Impact of rifampicin-inhibitable transport on the liver distribution and tissue kinetics of erlotinib assessed with PET imaging in rats

Erlotinib is a reversible inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity, and it is approved for treatment of advanced and metastatic non-small cell lung cancer (NSCLC) with mutated EGFR [1]. However, nearly 30% of patients who are carriers of the somatic EGFR mutation do not respond adequately to therapy with tyrosine kinase inhibitors [2]. NSCLC patients, receiving the therapeutic dose of 150 mg erlotinib once daily, showed a marked interpatient variability in trough plasma concentrations and area under the curve (AUC) of 64% and 51%, respectively [3]. Variability in the pharmacokinetics (PK) of erlotinib is therefore assumed to contribute to variability in therapeutic response [4]. Furthermore, a correlation of erlotinib plasma PK parameters and treatment efficacy and occurrence of side effects was found [5, 6].

The molecular determinants for this PK variability are not fully understood. ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2), were shown to play pivotal roles in restricting the intestinal absorption of erlotinib [7]. Erlotinib is mainly eliminated through hepatobiliary clearance and undergoes hepatic metabolism by cytochrome P450 (CYP) 3A4/3A5 and, to a lesser extent, by CYP1A1/2 [8]. Thus, CYP3A4 inducers and inhibitors are likely to affect erlotinib PK and may be responsible for drug-drug interactions (DDIs) [9, 10]. In addition to metabolism considerations, erlotinib is also a substrate of several transporters of importance for PK [11]. P-gp and BCRP are now recognized as major determinants for tissue distribution and clearance erlotinib [7, 12].

Recent positron emission tomography (PET) studies using carbon-11-labeled erlotinib provided novel information regarding the clearance of this drug. In baboons, injection of a high dose of unlabeled erlotinib decreased the plasma clearance of ¹¹C-erlotinib, pointing to non-linear PK [13]. Similarly, a high dose of erlotinib in mice was associated with a decreased liver uptake of ¹¹C-erlotinib, suggesting a saturable and possibly carrier-mediated transport mechanism for erlotinib at the basolateral membrane of hepatocytes [14]. This observation was recently confirmed in healthy human volunteers using a similar saturation approach [15].

The hepatic uptake of various xenobiotics is mediated by sinusoidal membrane transporters, mainly solute carrier (SLC) transporters, as a prerequisite for hepatobiliary elimination [16]. Three members of the organic anion-transporting polypeptide (OATP) family (SLCO gene), namely OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3), and OATP2B1 (SLCO2B1), are expressed in hepatocytes and are considered of importance for PK [11]. Recent in vitro studies demonstrated that erlotinib is selectively transported by OATP2B1 among the other OATP transporters expressed in hepatocytes [15]. OATP2B1 expression is not restricted to the liver and may also play a role in the uptake of its substrates into other organs [17]. OATP2B1 function may thus account for variability in the distribution of erlotinib to the liver and to target/vulnerable tissues, with consequences for treatment efficacy and safety. OATP function was thus hypothesized to control the liver uptake of erlotinib with consequences for plasma and tissue kinetics in vivo.

In the present study, ¹¹C-erlotinib PET imaging was performed in anesthetized rats in the absence and the presence of the broad spectrum OATP inhibitor rifampicin to understand whether rifampicin-inhibitable uptake transport may be responsible for the pharmacokinetic variability of erlotinib in patients.

Figure 1 shows representative PET images centered on the abdominal region for a control and a rifampicin-pretreated rat. Radioactivity was primarily localized in the liver within the first minutes after ¹¹C-erlotinib injection, and then it gradually moved to the intestine, thus dynamically highlighting the predominantly hepatobiliary elimination of radioactivity (Fig. 1). Radioactivity also accumulated in the kidneys, which were best visualized on late summation PET images. The lungs were visible in the very first frames only, probably due to their high vascular content (Fig. 1).

From time points > 3 min after injection, hepatic accumulation of radioactivity was lower in rifampicin-pretreated rats than in control animals (Fig. 1). SUV_max in the liver was significantly lower in rifampicin-treated animals (7.3 ± 1.2) as compared with control animals (9.9 ± 0.7, p < 0.05). Rifampicin pretreatment caused a significant ~ 27% decrease in AUC_liver (p < 0.001; Fig. 2). In animals sacrificed at 15 min after injection, the percentage of unmetabolized ¹¹C-erlotinib in the liver ranged from 96.4 to 100% in the control group and from 96.0 to 100% in the rifampicin-treated group, respectively. This shows a limited contribution of radiolabeled metabolites to the PET signal in the liver within the first 15 min of scanning. AUC_liver from 0 to 15 min was significantly lower in rifampicin-treated animals (103.6 ± 15.2 SUV.min) as compared with the control animals (129.4 ± 10.1 SUV.min, p < 0.05).

Radioactivity concentrations in the kidney cortex were also lower in the rifampicin-treated group, and the difference tended to increase with time (Fig. 2). AUC_kidney was markedly reduced in rifampicin-treated animals (p < 0.001). The lungs showed a much lower exposure to radioactivity compared to the liver and the kidneys (Fig. 2). No significant differences in AUC_lung were observed between the two groups (Fig. 2).

Unexpectedly, rifampicin did not impact the image-derived blood kinetics. AUC_aorta (80.4 ± 24.7 SUV.min) was not increased by rifampicin treatment (86.6 ± 23.8 SUV.min; p > 0.05). Radiotracer metabolism and binding to blood cells in either the presence or the absence of rifampicin may interfere with the estimation of the transfer of parent ¹¹C-erlotinib from plasma to tissues. The plasma kinetics of parent ¹¹C-erlotinib was therefore measured in one animal of each condition, from repeated arterial blood sampling, in order to provide a representative metabolite-corrected arterial input function and calculate k_uptake in tissues. The percentage of parent ¹¹C-erlotinib versus time was similar in the control and the rifampicin-pretreated rat (Fig. 3a). At 1 h after radiotracer injection, the percentage of unmetabolized ¹¹C-erlotinib was 61% and 69% without and with rifampicin pretreatment, respectively. ¹¹C-erlotinib plasma concentrations peaked rapidly after i.v. injection, followed by a rapid wash out (Fig. 3b). A plateau was reached after 15 min (SUV_15 min = 0.34) with SUV_60 min = 0.22 and 0.24 for the baseline and the rifampicin pretreated rats, respectively.

The lower liver and kidney exposures to radioactivity observed in the rifampicin-pretreated animals were consistent with significant decreases in k_{uptake, liver} and k_{uptake, kidney} (Fig. 4). There was no effect of rifampicin on k_{uptake, lung}, which was consistent with the absence of an effect of rifampicin on AUC_lung (Fig. 2).

PET imaging is an appealing strategy to study the impact of OATP transporters on the tissue distribution of radiolabeled drugs [23]. In vitro studies using transfected cells showed that erlotinib is specifically transported by the human OATP2B1 [15]. Recent PET studies have demonstrated saturable liver uptake of ¹¹C-erlotinib in humans [15]. To the best of our knowledge, the transport of erlotinib by the rodent rOATP2B1 has not been addressed but a similar saturation of the liver uptake of ¹¹C-erlotinib was also observed in mice [14]. We therefore hypothesized that OATP inhibition with rifampicin may decrease the liver uptake of ¹¹C-erlotinib with consequences for drug exposure to the lungs, as the therapeutic target tissue of erlotinib.

OATP transporters are recognized as the key determinants for the PK of many drugs [11]. They are expressed in the sinusoidal membrane of hepatocytes and were shown to control the uptake of their substrates from the blood into the liver. Conventional PK studies, based on the determination of drug concentrations in the plasma, have shown how alterations in hepatic OATP1B1 and OATP1B3 function may influence the plasma PK of diverse drugs. Genetic polymorphisms of the SLCO genes [24] as well as DDIs involving inhibitors of OATP transporters [25] have been deemed of clinical importance for PK variability. The importance of OATP2B1 in the liver is less understood, probably due to its overlapping substrate specificity with OATP1B1 and OATP1B3 [25]. Expression of OATP2B1 has been detected at many other blood-tissue interfaces than the sinusoidal membrane of hepatocytes, suggesting that OATP2B1 may control the target/vulnerable tissue exposure to its substrates in addition to its impact on hepatobiliary clearance [17].

Rifampicin is a prototypical OATP inhibitor used in clinical studies in drug development to assess the PK importance of OATP function [11, 26]. In vitro, rifampicin is a potent inhibitor of the human OATP1B1 (IC₅₀ = 1.9 ± 0.16 μM) and OATP1B3 (IC₅₀ = 6.4 ± 0.5 μM) and a weaker inhibitor of OATP2B1 (IC₅₀ = 91.0 ± 14.6 μM) [27, 28]. Rifampicin was also shown to inhibit several rat OATPs including the liver-specific rOATP1B2 (K_i = 0.79 μM). Rifampicin also inhibits rOATP1A1 (K_i = 18 μM) and rOATP1A4 (K_i = 1.4–2.9 μM) [29‐31]. The inhibition potency of rifampicin for other SLC transporters of PK importance has been studied in vitro and suggests a relatively good specificity for OATP transporters. Indeed, rifampicin inhibited neither organic cation transporter 1 (OCT1, SLC22A1, K_i > 100 μM) nor organic anion transporter 2 (OAT2, SLC22A7), which are both expressed in the sinusoidal hepatocyte membrane. Rifampicin most likely does not inhibit organic cation transporter 2 (OCT2, SLC22A2) and organic anion transporter 3 (OAT3, SLC22A8) function in vivo [32]. Plasma concentrations of rifampicin, when administered to rats at a dose of 40 mg/kg i.v., ranged from 100 to 300 μM, which exceeded the reported in vitro K_i values for inhibition of OATP transporters [20, 22]. The selected rifampicin dose was previously shown to inhibit the rOATP-mediated liver uptake of ¹¹C-dehydropravastatin and ¹¹C-rosuvastatin, as assessed with PET imaging in rats [20, 22]. In our study, we chose a simple i.v. bolus administration protocol, which did not require constant infusion during PET scanning.

Our PET data showed a predominant liver accumulation of radioactivity, which was consistent with hepatobiliary elimination of erlotinib [33]. A high hepatic accumulation of ¹¹C-erlotinib has also been observed in mice and in humans [14, 15]. Rifampicin pretreatment significantly decreased the liver uptake and subsequent liver exposure to ¹¹C-erlotinib. This suggested a role of sinusoidal rOATP transporters in controlling the hepatobiliary elimination of erlotinib. Radioactivity in the liver, analyzed at 15 min after injection, mainly consisted of unmetabolized ¹¹C-erlotinib, regardless of the presence or absence of rOATP inhibition. This suggested that the quantification of k_{uptake, liver} values was not influenced by radiotracer metabolism and accurately reflected the transfer constant of parent ¹¹C-erlotinib from the blood into the liver.

Rifampicin is also a potent inhibitor of MRP2 (ABCC2) expressed at the canalicular side of hepatocytes [19]. Erlotinib is not a substrate of MRP2 [7]. Consistently, the slopes of ¹¹C-erlotinib elimination from the liver were similar in the control and rifampicin-treated animals suggesting a predominant impact of rifampicin in reducing the basaloteral uptake of ¹¹C-erlotinib by hepatocytes [19].

Control ¹¹C-erlotinib PET images showed a relatively high uptake of the radioactivity in the kidney cortex. Unexpectedly, rifampicin also decreased the uptake of ¹¹C-erlotinib into the kidneys, which led to a significant 30.4% decrease in kidney exposure. This effect is consistent with the saturable uptake of ¹¹C-erlotinib by the kidneys reported in mice [14]. A similar phenomenon has been observed for the OATP substrate ¹¹C-rosuvastatin, which kidney uptake was also decreased after rifampicin administration [20]. This suggested a role for rifampicin-inhibitable transporter(s) expressed in rat kidney epithelial cells in controlling the uptake and subsequent kidney exposure to erlotinib. The decrease in the kidney exposure was related to the decrease in k_{uptake, kidneys}. This suggests a role for a basolateral blood-to-kidney transport rather than secretion or reabsorption process at the tubular level, as previously reported for ¹¹C-rosuvastatin [20]. Rifampicin is an inhibitor of OAT1 and OAT2 but not OAT3 expressed at the basolateral membrane of kidney proximal tubules [20]. However, erlotinib is neither transported by OAT1 nor OAT2 but is a substrate of OAT3 [12]. Erlotinib is also a substrate of OCT2 which is not inhibited by rifampicin [12, 27]. Rifampicin is not an inhibitor of OATP4C1 [34] which importance for erlotinib transport is not known. Previous mice studies have shown that parent unmetabolized ¹¹C-erlotinib could not be detected in urines [14]. It is therefore difficult to address whether changes in the ¹¹C-erlotinib uptake by the kidneys translated into changes in the urinary clearance of the tracer. Species differences in the expression and function of membrane transporters may occur between rats and humans. Clinical experiments using ¹¹C-erlotinib along with urine sampling would be useful to finally address the putative impact of these rifampicin-inhibitable transporter(s) in the uptake by the kidney tissue and whether it could drive any change in the renal elimination of erlotinib and its metabolites.

The lungs are the main target tissue for erlotinib therapy in patients. A decrease in the distribution of ¹¹C-erlotinib to the major clearance organs liver and kidneys was expected to lead to an increase in plasma concentrations and a concomitant increase in lung exposure. Increase in plasma concentrations has been observed for ¹¹C-rosuvastatin, as a consequence of decreased hepatic and renal uptake by rifampicin [20]. Interestingly, our results indicated that rifampicin decreased the distribution of erlotinib into the main elimination organs of the body with no or negligible consequences for blood and lung exposure. The lungs contain a high vascular fraction, and the concentration of radioactivity measured with PET in the lungs can therefore be expected to be influenced by the plasma kinetics of ¹¹C-erlotinib, which did not differ between the control and rifampicin-treated rats.

The lack of an effect of rifampicin on systemic exposure suggested a limited role for rifampicin-inhibitable transporter(s) in controlling the total clearance of erlotinib. First, it can be hypothesized that a larger decrease in the uptake of ¹¹C-erlotinib by the liver or kidneys would be necessary to detect changes into plasma PK. Indeed, saturation of the liver and kidney transport by unlabeled erlotinib led to significant changes in the plasma PK of ¹¹C-erlotinib [14]. This suggests that substantial changes into the uptake of drugs by elimination organs may not directly translate into changes in plasma PK. This phenomenon could also be explained by compensatory mechanisms, such as a shift from hepatobiliary elimination to another (probably urinary) elimination pathway, which was previously reported for ¹¹C-erlotinib in mice [14, 35]. It may also be hypothesized that efficient reabsorption of ¹¹C-erlotinib at the enterohepatic level, which may not be inhibited by rifampicin, may counteract the decreased transport of ¹¹C-erlotinib throughout hepatocytes. The impact of transporter function on the enterohepatic circulation of drugs is difficult to assess using PET. Nevertheless, such phenomenon would probably translate into changes in the early plasma kinetics [36], which was not observed in our experiments.

Rifampicin had no significant impact on k_{uptake, lung}, suggesting that erlotinib uptake by the lungs is independent of rOATP transporter function. This is consistent with previous studies showing that rOATP1A1, rOATP1A4, and rOATP1B2, which can be inhibited with rifampicin, are not expressed in the lungs [37].

Imaging data obtained in the present animal study should be interpreted with respect to a clinical extrapolation. First, the expression, function, and tissue distribution of OATP transporters differ between rats and humans [38]. Second, the rifampicin dose used in this study was considerably higher than the rifampicin dose used to test OATP-mediated DDIs in humans (10 mg/kg). Third, we used in the present study a microdose of erlotinib, on a relatively short time, which may not reflect the PK behavior of a pharmacological dose of erlotinib. In fact, a previous ¹¹C-erlotinib PET study in healthy volunteers suggested that OATP2B1-mediated liver uptake of erlotinib was negligible at pharmacological doses, due to saturation of transport activity [15]. Nevertheless, our results demonstrate that discrepancies may exist between the PK of drugs in elimination organs, their systemic PK, and subsequent exposure to target tissues. Our data suggest that OATP-mediated DDIs, with consequences for liver and kidney exposure, may not necessarily translate into changes in systemic PK. Previous studies have demonstrated the feasibility of ¹¹C-erlotinib PET imaging in healthy volunteers [15], which may be combined with rifampicin administration [39]. A clinical translation of our results is thus possible to validate ¹¹C-erlotinib as a PET probe for imaging of OATP2B1 function and to assess the impact of OATP inhibition on the liver distribution and PK of erlotinib in humans.

Passive diffusion is often assumed to be the main mechanism for erlotinib to enter cancer cells and reach the EGFR kinase domains. However, it is not known whether TKIs such as erlotinib can freely cross membranes by passive diffusion or whether SLC transporters play a role in the tumor uptake [40]. Such phenomenon may control drug delivery to the tumor and account for therapeutic efficiency, with limited impact on the plasma PK. ¹¹C-erlotinib PET imaging, performed with and without transporter inhibitors such as rifampicin, could be useful to address the impact of membrane transporters in controlling the uptake of erlotinib by tumor cells in vivo [41, 42].

In this study, the impact of rifampicin on erlotinib uptake by the liver and kidneys was assessed using ¹¹C-erlotinib PET imaging in rats. Unexpectedly, despite significant decreases in liver and kidney exposure, rifampicin did not affect systemic exposure and subsequent exposure to the lungs as the main therapeutic target tissue of erlotinib. The consequences for erlotinib PK of these newly reported rifampicin-inhibitable transports, possibly OATP, should be addressed in humans using ¹¹C-erlotinib PET imaging [39].