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.

Challenges remain in understanding the impact of hepatic uptake transporter, OATPs, on plasma and liver concentrations for OATP substrate drugs with distinct pharmacokinetic (PK) profiles and elimination routes.

WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

How do hepatic active transport, metabolism, biliary excretion, and passive permeability impact the systemic and liver exposure?

WHAT QUESTION DID THIS STUDY ADDRESS?

This study elucidates the important roles of hepatic active transport in determining drug plasma and liver concentrations and the translation of in vitro data to in vivo using the physiologically‐based PK modeling approach.

WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

The disconnection between plasma concentration and liver exposure followed by OATP activity reduction can explain the PK/pharmacodynamic relationship for the liver‐targeted drugs.

HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

INTRODUCTION

The organic anion‐transporting polypeptide (OATP) is a family of membrane proteins that plays a significant role in the disposition of many endogenous and exogenous substances. OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are the two members in the family predominantly expressing on the sinusoidal membrane of hepatocytes and are involved in taking up the body essentials and metabolic waste into liver. For example, OATP1B1/1B3 transport unconjugated and conjugated bilirubin into the liver. Loss of OATP functions caused by homozygous mutation of SLCO1B1 and SLCO1B3 genes can lead to hyperbilirubinemia, a genetic disease known as Rotor syndrome. Additionally, OATP transporters facilitate the uptake of certain bile acids, such as cholic acid, chenodeoxycholic acid, and deoxycholic acid, into hepatocytes for further metabolism and contribute significantly to the bile salt enterohepatic recirculation. , Recent publications also demonstrated that OATPs transport endogenous molecules coproporphyrin I (CP‐I) and CP‐III, which can be used as biomarkers to monitor OATPs function. ,

Many drugs are transported into the liver by OATPs and subsequentially undergo biotransformation and biliary excretion. Cholesterol lowering statin drugs have been reported to be substrates of OATP1B1/1B3. Therefore, OATP‐mediated hepatic uptake can be the rate limiting step of systemic clearance for statins with low passive membrane permeability. As a result, the systemic exposure of these drugs can be affected by the activity of the transporters due to either genetic variations or the presence of the inhibitors. Drugs known to be OATP1B1 substrates have shown an increased plasma exposure and reduced hepatic clearance in subjects carrying OATP1B1 *5 or *15. When co‐administered orally with rifampin (RIF), a known OATP pan inhibitor, the systemic exposure of pitavastatin was increased 5.7‐fold in humans. Similar drug‐drug interactions (DDIs) have been reported for many OATP1B1/1B3 inhibitors and their substrates.

Although the impact of OATP1B1/1B3 on the drug plasma exposures has been well‐characterized, only a few studies have been conducted to elucidate the relationship between transporter functions and the liver exposure of their substrate drugs. , The drug concentration in the liver is determined by several processes, including passive diffusion, transporter (i.e., OATP1B1/1B3) mediated sinusoidal uptake and efflux, metabolism, and biliary excretion. Over the past a few years, limited studies revealed that OATP inhibition has a minimal impact on the liver exposure. As evaluated using positron emission tomography, the total radioactivity in the liver was not significantly changed in the first 30 min following a single IV dose of 11C‐rosuvastatin in subjects co‐administered with an OATP inhibitor, cyclosporin A. In addition to rosuvastatin, further evaluation is necessary to fully understand the impact of OATP transporter inhibition on liver exposures of other drugs with different metabolic/transport pathways.

OATP1B1 and OATP1B3 have both been identified in cynomolgus monkey and their gene sequences are 91.9% and 93.5% identical to the human orthologs. In the transporter recombinantly overexpressing HEK293 cells, the ability of OATP1B1/1B3 to uptake known substrates is very similar between monkeys and humans. Several other studies also indicated that the cynomolgus monkey is a good animal model to characterize OATP function. , Thus, we used the cynomolgus monkey to investigate the liver exposure of rosuvastatin, carotegrast, and bromfenac in the absence and presence of RIF, an OATP inhibitor. In this study, blood samples were collected up to 24 h and liver tissues were sampled up to 6 h post drug administration, to capture the distribution and elimination phase of pharmacokinetics (PKs) for each drug in plasma and liver. In addition, a physiologically‐based PK (PBPK) model was built to incorporate factors, such as in vitro hepatic uptake, biliary excretion, and metabolism processes for the understanding of PK changes of these three drugs in both plasma and the liver.

MATERIALS AND METHODS

Chemicals

Rosuvastatin, bromfenac, RIF, RIF SV, and midazolam were purchased from Sigma‐Aldrich, Inc (St. Louis, MO). Carotegrast was synthesized at Gilead Sciences. CP‐I and CP‐III reference standards and stable isotope labeled CP‐I (N4‐CP‐I) and CP‐III (D8‐CP‐III) were purchased from Toronto Research Chemicals (North York, ON, Canada). Cryopreserved monkey hepatocytes (lot #UHK) were obtained from BioIVT (Hicksville, NY). All other reagents and solvents were of analytical grade.

Pharmacokinetic study in cynomolgus monkeys

The PKs and liver exposure of intravenously dosed rosuvastatin, carotegrast, and bromfenac were characterized in cynomolgus monkeys in the presence and absence of the oral administration of OATP inhibitor, RIF, at 20 mg per kilogram body weight. The study was conducted at WuXi AppTec Co., Ltd., a contract research organization certified by the Association for Assessment and Accreditation of Laboratory Animal Care. The study protocol and experimental procedures were approved by the Institutional Animal Care and Use Committee. All animals were housed in rooms with controlled humidity (40% to 70%), temperature (18°C to 26°C), air changes (10–20 per hour), and light/dark cycle (12 h). Non‐naïve male cynomolgus monkeys (3–5 kg and 2.5–6 years old) were acclimated for at least 2 weeks. Prior to the study, a physical examination for general health was conducted. All animals were then fasted overnight, and food was returned 4 h post oral dose. Water was available ad libitum throughout the whole study.

Eighteen animals were randomly assigned to two groups. Both groups received a single oral dose of either RIF or vehicle (100% PEG300) 3 h before the IV infusion (30 min) of rosuvastatin, carotegrast, and bromfenac. RIF was prepared in 100% PEG300 (20 mg/ml; dose volume of 1 ml/kg for 20 mg/kg dose). The cassette dose of rosuvastatin, carotegrast, and bromfenac was co‐formulated at the final dose of 0.5 mg/kg for each testing article in 5% dextrose solution (pH 9.4, 2 ml/kg, 0.25 mg/ml). The first group was designated to evaluate PKs of rosuvastatin, carotegrast, and bromfenac, in predosed with vehicle or with RIF (N = 3). Blood was collected into commercially available tubes (Jiangsu Kangjian Medical Supplies Co., LTD.) containing Potassium (K2) EDTA*2H2O (0.85–1.15 mg) on wet ice at 0, 0.25, 0.5, 0.583, 0.75, 1, 1.5, 2.5, 4, 6, 8, 12, and 24 h. Blood samples were processed for plasma within 30 min of collection. An aliquot of plasma (150 µl) was transferred to amber micro‐centrifuge tubes preloaded with 3 µl of formic acid (final formic acid concentration 2%). The second group was to determine liver exposure in the absence (N = 6) and presence (N = 6) of RIF. At 1, 2.5, and 6 h post the IV infusion, animals (N = 2 per time point) were anesthetized with IV injection of pentobarbital sodium (60 mg/kg). Aliquots (~ 1 g) were sampled from the left liver lobe and frozen immediately in liquid nitrogen. Plasma samples were also collected in a similar method for each monkey up to the time of liver sampling. All plasma and liver samples were protected from light and stored at −60°C or below until analysis.

The detailed method for sample preparation and liquid chromatography‐tandem accurate mass spectrometry (LC‐MS/MS) analysis to quantify rosuvastatin, carotegrast, bromfenac, CP‐I, CP‐III, and RIF are described in the Supplementary Material. The PK parameters of rosuvastatin, carotegrast, bromfenac, CP‐I, CP‐III, and RIF were derived from plasma concentrations versus time with a noncompartmental analysis (NCA) model using Phoenix software (Certara, Princeton, NJ). Statistical tests were conducted using Student’s t‐test and differences were considered significant when p < 0.05.

In vitro hepatic uptake and biliary clearance

The hepatic uptake clearance of rosuvastatin, carotegrast, and bromfenac was evaluated in vitro using suspension hepatocytes as described previously with modifications. , Briefly, intracellular accumulation of test compound was determined in suspension hepatocyte incubated at 4°C and 37°C up to 5 min. The OATP activity was also assessed at 37°C in the presence of 200 µM rifamycin‐SV. The hepatocytes were diluted to 2 million cells/ml in KHB with 10% (v/v) cynomolgus monkey serum. The detailed method for sample preparation and LC‐MS/MS analysis are described in the Supplementary Material. The active transporter‐mediated uptake was assessed at 37°C and passive diffusion was assessed at 4°C, assuming minimal transporter activities at low temperature. The total hepatic uptake and passive clearance were obtained from the initial uptake rates at 37°C or 4°C, respectively. The initial uptake rates were estimated from the slopes of linear uptake phase using linear regression analysis. Active uptake clearance was calculated by subtracting the passive uptake clearance from the total clearance. The unbound active and passive uptake clearance (CLu,int,active, and CLu,int,passive) were calculated by dividing the initial uptake velocity by the unbound fraction of nominal substrate concentration. The detail of calculation is described in the Supplementary Material.

In vitro biliary clearance was estimated in sandwich‐cultured cynomolgus monkey hepatocytes (SCCHs). Cryopreserved monkey hepatocytes were thawed into plating medium (INVITROGRO CP, BioIVT). The cells were seeded at 4 × 105 cells per well in 24‐well collagen‐coated plates. After 4‐h attachment, cells were washed once with cold incubation medium (INVITROGRO HI, BioIVT) and then overlaid with Matrigel (Corning, Tewksbury, MA) in incubation medium at 0.25 mg/ml on ice. The cells were then incubated overnight at 37°C in a humidified 5% CO2 incubator. Incubation media was changed every day for a total of 4 days. On day 4, hepatocytes were washed twice and preincubated for 10 min at 37°C in either HBSS buffer, or Ca2+/Mg2+‐free HBSS containing 1 mM EGTA. Compounds dissolved in protein free HBSS buffer were added at final concentration of 1 μM and incubated at 37°C up to 10 min. Midazolam was used as negative control. The hepatocytes were collected in duplicate for each condition. The detailed method of LC‐MS/MS quantification and the in vitro intrinsic clearance calculation are described in the Supplementary Material.

Physiologically‐based pharmacokinetic modeling

A PBPK model was built to understand the impact of OATP inhibition on the PKs of rosuvastatin, carotegrast, and bromfenac in monkeys. The PBPK model was structured using SAAM II software (the Epsilon Group, Charlottesville, VA) with 14 compartments representing major organs, including the lungs, kidneys, brain, muscles, adipose, heart, skin, bones, spleen, gut, liver, and the rest of body, which were connected by the arterial and venous circulating system (Figure S1). All organs were treated as perfusion limited compartments except for the liver, which was permeability limited. A five‐compartmental liver model was adapted from previously publications. , , Briefly, the extracellular compartment (EC) and intracellular compartment (IC) of the liver were divided equally in five compartments and every drug was cleared through each compartment in a sequential manner. A Kp scalar was included in the model, which was equally applied to all predicted Kp values for non‐liver tissues. The Kp scalar was used to optimize and improve the model to capture in vivo distribution profile. The rest of the body compartment lumping other tissues (i.e., eyes, carcass, deep tissue compartments, etc.), in addition to the organs listed above, serviced as a slow distribution organ in the model for better capturing the drug distribution profile. The differential equations describing each compartment and parameter applied in the PBPK model were included in Supplementary Methods and Table S1, respectively.

Biliary clearance was assumed to be the major route of elimination for rosuvastatin and carotegrast, , whereas metabolism in the liver was assumed to be the major pathway of elimination for bromfenac in the model fitting. Urinary excretion of rosuvastatin in monkeys was reported to be ~ 20% of the absorbed dose. , In the rosuvastatin model, the renal clearance was fixed to 20% of total CL (4.4 ml/min/kg), which is comparable to the previous reported value. Urinary excretion of carotegrast was estimated to be 7.5% of total dose from an in‐house bile duct cannulation (BDC) monkey study (data not shown). For bromfenac, no urinary excretion was assumed. The data of plasma and liver concentration was simultaneously fitted to estimate the liver intrinsic passive uptake clearance (CLu,int,passive), active uptake clearance (CLu,int,active), metabolic clearance (CLu,int,meta), or biliary excretion clearance (CLu,int,bile), Kp scalar, and the partition coefficient for the rest of the body (Kp,rest). The initial input of intrinsic CLu,int,active, CLu,int,passive, CLu,int,meta, and CLu,int,bile in the model were scaled from the in vitro intrinsic clearance (Table S3). All of the above parameters were first estimated based on the drug profiles in the absence of RIF. The estimated Kp,rest and Kp scalar values were then fixed in the model to estimate the clearance parameters in the presence of RIF.

RESULTS

In vitro hepatic uptake and biliary excretion of rosuvastatin, carotegrast, and bromfenac

The uptake of rosuvastatin, carotegrast, and bromfenac in suspension monkey hepatocytes are shown in Figure 1. The intracellular accumulation of all three drugs increased in a time‐dependent manner at 37°C incubation. The hepatocyte uptake was almost abolished at 4°C. In the presence of 200 µM of rifamycin SV, the uptake of rosuvastatin and carotegrast were greatly inhibited and appeared to be comparable to the uptake at 4°C. However, rifamycin SV was only able to partially inhibit the hepatocyte uptake of bromfenac. The derived in vitro CL values of hepatic uptake are summarized in Table S2.

FIGURE 1

In vitro evaluation of hepatic uptake and biliary excretion of rosuvastatin, carotegrast, and bromfenac. Intracellular accumulation of rosuvastatin (a, mean ± SD in triplicates), carotegrast (b, mean from duplicates), and bromfenac (c, mean ± SD in triplicates) in suspension hepatocytes and biliary excretion of rosuvastatin and carotegrast in sandwich‐cultured cynomolgus monkey hepatocyte (SCCH), (d, mean ± SD in triplicates)

For both rosuvastatin and carotegrast, biliary clearance is the major elimination path in humans and animals. As expected, higher accumulation was shown in the buffer with Ca2+ compared to the Ca2+ free buffer, suggesting significant biliary excretion detected in SCCH; whereas there was no difference in the accumulation between Ca2+ buffer and Ca2+ free buffer for the negative control, midazolam (Figure 1d). Rosuvastatin was used as a positive control in our in‐house assay and the data aligned with our historical data. As previously published, the major routes of elimination for bromfenac were glucuronidation and P450 oxidation ; therefore bromfenac was not assayed in SCCH.

Pharmacokinetic of rifampin

Following oral administration, the mean plasma concentration of RIF was 20.8 µM at 3.25 h postdosing (0.25 h post IV dose) in group 1 and group 2 (Figure S2). Time to maximum plasma concentration (Tmax) was observed at 3.5–4.5 h post‐RIF oral dose and the liver to plasma ratio (Kp,liver) was about 10. The plasma concentration was above 10 µM 6 h after the IV doses of OATP substrates.

CP‐I and CP‐III exposure in plasma and the liver in the presence and absence of RIF

In the presence of RIF, plasma concentrations of both endogenous CP‐I and CP‐III were increased (Figure 2). Accordingly, the plasma exposure (area under the concentration‐time curve from time of administration up to the time of the last quantifiable concentration [AUClast]) was increased 5.4 and 8.8‐fold for CP‐I and CP‐III, respectively (Table 1). In contrast, the liver concentration was minimally changed at 1 h but increased on average of 3.2 and 3.7‐fold at 6 h for CP‐I and CP‐III, respectively.

FIGURE 2

Plasma and liver concentrations of coproporphyrin CP‐I (a, c) and CP‐III (b, d) in cynomolgus monkeys dosed with a single IV infusion (30 min) of three drug combination solution (0.5 mg/kg each) pretreated with oral amination of vehicle or rifampin (20 mg/kg). Plasma concentrations from group 1 (a, b) are presented as mean ± SD (N = 3); Comparisons between plasma and liver concentrations (c, d) at 1, 2.5, and 6 h from group 2 are presented as mean values (N = 2)

TABLE 1

Pharmacokinetics of rosuvastatin, carotegrast and bromfenac following a single IV infusion (30 min) of three drug combination solution (0.5 mg/kg each) to cynomolgus monkeys pretreated with vehicle or 20 mg/kg rifampin orally (n = 3, mean ± SD)

	Rosuvastatin		Carotegrast		Bromfenac		CP‐I		CP‐III
	Vehicle	Rifampin	Vehicle	Rifampin	Vehicle	Rifampin	Vehicle	Rifampin	Vehicle	Rifampin
T 1/2 (h)	4.21 ± 0.61	4.63 ± 1.03	0.82 ± 0.51	1.49 ± 0.11	1.10 ± 0.46	1.14 ± 0.02	–	–	–	–
Plasma CL (ml/min/kg)	22.02 ± 1.91	9.96 ± 3.18*	6.91 ± 4.00	0.69 ± 0.25*	9.97 ± 1.36	4.72 ± 0.88*	–	–	–	–
V (ml/kg)	489 ± 75	452 ± 210	114 ± 63	45 ± 16*	192 ± 21	138 ± 31	–	–	–	–
Cmax (nM)	1598 ± 155	3302 ± 1061*	4717 ± 1827	22806 ± 6572*	4488 ± 696	8408 ± 2495*	0.77 ± 0.15	4.83 ± 0.26*	0.25 ± 0.06	2.03 ± 0.38*
AUClast (h*nM)	786 ± 64	1842 ± 610*	2625 ± 1209	23770 ± 8626*	2522 ± 335	5401 ± 1087*	12.6 ± 1.00	68.6 ± 18.71*	3.38 ± 0.36	29.7 ± 0.95*

Abbreviations: AUClast, area under the concentration‐time curve from time of administration up to the time of the last quantifiable concentration; CL, clearance; Cmax, peak plasma concentration; CP, coproporphyrin; T 1/2, terminal half‐life; V, volume.

p < 0.05 as compared to the vehicle group.

Pharmacokinetics of rosuvastatin, carotegrast, and bromfenac

In the group 1, the treatment of RIF (20 mg/kg) increased the plasma exposure of rosuvastatin, carotegrast, and bromfenac (Figure 3). The changes were the most prominent for carotegrast with maximum plasma concentration (Cmax) and AUClast increasing 4.8 and 9.1‐fold, respectively (Table 1). The CL of carotegrast correspondingly decreased about 10‐fold in the presence of RIF, whereas the volume of distribution (V) was also reduced about a half from 0.11 to 0.05 L/kg. RIF also increased systemic exposures of rosuvastatin and bromfenac, with the AUClast changes of 2.3 and 2.1‐fold, respectively. The systemic CLs of rosuvastatin and bromfenac were reduced by 2.2‐fold, and 2.1‐fold, respectively. The volume distributions of rosuvastatin and bromfenac were also reduced.

FIGURE 3

Plasma and liver concentrations of rosuvastatin (a, d), carotegrast (b, e) and bromfenac (c, f) following a single IV infusion (30 min) of three drug combination solution (0.5 mg/kg each) in cynomolgus monkeys in the absence or presence of rifampin (20 mg/kg) oral administration. Plasma concentrations from group 1 (a, b, c) are presented as mean ± SD (N = 3); comparisons between plasma and liver concentrations (d, e, f) at 1, 2.5, and 6 h from group 2 are presented as mean values (N = 2)

The group 2 was designed to characterize the impact of OATP inhibition on liver exposure. Although similar increases of plasma exposures for rosuvastatin, carotegrast, and bromfenac were observed by the treatment of RIF, the impact of OATP inhibition on the liver drug exposures appeared to be different. The liver concentrations overlapped at 1, 2.5, and 6 h for rosuvastatin and bromfenac between the vehicle and RIF pretreated groups (Figure 3d,f). For carotegrast, the liver concentrations were comparable at 1 h but were higher at 2.5 h and 6 h in the RIF‐treated group than the vehicle group (Figure 3e and Figure S3). As shown in Figure S3, the average of Kp,liver values were about 100, 17, and 2 for rosuvastatin, carotegrast, and bromfenac, respectively, and were reduced by the treatment of RIF.

PBPK modeling

In order to mechanistically describe the changes of plasma and liver concentrations following the treatment of OATP inhibitor, a monkey PBPK model was constructed to incorporate the in vitro parameters to interpret in vivo monkey PK and measured liver exposure. Empirical scaling factors were assigned to compensate for the discrepancy from in vitro scaled intrinsic clearance measured in hepatocyte models to in vivo parameters yielded from curve fitting (Table S3). The observed and model fitted plasma and liver concentrations are shown in Figure 4. With scaling factors applied to the active hepatic uptake, biliary, and passive diffusion, the model reasonably described the systemic and liver concentrations of all three drugs. The reliability of in vivo parameters obtained from the curve fitting in the model was further verified by the DDI data. The model was able to accurately fit the observed plasma concentration time profile of rosuvastatin, carotegrast, and bromfenac in RIF‐treated monkeys, in which the hepatic uptake clearance was reduced while the passive permeability (CL,u,int,passive) was not changed for all three drugs. As such, the model captured the liver exposures of carotegrast and bromfenac. In contrast, the model overpredicted the liver concentration of rosuvastatin at early time points (1 and 2.5 h). The model fitted parameters and percent of the coefficients of variation (%CV) on parameter estimates are summarized in Table 2. Furthermore, the sensitivity analysis was conducted to elucidate the interplays among CLu,int,passive, CLu,int,active, CLu,int,bile, or CLu,int,met and the plasma and live exposure. As shown in Figure S4, generally, the CLu,int,passive and CLu,int,active are more sensitive parameters to affect plasma concentration, whereas the CLu,int,bile or CLu,int,met are more sensitive to regulate liver exposure of the three OATP substrates.

FIGURE 4

Observed and fitted plasma and liver concentration of rosuvastatin (a, b), carotegrast (c, d), and bromfenac (e, f) in cynomolgus monkeys pretreated with oral amination of vehicle or rifampin (20 mg/kg)

TABLE 2

Estimated parameters from plasma and liver concentrations of rosuvastatin, carotegrast, and bromfenac in cynomolgus monkeys using PBPK model

	Rosuvastatin		Carotegrast		Bromfenac
	Vehicle	Rifampin	Vehicle	Rifampin	Vehicle	Rifampin
CLu,int,active (ml/min/kg)	331.8 (13.8%)	81.7 (8.5%)	1154.1 (5.2%)	95.2 (4.4%)	2300 (18.7%)	820 (10.8%)
CL,u,int,passive (ml/min/kg)	1.0 (22.5%)	0.5 (41%)	4.1(11.8%)	2.9 (32.4%)	13.1 (96.2%)	11.2 (95.3%)
CLu,int,bile (ml/min/kg)	2.8 (1.7%)	2.6 (19.7%)	16.3 (0.022%)	11.7 (0.1%)	NA	NA
CLu,int,meta (ml/min/kg)	NA	NA	NA	NA	49.7 (16.9%)	84.8 (3.8%)
Kp,rest	2.7 (4.5%)	2.7 (fixed)	0.85 (6.1%)	0.85 (fixed)	0.8 (6.3%)	0.8 (fixed)
Kp,scalar	1.1 (3.5%)	1.1 (fixed)	0.16 (29.4%)	0.16 (fixed)	0.5 (27%)	0.5 (fixed)
V (ml/kg)	394	404	132	116	121	114
CL (ml/min/kg)	18.3	11.7	8.3	1.2	6.7	3.3

Plasma from two groups of animals (n = 9) and liver concentrations (n = 2 per time point) were simultaneously fitted to estimate parameters and the coefficients of variation (%CV). All of the above parameters were first estimated in the absence of rifampin. The estimated K p,rest and K p scalar values were then applied in the model to estimate the clearance parameters in the presence of rifampin. The V and CL were calculated from simulated plasma PK with a noncompartmental model (NCA).

Abbreviations: CLu,int,bile, intrinsic biliary excretion clearance; CLu,int,active, intrinsic active uptake clearance; CLu,int,meta, intrinsic metabolic clearance; CL,u,int,passive, intrinsic passive uptake clearance; NA, not applicable; PBPK, physiologically‐based pharmacokinetic.

DISCUSSION

Hepatic transport, including passive diffusion and active sinusoidal membrane transport, is a major player in systemic clearance of a drug, and more importantly controls drug availability for the metabolism and biliary clearance in the liver. Although the passive permeation is a compound‐dependent parameter that relates to physiochemical properties, such as logD and pKa, etc., active hepatic uptake is attributed to membrane transporter(s) expressed on the sinusoid membrane of hepatocytes. On many occasions, the active uptake can be attributed by more than one transporter due to the overlapping of substrate specificity. Therefore, three drugs with different chemotype and metabolic/excretion profiles were studied here to comprehensively evaluate the impact of reduction of OATP activity on their plasma PK and liver exposures in cynomolgus monkeys. In addition, the changes followed by the administration of the OATP inhibitor RIF offer the best case to construct a PBPK model to understand interplays among hepatic uptake, metabolism, and biliary excretions.

Rosuvastatin, bromfenac, and carotegrast are all known to be OATP substrates but have different elimination profiles on metabolism and additional transporter‐mediated uptake and biliary efflux. All three OATP transporters (OATP1B1, 1B3, and 2B1) in the liver are known to contribute to the liver uptake of rosuvastatin. Rosuvastatin is mainly cleared in the bile as the parent drug, with less than 10% cleared through renal excretion in humans. Bromfenac is a nonsteroidal anti‐inflammatory drug, which is a substrate for both OATPs and OAT2. , Bromfenac is mainly eliminated by hepatic biotransformation via a combination of oxidative and conjugative mechanisms as bromfenac glucuronide is excreted in urine and accounts for 82.5% of the administered dose in healthy subjects. Carotegrast, a parent drug of carotegrast methyl developed for the treatment of active ulcerative colitis, is a substrate for OATP1B1 and OATP1B3. Carotegrast is minimally excreted in urine but primarily eliminated in bile via MRP2 in humans. The results observed in the suspension hepatocyte uptake assays are in good agreement with the above literature publications. In vitro hepatocyte uptake of rosuvastatin and carotegrast were almost abolished by 200 µM rifamycin SV in the current study, which is much higher than the reported Ki values of 2 and 3 µM against OATP1B1 and OATP1B3, respectively. On the other hand, the uptake of bromfenac was only partially inhibited by rifampicin SV. This is consistent with earlier report that the half‐maximal inhibitory concentration (IC50) of rifamycin SV for OAT2 was around 200 µM, and the OAT2 activity was not fully inhibited by rifamycin SV in the current assay condition.

The impact of OATP inhibition on plasma and liver exposures was investigated in monkeys receiving a single IV cassette dose of rosuvastatin, bromfenac, and carotegrast at 0.5 mg/kg each. The selection of cassette dosing was to minimize the animal use and intersubject variations. The plasma exposure (AUC) of rosuvastatin, when normalizing to the dose, and the OATP biomarkers CP‐I and CP‐III in the presence and absence of rifampin in our study were consistent with literature data reported in monkeys, , , therefore the data confirmed the lack of interactions among these three compounds in a cassette dose. In addition, the large change of plasma AUC in CP‐III can be due to an additive effect of hepatic and renal CL inhibition of CP‐III, as reported by Shen et al. Although urine sample collection was included in the study design, no meaningful urine excretion data were obtained probably due to sample handling errors. A previous study showed that RIF increased the amount of rosuvastatin excreted in urine due to increase plasma exposure, but the renal CL remains unchanged.

The exposure of RIF dosed at 20 mg/kg in the monkeys was above the threshold of OATP inhibition (Figure S2). Therefore, it is expected that the plasma exposures of rosuvastatin, bromfenac, and carotegrast would increase followed by RIF treatment, although the magnitudes of AUC change are different among the three drugs. Although rifamycin SV fully inhibited hepatic uptake of rosuvastatin in hepatocytes in vitro, RIF treatment only increased rosuvastatin AUC about twofold. One of the explanations is that the clearance of rosuvastatin in monkeys was close to liver blood flow, and thus, the CL of rosuvastatin could be hepatic blood flow‐limited when dosed intravenously. Additionally, rosuvastatin is a substrate of monkey NTCP, , and under the in vitro condition with high concentration of rifamycin SV, the NTCP‐mediated active uptake can be inhibited. The disconnection of in vitro OATP inhibition to in vivo observation likely reflected the contribution of NTCP to rosuvastatin uptake that was not fully inhibited by RIF in vivo. For bromfenac, an approximately twofold AUC increase was also observed in monkeys treated with rifampicin. The smaller DDI could be due to the involvement of OAT2, which is not inhibited by rifampin in the current study. In contrast, AUC increased more extensively with OATP inhibition for carotegrast, indicating that OATP‐mediated transport could be the predominant contributor to the hepatic uptake clearance.

The impacts of RIF inhibition on liver concentrations were different when compared with plasma exposures. The Kp,liver was decreased for all three drugs by the treatment of RIF, but the liver concentrations were not reduced. With incorporation of in vitro clearances obtained from hepatocyte assays, the monkey PBPK model was able to simultaneously fit the exposures of plasma and liver in the presence and absence of OATP inhibitor, RIF. Consistent with literature reports, scaling factors are needed to translate parameter values from in vitro to in vivo. , Based on the PBPK model, both passive permeability and OATP‐mediated active uptake, as well as the involvement of other transporters, determine the Kp,liver. A reduction of 75% was modeled for rosuvastatin active uptake clearance with rifampin inhibition. The average Kp,liver of rosuvastatin in the presence of rifampin was still greater than 1, suggesting that other transporters (e.g., NTCP) can also contribute to the liver uptake in monkeys. The active uptake of bromfenac was only reduced by 64% with RIF inhibition, consistent with the observation in suspension hepatocytes. Given the extensive metabolism of bromfenac, the influence of RIF on its metabolic clearance and biliary excretion are negligible, and therefore the unchanged liver concentration is due to passive permeability and OAT2 activity. Interestingly, the model analysis suggested that biliary excretion of carotegrast was reduced slightly in the presence of RIF while passive permeability remained unchanged, leading to the liver accumulation. Carotegrast was determined to be a substrate of MRP2 in vitro (unpublished in‐house data). Similar profiles were also observed for CP‐I and CP‐III, which were also reported to be MRP2 substrates. Previous studies suggested that RIF could inhibit MRP2 in addition to OATPs in humans. , As shown in Figure S4, the sensitivity analysis suggested that both hepatic uptake inhibition and impaired monkey MPR2 activity by rifampin treatment can partially explain the higher liver exposure of carotegrast, CP‐I, and CP‐III. Aligned with the above discussions, the model sensitivity analysis confirmed that the active uptake clearance was the key factor contributing to the plasma exposure, whereas the metabolism or biliary excretion was critical for regulating liver concentration. One of the limitations of the current PBPK model was that it does not incorporate the enterohepatic circulation (EHC). In humans, rosuvastatin has a long terminal half‐life resulting from EHC. In the current study, the plasma concentrations were able to detect up to 12 h in monkeys. The EHC unlikely occurred for carotegrast as it has low intestinal absorption and therefore is dosed as prodrug to boost the absorption. Therefore, the EHC component may have minimal impact on the current PBPK modeling.

Overall, inhibition of OATP activity increased systemic exposure and reduced the liver to plasma ratio of all three drugs, but the liver exposure of the drugs remained unchanged. Our findings of rosuvastatin in monkeys agree with the earlier report that total radioactivity of 11C‐rosuvastatin was not significantly changed in the human liver in the first 30 min with OATP function attenuated. Because we were able to quantify the rosuvastatin in plasma and the liver for up to 6 h, rather than the total radioactivity (rosuvastatin and its metabolites), which allowed us to construct the PBPK model to understand the interactions among liver OATP uptake, biliary excretion and metabolism based on observed data. In addition, a recent clinical trial data indirectly supported our findings and modeling analysis in monkeys. The pharmacodynamic (PD) effect of GS‐0976, a liver‐targeted ACC inhibitor under clinical development, was not altered by potent OATP inhibitor, although its plasma concentration was increased significantly. Most likely the liver exposure of GS‐0976 was not changed by OATP inhibition. Extending this finding to the liver disease population, such as nonalcoholic fatty liver disease and patients with genetic variants in hepatic transporters, the transporter function can be reduced; however, the liver drug exposure may not be affected in these populations. Understanding factors that regulate the liver drug exposure is critical to estimate the efficacious dose in patients, especially for liver‐targeting therapeutic reagents. It should be noted that OATPs are only one class of transporters in the liver, and there are many other transporters expressed on the sinusoidal and canalicular membrane of hepatocytes. When other extrahepatic CL pathways playing a significant role in overall CL, the inhibition of hepatic uptake (e.g., OATP inhibition) can result in the reduction of liver exposure, which had been showed in the model simulation by Patilea‐Vrana et al. Therefore, it is important to consider all these factors together to understand the tissue exposure of a drug in various clinical scenarios. More importantly, species differences regarding the function of OATP and other transporters between monkeys and humans must be noted and the drug metabolic profiles could also be different. Further investigations are necessary to translate the current observations to humans.

To the best of our knowledge, this is the first study evaluating the impact of OATP inhibition on the plasma and liver exposure of three OATP substrates with distinct elimination routes and PK profiles in monkeys. The study demonstrates that inhibition of OATP activities can increase the plasma exposure of OATP substrates but have minimum impact on liver drug exposure in monkeys. The PBPK model using in vitro parameters in combination with scaling factors is a viable approach to model the interplay of in vivo processes of hepatic transport, drug metabolism, and biliary excretion, and elucidate the differential changes of plasma and liver concentrations.

CONFLICT OF INTEREST

All authors were employees of Gilead Sciences Inc. during this research.

AUTHOR CONTRIBUTIONS

Y.C., X.L., and Y.L. wrote the manuscript. Y.C., and Y.L. designed the research. X.L., J.H., and C.N. performed the research. Y.C., X.L., J.H., C.N., and Y.L. analyzed the data.

Supplementary Material

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