Pharmacodynamics, and Toxicology.

Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid present endogenously in the brain and used therapeutically for the treatment of narcolepsy, as sodium oxybate, and for alcohol abuse/withdrawal. GHB is better known however as a drug of abuse and is commonly referred to as the "date-rape drug"; current use in popular culture includes recreational "chemsex," due to its properties of euphoria, loss of inhibition, amnesia, and drowsiness. Due to the steep concentration-effect curve for GHB, overdoses occur commonly and symptoms include sedation, respiratory depression, coma, and death. GHB binds to both GHB and GABAB receptors in the brain, with pharmacological/toxicological effects mainly due to GABAB agonist effects. The pharmacokinetics of GHB are complex and include nonlinear absorption, metabolism, tissue uptake, and renal elimination processes. GHB is a substrate for monocarboxylate transporters, including both sodium-dependent transporters (SMCT1, 2; SLC5A8; SLC5A12) and proton-dependent transporters (MCT1-4; SLC16A1, 7, 8, and 3), which represent significant determinants of absorption, renal reabsorption, and brain and tissue uptake. This review will provide current information of the pharmacology, therapeutic effects, and pharmacokinetics/pharmacodynamics of GHB, as well as therapeutic strategies for the treatment of overdoses. Graphical abstract.

Predicting Clinical Effects of CYP3A4 Modulators on Abemaciclib and Active Metabolites Exposure Using Physiologically Based Pharmacokinetic Modeling.

Abemaciclib, a selective inhibitor of cyclin-dependent kinases 4 and 6, is metabolized mainly by cytochrome P450 (CYP)3A4. Clinical studies were performed to assess the impact of strong inhibitor (clarithromycin) and inducer (rifampin) on the exposure of abemaciclib and active metabolites. A physiologically based pharmacokinetic (PBPK) model incorporating the metabolites was developed to predict the effect of other strong and moderate CYP3A4 inhibitors and inducers. Clarithromycin increased the area under the plasma concentration-time curve (AUC) of abemaciclib and potency-adjusted unbound active species 3.4-fold and 2.5-fold, respectively. Rifampin decreased corresponding exposures 95% and 77%, respectively. These changes influenced the fraction metabolized via CYP3A4 in the model. An absolute bioavailability study informed the hepatic and gastric availability. In vitro data and a human radiolabel study determined the fraction and rate of formation of the active metabolites as well as absorption-related parameters. The predicted AUC ratios of potency-adjusted unbound active species with rifampin and clarithromycin were within 0.7- and 1.25-fold of those observed. The PBPK model predicted 3.78- and 7.15-fold increases in the AUC of the potency-adjusted unbound active species with strong CYP3A4 inhibitors itraconazole and ketoconazole, respectively; and 1.62- and 2.37-fold increases with the concomitant use of moderate CYP3A4 inhibitors verapamil and diltiazem, respectively. The model predicted modafinil, bosentan, and efavirenz would decrease the AUC of the potency-adjusted unbound active species by 29%, 42%, and 52%, respectively. The current PBPK model, which considers changes in unbound potency-adjusted active species, can be used to inform dosing recommendations when abemaciclib is coadministered with CYP3A4 perpetrators.

Pharmacokinetics of Organic Cation Transporter 1 (OCT1) Substrates in Oct1/2 Knockout Mice and Species Difference in Hepatic OCT1-Mediated Uptake.

Organic cation transporter 1 (OCT1) plays a role in hepatic uptake of drugs, affecting in vivo exposure, distinguished primarily through pharmacogenetics of the SLC22A1 gene. The role of OCT1 in vivo has not been confirmed, however, via drug-drug interactions that similarly affect exposure. In the current research, we used Oct1/2 knockout mice to assess the role of Oct1 in hepatic clearance and liver partitioning of clinical substrates and assess the model for predicting an effect of OCT1 function on pharmacokinetics in humans. Four OCT1 substrates (sumatriptan, fenoterol, ondansetron, and tropisetron) were administered to wild-type and knockout mice, and plasma, tissue, and urine were collected. Tissue transporter expression was evaluated using liquid chromatography-mass spectrometry. In vitro, uptake of all compounds in human and mouse hepatocytes and human OCT1- and OCT2-expressing cells was evaluated. The largest effect of knockout was on hepatic clearance and liver partitioning of sumatriptan (2- to 5-fold change), followed by fenoterol, whereas minimal changes in the pharmacokinetics of ondansetron and tropisetron were observed. This aligned with uptake in mouse hepatocytes, in which inhibition of uptake of sumatriptan and fenoterol into mouse hepatocytes by an OCT1 inhibitor was much greater compared with ondansetron and tropisetron. Conversely, inhibition of all four substrates was evident in human hepatocytes, in line with reported clinical pharmacogenetic data. These data confirm the role of Oct1 in the hepatic uptake of the four OCT1 substrates and elucidate species differences in OCT1-mediated hepatocyte uptake that should be considered when utilizing the model to predict effects in humans. SIGNIFICANCE STATEMENT: Studies in carriers of SLC22A1 null variants indicate a role of organic cation transporter 1 (OCT1) in the hepatic uptake of therapeutic agents, although OCT1-mediated drug-drug interactions have not been reported. This work used Oct1/2 knockout mice to confirm the role of Oct1 in the hepatic clearance and liver partitioning in mice for OCT1 substrates with reported pharmacogenetic effects. Species differences observed in mouse and human hepatocyte uptake clarify limitations of the knockout model for predicting exposure changes in humans for some OCT1 substrates.

Physiologically-Based Pharmacokinetic Modeling of Atorvastatin Incorporating Delayed Gastric Emptying and Acid-to-Lactone Conversion.

The drug-drug interaction profile of atorvastatin confirms that disposition is determined by cytochrome P450 (CYP) 3A4 and organic anion transporting polypeptides (OATPs). Drugs that affect gastric emptying, including dulaglutide, also affect atorvastatin pharmacokinetics (PK). Atorvastatin is a carboxylic acid that exists in equilibrium with a lactone form in vivo. The purpose of this work was to assess gastric acid-mediated lactone equilibration of atorvastatin and incorporate this into a physiologically-based PK (PBPK) model to describe atorvastatin acid, lactone, and their major metabolites. In vitro acid-to-lactone conversion was assessed in simulated gastric fluid and included in the model. The PBPK model was verified with in vivo data including CYP3A4 and OATP inhibition studies. Altering the gastric acid-lactone equilibrium reproduced the change in atorvastatin PK observed with dulaglutide. The model emphasizes the need to include gastric acid-lactone conversion and all major atorvastatin-related species for the prediction of atorvastatin PK.

Hepatic Organic Anion Transporting Polypeptide-Mediated Clearance in the Beagle Dog: Assessing In Vitro-In Vivo Relationships and Applying Cross-Species Empirical Scaling Factors to Improve Prediction of Human Clearance.

In the present study, the beagle dog was evaluated as a preclinical model to investigate organic anion transporting polypeptide (OATP)-mediated hepatic clearance. In vitro studies were performed with nine OATP substrates in three lots of plated male dog hepatocytes ± OATP inhibitor cocktail to determine total uptake clearance (CLuptake) and total and unbound cell-to-medium concentration ratio (Kpuu). In vivo intrinsic hepatic clearances (CLint,H) were determined following intravenous drug administration (0.1 mg/kg) in male beagle dogs. The in vitro parameters were compared with those previously reported in plated human, monkey, and rat hepatocytes; the ability of cross-species scaling factors to improve prediction of human in vivo clearance was assessed. CLuptake in dog hepatocytes ranged from 9.4 to 135 µl/min/106 cells for fexofenadine and telmisartan, respectively. Active process contributed >75% to CLuptake for 5/9 drugs. Rosuvastatin and valsartan showed Kpuu > 10, whereas cerivastatin, pitavastatin, repaglinide, and telmisartan had Kpuu < 5. The extent of hepatocellular binding in dog was consistent with other preclinical species and humans. The bias (2.73-fold) obtained from comparison of predicted versus in vivo dog CLint,H was applied as an average empirical scaling factor (ESFav) for in vitro-in vivo extrapolation of human CLint,H The ESFav based on dog reduced underprediction of human CLint,H for the same data set (geometric mean fold error = 2.1), highlighting its utility as a preclinical model to investigate OATP-mediated uptake. The ESFav from all preclinical species resulted in comparable improvement of human clearance prediction, in contrast to drug-specific empirical scalars, rationalized by species differences in expression and/or relative contribution of particular transporters to drug hepatic uptake.

Food Effect on Rosuvastatin Disposition and Low-Density Lipoprotein Cholesterol.

Rosuvastatin is commonly prescribed for the treatment of hypercholesterolemia and hepatic transporter-mediated accumulation in the liver enhances its efficacy. Current guidelines indicate no preference for fed or fasted rosuvastatin administration. We investigated the association between food intake and rosuvastatin disposition in healthy subjects and low-density lipoprotein cholesterol (LDL-C)-lowering effects among patients taking rosuvastatin. We demonstrate that administration with food resulted in a near 40% reduction of rosuvastatin exposure in healthy Asian (n = 12) and Caucasian (n = 11) subjects. Higher rosuvastatin concentrations in Asian subjects also correlated with higher allele frequency of ABCG2 c.421C>A. In mice, a greater rosuvastatin liver:plasma ratio was noted when administered with food. Among rosuvastatin patients (n = 156), there was no difference in dose needed to reach target LDL-C, measured LDL-C, or lathosterol concentrations, when administered in a fed or fasting state. Therefore, taking rosuvastatin with food could reduce systemic concentrations, and subsequent myopathy risk, without compromising LDL-C-lowering benefit.

The Drug of Abuse Gamma-Hydroxybutyric Acid Exhibits Tissue-Specific Nonlinear Distribution.

The drug of abuse γ-hydroxybutyric acid (GHB) demonstrates complex toxicokinetics with dose-dependent metabolic and renal clearance. GHB is a substrate of monocarboxylate transporters (MCTs) which are responsible for the saturable renal reabsorption of GHB. MCT expression is observed in many tissues and therefore may impact the tissue distribution of GHB. The objective of the present study was to evaluate the tissue distribution kinetics of GHB at supratherapeutic doses. GHB (400, 600, and 800 mg/kg iv) or GHB 600 mg/kg plus L-lactate (330 mg/kg iv bolus followed by 121 mg/kg/h infusion) was administered to rats and blood and tissues were collected for up to 330 min post-dose. K p values for GHB varied in both a tissue- and dose-dependent manner and were less than 0.5 (except in the kidney). Nonlinear partitioning was observed in the liver (0.06 at 400 mg/kg to 0.30 at 800 mg/kg), kidney (0.62 at 400 mg/kg to 0.98 at 800 mg/kg), and heart (0.15 at 400 mg/kg to 0.29 at 800 mg/kg), with K p values increasing with dose consistent with saturation of transporter-mediated efflux. In contrast, lung partitioning decreased in a dose-dependent manner (0.43 at 400 mg/kg to 0.25 at 800 mg/kg) suggesting saturation of active uptake. L-lactate administration decreased K p values in liver, striatum, and hippocampus and increased K p values in lung and spleen. GHB demonstrates tissue-specific nonlinear distribution consistent with the involvement of monocarboxylate transporters. These observed complexities are likely due to the involvement of MCT1 and 4 with different affinities and directionality for GHB transport.

Physiologically Based Pharmacokinetic Modeling of Transporter-Mediated Hepatic Clearance and Liver Partitioning of OATP and OCT Substrates in Cynomolgus Monkeys.

In the present investigations, we evaluate in vitro hepatocyte uptake and partitioning for the prediction of in vivo clearance and liver partitioning. Monkeys were intravenously co-dosed with rosuvastatin and bosentan, substrates of the organic anion transporting polypeptides (OATPs), and metformin, a substrate of organic cation transporter 1 (OCT1). Serial plasma and liver samples were collected over time. Liver and plasma unbound fraction was determined using equilibrium dialysis. In vivo unbound partitioning (Kpu,u) for rosuvastatin, bosentan, and metformin, calculated from total concentrations in the liver and plasma, were 243, 553, and 15, respectively. A physiologically based pharmacokinetic monkey model that incorporates active and passive hepatic uptake was developed to fit plasma and liver concentrations. In addition, a two-compartment model was used to fit in vitro hepatic uptake curves in suspended monkey hepatocyte to determine active uptake, passive diffusion, and intracellular unbound fraction parameters. At steady-state in the model, in vitro Kpu,u was determined. The results demonstrated that in vitro values under-predicted in vivo active uptake for rosuvastatin, bosentan, and metformin by 6.7-, 28-, and 1.5-fold, respectively, while passive diffusion was over-predicted. In vivo Kpu,u values were under-predicted from in vitro data by 30-, 79-, and 3-fold. In conclusion, active uptake and liver partitioning in monkeys for OATP substrates were greatly under-predicted from in vitro hepatocyte uptake, while OCT-mediated uptake and partitioning scaled reasonably well from in vitro, demonstrating substrate- and transporter-dependent scaling factors. The combination of in vitro experimental and modeling approaches proved useful for assessing prediction of in vivo intracellular partitioning.

Effect of chronic γ-hydroxybutyrate (GHB) administration on GHB toxicokinetics and GHB-induced respiratory depression.

BACKGROUND: γ-hydroxybutyrate (GHB) has a high potential for illicit use; overdose of this compound results in sedation, respiratory depression and death. Tolerance to the hypnotic/sedative and electroencephalogram effects of GHB occurs with chronic GHB administration; however, tolerance to respiratory depression has not been evaluated. GHB toxicodynamic effects are mediated predominantly by GABAB receptors. Chronic treatment may affect monocarboxylate transporters (MCTs) and alter the absorption, renal clearance and brain uptake of GHB. OBJECTIVES: To determine effects of chronic GHB dosing on GHB toxicokinetics, GHB-induced respiratory depression, and MCT expression. METHODS: Rats were administered GHB 600 mg/kg intravenously daily for 5 days. Plasma, urine and tissue samples and respiratory measurements were obtained on days 1 and 5. Plasma and urine were analyzed for GHB by LC/MS/MS and tissue samples for expression of MCT1, 2 and 4 and their accessory proteins by QRT-PCR. RESULTS: No differences in GHB pharmacokinetics or respiratory depression were observed between days 1 and 5. Opposing changes in MCT1 and MCT4 mRNA expression were observed in kidney samples on day 5 compared to GHB-naïve animals, and MCT4 expression was increased in the intestine. CONCLUSIONS: The lack of tolerance observed with GHB-induced respiratory depression, in contrast to the tolerance reported for the sedative/hypnotic and electroencephalogram effects, suggests that different GABAB receptor subtypes may be involved in different GABAB-mediated toxicodynamic effects of GHB. Chronic or binge users of GHB may be at no less risk for fatality from respiratory arrest with a GHB overdose than with a single dose of GHB.

Identification and Characterization of Trimethylamine-N-oxide Uptake and Efflux Transporters.

Trimethylamine-N-oxide (TMAO) is a recently identified predictor of cardiovascular and chronic kidney disease. TMAO is primarily generated through gut-microbiome mediated conversion of dietary choline and carnitine to TMA, which is converted to TMAO by hepatic flavin monooxygenase 3 (FMO3) and subsequently undergoes renal elimination. We investigated the role of uptake and efflux drug transporters in TMAO disposition in vitro and in vivo. After screening a large array of uptake transporters, we show organic cation transporter 2 (OCT2) is the key transporter for TMAO cellular uptake. In Oct1/2 knockout mice, we observed increased plasma TMAO levels with reduced renal retention, suggesting the importance of Oct2 in facilitating the uptake of TMAO into renal tubular cells in vivo. Multiple transporters of the ATP-binding cassette (ABC) family, including ABCG2 (BCRP) and ABCB1 (MDR1), were capable of TMAO efflux. In human subjects, clinical, dietary, and pharmacogenetic covariates were evaluated for contribution to TMAO levels in a cohort of dyslipidemic patients (n = 405). Interestingly, genetic variation in ABCG2, but not other transporters, appeared to play a role in modulating TMAO exposure.

Advanced chronic kidney disease populations have elevated trimethylamine N-oxide levels associated with increased cardiovascular events.

Cardiovascular disease is more common in patients with chronic kidney disease (CKD), and traditional risk factors do not adequately predict those at risk for cardiovascular (CV) events. Recent evidence suggests elevated trimethylamine N-oxide (TMAO), created by gut microflora from dietary L-carnitine and choline, is associated with CV events. We investigated the relationship of TMAO levels in patients with stages 3b and 4 CKD to ischemic CV events using the CanPREDDICT cohort, a Canada-wide observational study with prospective 3-year follow-up of adjudicated CV events. Baseline samples were obtained for 2529 CKD patients. TMAO, choline, and L-carnitine levels were measured using tandem mass spectrometry. Baseline median TMAO level was high for the whole cohort (20.41 µM; interquartile range [IQR]: 12.82-32.70 µM). TMAO was independently associated with CV events (hazard ratio 1.23; 95% confidence interval: 1.06-1.42 / 1 SD lnTMAO) after adjusting for all potential CV risk factors. Those in the highest TMAO quartile had significantly higher risk of CV events (adjusted hazard ratio 1.59; 95% confidence interval: 1.04-2.43; P = 0.0351) in the analysis of recurring ischemic events. Among those with stage 3b CKD (hazard ratio 1.45; 95% confidence interval: 1.12-1.87 / 1 SD lnTMAO), independent of kidney function, TMAO levels identified those at highest risk for events. Our results suggest that TMAO may represent a new potentially modifiable CV risk factor for CKD patients. Further studies are needed to determine sources of variability and if lowering of TMAO reduces CV risk in CKD.

Rosuvastatin Liver Partitioning in Cynomolgus Monkeys: Measurement In Vivo and Prediction Using In Vitro Monkey Hepatocyte Uptake.

Unbound plasma concentrations may not reflect those in target tissues, and there is a need for methods to predict tissue partitioning. Here, we investigate the unbound liver partitioning (Kpu,u) of rosuvastatin, a substrate of hepatic organic anion transporting peptides, in cynomolgus monkeys and compare it with that determined using hepatocytes in vitro. Rosuvastatin (3 mg/kg) was administered orally to monkeys and plasma and liver (by ultrasound-guided biopsy) collected over time. Uptake into monkey hepatocytes was evaluated up to steady state. Binding in monkey plasma, liver, and hepatocytes was determined using equilibrium dialysis. Mean in vivo Kpu,u was 118 after correcting total liver partitioning by plasma and liver binding. In vitro uptake data were analyzed by compartmental modeling to determine active uptake clearance, passive diffusion, the intracellular unbound fraction, and Kpu,u. In vitro Kpu,u underpredicted that in vivo, resulting in the need for an empirical in vitro to in vivo scaling factor of 10. Adjusting model parameters using hypothetical scaling factors for transporter expression and surface area or assuming no effect of protein binding on active transport increased partitioning values by 1.1-, 6-, and 9-fold, respectively. In conclusion, in vivo rosuvastatin unbound liver partitioning in monkeys was underpredicted using hepatocytes in vitro. Modeling approaches that allow integrating corrections from passive diffusion or protein binding on active uptake could improve the estimation of in vivo intracellular partitioning of this organic anion transporting peptide substrate. A similar assessment of other active hepatic transport mechanisms could confirm and determine the extent to which limited accumulation in isolated hepatocytes needs to be considered in drug development.

Trimethylamine-N-oxide: A Novel Biomarker for the Identification of Inflammatory Bowel Disease.

BACKGROUND: The gastrointestinal (GI) microbiome is recognized for potential clinical relevance in inflammatory bowel disease (IBD). Data suggest that there is a disease-dependent loss of microbial diversity in IBD. Trimethylamine-N-oxide (TMAO) is generated by GI anaerobes through the digestion of dietary phosphatidylcholine and carnitine in a microbial-mammalian co-metabolic pathway. IBD-related changes in the gut microbiome may result in disease-specific changes in TMAO plasma concentrations. AIM: To determine whether TMAO plasma levels in IBD are altered compared to controls and whether they correlate with disease presence or activity. METHODS: Liquid chromatography-tandem mass spectrometry was used to measure TMAO, choline, and carnitine plasma levels in 479 subjects (373 non-IBD controls, 106 IBD). Subjects were also genotyped for the flavin monooxygenase (FMO)3 variants, E158K and E308G. RESULTS: Plasma TMAO levels were 2.27 µM lower in the IBD population compared to the control population (p = 0.0001). Lower TMAO levels were similarly seen in active ulcerative colitis (UC) (1.56 µM) versus inactive disease (3.40 µM) (p = 0.002). No difference was seen in active Crohn's disease (CD) versus inactive CD. No intergroup variation existed in plasma TMAO levels based on FMO3 genotype. Choline levels were higher in IBD, while carnitine levels were similar between the two groups, suggesting that lower TMAO levels in IBD were not due to dietary differences. CONCLUSIONS: Decreased TMAO levels are seen in IBD compared to a non-IBD population. These data suggest that TMAO may have potential as a biomarker to support IBD diagnosis as well as to assess disease activity in UC.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression.

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine.