Volume 78, Issue 3 p. 587-598
Drug metabolism
Free Access

Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin

Thomayant Prueksaritanont

Thomayant Prueksaritanont

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Xiaoyan Chu

Xiaoyan Chu

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Raymond Evers

Raymond Evers

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Stephanie O. Klopfer

Stephanie O. Klopfer

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Luzelena Caro

Luzelena Caro

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Prajakti A. Kothare

Prajakti A. Kothare

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Cynthia Dempsey

Cynthia Dempsey

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Scott Rasmussen

Scott Rasmussen

Quintiles, Durham, NC, USA

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Robert Houle

Robert Houle

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Grace Chan

Grace Chan

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Xiaoxin Cai

Xiaoxin Cai

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Robert Valesky

Robert Valesky

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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Iain P. Fraser

Iain P. Fraser

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

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S. Aubrey Stoch

Corresponding Author

S. Aubrey Stoch

Merck Research Laboratories, Merck & Co., Inc., Whitehouse Station, NJ, USA

Correspondence

Dr S. Aubrey Stoch, Merck Research Laboratories, PO Box 2000, RY34-A254, 126 East Lincoln Avenue, Rahway, NJ 07065-0900, USA.

Tel.: +1 732 594 4405

Fax: +1 732 594 5512

E-mail: [email protected]

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First published: 11 March 2014
Citations: 138

Abstract

Aims

Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion-transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters.

Methods

The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems.

Results

Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration–time curve were 5.7- and 7.6-fold for pitavastatin and 4.4- and 3.3-fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3.

Conclusions

The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose.

What is Already Known about this Subject

  • Pitavastatin and rosuvastatin are known as substrates of organic anion-transporting polypeptide 1B (OATP1B) and several other transporters in vitro. Both have been employed as in vivo OATP1B probe substrates for clinical assessment of the drug interaction potential of OATP1B inhibitors.
  • Rifampicin is a potent OATP1B inhibitor. A single intravenous (IV) or oral (PO) dose of rifampicin increases exposure of OATP1B substrates.

What this Study Adds

  • The pharmacokinetics of pitavastatin is more sensitive than that of rosuvastatin to inhibition by a single dose of rifampicin. Unlike pitavastatin, PO rifampicin affects the pharmacokinetics of rosuvastatin more significantly than does IV rifampicin.
  • Rifampicin is an inhibitor not only of OATP1B, but also of gut efflux transporters, such as breast cancer resistance protein and multidrug resistance protein 2, especially when given PO.
  • The results provide a rationale for recommending pitavastatin over rosuvastatin as a more sensitive and selective clinical OATP1B probe, and rifampicin IV over PO as a more selective OATP1B inhibitor. Additionally, clinical studies with single IV and PO doses of rifampicin can be applied to provide an insight into the in vivo relevance of OATP1B versus gut breast cancer resistance protein, P-glycoprotein and multidrug resistance protein 2.

Introduction

The organic anion-transporting polypeptides 1B1, 1B3 and 2B1 (OATP1B1, OATP1B3 and OATP2B1) are major uptake transporters expressed in the sinusoidal membrane of human hepatocytes shown to be involved in the uptake of a range of endogenous substrates and drugs, most notably statins 1-4. The important role of OATP1B1 in the disposition of statins in vivo has been confirmed by pharmacogenetic studies. Compared with the wild-type allele, patients expressing the SLCO1B1 (encoding OATP1B1) variant c.521T>C, a single-nucleotide polymorphism (SNP) associated with reduced uptake activity in vitro, showed plasma exposure increases of 1.5- to 1.7-fold for atorvastatin, rosuvastatin and pravastatin and 2.6- to 3-fold for pitavastatin and simvastatin acid 5. In addition, clinically significant drug interactions have been reported for a number of OATP1B substrates, including statins, with drugs known to inhibit OATP1B activity often resulting in markedly increased plasma exposure of the probe substrates of ≥5-fold 2, 6. To mitigate potential consequences to drug efficacy and safety, the US Food and Drug Administration (FDA) has issued a draft drug–drug interaction (DDI) Guidance 7 recommending a new investigational drug be evaluated as an OATP1B inhibitor/substrate. In the draft Guidance, pitavastatin, rosuvastatin and pravastatin have been proposed as probe substrates to assess interactions with OATP1B1 and OATP1B3 inhibitors. However, limited data are available to address which of these probes is most sensitive for clinical assessment of drug interaction potential of OATP1B inhibitors. In addition, owing to the promiscuous nature of drug transporters, there are currently no known specific probe substrates for transporters. It is well known that a drug, including a statin, is capable of interacting with many drug transporters 8, 9, at least in vitro (Table 1). In this regard, both pravastatin and rosuvastatin have been shown in vitro to be substrates for the renally expressed, basolaterally localized organic anion transporter 3 (OAT3) 10. Organic anion transporter 3 is believed to play an important role in the renal elimination of pravastatin and rosuvastatin, which amounts to ∼50 and ∼25% of total clearance, respectively 2, 10, 11. In vitro, pitavastatin, pravastatin and rosuvastatin are also substrates of the apically localized efflux transporters multidrug resistance protein (MDR1) P-glycoprotein (Pgp; ABCB1), multidrug resistance protein 2 (MRP2; ABCC2) and the breast cancer resistance protein (BCRP; ABCG2) 11. Studies comparing homozygous individuals expressing the c.421C>A SNP in BCRP with those expressing the wild-type allele 5, 12 show a 140% increase in rosuvastatin exposure but no such effect for pitavastatin or pravastatin 13, 14. Additionally, these statins have been reported to be in vitro substrates of many other drug transporters, including the sodium-dependent taurocholate cotransporter (NTCP) 6; the in vivo relevance of these transporters in the disposition of statins has yet to be confirmed. Further complicating the matter, these statins are also substrates of various drug-metabolizing enzymes to a variable extent (Table 1). Currently, unlike drug-metabolizing enzymes, the lack of specific probe substrates for drug transporters in general and OATP1B in particular is a significant barrier to a meaningful assessment of OATP1B-mediated DDI potential for new drugs 8.

Table 1. Drug transporters and drug-metabolizing enzymes shown (in vitro and/or in vivo) to be involved in disposition of pitavastatin and rosuvastatin
Transporters Transporters Enzymes
Liver/intestine Kidney Liver/intestine
Basal Apical Basal CYPs/UGTs
Pravastatin OATP1B1/1B3/2B1/NTCP Pgp/MRP2/BCRP OAT3 CYP3A (minor)
Pitavastatin OATP1B1/1B3/2B1/NTCP Pgp/MRP2/BCRP UGTs (lactonization); CYP2C9 (minor)
Rosuvastatin OATP1B1/1B3/2B1/NTCP Pgp/MRP2/BCRP OAT3 CYP2C9/19; UGTs
  • Modified from the Supplementary Material of Yoshida et al. 6 with permission from the authors. Prueksaritanont et al. 8, 39 and references therein. BCRP, Breast Cancer Resistance Protein; MRP2, multidrug resistance protein 2; NTCP, Na-dependent taurocholate co-transporter; OAT3, organic anion transporter 3; OATP1B1, organic anion transporting polypeptide 1B1; OATP1B3, organic anion transporting polypeptide 1B3; OATP2B1, organic anion transporting polypeptide 2B1; Pgp, P-glycoprotein.

This issue is further compounded by lack of availability of specific inhibitors. Ciclosporin, commonly referred to as a potent OATP1B inhibitor, is an inhibitor of many other transporters as well as drug-metabolizing enzymes 8. Rifampicin, another potent inhibitor of OATP1B 3, 15, is also widely known as a potent inducer of many drug-metabolizing enzymes and drug transporters, including Cytochrome P450 (CYP), UDP-glucuronosyltransferases (UGTs) and Pgp 16, 17. Although the inductive effects of rifampicin can be minimized with single administration, a systematic evaluation of its specificity with regard to the inhibition of other transporters implicated in the disposition of statins is lacking, thereby limiting mechanistic interpretations from such studies. Recently, Reitman et al. have demonstrated that rifampicin, given acutely, is also an inhibitor of Pgp at the gut but not the systemic level 18. This suggests the potential utility of a single dose of rifampicin administered orally (PO) vs. intravenously (IV) to provide additional information on selectivity and decoupling the role of intestinal vs. liver (and other nongut) transporters. To date, a single PO dose of rifampicin has been employed in a clinical setting to assess the significance of OATP1B in disposition of drugs, including atorvastatin and pravastatin 19, 20. Interestingly, the effect of rifampicin on pravastatin plasma exposure was relatively small (∼2-fold) 20, indicating that pravastatin may not be a sensitive probe of OATP1B despite being a good substrate of this transporter in vitro. This finding, although not unexpected given that pravastatin is eliminated significantly (∼50%) via the renal route, highlights the need for a holistic understanding of the relative in vivo contributions of each key determinant impacting drug disposition. Thus far, there have been no reports comparing the effects of PO and IV rifampicin in the same study on exposure of OATP1B substrates, including rosuvastatin and pitavastatin.

The aim of this work, therefore, was to investigate the relative sensitivity and selectivity of pitavastatin and rosuvastatin as in vivo OATP1B probe substrates for clinical assessment of OATP1B-mediated DDIs using a combination of a clinical study with single PO and IV doses of rifampicin, together with a comprehensive in vitro assessment of the inhibitory effect of rifampicin on transporters known to transport these statins. Availability of a selective and sensitive OATP1B probe, if established, should be helpful in assessing the OATP1B-mediated DDI potential of new drugs.

Methods

Detailed bioanalysis and in vitro studies are presented in Appendix S1.

Clinical study design

This was an open-label, two-panel, randomized, three-period crossover study conducted in healthy nonsmoking male and female (nonchildbearing potential) subjects of 19–55 years of age with a body mass index of 19.0–32.0 kg m−2, inclusive. The study protocol and informed consent were reviewed and approved by an Institutional Review Booard (IRB#00000790; Chesapeake Research Review, Inc., Columbia, MD, USA), and the study was conducted at a single clinical site (Celerion, Lincoln, NE, USA). Subjects agreed to refrain from the use of any drugs or substances known to be significant inhibitors/inducers of CYP enzymes, Pgp and/or OATP1B within 14 days or five times the t1/2 (half-life) of the product (whichever is longer) prior to the first dose of study drug(s). Each subject provided written informed consent prior to initiation of study procedures.

In each of the two panels, eight subjects were randomized to one of six sequences, each consisting of three treatments (A, B and C), with a minimal 7 day washout between each treatment period. Different subjects participated in each of the two panels. Treatment A consisted of a single oral dose of 1 mg pitavastatin (panel 1) or 5 mg rosuvastatin (panel 2); treatment B consisted of a single oral dose of 1 mg pitavastatin (panel 1) or 5 mg rosuvastatin (panel 2) together with a single oral dose of 600 mg rifampicin; and treatment C consisted of a single oral dose of 1 mg pitavastatin (panel 1) or 5 mg rosuvastatin (panel 2) together with a single 600 mg IV dose of rifampicin. Intravenous rifampicin was administered over a 30 min period, and pitavastatin or rosuvastatin was given immediately at the end of the 30 min infusion.

All study drugs, besides the rifampicin IV formulation, were administered as single oral doses, after an overnight fast, with ∼240 ml of water. Blood samples (4–7 ml) for the assessment of plasma pitavastatin, pitavastatin lactone and rosuvastatin pharmacokinetics were collected predose on day 1 and until 72 h postdose for pitavastatin and until 120 h postdose for rosuvastatin in each period. Safety and tolerability were assessed with clinical evaluations, which included a physical examination and laboratory assessments. Adverse experiences were monitored throughout the study.

Pharmacokinetic analysis

Noncompartmental pharmacokinetic parameters were calculated using the software Phoenix® WinNonlin® (St. Louis, MO, USA) (version 6.3). In addition, the parameter C24 h for pitavastatin, pitavastatin lactone, rosuvastatin and rifampicin was calculated using SAS® (SAS Institute Inc., Cary, NC, USA) (version 9.1). Following each dose, the maximal concentration (Cmax) and concentrations at 24 h postdose (C24h) and their corresponding times were reported from the observed data. The area under the plasma concentration–time curve (AUC) was calculated using the log–linear trapezoidal method. The AUC0–∞ (AUC from time 0 to infinity) was calculated as the sum of AUC0–last and Ct/λ, where λ is the apparent terminal rate constant and Ct is the predicted concentration at Tlast (last time point with measureable concentrations). For each subject, λ was calculated by regression of the terminal log–linear portion of the plasma concentration–time profile, and the apparent terminal t1/2 (half-life) was calculated as the quotient of the natural log of 2 (ln [2]) and λ. Vdss/F (steady-state volume of distribution/bioavailability) was calculated as the quotient of dose and the product of AUC0–∞ and λ (dose/AUC0–∞ × λ).

Statistical analysis

The pharmacokinetic parameters were analysed using a linear mixed-effects model, which included terms for treatment and period. The pharmacokinetic parameters AUC0–24 h and Cmax were natural logarithmically transformed to satisfy assumptions of the statistical model; the rank transformation was applied to Tmax. The geometric mean ratio (GMR) and its 90% confidence interval (CI) were calculated by determining the arithmetic difference of the log means and its associated 90% CIs and then exponentiating these results back to the original (GMR) scale. The t-distribution was assumed in the calculation of all CIs.

A significant effect of rifampicin on the statin was inferred if the lower bound of the 90% CI for the AUC GMR (statin + rifampicin/statin alone) of the statin exceeded 2.0. In contrast, a differential effect of PO and IV dosing on the statin was inferred if the lower bound of the 90% CI for the AUC GMR (statin + PO rifampicin/statin + IV rifampicin) of the statin AUC values exceeded 1.0.

Bioanalysis

Plasma pitavastatin, pitavastatin lactone, rosuvastatin and rifampicin concentrations were determined using a validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method by PPD Bioanalytical Laboratories (Wilmington, NC, USA). The lower limits of quantitation (LLOQ) for pitavastatin, pitavastatin lactone, rosuvastatin, and rifampicin were 0.960 ng ml−1, 1.00 ng ml−1, 0.1 ng ml−1 and 0.1 μg ml−1, respectively. The linear calibration ranges in plasma for pitavastatin, pitavastatin lactone, rosuvastatin and rifampicin were 0.960–192 ng ml−1, 1.00–200 ng ml−1, 0.100–1.00 ng ml−1 and 0.10–10.0 μg ml−1, respectively. The assays were evaluated for possible interference before study samples were analysed.

In vitro transporter studies

The inhibitory effect of rifampicin on the initial uptake rate of [3H]pitavastatin (0.1 μm) and [3H]rosuvastatin (0.1 μm) was evaluated in cryopreserved human hepatocytes, as well as SLCO1B1-, SLCO1B3-, SCLO2B1- or SLC22A8-transfected cells as described previously 21, 22. The inhibitory effect of rifampicin on uptake of [3H]taurocholic acid ( 1 μm) by NTCP-transfected cells was also determined, while that on uptake of [3H]rosuvastatin (0.5 μm) and [14C]ethacrinic acid–glutathione conjugate (2 μm) by BCRP and MRP2 was conducted in membrane vesicles containing BCRP and MRP2, respectively 23.

Results

Safety evaluations

Single doses of pitavastatin and rosuvastatin administered either alone or co-administered with PO or IV rifampicin were generally well tolerated in the healthy subjects. No serious adverse experiences, deaths, laboratory adverse experiences or events of clinical interest were reported. No subjects were discontinued by the Investigator. Adverse experiences were all of mild intensity (with the exception of one incidence of moderate headache in the rosuvastatin panel), transient in duration, and resolved by study completion.

Pharmacokinetics of pitavastatin and pitavastatin lactone

A single 600 mg dose of rifampicin (PO and IV) markedly increased the area under plasma concentration–time curve from 0 to 4 h (AUC0–4 h) or to infinity (AUC0–∞) and maximal plasma concentrations (Cmax) of pitavastatin (GMR ∼5–6 and 4 for PO, and ∼7 and 6 for IV, respectively), compared with the control phase (Figure 1A and Table 2). Time to maximal concentration (Tmax) and apparent half-life (t1/2) appeared unaltered. However, given that the full terminal phase was not captured in all subjects, the t1/2 values are likely to be underestimated and the steady-state volume of distribution (Vdss/F) values may not be accurate. Nevertheless, marked reduction in Vdss/F was apparent with both PO and IV rifampicin (Table 2). The increase in pitavastatin AUC and Cmax following PO rifampicin was slightly (∼25%), although statistically significantly, lower than following IV rifampicin (Figure 2A and Table 2). For all subjects, neither PO nor IV rifampicin altered pitavastatin lactone pharmacokinetics (Figure 1B and Table 2). As a result, the values for AUC or Cmax ratios for pitavastatin lactone to pitavastatin were significantly reduced by >80% by both PO and IV rifampicin (Table 2).

figure

Mean plasma concentration–time profiles of pitavastatin (A) and pitavastatin lactone (B) following administration of a single oral (PO) dose of 1 mg pitavastatin, and of rosuvastatin (C) following administration of a single oral dose of 5 mg rosuvastatin, with or without co-administration of a single oral (PO) or intravenous (IV) dose of 600 mg rifampicin in healthy adult subjects. Bars represent SD. (image) control, (image) PO rifampicin, (image) IV rifampicin

Table 2. Pharmacokinetic parameters of pitavastatin and pitavastatin lactone following administration of a single oral (PO) dose of 1 mg pitavastatin with or without co-administration of a single oral or IV dose of 600 mg rifampicin in healthy adult subjects
Parameters Control phase IV rifampicin phase PO rifampicin phase Ratio of IV rifampicin to control (90% CI) Ratio of PO rifampicin to control (90% CI) Ratio of PO rifampicin/IV rifampicin (90% CI)
Pitavastatin
AUC0–4 h (ng h ml−1) 21.0 ± 7.1 145.7 ± 44.1 118.0 ± 53.0 7.08 (6.47, 7.74) 5.21 (4.16, 6.52) 0.74 (0.58, 0.94)
AUC0–∞ (ng h ml−1) 29.0 ± 20.6 193 ± 62.3 157 ± 62.2 7.55 (6.65, 8.58) 5.70 (4.65, 6.99) 0.75 (0.60, 0.95)
Cmax (ng ml−1) 16.5 ± 9.32 91.3 ± 26.6 78.8 ± 45.3 6.00 (5.25, 6.86) 4.36 (3.30, 5.76) 0.73 (0.54, 0.98)
Tmax (h) 1.0 (0.5–1.0) 1.0 (0.5–1.5) 1.0 (0.5–2.0)
t1/2 (h) 4.0 ± 6.7 3.1 ± 0.4 3.0 ± 0.8
Vdss/F (l) 137 ± 110 24.8 ± 7.62 32.3 ± 19.6 0.22 (0.11, 0.43) 0.28 (0.14, 0.56) 1.28 (1.08, 1.51)
Pitavastatin lactone
AUC0–24 h (ng h ml−1) 81.4 ± 42.4 83.0 ± 33.4 79.4 ± 25.1 1.11 (0.98, 1.27) 1.06 (0.80, 1.42) 0.96 (0.76, 1.20)
AUC0–∞ (ng h ml−1) 98.6 ± 51.3 96.5 ± 39.3 91.6 ± 29.5 1.07 (0.94, 1.23) 1.02 (0.76, 1.38) 0.95 (0.76, 1.19)
Cmax (ng ml−1) 9.30 ± 4.01 9.23 ± 2.12 9.54 ± 2.11 1.05 (0.91, 1.22) 1.07 (0.89, 1.27) 1.01 (0.89, 1.15)
Tmax (h) 2.0 (1.5–2.1) 3.0 (2.0–4.0) 2.0 (1.5–4.0)
t1/2 (h) 7.8 ± 2.7 6.6 ± 1.9 6.7 ± 1.6
Pitavastatin lactone/pitavastatin ratio
AUC0–∞ ratio 3.92 ± 1.81 0.51 ± 0.10 0.67 ± 0.30 0.14 (0.13, 0.15) 0.18 (0.15, 0.22) 1.27 (1.06, 1.52)
  • Pharmacokinetic parameters are expressed as arithmetic means ± SD, except for Tmax, for which values are expressed as the median and range. The AUC, Cmax and Vdss/F ratios relative to the control phase are geometric means (90% confidence intervals, CI). AUC0–∞, area under plasma concentration-time curve from time 0 to infinity; AUC0–4 h, area under plasma concentration-time curve from time 0 to 4 h; AUC0–24 h, area under plasma concentration-time curve from time 0 to 24 h; Cmax, maximum plasma concentration; IV, intravenous; PO, oral; t1/2, apparent half-life; Tmax, time to maximal concentration; Vdss/F, steady-state volume of distribution/oral bioavailability.
figure

The individual area under the plasma–concentration time curve (AUC) and maximal concentration (Cmax) ratio between IV rifampicin and PO rifampicin phases for pitavastatin (A) and rosuvastatin (B). In (A), (image) subject 1, (image) subject 2, (image) subject 3, (image) subject 4, (image) subject 6, (image) subject 7, (image) subject 8, (image) subject 105. In (B), (image) subject 21, (image) subject 22, (image) subject 23, (image) subject 24, (image) subject 25, (image) subject 26, (image) subject 27, (image) subject 28

Pharmacokinetics of rosuvastatin

Likewise, rifampicin significantly increased rosuvastatin exposure, although with lower GMR values for AUC0–∞ (∼4 and 3 for PO and IV rifampicin, respectively) or AUC0–24 h (∼5 for both PO and IV rifampicin), compared with pitavastatin exposure (Figure 1C and Table 3). Significant increases in rosuvastatin Cmax were also observed after both PO and IV rifampicin, with GMR values of ∼9 and 6 for PO and IV rifampicin, respectively (Figure 1C and Table 3). However, unlike the effect on pitavastatin, PO rifampicin had a statistically greater impact on rosuvastatin AUC and Cmax, with GMR values of 1.3 and 1.8, respectively (Figure 2B and Table 3). Rifampicin, either PO or IV, also caused reductions in Tmax, t1/2 and Vdss/F of rosuvastatin compared with the control phase (Table 3 and Figure S1).

Table 3. Pharmacokinetic parameters of rosuvastatin following administration of a single oral dose of 5 mg rosuvastatin with or without co-administration of a single oral or IV dose of 600 mg rifampicin in healthy adult subjects
Parameters Control phase IV rifampicin phase PO rifampicin phase Ratio of IV rifampicin to control (90% CI) Ratio of PO rifampicin to control (90% CI) Ratio of PO rifampicin/IV rifampicin (90% CI)
Rosuvastatin
AUC0–24 h (ng h ml−1) 19.2 ± 7.7 68.5 ± 26.4 88.5 ± 16.7 4.55 (2.95, 7.02) 5.24 (3.66, 7.49) 1.15 (0.65, 2.03)
AUC0–∞ (ng h ml−1) 23.5 ± 11.9 71.3 ± 24.9 96.1 ± 18.1 3.30 (2.42, 4.50) 4.37 (3.18, 6.01) 1.32 (1.15, 1.53)
Cmax (ng ml−1) 2.79 ± 1.06 15.2 ± 5.41 26.2 ± 7.69 5.51 (4.38, 6.93) 9.93 (7.25, 13.60) 1.80 (1.40, 2.31)
Tmax (h) 4.0 (2.0–5.0) 2.0 (1.0–3.0) 1.5 (1.0–3.0)
t1/2 (h) 12.8 ± 12.1 4.4 ± 4.3 3.1 ± 1.1
Vdss/F (l) 3540 ± 1760 471 ± 452 229 ± 68 0.12 (0.07, 0.18) 0.07 (0.04, 0.10) 0.58 (0.35, 0.95)
  • Pharmacokinetic parameters are expressed as arithmetic means ± SD, except for Tmax, for which values are expressed as the median and range. The AUC, Cmax and Vdss/F ratios relative to the control phase are geometric means (90% confidence intervals, CI). AUC0–∞, area under plasma concentration-time curve from time 0 to infinity; AUC0–24 h, area under plasma concentration-time curve from time 0 to 24 h; Cmax, maximum plasma concentration; IV, intravenous; PO, oral; t1/2, apparent half-life; Tmax, time to maximal concentration; Vdss/F, steady-state volume of distribution/oral bioavailability.

Pharmacokinetics of rifampicin

The mean rifampicin plasma concentration vs. time profiles following the administration of a single dose of 600 mg PO or IV (infused over a 30 min period) rifampicin to healthy adult subjects with pitavastatin were similar to those observed with rosuvastatin (Figure 3). Rifampicin concentrations exhibited similar terminal phases following PO or IV administration, but the Cmax was ∼2-fold higher following IV (∼20 μg ml−1) than PO administration (∼10 μg ml−1). In most subjects across all phases, plasma concentrations of rifampicin were >1 μg ml−1 by 12 h postdose and reached the limit of assay quantification of 0.1 μg ml−1 by 24 h postdose. These IV results were similar to those reported previously by Lau et al. 19. However, the oral exposure was somewhat lower than that reported in Japanese subjects by Maeda et al. 24.

figure

Mean plasma concentration–time profiles of rifampicin following the administration of a single PO or IV dose (infused over a 30 min period) of 600 mg rifampicin given simultaneously with a single PO dose of 1 mg pitavastatin or 5 mg rosuvastatin in healthy adult subjects. Bars represent SD. (image) PO rifampicin with pitavatatin, (image) PO rifampicin with rosuvastatin, (image) IV rifampicin with pitavatatin, (image) IV rifampicin with rosuvastatin

In vitro transporter studies

Given that substrate-dependent inhibition has been reported for some transporters 8, 25, the studies were conducted using pitavastatin and rosuvastatin as probe substrates where possible. The results are summarized in Table 4 and Figures S2–4. In general, rifampicin showed similarly potent inhibition of pitavastatin and rosuvastatin uptake in human hepatocytes (IC50 = 1.2–2.2 μm) and OATP1B1 (IC50 = 1.1–1.6 μm) and OATP1B3 transfected cells (IC50 = 0.3–0.5 μm). In addition, rifampicin inhibited transport of rosuvastatin in BCRP-containing vesicles (IC50 =14 μm), showed weak inhibition on uptake of rosuvastatin in OATP2B1-transfected cells (IC50 = 81 μm) and minimally inhibited OAT3-mediated renal uptake of rosuvastatin (IC50 > 300 μm). Similar studies on these transporters were not conducted for pitavastatin due to either a low contribution to hepatic uptake (OATP2B1) 3 or low and/or minimal transporter activity (BCRP, OAT3) with pitavastatin in the tested systems. Transport activity of pitavastatin and rosuvastatin in Pgp- or MRP2-containing vesicles and MDR1 Pgp- or NTCP-transfected cells also was not observed or was too low to enable reliable determination of inhibition potency (data not shown). Notably, pitavastatin and rosuvastatin are reportedly substrates of NTCP, MDR1 Pgp and MRP2 in human hepatocytes, as well as in OATP1B1 + Pgp and OATP1B1 + MRP2 double-transfected cell monolayers 3, 26, 27. Thus, the inhibitory effect of rifampicin on NTCP and MRP2 in the present study was measured using taurocholic acid and ethacrinic acid–glutathione conjugate as probe substrates, with obtained IC50 values of 277 μm and 14.7 μm, respectively. The inhibitory effect of rifampicin on Pgp-mediated digoxin efflux was previously determined in this laboratory, with an IC50 value of 169 μm 18.

Table 4. Values of IC50 for inhibitory effects of rifampicin on transport of pitavastatin and rosuvastatin by several transporters
Transporters In vitro systems IC50m)
Pitavastatin (0.1 μm) Rosuvastatin (0.1 μm)
Hepatic uptake transporters Cryopreserved human hepatocyte suspension 2.2 ± 0.3 1.2 ± 0.1
OATP1B1 OATP1B1-transfected MDCKII cells 1.6 ± 0.1 1.1 ± 0.2
OATP1B3 OATP1B3-transfected MDCKII cells 0.5 ± 0.1 0.3 ± 0.06
OATP2B1 OATP2B1-transfected MDCKII cells ND 81 ± 34
NTCP NTCP-transfected CHO-K1 cells 277 ± 41* 277 ± 41*
MDR1 Pgp MDR1 Pgp-transfected LLC-PK1 cells 169 ± 18 169 ± 18
BCRP BCRP-containing vesicles NA 14.0 ± 3.4
MRP2 MRP2-containing vesicles 14.7 ± 0.8 14.7 ± 0.8
OAT3 OAT3-transfected MDCKII cells ND 19% inhibition at 300 μm
  • *Using [3H]taurocholic acid (1 μm) as probe substrate. †Obtained from Reitman et al. 18 using [3H]digoxin (0.1 μm) as probe substrate. ‡Using [14C]ethacrinic acid–glutathione conjugate (2 μm) as probe substrate. Abbreviations are as follows: BCRP, Breast Cancer Resistance Protein; MRP2, multidrug resistance protein 2; NA, not available due to low transport activity in in vitro system tested; ND, not determined (see main text); NTCP, Na-dependent taurocholate co-transporter; OAT3, organic anion transporter 3; OATP1B1, organic anion transporting polypeptide 1B1; OATP1B3, organic anion transporting polypeptide 1B3; OATP2B1, organic anion transporting polypeptide 2B1; Pgp, P-glycoprotein.

Discussion

This study demonstrates the utility of single IV and PO doses of rifampicin, together with in vitro transporter studies, to help determine the relative selectivity and sensitivity of pitavastatin vs. rosuvastatin pharmacokinetics to OATP1B and other transporters, and thus defining their relative suitability as OATP1B probe substrates for clinical DDI studies. The results for both routes of rifampicin administration showed a marked effect on pitavastatin exposure, suggesting the sensitivity and selectivity of pitavastatin to OATP1B, with minimal involvement of gut transporters. In contrast, a greater differential impact of PO vs. IV rifampicin on rosuvastatin pharmacokinetics was evident, indicating that gut efflux transporters play an important role in the oral pharmacokinetics of rosuvastatin. These data are in agreement with the pharmacogenetic data, which showed that the pharmacokinetics of pitavastatin was susceptible to SNPs in SLCO1B1 (encoding OATP1B1), but not BCRP, while that of rosuvastatin was susceptible to SNPs in both SLCO1B1 and BCRP, with pitavastatin being more sensitive than rosuvastatin to SLCO1B1 variants 5. These clinical results are also consistent with the in vitro data indicating that rifampicin has the potential to inhibit not only the hepatic transporter OATP1B but also efflux transporters, such as BCRP, Pgp and MRP2, all of which are known to be present in the intestine.

Co-administration of IV rifampicin with pitavastatin markedly increased the AUC and Cmax of pitavastatin by 7.6- and 6-fold, respectively. Interestingly, the impact of IV rifampicin on pitavastatin appeared to be slightly but significantly greater than that of PO rifampicin (5.7- and 4.4-fold for AUC and Cmax, respectively). This finding, although unanticipated, suggests minimal involvement of gut transporters, such as BCRP or Pgp, in the disposition of pitavastatin. It is explainable on the basis of ∼2-fold higher rifampicin Cmax following IV than PO dosing, and thus greater inhibitory impact on pitavastatin uptake by OATP1B. It is noteworthy that the calculated unbound plasma concentrations of rifampicin (based on 20% unbound fraction) 28 were above or close to the IC50 values for the OATP1B-mediated uptake of pitavastatin for ∼12 h post PO and IV doses, covering the majority of the pitavastatin plasma concentration–time profile in most subjects. Although pitavastatin is reportedly an in vitro substrate for NTCP and efflux transporters BCRP, MRP2 and Pgp, the findings could not be reproduced in our in vitro systems, suggesting that these transporters are less likely to play a significant role in pitavastatin disposition. Likewise, Hirano et al. have shown that OATP2B1 contributed minimally to the hepatic uptake of pitavastatin 3. Together with the observed weak in vitro inhibitory profile of rifampicin (relative to its systemic exposure) on OATP2B1, NTCP and Pgp, inhibition of OATP1B-mediated hepatic uptake of pitavastatin was primarily responsible for the pronounced increase in pitavastatin exposure in the presence of rifampicin, after both IV and PO dosing. While potential involvement of MRP2 in the rifampicin–pitavastatin interaction cannot be excluded completely, its contribution is likely to be small because MRP2 is an efflux transporter present in both liver and gut, and there was no additional impact of PO vs. IV rifampicin on pitavastatin pharmacokinetics.

The absolute magnitude of change in pitavastatin exposure in the present rifampicin inhibition study (6- to 7-fold) was much greater than that reported with SLCO1B1 polymorphisms (∼2.5-fold) 5. While this is consistent with the notion that genetic polymorphisms in transporters only partly affect and do not completely abolish transport function, we cannot rule out the potential involvement of another, yet to be identified transporter in the rifampicin–pitavastatin interaction, considering that only a fraction of transporter gene products have been characterized to date. The magnitude of the PO rifampicin impact on pitavastatin AUC observed in the present study was in a similar range to that recently reported in a clinical drug interaction study with a single 600 mg oral dose of rifampicin and a 4 mg dose of pitavastatin 29. In that earlier study 29, the effect of IV rifampicin was not examined. A comparable magnitude of the AUC change was also reported with a single 600 mg IV dose of rifampicin and a 40 mg dose of atorvastatin, another OATP1B substrate 19. In all reported studies, including ours, subjects were not genotyped for SLCO1B1 variants. In a separate clinical study, a single dose of rifampicin has been shown to influence atorvastatin pharmacokinetics in a SLCO1B1 polymorphism-dependent manner, with ∼2-fold higher impact in c.521TT than c.521TC or c.521CC individuals 30.

Rifampicin, both IV and PO, had no impact on either AUC or Cmax of pitavastatin lactone, but markedly decreased the pitavastatin lactone/pitavastatin AUC ratio in all subjects. This is indicative of rifampicin impacting metabolism of pitavastatin, considering that pitavastatin lactone is formed following spontaneous lactonization of pitavastatin glucuronide mediated by UGTs 21. However, given that rifampicin is not an inhibitor of UGTs 31, the apparent decreased pitavastatin metabolism was probably secondary to the impact of rifampicin on the OATP1B-mediated hepatic uptake of pitavastatin preventing its subsequent metabolism. In this regard, it is worth noting that a common feature of drug interaction studies with a single dose of rifampicin and OATP1B substrates, such as atorvastatin 19 and glyburide 32, has been a decrease in t1/2 of the substrates during the rifampicin phase, attributable partly to their impaired tissue uptake. Unfortunately, due to limits of analytical sensitivity, the t1/2 of pitavastatin (and therefore Vdss/F) in the present control phase could not be determined accurately. In the clinical study with the higher dose (4 mg) of pitavastatin, with better characterization of t1/2, a 2-fold reduction in pitavastatin t1/2 has been observed 29, in line with previous observations with other OATP1B substrates.

In contrast to the observation with pitavastatin, rifampicin, either IV or PO, had a greater impact on Cmax (5.5- to 9.3-fold) than AUC (3.3- to 5.2-fold) of rosuvastatin, suggesting that the impact of rifampicin on rosuvastatin is primarily at the presystemic level. The finding of a greater impact of PO than IV rifampicin on rosuvastatin pharmacokinetics indicates an additional contributory factor at the gut level. Similar to pitavastatin, rosuvastatin has been shown in vitro to be a substrate of transporters other than OATP1B1, OATP1B3 and OATP2B1, such as NTCP, BCRP, MRP2 and Pgp, and additionally OAT3 for its renal elimination 6. Our in vitro studies with rifampicin suggest a potential for inhibition of rosuvastatin transport mediated by OATP1B1, OATP1B3, BCRP and MRP2, but probably not by OAT3, OATP2B1 or NTCP at the systemic level. When given PO, rifampicin lumenal concentrations are expected to be significantly higher than systemic concentrations and thus it has the potential to inhibit rosuvastatin transport by gut BCRP, MRP2 and Pgp. The ability of a single oral dose of rifampicin to inhibit gut Pgp has been demonstrated in an earlier clinical drug interaction study with digoxin 18. Based on a comparable value between the BCRP IC50 (14 μm; Table 4) and theoretical maximal unbound rifampicin concentration at the hepatic inlet (∼9 μm after PO rifampicin) 33, it is possible that hepatic BCRP was also involved in the disposition and the observed increase in AUC and Cmax of rosuvastatin by rifampicin. On the contrary, considering that the unbound Cmax of rifampicin (<1 μm) is many times lower than the IC50 value, rifampicin is not expected to have a significant impact on BCRP-mediated transport of rosuvastatin at the kidney level. To our knowledge, this is the first study to demonstrate an inhibitory potential of rifampicin on BCRP, both in vitro and in vivo. Additionally, given the in vitro results with MRP2 and the clinical observations suggesting a presystemic effect of rifampicin, we cannot rule out the potential involvement of MRP2 on the absorption and disposition of rosuvastatin at both the gut and liver level. We also cannot exclude the possibility for involvement of other transporters, such as MRP4, a hepatic sinusoidal and renal apical efflux, in this interaction, considering a recent study demonstrating that rosuvastatin is an in vitro substrate of MRP4 34. We were unable to detect significant rosuvastatin transport activity in NTCP-transfected CHO-K1 cells to support a recent analysis by Elsby et al. of ∼20% contribution of NTCP to rosuvastatin hepatic uptake 35. In keeping with other drug-interaction observations with a single IV or PO dose of rifampicin, the reduction in rosuvastatin t1/2 suggests that rifampicin may impact transporter-mediated distribution of rosuvastatin.

Recently, there has been a growing interest in a quantitative prediction of transporter-mediated drug interactions based on in vitro data. In this regard, a mechanistic static model approach, which takes into account the relative contribution of multiple elimination pathways of victim drugs by various transporters, has been proposed and applied to DDI prediction for statins 35, 36. Using this approach, a significant underprediction was observed, suggesting that the proposed input parameters (e.g. fraction eliminated via each pathway) would require some adjustments. For pitavastatin, using the estimated fe (fraction excreted via certain transporter) of 0.78, 0.70 and 0.08 for OATP1Bs, OATP1B1 and OATP1B3, respectively 35 together with in vitro IC50 values determined in the present study, the predicted change in AUC by inhibition of both OATP1B1 and OATP1B3 following IV and PO rifampicin was 2.1- and 2.7-fold, which is 3.6- and 2.1-fold lower than observed values. Likewise, for rosuvastatin, using the assumed fe values (0.70, 0.38, 0.21 and 0.11 for total active uptake, OATP1B1, NTCP and OATP1B3, respectively), the predicted fold-changes of AUC for inhibition of overall hepatic uptake are 1.6 and 1.7 for IV and PO rifampicin, respectively, which is 2.1- to 2.6-fold lower than the observed values. Only when taking into account gut BCRP-mediated efflux and assuming complete inhibition by PO rifampicin as the worst case scenario is the fold-change of AUC (3.9) within the observed range. It is important to note that this static modelling prediction does not take into account the potential inhibition of rifampicin of active biliary excretion of pitavastatin or rosuvastatin by BCRP, MRP2 or Pgp. While unlikely to be a reason for the significant underprediction for pitavastatin, this may contribute partly to the underprediction for rosuvastatin with rifampicin. A physiologically based pharmacokinetic model incorporating our current clinical and in vitro parameters is currently being developed to characterize pitavastatin and rosuvastatin disposition better.

Finally, on the basis of our clinical data suggesting pitavastatin as a more sensitive and selective OATP1B probe, we searched for clinical drug-interaction reports conducted with pitavastatin. Thus far, only ciclosporin, erythromycin and gemfibrozil have been shown to increase exposure of pitavastatin, with gemfibrozil showing the least impact (∼40% increase in AUC; Table 5). Neither ciclosporin nor erythromycin is an inhibitor of UGTs; therefore, their impact on pitavastatin exposure should be due primarily to OATP1B inhibition. Despite the recent finding that ciclosporin exhibited long-lasting inhibitory effects on rat Oatp and human OATP-mediated uptake 37, 38, the in vivo impact of ciclosporin on pitavastatin exposure was 4.5-fold, which was less than that of rifampicin (6- to 8-fold), suggesting that at clinically relevant exposures, ciclosporin is not as potent an inhibitor as rifampicin of OATP1B-mediated liver uptake of pitavastatin. Ciclosporin has been commonly referred to as a broad inhibitor of transporters and CYPs, and the results from clinical DDI studies with ciclosporin have been accepted by regulatory agencies as the worst case scenario for substrates of transporters and enzymes 7. In addition, this 4.5-fold magnitude observed with ciclosporin has been used to estimate a theoretical maximal increase of pitavastatin due to OATP inhibition 35. Evidently, these notions and associated assumptions should be revisited. Unlike ciclosporin and erythromycin, gemfibrozil has been shown to be an inhibitor of statin glucuronidation as well as an inducer of CYPs and UGTs 39, 40. Thus, it is unclear whether the magnitude of increase in pitavastatin exposure following multiple dosing of gemfibrozil can be attributable primarily to its inhibitory effect on OATP1B. Nevertheless, gemfibrozil (and its glucuronide) is likely to be a weak OATP1B inhibitor in vivo based on its in vitro IC50 value in relationship to its anticipated unbound maximal portal concentration (Iin,max,u) 3 or reflected in the ‘R’ value, criteria used by the European Medicines Agency and US FDA, respectively, to indicate potential in vivo impact due to OATP1B inhibition 8. Interestingly, there appeared to be a rank-order correlation between these values and magnitudes of pitavastatin AUC increases across compounds presented in Table 5. This apparent qualitative in vitro–in vivo correlation may be viewed as independent support for pitavastatin as a clinical OATP1B probe. Data on additional compounds are, however, needed to confirm this.

Table 5. Effects of OATP1B inhibitors on pharmacokinetics of pitavastatin in healthy subjects
Perpetrator (dose, regimen) IC50m)* OATP1B1 R value (FDA) (≥1.25) Iin,max,u/IC50 (EMA) (≥0.04) Fold change in pitavastatin AUC
Rifampicin (600 mg SD IV) 1.6 15§ 2.3§ 8
Rifampicin (600 mg SD PO) 1.6 6.7 5.7 6.9
Ciclosporin (2 mg kg−1 SD) 0.3 3.1 2.1 4.5§
Erythromycin (500 mg QID) 11 2.9 1.9 2.8
Gemfibrozil (600 mg, QID) 90 1.1 0.1 1.4**
  • *Most are in-house data obtained using pitavastatin (0.1 μm) as a probe substrate and OATP1B1-transfected MDCKII cells; the IC50 value for erythromycin was from Hirano et al. 3. †FDA and ‡EMA Guidance criteria with a cut-off value of 1.25 for R, and 0.04 for Iu,max/IC50 indicating potential for DDIs 6, 8. §Values are Cmax,total/IC50 for FDA and unbound Cmax/IC50 for EMA. §Prueksaritanont et al. 8. ¶Product label. **Matthew et al. 41. Abbreviations are as follows: Iin,max,u, maximum unbound inhibitor concentration at the inlet to the liver; QID, four times a day; SD, single dose.

Collectively, the present clinical and in vitro drug interaction studies provided a mechanistic support for pitavastatin over rosuvastatin as the relatively selective and sensitive OATP1B probe substrate for assessment of drug interaction potential of an in vitro OATP1B inhibitor, providing that potential transporter interplay with UGTs/CYPs, enzymes catalysing pitavastatin metabolism, is taken into consideration. Additionally, a single IV dose of rifampicin may be preferred over a PO dose to aid in assessment of in vivo relevance of OATP1B in the disposition of OATP1B substrates to minimize potential complications from gut efflux transporters. Furthermore, clinical studies with single IV and PO doses of rifampicin can be applied to provide an insight into the in vivo relevance of OATP1B vs. gut BCRP/Pgp/MRP2. Lastly, IV rifampicin, although unlikely to inhibit OATP2B1, NTCP and OAT3, has the potential to inhibit hepatic BCRP and MRP2, in addition to OATP1B, and thus caution should be exercised when interpreting clinical drug interactions of substrates of multiple transporters. Overall, pitavastatin and IV rifampicin, when appropriately applied, should provide a more sensitive and selective assessment of OATP1B activity than other combinations of probe substrate (such as rosuvastatin or pravastatin) and perpetrator (such as ciclosporin).

Competing Interests

All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare support from Merck & Co. for the submitted work. All authors except Dr Rasmussen (Quintiles) are employees of Merck & Co. and may own stock and/or have options in the company.

We thank the Celerion staff (Megan Kozisek, John Brejda, Carrie Mitchell and Mike Di Spirito), PPD staff (Sadie Erickson, Jack Steiner, Laura Schweitzer, Brendan Laing, Michelle Kirby and Nichole Barden), Dr Man-Wai Lo and Ms Jin Zhang for clinical support, and Drs Larrissa Wenning, Ronda Rippley, Daniel Tatosian, Nancy Agrawal, David Cutler, Jane Harrelson and Lisa Shipley for helpful discussions and suggestions.