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Research ArticleArticle

Differential Effects of Rifampin and Ketoconazole on the Blood and Liver Concentration of Atorvastatin in Wild-Type and Cyp3a and Oatp1a/b Knockout Mice

Jae H. Chang, Justin Ly, Emile Plise, Xiaolin Zhang, Kirsten Messick, Matthew Wright and Jonathan Cheong
Drug Metabolism and Disposition June 2014, 42 (6) 1067-1073; DOI: https://doi.org/10.1124/dmd.114.057968

Abstract

Atorvastatin is eliminated by CYP3A4 which follows carrier-mediated uptake into hepatocytes by OATP1B1, OATP1B3, and OATP2B1. Multiple clinical studies demonstrated that OATP inhibition by rifampin had a greater impact on atorvastatin systemic concentration than itraconazole-mediated CYP3A4 inhibition. If it is assumed that the blood and hepatocyte compartments are differentiated by the concentration gradient that is established by OATPs, and if the rate of uptake into the hepatocyte is rate-determining to the elimination of atorvastatin from the body, then it is hypothesized that blood concentrations may not necessarily reflect liver concentrations. In wild-type mice, rifampin had a greater effect on systemic exposure of atorvastatin than ketoconazole, as the blood area under the blood concentration-time curve increased 7- and 2-fold, respectively. In contrast, liver concentrations were affected more by ketoconazole than by rifampin, as liver levels increased 21- and 4-fold, respectively. Similarly, in Cyp3a knockout animals, 39-fold increases in liver concentrations were observed despite insignificant changes in the blood area under the blood concentration-time curve. Interestingly, blood and liver levels in Oatp1a/b knockout animals were similar to wild types, suggesting that Oatp1a/b knockout may be necessary but not sufficient to completely describe atorvastatin uptake in mice. Data presented in this work indicate that there is a substantial drug interaction when blocking atorvastatin metabolism, but the effects of this interaction are predominantly manifested in the liver and may not be captured when monitoring changes in the systemic circulation. Consequently, there may be a disconnect when trying to relate blood exposure to instances of hepatotoxicity because a pharmacokinetic-toxicity relationship may not be obvious from blood concentrations.

Introduction

Organic anion-transporting polypeptides (OATPs) are a family of drug transporters, encoded by SLCO genes that mediate uptake into various compartments within the body. One of the main physiologic function of OATPs is the elimination of bilirubin (Scharschmidt et al., 1975; Cui et al., 2001), but they also contribute to the disposition of a wide array of xenobiotics, including statins. OATPs are ubiquitously expressed (e.g., OATP4A1) or localized to a particular tissue such as OATP1B1 in the liver (Hagenbuch and Meier, 2004). In addition to OATP1B1, OATP1B3 and OATP2B1 are found at the sinusoidal membrane of hepatocytes (Kullak-Ublick et al., 2001). There is increasing evidence of drug interactions involving these liver-bound OATPs, as loss of function due to chemical inhibition or genetic polymorphisms has been associated with clinically relevant drug interactions (Kalliokoski and Niemi, 2009; Shitara et al., 2013) and hyperbilirubinemia (van de Steeg et al., 2012; Chang et al., 2013).

Atorvastatin (Lipitor) is a widely prescribed lipid-lowering statin that targets HMG-CoA reductase. Metabolism consists of hydroxylation and beta-oxidation; and in particular, the role of CYP3A4 has been well-documented as it catalyzes the formation of two major active metabolites, 2-hydroxy- and 4-hydroxy-atorvastatin (Lea and McTavish, 1997). However, before atorvastatin can get metabolized, it needs to enter the liver, which is mediated by OATP1B1 (Kameyama et al., 2005; Lau et al., 2007), OATP1B3 (Schwarz et al., 2011), and OATP2B1 (Grube et al., 2006). Among them, OATP2B1 exhibits one of the highest affinities toward atorvastatin with Km of 200 nM (Grube et al., 2006).

Multiple clinical studies have attempted to discriminate the relative contributions of CYP3A4 and OATPs to the disposition of atorvastatin and for assessing drug interaction liabilities. When rifampin was given as a single dose to inhibit OATPs, 10- to 12-fold increases in atorvastatin plasma concentrations were observed (Lau et al., 2007; Maeda et al., 2011). Interestingly, a CYP3A4 blocker itracontazole was less effective to yield only 2- to 3-fold increases (Kantola et al., 1998; Mazzu et al., 2000), and in one study no effect (Maeda et al., 2011), in atorvastatin plasma concentrations. These results suggest that hepatic uptake via OATPs may be the critical step in the disposition of atorvastatin. Indeed, studies have shown that uptake of atorvastatin into hepatocytes was rate-determining, and, therefore, limiting OATP function would have a bigger impact than inhibiting CYP3A4 on the systemic exposure of atorvastatin (Lau et al., 2007; Watanabe et al., 2010). However, although only modest changes were observed with atorvastatin in the plasma, modulating CYP3A4 activity may have a profound effect on liver concentrations because the liver is where atorvastatin is eliminated from the body (Watanabe et al., 2009).

The objective of this study was to investigate changes in blood and liver concentrations of atorvastatin when blocking uptake (via single-dose rifampin administration or with Oatp1a/b knockout mice) or metabolism (via ketoconazole administration, which is also a CYP3A inhibitor, or with Cyp3a knockout mice). In the vehicle-treated wild-type animals, the liver-to-blood concentration ratio (Kp) would be greater than 1 because uptake into the liver governs the elimination of atorvastatin (Fig. 1A). It is hypothesized that in the presence of rifampin or in the Oatp1a/b knockout animals (Fig. 1B), blood concentrations would rise because Oatps directly modulate blood atorvastatin levels. Meanwhile, the liver concentrations would rise or fall depending on the magnitude of variation in the blood compartment, as atorvastatin will enter the liver via passive process. Overall, Kp would be reduced to a value less than 1. Conversely, in the presence of ketoconazole or in the Cyp3a knockout group (Fig. 1C), both the blood and liver concentrations would be higher than the vehicle-treated wild-type group, yielding large increases in Kp. But it is expected that constraining metabolism will have a more profound effect on the liver because metabolism directly modulates liver atorvastatin levels. Therefore, for compounds whose clearance is dependent on uptake into eliminating organs, drug interaction involving drug transporters and drug-metabolizing enzymes may manifest differently in the systemic circulation compared with the organs. Although atorvastatin is typically dosed as oral tablets, the current study used i.v. administration to focus the study to the liver. FVB mouse strains were employed because our preliminary data showed that rifampin and ketoconazole also showed differential effect on atorvastatin blood levels as observed in the clinic, and because FVBs were the background strain of Cyp3a and Oatp1a/1b knockout mice that were used to further investigate the varying contribution of metabolism and uptake in atorvastatin disposition.

Fig. 1.
Fig. 1.

Illustration detailing the proposed hypothesis in which the blood and the hepatocyte compartment do not reach rapid equilibrium because the rate of uptake into the liver dictates the elimination of atorvastatin from the body. (A) In the absence of any inhibitors or loss of proteins, liver concentration of atorvastatin is higher than the blood to yield Kp value >1. (B) In the presence of rifampin or the loss of all OATP proteins, large increases would be observed in the blood, whereas modulation of liver levels would be dependent on the extent of change in the blood compartment as entry into the liver will be a passive process to yield Kp value <1. (C) In the presence of ketoconazole or the loss of CYP3a protein, extensive rise in liver concentrations would yield Kp value that is greater than the control; however, continued function of OATPs may limit the increases in the blood compartment.

Materials and Methods

Chemicals.

Ketoconazole, rifampin, and cyclosporine were purchased from Fisher Scientific (Waltham, MA). Atorvastatin calcium was purchased from Crescent Chemical (Islandia, NY). Pitavastatin and rosuvastatin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). O-hydroxy atorvastatin lactone d5 was purchased from US Biologic (Salem, MA). Polyethylene glycol 400 (PEG400) was purchased from Avantor Performance Materials (Center Valley, PA). The 0.9% sodium chloride (saline) was purchased from Hospira (Providence, RI). Dulbecco’s modified Eagle’s medium was purchased from Invitrogen (Carlsbad, CA). All other chemicals and reagents were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO). Multiscreen HTS 96-well filter plates and HTS Vacuum Manifold were obtained from EMD Millipore (Darmstadt, Germany). Parent Human Embryonic Kidney (HEK) cells and OATP1B1-, OATP1B3-, and OATP2B1-transfected HEK cells were obtained from Y. Sugiyama (University of Tokyo, Tokyo, Japan).

OATP1B1, OATP1B3, and OATP2B1 Inhibition Assay.

OATP1B1, OATP1B3, and OATP2B1 inhibition assays were run as previously described (Chang et al., 2013). Briefly, the parental HEK cells and OATP1B1-, OATP1B3-, and OATP2B1-transfected HEK cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 400 µg/mL G418 at 37°C with 5% CO2 and 95% humidity. The inhibition studies were carried out between 48 and 72 hours after seeding the cells at a density of 5.0 × 105 cells/well in 24-well tissue culture plates coated with poly-d-lysine and laminin. Cells were preincubated at 37°C in Hanks’ balanced salt solution (HBSS) for 10 minutes. The uptake inhibition studies were initiated with 0.1–200 μM ketoconazole or rifampin along with 0.1 µM atorvastatin. For the positive control, inhibition of uptake of 0.1 µM pitavastatin by rifampin was measured for OATP1B1 and OATP1B3 (Matsushima et al., 2008), whereas the inhibition of uptake of 0.1 µM rosuvastatin by cyclosporine was measured for OATP2B1 (Ho et al., 2006). Following 5-minute incubation at 37°C, the reaction was terminated by rinsing the cells twice with 1 mL ice-cold HBSS buffer. The cells were then sonicated in 200 µL HBSS for 5 minutes and precipitated with an equal volume of acetonitrile. Each sample was centrifuged, and the supernatant was injected onto liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to monitor for atorvastatin.

IC50 values are presented as mean ± S.D. with a minimum of three wells used to generate each data point. The IC50 was estimated by a sigmoidal inhibition model and was fit to:Embedded Imagewhere V is the uptake of atorvastatin (or pitavastatin and rosuvastatin for the positive control) in the presence of the test inhibitors, V0 is the uptake of atorvastatin (or pitavastatin and rosuvastatin for the positive control) in the absence of the inhibitor, I is the inhibitor concentration, and n is the hillslope.

In Vivo Pharmacokinetics Studies.

Female FVB (wild-type), Cyp3a knockout, and Oatp1a/b knockout mice (Taconic Farms, New York, NY) were housed at controlled temperature and humidity in an alternating 12-hour light/dark cycle with free access to food and water before the study. All in vivo studies were performed in accordance with Institutional Animal Care and Use Committee guidelines and in harmony with the Guide for Laboratory Animal Care and Use. Six animals from each mouse model were randomly divided into two groups. One group was dedicated to collecting blood pharmacokinetics, whereas the other group was used to collect blood and liver at 15 minutes postdose. Six animals from each mouse model were pretreated either with 35 mg/kg ketoconazole (60% PEG400/40% saline) given orally 30 minutes prior to atorvastatin administration (Lau et al., 2006b), or with 20 mg/kg rifampin (10% DMSO/10% PEG400/80% saline) given i.v. via tail vein 5 minutes prior to atorvastatin administration (van Waterschoot et al., 2009), or with the appropriate vehicle control. Following pretreatments, all animals were given a single 5 mg/kg i.v. dose of atorvastatin (25% PEG400/75%). In the blood pharmacokinetics group, each animal was serially bled via tail nick at 0.033, 0.083, 0.25, 0.5, 1, 2, and 4 hours postdose. A total of 15 µL blood was collected from each time point and diluted with 60 µL water containing 1.7 mg/mL EDTA. In the second group, blood was collected and the livers were excised at 15 minutes postdose. This time point was chosen because previous work with pravastatin showed that liver levels may quickly decline and Kp ratios may plateau after 30 minutes in some strains (Iusuf et al., 2012). The 15-minute time point was the earliest at which it was technically feasible. All blood and liver samples were stored at approximately −80°C until thawed for LC-MS/MS analysis.

Sample Analysis.

Atorvastatin concentrations in blood and liver were determined by LC-MS/MS. The LC-MS/MS system includes a Shimadzu SBM-20A controller with LC-30AD pumps, a Shimadzu Nexera ×2 autosampler with a CTO-20AC column oven (Shimadzu, Columbia, MD), and an AB Sciex API 5500 quadrupole ion trap mass spectrometer (Applied Biosystems, Foster City, CA). Blood was diluted 5-fold with water containing 0.05% formic acid, and the liver was homogenized by using 5-fold volume of water containing 0.05% formic acid. Following protein precipitation (with 3× volume of acetonitrile containing o-hydroxy atorvastatin lactone d5 as the internal standard), the supernatant was injected onto a Phenomenex Kinetex XC18 column (50 × 2 mm, 2.7 µm particle size). The aqueous mobile phase was water with 0.1% formic acid (A), and the organic mobile phase was acetonitrile with 0.1% formic acid (B). The gradient was the following: 20% B for the first 0.2 minutes, increased to 80% B from 0.2 to 2 minutes, maintained at 80% B for 0.5 minutes, and decreased to 20% B within 0.03 minutes. The total run time was 3 minutes with flow rate at 1 ml/min. The ionization was conducted in the positive electrospray ionization ion mode using m/z 559.1->440.1 for atorvastatin and 562.1->453.1 for 2-hydroxy atorvastatin lactone d5 as the internal standards for quantitation. The injection volume was 10 µL.

Atorvastatin concentrations were determined by using a 12-point calibration curve. Peak area ratio of parent/internal standard was plotted against nominal analyte concentrations, which was fit with quadratic regression using 1/x weighting. The R2 was between 0.98 and 0.99, with nominal concentration range between 0.193 and 11940 nM for blood and 0.609 and 3974 nM for liver. The limit of quantitation was 0.193 nM for blood and 0.609 nM for the liver.

Data Analysis.

Area under the blood concentration-time curve (AUC) was determined by noncompartmental methods using Phoenix WinNonlin, version 6.3 (Pharsight, Mountain View, CA). AUCs were transformed to natural logarithm, and analysis of variance (GraphPad Prism 5, San Diego, CA) was used to analyze differences between the vehicle-, ketoconazole-, and rifampin-treated groups within a particular animal model. The significance of difference was determined using Tukey’s post hoc test, and Student’s paired t test was used to determine the differences between rifampin- and ketoconazole-treated groups with vehicle. Mean (±S.D.) was reported, and P < 0.05 was considered statistically significant.

Results

Pharmacokinetics of Atorvastatin.

Atorvastatin was rapidly eliminated from the blood following i.v. administration with an apparent half life ranging between 30 and 50 minutes. Figure 2 shows that, in the wild-type and Oatp1a/1b knockout groups, atorvastatin levels were below the limit of quantitation at 2 hours, whereas atorvastatin was measurable out to 4 hours for the remaining groups. Table 1 summarizes the AUC of atorvastatin in the absence or presence of rifampin and ketoconazole to inhibit Oatps and Cyp3a, respectively. AUC of atorvastatin in the vehicle-treated animals was 0.957 ± 0.385 µM × hour. When coadministered with rifampin, the exposure of atorvastatin increased 7.3-fold to 7.01 ± 4.89 µM × hour. Changes associated with ketoconazole were not significant with AUC of 2.07 ± 1.47 µM × hour. This differential modulation of atorvastatin systemic concentration was consistent with multiple observations in the clinic (Kantola et al., 1998; Mazzu et al., 2000; Lau et al., 2007; Maeda et al., 2011).

Fig. 2.
Fig. 2.

Summary of blood concentration-time profile of atorvastatin following 5 mg/kg i.v. administration in (A) wild-type FVB (WT), (B) Oatp1a/b knockout (Oatp KO), and (C) Cyp3a knockout (Cyp3a KO) animals (N = 3). In each panel representing the various mouse models, the circle, square, and triangle symbols represent vehicle-treated (Veh), rifampin-treated (Rif), and ketoconazole-treated (Keto) groups, respectively. Data are presented as mean ± S.D.

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TABLE 1

Summary of mean atorvastatin blood AUC (±S.D.) when administered 5 mg/kg i.v. in the absence and presence of 20 mg/kg i.v. rifampin or 35 mg/kg oral ketoconazole in various animal models (N = 3); *P < 0.05

To further probe the contribution of uptake and metabolism, the disposition of atorvastatin was evaluated in Oatp1a/b and Cyp3a knockout mice. The Cyp3a knockout mice are deficient for all eight-cluster Cyp3a genes, which most likely represent all the human CYP3A in the liver, kidney, and intestines (van Herwaarden et al., 2007). Further analyses have shown that UDP-glucuronosyltransferase (UGT) mRNA levels were not different than the wild-type animals (N. Scheer, personal communication). The exposure of atorvastatin in the vehicle-treated Cyp3a knockout mice was 1.27 ± 0.30 µM × hour. As reported in the clinical microdosing study by Maeda et al. (2011), the exposure of atorvastatin between the wild-type and the Cyp3a knockout group was not statistically different. Coadministration of ketoconazole had no further effect, but rifampin caused 2.7-fold elevation of atorvastatin exposure to yield 3.45 ± 0.66 µM × hour.

The Oatp1a/b knockout mice are devoid of the Slco1a/1b cluster genes, which most likely represent human OATP1B1 and OATP1B3, but still express Oatp2b1 (van de Steeg et al., 2010). Further analyses have shown that Cyp3a and UGT mRNA levels were not different than the wild-type animals (L. Salphati, personal communication). In the vehicle-treated Oatp1a/b knockout animals, the AUC was 1.66 ± 0.56 µM × hour. As observed in the Cyp3a knockout group, just a modest elevation of atorvastatin exposure was observed with ketoconazole at 3.13 ± 0.36 µM × hour. Interestingly, although there was little change in atorvastatin exposure in the Oatp1a/b knockout compared with vehicle-treated wild-type animals, rifampin enhanced atorvastatin levels by 5.2-fold to 8.56 ± 6.3 µM × hour, suggesting that atorvastatin uptake into the liver may be mediated by additional transporters such as Oatp2b1.

Liver Concentration of Atorvastatin.

Atorvastatin concentrations in the liver following i.v. administration were monitored at 15 minutes postdose. Table 2 summarizes the liver concentrations of atorvastatin in the absence or presence of rifampin and ketoconazole in various animal models. Overall, unlike the systemic circulation, blocking metabolism had a bigger impact than uptake on the liver levels. In the wild-type animals, atorvastatin liver concentration was 1.15 ± 0.61 nmol/g tissue. This was associated with liver-to-blood concentration ratio (Kp) of 3.3, as shown in Figure 3, suggesting that active uptake allows atorvastatin to preferentially cross the sinusoidal barrier to reside in hepatocytes. In the presence of rifampin, the Kp value fell below unity to 0.33, as 3.7-fold increase in liver concentration was accompanied by 37-fold increases in the blood, supporting the view that active uptake is required for atorvastatin to cross the blood-hepatocyte barrier. Conversely, Kp increased to 34 with ketoconazole, which is probably driven by the 21-fold boost in the liver concentration to 24.3 ± 5.5 nmol/g tissue. In the vehicle-treated Cyp3a knockout group, considerable elevations in liver concentration were observed at 44.5 ± 15.8 nmol/g tissue, resulting in Kp value of 82. Coadministration of rifampin or ketoconazole did not have a significant impact on the liver levels, and the Kp values ranged between 13 and 82. In the vehicle-treated Oatp1a/b knockout group, liver levels were unchanged compared with the vehicle-treated wild-type group. However, rifampin did augment the liver levels by 2-fold to 3.72 ± 0.30 nmol/g tissue and Kp dropped below 1, which supported the observation that atorvastatin uptake into the liver may be mediated by additional transporters sensitive to rifampin. In the presence of ketoconazole, Kp increased as the liver concentration reached 31.6 ± 8.2 nmol/g tissue.

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TABLE 2

Summary of mean atorvastatin liver and blood concentration (±S.D.) when administered 5 mg/kg i.v. in the absence and presence of 20 mg/kg i.v. rifampin or 35 mg/kg ketoconazole in various animal models

Livers were sampled at 15 minutes postdose (N = 3); *P < 0.05.

Fig. 3.
Fig. 3.

Summary of liver-to-blood concentration ratio (Kp) of atorvastatin following 5 mg/kg i.v. administration (N = 3). Wild-type FVBs are represented by solid black bars, Cyp3a knockouts are represented by checkered bars, and Oatp1a/b knockout mice are represented by solid gray bars. Each animal group were treated with either vehicle (Veh), rifampin (Rif) or ketoconazole (Keto). Data are presented as mean ± S.D. *P < 0.05.

Inhibition of Atorvastatin Uptake of OATP1B1, OATP1B3, and OATP2B1.

The inhibition of atorvastatin uptake by rifampin or ketoconazole was assessed in HEK cells expressing OATP1B1, OATP1B3, and OATP2B1. Inhibition, measured as IC50, is summarized in Table 3. Rifampin block of 0.1 µM pitavastatin uptake was run as a positive control for OATP1B1 and OATP1B3 to yield IC50 values of 1.4 ± 0.1 µM and 1.2 ± 0.6 µM, respectively, which are within range of published Ki values (Matsushima et al., 2008). Although rifampin was more potent than ketoconazole, the uptake of atorvastatin by OATP1B1 and OATPY1B3 was also reduced at higher ketoconazole concentrations. Specifically, rifampin IC50 values against atorvastatin uptake in OATP1B1 and OATP1B3 were 2.0 ± 0.6 μM and 1.4 ± 0.1 μM, respectively, whereas ketoconazole IC50 values in OATP1B1 and OATP1B3 were 23.9 ± 3.2 μM and 28.4 ± 5.7 μM, respectively. IC50 of cyclosporine against the uptake of 0.1 µM rosuvastatin was run as a positive control for OATP2B1 to yield values of 0.10 ± 0.01 μM, which is consistent with published values (Ho et al., 2006). Both rifampin and ketoconazole were less effective in inhibiting OATP2B1 uptake of atorvastatin, as the IC50 values were 160 ± 48 μM and 211 ± 58 μM, respectively. When administered to mice in vivo, the blood concentration of rifampin and ketoconazole at 15 minutes postdose was approximately 19 μM and 4 μM, respectively (data not shown). The Ki of ketoconazole against CYP3A4 was previously reported at 14.9 nM (Gibbs et al., 1999), whereas rifampin did not have an effect on atorvastatin metabolism (Lau et al., 2006a). These data suggest that rifampin may be affecting only OATP1B1 and OATP1B3 activity, whereas ketoconazole may be specific to only blocking Cyp3A.

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TABLE 3

Summary of mean IC50 ± S.D. of ketoconazole or rifampin against atorvastatin uptake in HEK-transfected OATP1B1, OATP1B3, and OATP2B1 cells (N = 3)

Discussion

As combination drug therapy becomes more common, the need to better understand drug interactions is growing. Once believed to be most applicable for drug-metabolizing enzymes such as CYP3A4, interpretation of drug interactions has more frequently included drug transporters. Consequently, Food and Drug Administration guidance on drug interaction encompasses scenarios for both metabolizing enzymes and drug transporters (FDA, 2012). It is assumed that the systemic concentration will mirror changes in the organs and tissues. However, depending on the mechanisms controlling drug disposition, drug interactions may manifest differently in the blood compared with other compartments within the body (Watanabe et al., 2009). The objective of this study was to investigate changes in blood and liver concentrations of atorvastatin when blocking uptake (via single-dose rifampin administration and with Oatp1a/b knockout mice) or metabolism (via ketoconazole administration, which is also a CYP3A inhibitor, and with Cyp3a knockout mice).

Atorvastatin is extensively metabolized with contributions from CYP3A4 (Lea and McTavish, 1997), but a prerequisite to metabolism is active uptake into the liver, which is mediated by OATP1B1 (Kameyama et al., 2005; Lau et al., 2007), OATP1B3 (Schwarz et al., 2011), and OATP2B1 (Grube et al., 2006). Multiple clinical studies have shown that rifampin had a larger effect on atorvastatin plasma concentration than itraconazole (Kantola et al., 1998; Mazzu et al., 2000; Lau et al., 2007; Maeda et al., 2011). These observations may initially indicate that atorvastatin drug interactions involving OATPs may be of greater consequence than with CYP3A. In fact, the prescribing recommendation to avoid the risk of myalgia and rhabdomyolysis (LIPITOR, 2012), which is driven by systemic concentrations, is more cautious for OATP inhibitors such as cyclosporine (Ho et al., 2006) and protease inhibitors (Chang et al., 2013) than for itraconazole. However, if it is assumed that the blood and the hepatocyte compartments are differentiated by the concentration gradient that is established by OATPs, and if the rate of uptake into the hepatocyte is rate-determining to the elimination of atorvastatin from the body, it is hypothesized that the systemic concentrations may not necessarily reflect liver concentrations, especially in the presence of various inhibitors. Specifically, big increases in the systemic concentration are expected with rifampin because active uptake directly influences the depletion of atorvastatin from the blood (Fig. 1B). When OATPs are removed, atorvastatin will rely on passive diffusion to enter the hepatocytes, and, therefore, liver levels would depend on the concentration gradient that is established between the blood and hepatocyte compartment. In this study, Kp value would decrease and fall below unity. Conversely, big increases in the liver are expected with itraconazole or ketoconazole to yield a higher Kp value because metabolism directly influences the depletion of atorvastatin in the liver (Fig. 1C). Although the systemic concentration may rise if there is a big enough modulation in the liver, elevation of atorvastatin levels in the liver does not necessarily imply that equivalent changes will be observed in the blood compartment because the concentration gradient established by OATPs remains intact. This effect of reducing metabolism is in contrast to compounds like midazolam whose elimination is not limited by uptake into an organ of elimination, and whose systemic concentration would reflect the liver concentration. Therefore, for compounds whose clearance is dependent on uptake into eliminating organs, drug interaction involving drug transporters and drug-metabolizing enzymes may manifest differently in the systemic circulation than in organs and tissues.

Overall, Figure 2 shows that the elimination of atorvastatin was rapid, as evidenced by concentrations falling below the limit of quantitation after 4 hours postdose for most groups of animals, which was consistent with previous observations (DeGorter et al., 2012). Following concomitant administration of rifampin or ketoconazole, Tables 1 and 2 showed that these inhibitors produced differential effects on the blood and liver concentrations of atorvastatin in the wild-type animals. Specifically, rifampin had a greater effect on atorvastatin systemic exposure than ketoconazole. In contrast, ketoconazole had a greater effect on the liver levels than with rifampin, as hypothesized. The divergent effect in the blood and the liver is apparent in the Kp ratio, as shown in Figure 3. In the vehicle-treated wild-type group, Kp ratio was greater than unity at 3.3, confirming that atorvastatin requires active uptake into the hepatocytes. This value is within range of what was described previously (Chen et al., 2007; DeGorter et al., 2012; Higgins et al., 2014). Kp values declined to 0.33 with rifampin, further supporting the claim that uptake is required for atorvastatin. Meanwhile, ketoconazole augmented the Kp value to 34, which corresponds to the large accumulation of atorvastatin observed in the liver.

Although chemical inhibitors are valuable tools to investigate the function of a specific enzyme or transporter, data interpretation and analysis can be confounded by the possibility of cross-reactivity due to nonspecificity. Table 3 shows that, whereas rifampin was at least 10-fold more potent than ketoconazole against OATP1B1 and OATP1B3, higher concentrations of ketoconazole blocked the uptake of atorvastatin. To further differentiate contributions from uptake and metabolism for atorvastatin disposition, Cyp3a and Oatp1a/b knockout animals were used. As observed in the ketoconazole-treated wild-type animals, Cyp3a knockout group exhibited extensive changes only in the liver. Rifampin increased the blood exposure, but ketoconazole did not produce additional effects. As illustrated in Figure 1C, these data suggest that there is a substantial drug interaction when opposing atorvastatin metabolism, but this is only manifested in the liver and may not be captured if monitoring changes in the systemic concentration. Therefore, there may be disconnects when trying to relate exposure to instances of hepatotoxicity, because defining a pharmacokinetic-toxicity relationship may not be obvious when using plasma concentrations. It should be noted that the translatability of the aforementioned drug interaction in the preclinical models to the clinic is unknown, especially because the metabolism profile may be different between mice and humans (Black et al., 1998). Moreover, the implication of such drug interaction in the liver may seem inconsequential because the frequency of reported hepatotoxicity with atorvastatin is rare considering its prevalent use. However, this may be attributed to the fact that atorvastatin is already contraindicated with strong CYP3A4 inhibitors (LIPITOR, 2012); and indeed, hepatotoxicity was observed when atorvastatin was administered with CYP3A4 inhibitor diltiazam (Lewin et al., 2002; Liu et al., 2010). In addition, data presented in this study provide the first set of evidence on the potential risk of hepatotoxicity when blocking metabolism, despite only small changes in the plasma compartment. Nonetheless, experiments in humanized CYP3A animal models to monitor for both atorvastatin and its metabolites are planned to further investigate CYP3A-atorvastatin drug interaction.

There are differing reports with atorvastatin disposition in Oapt1a/1b knockout animals. Following i.v. administration, one study saw no alterations of atorvastatin plasma levels (DeGorter et al., 2012), whereas another showed 17-fold increase in blood AUC (Higgins et al., 2014). These discrepancies may be attributed to differences in study design such as dose and sex/strain differences. Data presented in this work are more aligned with DeGorter et al. (2012), because no differences were observed between wild-type and Oatp1a/b knockout animals. It is not clear why there are discrepancies between various laboratories, but it is noteworthy to mention that, in addition to atorvastatin, data in Higgins et al. (2014) were different for simvastatin (DeGorter et al., 2012) and also for pravastatin in the FVB mice (Iusuf et al., 2012). Interestingly, coadministration of rifampin in the Oatp1a/b knockouts generated an additional increase in atorvastatin concentration, as illustrated in Figure 1B, suggesting that removing Oatp1a/b may be necessary but not sufficient to prevent atorvastatin uptake. Although atorvastatin is also a good substrate for OATP2B1 (Grube et al., 2006), Table 3 shows that rifampin is unlikely to impact Oatp2b1 activity, suggesting that there may be additional rifampin-sensitive transporters in the mice that can modulate the uptake of atorvastatin into liver. These data with atorvastatin show that, unlike the Cyp3a knockout models, the Oatp1a/b knockout models were not able to mimic the observed clinical interaction. These results demonstrate the need to further characterize and validate the drug transporter mouse models. In particular, for well-studied drugs such as simvastatin, which is not a substrate for Oatp2b1 (Grube et al., 2006), the Oatp1a/b knockout or the humanized OATP1B1/OATP1B3 models may be useful tools to understand human disposition and to assess clinical drug interactions. However, the utility of animal models focusing on a specific transporter may be limited for compounds whose distribution depends on multiple transporters such as with atorvastatin.

In summary, atorvastatin concentrations in the blood and liver were investigated when uptake mediated by OATP or metabolism catalyzed by CYP3A was blocked. Both chemical inhibitors and knockout animal models were used to better understand the disposition and drug interaction liability of atorvastatin. This is one of the first studies demonstrating that there are differential effects in the blood and liver concentration of atorvastatin, depending on which pathway is obstructed. Specifically, blood levels were most affected when the uptake pathway was restrained, whereas the liver levels were most affected when the metabolism pathway was reduced. These data indicate that important drug interaction may transpire in tissues and organs that may not necessarily be described by the systemic circulation.

Acknowledgments

The authors thank Shannon Stainton, Roxanne Vega, and the entire In Vivo Support group for outstanding support in the in vivo studies. We also thank Drs. Edna Choo and Laurent Salphati for insightful discussions and comments on the manuscript and Dr. Nico Scheer for helpful information regarding the Cyp3a knockout animals.

Authorship Contributions

Participated in research design: Chang, Ly.

Conducted experiments: Ly, Cheong, Plise, Zhang, Messick.

Performed data analysis: Chang, Ly, Cheong.

Wrote or contributed to the writing of the manuscript: Chang, Ly, Cheong, Wright, Zhang.

Footnotes

Abbreviations

AUC
area under the blood concentration-time curve
HBSS
Hanks’ balanced salt solution
HEK
Human Embryonic Kidney
LC-MS/MS
liquid chromatography-mass spectrometry/mass spectrometry
OATP
organic anion-transporting polypeptide
PEG400
polyethylene glycol 400
UGT
UDP-glucuronosyltransferase

References

  1. ↵
    1. Black AE,
    2. Sinz MW,
    3. Hayes RN, and
    4. Woolf TF
    (1998) Metabolism and excretion studies in mouse after single and multiple oral doses of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin. Drug Metab Dispos 26:755–763.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Chang JH,
    2. Plise E,
    3. Cheong J,
    4. Ho Q, and
    5. Lin M
    (2013) Evaluating the in vitro inhibition of UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP in predicting drug-induced hyperbilirubinemia. Mol Pharm 10:3067–3075.
    OpenUrlCrossRef
  3. ↵
    1. Chen C,
    2. Lin J,
    3. Smolarek T, and
    4. Tremaine L
    (2007) P-glycoprotein has differential effects on the disposition of statin acid and lactone forms in mdr1a/b knockout and wild-type mice. Drug Metab Dispos 35:1725–1729.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Cui Y,
    2. König J,
    3. Leier I,
    4. Buchholz U, and
    5. Keppler D
    (2001) Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. J Biol Chem 276:9626–9630.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. DeGorter MK,
    2. Urquhart BL,
    3. Gradhand U,
    4. Tirona RG, and
    5. Kim RB
    (2012) Disposition of atorvastatin, rosuvastatin, and simvastatin in oatp1b2−/− mice and intraindividual variability in human subjects. J Clin Pharmacol 52:1689–1697.
    OpenUrlCrossRefPubMed
  6. ↵
    1. FDA
    (2012) FDA Guidance for Industry: Drug Interaction Studies — Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations.
  7. ↵
    1. Gibbs MA,
    2. Thummel KE,
    3. Shen DD, and
    4. Kunze KL
    (1999) Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes: comparison of Ki values and impact of CYP3A5 expression. Drug Metab Dispos 27:180–187.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Grube M,
    2. Köck K,
    3. Oswald S,
    4. Draber K,
    5. Meissner K,
    6. Eckel L,
    7. Böhm M,
    8. Felix SB,
    9. Vogelgesang S,
    10. Jedlitschky G,
    11. et al.
    (2006) Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart. Clin Pharmacol Ther 80:607–620.
    OpenUrlCrossRefPubMed
  9. ↵
    Hagenbuch B and Meier PJ (2004) Organic anion transporting polypeptides of the OATP/SLC21 family: phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties. Pflugers Arch 447:653-665.
  10. ↵
    1. Higgins JW,
    2. Bao JQ,
    3. Ke AB,
    4. Manro JR,
    5. Fallon JK,
    6. Smith PC, and
    7. Zamek-Gliszczynski MJ
    (2014) Utility of Oatp1a/1b-knockout and OATP1B1/3-humanized mice in the study of OATP-mediated pharmacokinetics and tissue distribution: case studies with pravastatin, atorvastatin, simvastatin, and carboxydichlorofluorescein. Drug Metab Dispos 42:182–192.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Ho RH,
    2. Tirona RG,
    3. Leake BF,
    4. Glaeser H,
    5. Lee W,
    6. Lemke CJ,
    7. Wang Y, and
    8. Kim RB
    (2006) Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression, and pharmacogenetics. Gastroenterology 130:1793–1806.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Iusuf D,
    2. Sparidans RW,
    3. van Esch A,
    4. Hobbs M,
    5. Kenworthy KE,
    6. van de Steeg E,
    7. Wagenaar E,
    8. Beijnen JH, and
    9. Schinkel AH
    (2012) Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice. Mol Pharm 9:2497–2504.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Kalliokoski A and
    2. Niemi M
    (2009) Impact of OATP transporters on pharmacokinetics. Br J Pharmacol 158:693–705.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Kameyama Y,
    2. Yamashita K,
    3. Kobayashi K,
    4. Hosokawa M, and
    5. Chiba K
    (2005) Functional characterization of SLCO1B1 (OATP-C) variants, SLCO1B1*5, SLCO1B1*15 and SLCO1B1*15+C1007G, by using transient expression systems of HeLa and HEK293 cells. Pharmacogenet Genomics 15:513–522.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kantola T,
    2. Kivistö KT, and
    3. Neuvonen PJ
    (1998) Effect of itraconazole on the pharmacokinetics of atorvastatin. Clin Pharmacol Ther 64:58–65.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kullak-Ublick GA,
    2. Ismair MG,
    3. Stieger B,
    4. Landmann L,
    5. Huber R,
    6. Pizzagalli F,
    7. Fattinger K,
    8. Meier PJ, and
    9. Hagenbuch B
    (2001) Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver. Gastroenterology 120:525–533.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Lau YY,
    2. Huang Y,
    3. Frassetto L, and
    4. Benet LZ
    (2007) Effect of OATP1B transporter inhibition on the pharmacokinetics of atorvastatin in healthy volunteers. Clin Pharmacol Ther 81:194–204.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Lau YY,
    2. Okochi H,
    3. Huang Y, and
    4. Benet LZ
    (2006a) Multiple transporters affect the disposition of atorvastatin and its two active hydroxy metabolites: application of in vitro and ex situ systems. J Pharmacol Exp Ther 316:762–771.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Lau YY,
    2. Okochi H,
    3. Huang Y, and
    4. Benet LZ
    (2006b) Pharmacokinetics of atorvastatin and its hydroxy metabolites in rats and the effects of concomitant rifampicin single doses: relevance of first-pass effect from hepatic uptake transporters, and intestinal and hepatic metabolism. Drug Metab Dispos 34:1175–1181.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Lea AP and
    2. McTavish D
    (1997) Atorvastatin: a review of its pharmacology and therapeutic potential in the management of hyperlipidaemias. Drugs 53:828–847.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Lewin JJ 3rd.,
    2. Nappi JM, and
    3. Taylor MH
    (2002) Rhabdomyolysis with concurrent atorvastatin and diltiazem. Ann Pharmacother 36:1546–1549.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    LIPITOR. (2012) Package insert. Pfizer, New York.
  23. ↵
    1. Liu Y,
    2. Cheng Z,
    3. Ding L,
    4. Fang F,
    5. Cheng KA,
    6. Fang Q, and
    7. Shi GP
    (2010) Atorvastatin-induced acute elevation of hepatic enzymes and the absence of cross-toxicity of pravastatin. Int J Clin Pharmacol Ther 48:798–802.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Maeda K,
    2. Ikeda Y,
    3. Fujita T,
    4. Yoshida K,
    5. Azuma Y,
    6. Haruyama Y,
    7. Yamane N,
    8. Kumagai Y, and
    9. Sugiyama Y
    (2011) Identification of the rate-determining process in the hepatic clearance of atorvastatin in a clinical cassette microdosing study. Clin Pharmacol Ther 90:575–581.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Matsushima S,
    2. Maeda K,
    3. Ishiguro N,
    4. Igarashi T, and
    5. Sugiyama Y
    (2008) Investigation of the inhibitory effects of various drugs on the hepatic uptake of fexofenadine in humans. Drug Metab Dispos 36:663–669.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Mazzu AL,
    2. Lasseter KC,
    3. Shamblen EC,
    4. Agarwal V,
    5. Lettieri J, and
    6. Sundaresen P
    (2000) Itraconazole alters the pharmacokinetics of atorvastatin to a greater extent than either cerivastatin or pravastatin. Clin Pharmacol Ther 68:391–400.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Scharschmidt BF,
    2. Waggoner JG, and
    3. Berk PD
    (1975) Hepatic organic anion uptake in the rat. J Clin Invest 56:1280–1292.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Schwarz UI,
    2. Meyer zu Schwabedissen HE,
    3. Tirona RG,
    4. Suzuki A,
    5. Leake BF,
    6. Mokrab Y,
    7. Mizuguchi K,
    8. Ho RH, and
    9. Kim RB
    (2011) Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity. Pharmacogenet Genomics 21:103–114.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Shitara Y,
    2. Maeda K,
    3. Ikejiri K,
    4. Yoshida K,
    5. Horie T, and
    6. Sugiyama Y
    (2013) Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm Drug Dispos 34:45–78.
    OpenUrlCrossRefPubMed
  30. ↵
    1. van de Steeg E,
    2. Stránecký V,
    3. Hartmannová H,
    4. Nosková L,
    5. Hřebíček M,
    6. Wagenaar E,
    7. van Esch A,
    8. de Waart DR,
    9. Oude Elferink RP,
    10. Kenworthy KE,
    11. et al.
    (2012) Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J Clin Invest 122:519–528.
    OpenUrlCrossRefPubMed
  31. ↵
    1. van de Steeg E,
    2. Wagenaar E,
    3. van der Kruijssen CM,
    4. Burggraaff JE,
    5. de Waart DR,
    6. Elferink RP,
    7. Kenworthy KE, and
    8. Schinkel AH
    (2010) Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs. J Clin Invest 120:2942–2952.
    OpenUrlCrossRefPubMed
  32. ↵
    1. van Herwaarden AE,
    2. Wagenaar E,
    3. van der Kruijssen CM,
    4. van Waterschoot RA,
    5. Smit JW,
    6. Song JY,
    7. van der Valk MA,
    8. van Tellingen O,
    9. van der Hoorn JW,
    10. Rosing H,
    11. et al.
    (2007) Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism. J Clin Invest 117:3583–3592.
    OpenUrlCrossRefPubMed
  33. ↵
    1. van Waterschoot RA,
    2. Rooswinkel RW,
    3. Sparidans RW,
    4. van Herwaarden AE,
    5. Beijnen JH, and
    6. Schinkel AH
    (2009) Inhibition and stimulation of intestinal and hepatic CYP3A activity: studies in humanized CYP3A4 transgenic mice using triazolam. Drug Metab Dispos 37:2305–2313.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Watanabe T,
    2. Kusuhara H,
    3. Maeda K,
    4. Kanamaru H,
    5. Saito Y,
    6. Hu Z, and
    7. Sugiyama Y
    (2010) Investigation of the rate-determining process in the hepatic elimination of HMG-CoA reductase inhibitors in rats and humans. Drug Metab Dispos 38:215–222.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Watanabe T,
    2. Kusuhara H,
    3. Maeda K,
    4. Shitara Y, and
    5. Sugiyama Y
    (2009) Physiologically based pharmacokinetic modeling to predict transporter-mediated clearance and distribution of pravastatin in humans. J Pharmacol Exp Ther 328:652–662.
    OpenUrlAbstract/FREE Full Text
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Varying Effects of OATP and CYP3A Inhibition on Atorvastatin

Jae H. Chang, Justin Ly, Emile Plise, Xiaolin Zhang, Kirsten Messick, Matthew Wright and Jonathan Cheong
Drug Metabolism and Disposition June 1, 2014, 42 (6) 1067-1073; DOI: https://doi.org/10.1124/dmd.114.057968
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