Organic Anion Transporting Polypeptide Substrates Research Articles

Significant increases in detection capability for assessment of organic anion transporting polypeptide-mediated drug-drug interaction in cynomolgus monkeys by modifying pretreatment of Coproporphyrin I and III, and glycochenodeoxycholate-3-sulfate in plasma

BackgroundCoproporphyrin I (CP–I), Coproporphyrin III (CP-III), and glycochenodeoxycholate-3-sulfate (GCDCA-S) act as endogenous substrates of Organic Anion Transporting Polypeptide (OATP) 1B and have been considered for application in OATP1B-mediated drug‒drug interaction (DDI) risk assessments. Prior assays of the endogenous OATP substrates might exhibit reduced DDI detection capability and possibly overlook low DDI risk. We pioneered a simultaneous assay of the three substrates in monkey plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) and applied it to monkey studies to identify lower DDI risk. ResultsThe methodology development indicated that precursors of CP-I/III were oxidized to form CP-I/III, diminishing the detection capability in DDI risk assessments. A precursor eliminated analytical (PEA) method was developed to eliminate the precursors through solid-phase extraction. This method aimed to prevent the oxidation of CP-I/III precursors by incorporating edaravone. For comparison, a precursor oxidized analytical (POA) method was also developed, wherein the precursors of CP-I/III were fully oxidized to CP-I/III. The PEA method achieved high sensitivity for CP-I/III and GCDCA-S, with lower quantification limits of 0.01 ng mL−1 and 0.5 ng mL−1, respectively. Both methods ensured that the validation parameters met the acceptance criteria. The two methods were applied to a monkey study, with CP-I/III showcasing notably enhanced DDI detection capabilities through the novel PEA method in comparison to the POA method. SignificanceThis study's methodology has future implications for OATP-mediated DDI risk assessment using endogenous substrates. The novel PEA method can identify lower OATP-mediated DDI risks for drugs that the current methods cannot detect. Our method is likely applicable in clinical settings, and its utility should be assessed in clinical trials.

358 In Vitro Uptake of Harmful Algal Bloom Toxin Microcystin-LR in Human Placental Cells

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.

Pharmacokinetic Effects of Different Models of Nonalcoholic Fatty Liver Disease in Transgenic Humanized OATP1B Mice.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 (collectively, OATP1B) transporters encoded by the solute carrier organic anion transporter (SLCO) genes mediate uptake of multiple pharmaceutical compounds. Nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease (NAFLD), decreases OATP1B abundance. This research characterized the pathologic and pharmacokinetics effects of three diet- and one chemical-induced NAFLD model in male and female humanized OATP1B mice, which comprises knock-out of rodent Oatp orthologs and insertion of human SLCO1B1 and SLCO1B3. Histopathology scoring demonstrated elevated steatosis and inflammation scores for all NAFLD-treatment groups. Female mice had minor changes in SLCO1B1 expression in two of the four NAFLD treatment groups, and pitavastatin (PIT) area under the concentration-time curve (AUC) increased in female mice in only one of the diet-induced models. OATP1B3 expression decreased in male and female mice in the chemical-induced NAFLD model, with a coinciding increase in PIT AUC, indicating the chemical-induced model may better replicate changes in OATP1B3 expression and OATP substrate disposition observed in NASH patients. This research also tested a reported multifactorial pharmacokinetic interaction between NAFLD and silymarin, an extract from milk thistle seeds with notable OATP-inhibitory effects. Males showed no change in PIT AUC, whereas female PIT AUC increased 1.55-fold from the diet alone and the 1.88-fold from the combination of diet with silymarin, suggesting that female mice are more sensitive to pharmacokinetic changes than male mice. Overall, the humanized OATP1B model should be used with caution for modeling NAFLD and multifactorial pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Advanced stages of NAFLD cause decreased hepatic OATP1B abundance and increase systemic exposure to OATP substrates in human patients. The humanized OATP1B mouse strain may provide a clinically relevant model to recapitulate these observations and predict pharmacokinetic interactions in NAFLD. This research characterized three diet-induced and one drug-induced NAFLD model in a humanized OATP1B mouse model. Additionally, a multifactorial pharmacokinetic interaction was observed between silymarin and NAFLD.

A Pilot Study To Assess the Suitability of Riboflavin As a Surrogate Marker of Breast Cancer Resistance Protein in Healthy Participants.

We recently showed that riboflavin is a selected substrate of breast cancer resistance protein (BCRP) over P-glycoprotein (P-gp) and demonstrated its prediction performance in preclinical drug-drug interaction (DDI) studies. The aim of this study was to investigate the suitability of riboflavin to assess BCRP inhibition in humans. First, we assessed the substrate potential of riboflavin toward other major drug transporters using established transfected cell systems. Riboflavin is a substrate for organic anion transporter (OAT)1, OAT3, and multidrug and toxin extrusion protein (MATE)2-K, with uptake ratios ranging from 2.69 to 11.6, but riboflavin is not a substrate of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)2, and MATE1. The effects of BMS-986371, a potent in vitro inhibitor of BCRP (IC 50 0.40 μM), on the pharmacokinetics of riboflavin, isobutyryl carnitine, and arginine were then examined in healthy male adults (N = 14 or 16) after oral administration of methotrexate (MTX) (7.5 mg) and enteric-coated (EC) sulfasalazine (SSZ) (1000 mg) alone or in combination with BMS-986371 (150 mg). Oral administration of BMS-986371 increased the area under the plasma concentration-time curves (AUCs) of rosuvastatin and immediate-release (IR) SSZ to 1.38- and 1.51-fold, respectively, and significantly increased AUC(0-4h), AUC(0-24h), and C max of riboflavin by 1.25-, 1.14-, and 1.11-fold (P-values of 0.003, 0.009, and 0.025, respectively) compared with the MTX/SSZ EC alone group. In contrast, BMS-986371 did not significantly influence the AUC(0-24h) and C max values of isobutyryl carnitine and arginine (0.96- to 1.07-fold, respectively; P > 0.05). Overall, these data indicate that plasma riboflavin is a promising biomarker of BCRP that may offer a possibility to assess drug candidate as a BCRP modulator in early drug development. SIGNIFICANCE STATEMENT: Endogenous compounds that serve as biomarkers for clinical inhibition of breast cancer resistance protein (BCRP) are not currently available. This study provides the initial evidence that riboflavin is a promising BCRP biomarker in humans. For the first time, the value of leveraging the substrate of BCRP with acceptable prediction performance in clinical studies is shown. Additional clinical investigations with known BCRP inhibitors are needed to fully validate and showcase the utility of this biomarker.

Toward improved predictions of pharmacokinetics oftransported drugs in hepatic impairment: Insights fromthe extended clearance model.

Hepatic impairment (HI) moderately (<5-fold) affects the systemic exposure (i.e., area under the plasma concentration-time curve [AUC]) of drugs that are substrates of the hepatic sinusoidal organic anion transporting polypeptide (OATP) transporters and are excreted unchanged in the bile and/or urine. However, the effect of HI on their AUC is much greater (>10-fold) for drugs that are also substrates of cytochrome P450 (CYP) 3A enzymes. Using the extended clearance model, through simulations, we identified the ratio of sinusoidal efflux clearance (CL) over the sum of metabolic and biliary CLs as important in predicting the impact of HI on the AUC of dual OATP/CYP3A substrates. Because HI may reduce hepatic CYP3A-mediated CL to a greater extent than biliary efflux CL, the greater the contribution of the former versus the latter, the greater the impact of HI on drug AUC ratio (AUCRHI ). Using physiologically-based pharmacokinetic modeling and simulation, we predicted relatively well the AUCRHI of OATP substrates that are not significantly metabolized (pitavastatin, rosuvastatin, valsartan, and gadoxetic acid). However, there was a trend toward underprediction of the AUCRHI of the dual OATP/CYP3A4 substrates fimasartan and atorvastatin. These predictions improved when the sinusoidal efflux CL of these two drugs was increased in healthy volunteers (i.e., before incorporating the effect of HI), and by modifying the directionality of its modulation by HI (i.e., increase or decrease). To accurately predict the effect of HI on AUC of hepatobiliary cleared drugs it is important to accurately predict all hepatobiliary pathways, including sinusoidal efflux CL.

Quantitative Prediction of OATP-Mediated Disposition and Biliary Clearance Using Human Liver Chimeric Mice.

Drug biliary clearance (CLbile) in vivo is among the most difficult pharmacokinetic parameters to predict accurately and quantitatively because biliary excretion is influenced by metabolic enzymes, transporters, and passive diffusion across hepatocyte membranes. The purpose of this study is to demonstrate the use of Hu-FRG mice [Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice transplanted with human-derived hepatocytes] to quantitatively predict human organic anion transporting polypeptide (OATP)-mediated drug disposition and CLbile To predict OATP-mediated disposition, six OATP substrates (atorvastatin, fexofenadine, glibenclamide, pitavastatin, pravastatin, and rosuvastatin) were administered intravenously to Hu-FRG and Mu-FRG mice (FRG mice transplanted with mouse hepatocytes) with or without rifampicin as an OATP inhibitor. We calculated the hepatic intrinsic clearance (CLh,int) and the change of hepatic clearance (CLh) caused by rifampicin (CLh ratio). We compared the CLh,int of humans with that of Hu-FRG mice and the CLh ratio of humans with that of Hu-FRG and Mu-FRG mice. For predicting CLbile, 20 compounds (two cassette doses of 10 compounds) were administered intravenously to gallbladder-cannulated Hu-FRG and Mu-FRG mice. We evaluated the CLbile and investigated the correlation of human CLbile with that of Hu-FRG and Mu-FRG mice. We found good correlations between humans and Hu-FRG mice in CLh,int (100% within threefold) and CLh ratio (R2 = 0.94). Moreover, we observed a much better relationship between humans and Hu-FRG mice in CLbile (75% within threefold). Our results suggest that OATP-mediated disposition and CLbile can be predicted using Hu-FRG mice, making them a useful in vivo drug discovery tool for quantitatively predicting human liver disposition. SIGNIFICANCE STATEMENT: OATP-mediated disposition and biliary clearance of drugs are likely quantitatively predictable using Hu-FRG mice. The findings can enable the selection of better drug candidates and the development of more effective strategies for managing OATP-mediated DDIs in clinical studies.

11C]glyburide PET imaging for quantitative determination of the importance of Organic Anion-Transporting Polypeptide transporter function in the human liver and whole-body

Organic Anion-Transporting Polypeptides (OATPs) are known to control the liver uptake of many drugs. Non-hepatic expression of OATPs has been reported although functional importance for whole-body pharmacokinetics (WBPK) remains unknown. Glyburide is a well described substrate of several hepatic and non-hepatic OATPs. Dynamic whole-body positron emission tomography (DWB-PET) with [11C]glyburide was performed in humans for determination of the importance of OATPs for liver uptake and WBPK. Seven healthy male subjects (24.7 ± 3.2 years) underwent [11C]glyburide PET scan with concomitant blood sampling. All subjects underwent baseline [11C]glyburide PET scan. Five subjects underwent a subsequent [11C]glyburide PET scan after infusion of the potent OATP inhibitor rifampicin (9 mg/kg i.v.). The transfer constant (kuptake) of [11C]glyburide from blood to the liver was estimated using the integration plot method. The tissue exposure of [11C]glyburide was described by the area under the time-activity curve (AUC) and corresponding tissue/blood ratio (AUCR). [11C]glyburide was barely metabolized in both the baseline and rifampicin conditions. Parent (unmetabolized) [11C]glyburide accounted for > 90 % of the plasma radioactivity. Excellent correlation was found between radioactive counting in arterial blood samples and in the image-derived input function, in both the baseline and rifampicin conditions (R2 = 97.9 %, p < 0.01). [11C]glyburide predominantly accumulated in the liver. Rifampicin decreased liver kuptake by 77.3 ± 7.3 %, which increased exposure in blood, kidneys, spleen, myocardium and brain (p < 0.05). No significant change in AUCR was observed except in the liver (p < 0.01). [11C]glyburide benefits from metabolic stability and high sensitivity to OATP inhibition which enables quantitative determination of OATP function. DWB-PET suggests negligible role for non-hepatic OATPs in controlling the tissue distribution of [11C]glyburide.

Inhibitory Effects of Cranberry Juice and Its Components on Intestinal OATP1A2 and OATP2B1: Identification of Avicularin as a Novel Inhibitor.

Organic anion-transporting polypeptide (OATP) 1A2 and OATP2B1 mediate the intestinal absorption of drugs. This study aimed to identify fruit juices or fruit juice components that inhibit OATPs and assess the risk of associated food-drug interactions. Inhibitory potency was assessed by examining the uptake of [3H]estrone 3-sulfate and [3H]fexofenadine into HEK293 cells expressing OATP1A2 or OATP2B1. In vivo experiments were conducted using mice to evaluate the effects of cranberry juice on the pharmacokinetics of orally administered fexofenadine. Of eight examined fruit juices, cranberry juice inhibited the functions of both OATPs most potently. Avicularin, a component of cranberry juice, was identified as a novel OATP inhibitor. It exhibited IC50 values of 9.0 and 37 μM for the inhibition of estrone 3-sulfate uptake mediated by OATP1A2 and OATP2B1, respectively. A pharmacokinetic experiment revealed that fexofenadine exposure was significantly reduced (by 50%) by cranberry juice. Cranberry juice may cause drug interactions with OATP substrates.

In Vitro Assessment of the Drug-Drug Interaction Potential of Verinurad and Its Metabolites as Substrates and Inhibitors of Metabolizing Enzymes and Drug Transporters.

Verinurad is a selective uric acid transporter 1 (URAT1) inhibitor in development for the treatment of chronic kidney disease and heart failure. In humans, two major acyl glucuronide metabolites have been identified: direct glucuronide M1 and N-oxide glucuronide M8. Using in vitro systems recommended by regulatory agencies, we evaluated the interactions of verinurad, M1, and M8 with major drug-metabolizing enzymes and transporters and the potential for clinically relevant drug-drug interactions (DDIs). The IC50 for inhibition of CYP2C8, CYP2C9, and CYP3A4/5 for verinurad was ≥14.5 µM, and maximum free plasma concentration (Iu,max)/IC50 was <0.02 at the anticipated therapeutic Cmax and therefore not considered a DDI risk. Verinurad was not an inducer of CYP1A2, CYP2B6, or CYP3A4/5. Verinurad was identified as a substrate of the hepatic uptake transporter organic anion-transporting polypeptide (OATP) 1B3. Since verinurad hepatic uptake involved both active and passive transport, there is a low risk of clinically relevant DDIs with OATP, and further study is warranted. Verinurad was a substrate of the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and renal transporter organic anion transporter 1 (OAT1), although it is not considered a DDI risk in vivo because of dose-proportional pharmacokinetics (P-gp and BCRP) and limited renal excretion of verinurad (OAT1). M1 and M8 were substrates of multidrug resistance-associated protein (MRP) 2 and MRP4 and inhibitors of MRP2. Apart from verinurad being a substrate of OATP1B3 in vitro, the potential for clinically relevant DDIs involving verinurad and its metabolites as victims or perpetrators of metabolizing enzymes or drug transporters is considered low. SIGNIFICANCE STATEMENT: Drug transporters and metabolizing enzymes have an important role in the absorption and disposition of a drug and its metabolites. Using in vitro systems recommended by regulatory agencies, we determined that, apart from verinurad being a substrate of organic anion-transporting polypeptide 1B3, the potential for clinically relevant drug-drug interactions involving verinurad and its metabolites M1 and M8 as victims or perpetrators of metabolizing enzymes or drug transporters is considered low.

Application of a PBPK model to elucidate the changes of systemic and liver exposures for rosuvastatin, carotegrast, and bromfenac followed by OATP inhibition in monkeys.

The impact of organic anion‐transporting polypeptide (OATP) inhibition on systemic and liver exposures of three OATP substrates was investigated in cynomolgus monkeys. A monkey physiologically‐based pharmacokinetic (PBPK) model was constructed to describe the exposure changes followed by OATP functional attenuation. Rosuvastatin, bromfenac, and carotegrast were administered as a single intravenous cassette dose (0.5 mg/kg each) in monkeys with and without predosing with rifampin (RIF; 20 mg/kg) orally. The plasma exposure of rosuvastatin, bromfenac, carotegrast, and OATP biomarkers, coproporphyrin I (CP‐I) and CP‐III were increased 2.3, 2.1, 9.1, 5.4, and 8.8‐fold, respectively, when compared to the vehicle group. The liver to plasma ratios of rosuvastatin and bromfenac were reduced but the liver concentration of the drugs remained unchanged by RIF treatment. The liver concentrations of carotegrast, CP‐I, and CP‐III were unchanged at 1 h but increased at 6 h in the RIF‐treated group. The passive permeability, active uptake, and biliary excretion were characterized in suspended and sandwich‐cultured monkey hepatocytes and then incorporated into the monkey PBPK model. As demonstrated by the PBPK model, the plasma exposure is increased through OATP inhibition while liver exposure is maintained by passive permeability driven from an elevated plasma level. Liver exposure is sensitive to the changes of metabolism and biliary clearances. The model further suggested the involvement of additional mechanisms for hepatic uptakes of rosuvastatin and bromfenac, and of the inhibition of biliary excretion for carotegrast, CP‐I, and CP‐III by RIF. Collectively, impaired OATP function would not reduce the liver exposure of its substrates in monkeys.

Improving the Translation of Organic Anion Transporting Polypeptide Substrates using HEK293 Cell Data in the Presence and Absence of Human Plasma via Physiologically Based Pharmacokinetic Modeling.

Accurately predicting the pharmacokinetics of compounds that are transporter substrates has been notoriously challenging using traditional in vitro systems and physiologically based pharmacokinetic (PBPK) modeling. The objective of this study was to use PBPK modeling to understand the translational accuracy of data generated with human embryonic kidney 293 (HEK293) cells overexpressing the hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1/3 with and without plasma while accounting for transporter expression. Models of four OATP substrates, two with low protein binding (pravastatin and rosuvastatin) and two with high protein binding (repaglinide and pitavastatin) were explored, and the OATP in vitro data generated in plasma incubations were used for a plasma model, and in buffer incubations for a buffer model. The pharmacokinetic parameters and concentration-time profiles of pravastatin and rosuvastatin were similar and well predicted (within 2-fold of observed values) using the plasma and buffer models without needing an empirical scaling factor, whereas the dispositions of the highly protein bound repaglinide and pitavastatin were more accurately simulated with the plasma models than the buffer models. This work suggests that data from HEK293 overexpressing transporter cells corrected for transporter expression represent a valid approach to improve bottom-up PBPK modeling for highly protein bound OATP substrates with plasma incubations and low protein binding OATP substrates with or without plasma incubations. SIGNIFICANCE STATEMENT: This work demonstrates the bottom-up approach of using in vitro data directly without employing empirical scaling factors to predict the intravenous pharmacokinetic (PK) profiles reasonably well for four organic anion transporting polypeptide (OATP) substrates. Based on these results, using HEK293 overexpressing cells, examining the impact of plasma for highly bound compounds, and incorporating transporter quantitation for the lot in which the in vitro data were generated represents a valid approach to achieve more accurate prospective PK predictions for OATP substrates.

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.

In Vitro Hepatic Uptake in Human and Monkey Hepatocytes in the Presence and Absence of Serum Protein and Its In Vitro to In Vivo Extrapolation.

It is well documented that human hepatic clearance based on in vitro metabolism or transporter assays systematically resulted in underprediction; therefore, large empirical scalars are often needed in either static or physiologically based pharmacokinetic (PBPK) models to accurately predict human pharmacokinetics (PK). In our current investigation, we assessed hepatic uptake in hepatocyte suspension in Krebs-Henseleit buffer in the presence and absence of serum. The results showed that the unbound intrinsic active clearance (CLu,int,active) values obtained by normalizing the unbound fraction in the buffer containing 10% serum were generally higher than the CLu,int,active obtained directly from protein free buffer, suggesting "protein-facilitated" uptake. The differences of CLu,int,active in the buffer with and without protein ranged from 1- to 925-fold and negatively correlated to the unbound serum binding of organic anion transporting polypeptide substrates. When using the uptake values obtained from buffer containing serum versus serum-free buffer, the median of scaling factors (SFs) for CLu,int,active reduced from 24.2-4.6 to 22.7-7.1 for human and monkey, respectively, demonstrating the improvement of in vitro to in vivo extrapolation in a PBPK model. Furthermore, values of CLu,int,active were significantly higher in monkey hepatocytes than that in human, and the species differences appeared to be compound dependent. Scaling up in vitro uptake values derived in assays containing species-specific serum can compensate for the species-specific variabilities when using cynomolgus monkey as a probe animal model. Incorporating SFs calibrated in monkey and together with scaled in vitro data can be a reliable approach for the prospective human PK prediction in early drug discovery. SIGNIFICANCE STATEMENT: We investigated the protein effect on hepatic uptake in human and monkey hepatocytes and improved the in vitro to in vivo extrapolation using parameters obtained from the incubation in the present of serum protein. In addition, significantly higher active uptake clearances were observed in monkey hepatocytes than in human, and the species differences appeared to be compound dependent. The physiologically based pharmacokinetic model that incorporates scaling factors calibrated in monkey and together with scaled in vitro human data can be a reliable approach for the prospective human pharmacokinetics prediction.

Prediction of Cyclosporin-Mediated Drug Interaction Using Physiologically Based Pharmacokinetic Model Characterizing Interplay of Drug Transporters and Enzymes.

Uptake transporter organic anion transporting polypeptides (OATPs), efflux transporters (P-gp, BCRP and MRP2) and cytochrome P450 enzymes (CYP450s) are widely expressed in the liver, intestine or kidney. They coordinately work to control drug disposition, termed as “interplay of transporters and enzymes”. Cyclosporine A (CsA) is an inhibitor of OATPs, P-gp, MRP2, BCRP and CYP3As. Drug–drug interaction (DDI) of CsA with victim drugs occurs via disordering interplay of transporters and enzymes. We aimed to establish a whole-body physiologically-based pharmacokinetic (PBPK) model which predicts disposition of CsA and nine victim drugs including atorvastatin, cerivastatin, pravastatin, rosuvastatin, fluvastatin, simvastatin, lovastatin, repaglinide and bosentan, as well as drug–drug interactions (DDIs) of CsA with nine victim drugs to investigate the integrated effect of enzymes and transporters in liver, intestinal and kidney on drug disposition. Predictions were compared with observations. Most of the predictions were within 0.5–2.0 folds of observations. Atorvastatin was represented to investigate individual contributions of transporters and CYP3As to atorvastatin disposition and their integrated effect. The contributions to atorvastatin disposition were hepatic OATPs >> hepatic CYP3A > intestinal CYP3As ≈ efflux transporters (P-gp/BCRP/MRP2). The results got the conclusion that the developed PBPK model characterizing the interplay of enzymes and transporters was successfully applied to predict the pharmacokinetics of 10 OATP substrates and DDIs of CsA with 9 victim drugs.

A Pharmacokinetic Natural Product-Disease-Drug Interaction: A Double Hit of Silymarin and Nonalcoholic Steatohepatitis on Hepatic Transporters in a Rat Model.

Patients with nonalcoholic steatohepatitis (NASH) exhibit altered hepatic protein expression of metabolizing enzymes and transporters and altered xenobiotic pharmacokinetics. The botanical natural product silymarin, which has been investigated as a treatment of NASH, contains flavonolignans that inhibit organic anion-transporting polypeptide (OATP) transporter function. The purpose of this study was to assess the individual and combined effects of NASH and silymarin on the disposition of the model OATP substrate pitavastatin. Male Sprague Dawley rats were fed a control or a methionine- and choline-deficient diet (NASH model) for 8 weeks. Silymarin (10 mg/kg) or vehicle followed by pitavastatin (0.5 mg/kg) were administered intravenously, and the pharmacokinetics were determined. NASH increased mean total flavonolignan area under the plasma concentration-time curve (AUC0-120 min) 1.7-fold. Silymarin increased pitavastatin AUC0-120 min in both control and NASH animals approx. 2-fold. NASH increased pitavastatin plasma concentrations from 2 to 40 minutes, but AUC0-120 min was unchanged. The combination of silymarin and NASH had the greatest effect on pitavastatin AUC0-120 min, which increased 2.9-fold compared with control vehicle-treated animals. NASH increased the total amount of pitavastatin excreted into the bile 2.7-fold compared with control animals, whereas silymarin decreased pitavastatin biliary clearance approx. 3-fold in both control and NASH animals. This double hit of NASH and silymarin on hepatic uptake transporters is another example of a multifactorial pharmacokinetic interaction that may have a greater impact on drug disposition than each hit alone. SIGNIFICANCE STATEMENT: Multifactorial effects on xenobiotic pharmacokinetics are within the next frontier for precision medicine research and clinical application. The combination of silymarin and NASH is a probable clinical scenario that can affect drug uptake, liver concentrations, biliary elimination, and ultimately, efficacy and toxicity.

Evaluation of [18F]pitavastatin as a positron emission tomography tracer for in vivo organic transporter polypeptide function

IntroductionTo understand the pathways involved in drug clearance from the body, quantitative evaluations of the hepatobiliary transport of drugs are important. The organic anion transporting polypeptide (OATP) family transporter, particularly OATP1B1 and 1B3, are considered to play an important role in hepatic uptake of organic anion compounds. Pitavastatin is a substrate of OATP, and it includes a fluorine group. Therefore, it represents an acceptable positron-emission tomography (PET) tracer using fluorine-18 to image in vivo hepatic transporter functions. Method[18F]Pitavastatin was synthesized using the method we previously reported. To evaluate the potential of [18F]pitavastatin in PET imaging of in vivo OATP functions, we investigated the hepatic uptake with/without rifampicin as an OATP inhibitor after administration in normal SD rats. [18F]Pitavastatin metabolite was evaluated using reverse-phase thin-layer chromatography (TLC) autoradiography. We subsequently analyzed the PET image results and demonstrated that [18F]pitavastatin selectively accumulated in the liver post-administration.Result and discussionIn metabolite analysis using reverse-phase TLC, we found that the radioactivity detected in the plasma, liver (>90% intact), and bile mostly originated from the parent pitavastatin of the PET study (~40 min). [18F]pitavastatin's hepatic uptake decreased (approximately 76%) with rifampicin co-administration in PET analysis. Because [18F]pitavastatin has lower clearance in rats than other previously reported OATP1B PET s, it holds the potential of an imaging tracer that has a higher sensitivity in monitoring hepatic OATP1B function's changes. ConclusionCompared with the previously reported OATP imaging tracers, [18F]pitavastatin is more suitable for the sensitive detection of functional changes in OATP transporters. We believe that [18F]pitavastatin enables quantitative analysis of the hepatobiliary transport system for organic anion compounds.

Characterization of Hepatocytes Uptake Clearance of Organic Anion Transporting Polypeptide (OATPs) Substrates in Human and Cynomolgus Cryopreserved Hepatocytes

Transporter‐mediated clearance plays significant roles in drug bioavailability and elimination, and evaluation of potential transporter‐mediated drug‐drug interactions (DDI) is recommended by regulatory agencies during drug development. Currently, the direct extrapolation from in vitro to in vivo is a common approach for human clearance prediction for the compounds that are metabolized by hepatic cytochrome P450 enzymes; however, challenge remains when using the similar approaches in prediction of transporter‐mediated clearance. Recent publications attempted to establish the in‐vitro‐to‐ in vivo scaling based on hepatocyte uptake studies and in vivo pharmacokinetic (PK) data from the preclinical animals. However, the differences in homology of transporter proteins and the associated substrate specificity in transporters between human and preclinical animals complicate the prediction using the correcting factors obtained from the preclinical animals' in‐vitro‐to‐ in vivo scaling. In this study, we obtained the in vitro hepatic uptake clearance in cynomolgus monkey and human for 14 literature compounds that were known as OATPs substrates and had clinical DDI reported. Hepatic uptake was conducted using the oil‐spin method. For each selected substrate, the uptake assay was conducted in the time course from 0.25 minute to 15 minutes in KHB buffer with 10% serum. The uptake clearance was estimated from the linear uptake phase. For each selected substrate, the uptake clearance was characterized in three lots (including single donor and pooled donors lots) of human and monkey cryopreserved hepatocytes. In addition, we tested different lots of human (7 lots) and monkey (10 lots) cryopreserved hepatocytes using rosuvastatin as a control probe to examine the variability among lots and donors. In summary, rosuvastatin uptake clearance in cynomolgus monkey hepatocytes was about 2.5 folds higher on average compared to that in human hepatocytes. Generally, for other OATPs substrates studied, hepatic uptake clearance in cynomolgus monkey appeared to be higher than that in human. Furthermore, variability of uptake clearance was also observed among lots and donors. The results obtained in the current investigations are useful for human prediction through understanding the source of variations and establishing the correlations of in vitro‐in vivo scaling.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

The Presence of a Transporter-Induced Protein Binding Shift: A New Explanation for Protein-Facilitated Uptake and Improvement for In Vitro-In Vivo Extrapolation.

Accurately predicting hepatic clearance is an integral part of the drug-development process, and yet current in vitro to in vivo (IVIVE) extrapolation methods yield poor predictions, particularly for highly protein-bound transporter substrates. Explanations for error include inaccuracies in protein-binding measurements and the lack of recognition of protein-facilitated uptake, where both unbound and bound drug may be cleared, violating the principles of the widely accepted free drug theory. A new explanation for protein-facilitated uptake is proposed here, called a transporter-induced protein binding shift High-affinity binding to cell-membrane proteins may change the equilibrium of the nonspecific binding between drugs and plasma proteins, leading to greater cellular uptake and clearance than currently predicted. The uptake of two lower protein-binding organic anion transporting polypeptide substrates (pravastatin and rosuvastatin) and two higher binding substrates (atorvastatin and pitavastatin) were measured in rat hepatocytes in incubations with protein-free buffer versus 100% plasma. Decreased unbound K m values and increased intrinsic clearance values were seen in the plasma incubations for the highly bound compounds, supporting the new hypothesis and mitigating the IVIVE underprediction previously seen for highly bound transporter substrates.

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.