PET Tracers To Study Clinically Relevant Hepatic Transporters

Review pubs.acs.org/molecularpharmaceutics

PET Tracers To Study Clinically Relevant Hepatic Transporters Andrea Testa,† Matteo Zanda,† Charles S. Elmore,‡ and Pradeep Sharma*,§ †

Kosterlitz Centre for Therapeutics, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, U.K. AstraZeneca R&D, Pepparedsleden 1, Mölndal 431 83, Sweden § AstraZeneca R&D, Cambridge Science Park, Milton Road, Cambridge CB4 0WG, U.K. ‡

ABSTRACT: Transporter proteins expressed on the cell membranes of hepatocytes are directly involved in the hepatic clearance, mediating the transport of drugs and metabolites through the hepatocyte, from the bloodstream into the bile. Reduction of hepatic transporter activity (due to chemical inhibition, genetic polymorphism, or low expression) can increase systemic or liver exposure to potentially toxic compounds, causing adverse effects. Many clinically used drugs have been associated with inhibition of hepatic transporters in vitro, suggesting the potential involvement of liver transporters in drug−drug interactions (DDIs). Recently, radiolabeled hepatic transporter substrates have been successfully employed in positron emission tomography (PET) imaging to demonstrate inhibition of clinically relevant hepatic transporters. The present article briefly describes the clinical relevance of hepatic transporters followed by a review of the application of PET imaging for the determination of pharmacokinetic parameters useful to describe the transporter activity and the design, accessibility, and preclinical and clinical applications of available radiotracers. Finally, based on the analysis of the strengths and limitations of the available tracers, some criteria for the development of novel PET probes for hepatic transporters and new potential applications are suggested. KEYWORDS: transporters, hepatic, positron emission tomography (PET), imaging, drug−drug interactions (DDI)

1. INTRODUCTION Transporters are membrane-bound proteins that facilitate the vectorial movement of endogenous or exogenous substrates across biological membranes.1−3 Transporters expressed in the liver have been shown to play critical roles in pharmacokinetics,4 pharmocodynamics,5 drug− drug interactions (DDIs),6 targeted or site specific deliveries,7 toxicological effects,8 and therapeutic efficacy of drugs.9,10 Their impact on environmental toxicology is another burgeoning area of research.11 The study of drug transporters involves the determination of binding affinities, transporter efficiency, inhibition of transport, up/down regulation of transporter proteins, expression and/or distribution patterns of novel putative carrier proteins, molecular mechanistic studies of solute-carrier interaction, and structure activity relationships of the transporter. Various in silico,12 in situ,13 in vitro,14 and in vivo15 model systems are available to accomplish these wide varieties of tasks. Each of these methodologies has advantages and disadvantages. Traditional human pharmacokinetic mass balance studies, based on the determination of the drug concentration in biological fluids (blood, plasma, serum, urine), do not provide precise information about the actual drug concentration in tissues, such as the liver and kidneys. Employing a labeled drug, molecular imaging can be used for the noninvasive quantification of drug concentrations in clearance organs, © XXXX American Chemical Society

allowing the determination of pharmacokinetic parameters related to transporter activity. Positron emission tomography (PET), because of its exceptional sensitivity and good spatial resolution, is a powerful functional imaging technique that has been successfully used to study the molecular mechanisms behind numerous biological processes. The current article briefly reviews the application of PET imaging in the determination of pharmacokinetic parameters useful to describe the hepatic transporter activity and the design, accessibility, and preclinical and clinical applications of the available radiotracers. Finally, based on the analysis of the strengths and limitations of the available tracers, some criteria for the development of novel PET probes for hepatic transporters and new potential applications are suggested.

2. APPLICATION OF PET TO STUDY HEPATIC TRANSPORTERS The application of PET technology to study hepatic transporters could have certain distinct advantages when compared Received: January 19, 2015 Revised: May 22, 2015 Accepted: June 2, 2015

A

DOI: 10.1021/acs.molpharmaceut.5b00059 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics XH(t )

to conventional in vitro cell assays and in vivo preclinical or clinical PK studies. These are listed below: • PET imaging can be performed on small animals, large animals, and humans • No physical collimators are required for coincidence counting with PET, and it has a higher sensitivity than other optical/nuclear imaging techniques • Since a PET tracer is typically used in microdose, it is unlikely to have pharmacological or toxic effects • PET is a quantitative molecular imaging technique which can be used for kinetic modeling applications. Accurate determination of the anatomical location of the probe activity helps to identify areas of prime significance if the transporter is expressed at multiple organs • PET has very low tissue attenuation (which can be corrected) of signal and , hence, no depth limitation • The good spatial and temporal resolution of PET allows various physiological phenomena (metabolism, transport, and cell kinetics) to be imaged in a noninvasive manner in living organisms at multiple time points. Well-known substrates of hepatic transporters have been labeled and employed to study clinically relevant transporters. The biodistribution of these tracers has been studied with or without the coadministration of selective inhibitors of transporters. The following are the major reported applications of PET imaging in the area of hepatic transport. 2.1. Determination of the Uptake Hepatic Clearance and the Apparent Liver to Blood AUC. Mathematical models called “integration plots” have been designed, in order to calculate the hepatic clearance and the canalicular efflux clearance by measuring the concentration of the tracer in the plasma and in tissues of interest such as liver and intestine. The tissue compartments are generally assumed to be well stirred, with a rapid equilibrium between the capillary bed and the interstitial fluid.16 While the tracer concentration in the blood can be monitored by measuring the radioactivity in blood samples collected during the imaging experiment, the concentrations of the tracer in the liver and intestine can be monitored via noninvasive PET imaging. Liver and intestine are selected as volumetric regions of interest (ROIs), and the radioactivity concentration, as fractions of the total injected radioactivity, can be measured over time. As demonstrated by Takashima et al.,17 the sum of the radioactivity in gallbladder and intestine can be considered equal to the radioactivity in the bile secreted to these tissues. This was proved by comparing the radioactivity measured in the intestine via PET imaging with the radioactivity found in bile samples obtained from bile duct cannulas. To consider the contribution of the partial volume effect from the bile excreted to the intestine, the ROIs for this tissue were defined to include a portion of the nearby regions. As proposed by Takashima and Watanabe,18 the hepatic uptake rates of the radiotracer can be calculated from the measure of the radioactivity in the liver and in the blood. Within short periods of time after PET tracer injection (generally 1−3 min), the metabolism of the tracer can be considered negligible and the efflux can be assumed to be much smaller than the influx. The hepatic uptake clearance CLuptake is determined from the linear eq 1

C blood(t )

= CLuptake

AUC blood(0 − t ) C blood(t )

+ V0

(1)

where XH(t) is the amount of radioactivity found in the liver at time t, Cblood(t) is the amount of the radioactivity in the blood at time t, AUCblood(0−t) is the area under the blood concentration− time curve from 0 to t, and V0 is the initial distribution volume in the liver at time 0. Plotting [XH(t)/Cblood (t)] versus [AUCblood(0−t)/Cblood(t)], V0 is the y intercept and CLuptake the slope of the straight line. It is also possible to calculate the apparent liver-to-blood AUC ratio, Kp,liver (2), which represents the liver exposure of the radiotracer and is a reflection of the hepatic extraction ratio: K p ,liver =

AUCH(0 − t ) AUC blood(0 − t )

(2)

where AUCH(0−t) is the area under the hepatic concentration− time curve from time 0 to t. If Kp > 1, the tracer is likely to be a substrate of uptake transporters. The significant differences can be found for Kp and CLuptake values between control groups and hepatic transporter−inhibitor-treated groups. 2.2. Determination of the Biliary Clearance. The biliary clearance of the radiotracer can be determined in a noninvasive way as proposed by Takashima et al.18 by measuring the amount of radiotracer in the liver and the amount of the radiotracer excreted into the intestine through the bile. In fact, the accumulation of the radioactivity in the bile can be described by the linear eq 3: Xbile(t ) = CL int,bile·AUCH(0 − t ) + Ve

(3)

where Xbile(t) is the amount of radioactivity found in the bile (intestine), CLint, bile is the canalicular efflux clearance of the radiotracer, and Ve the y intercept of the straight line. If the radiotracer undergoes hepatic metabolism during the imaging experiment, the number of metabolites and their relative amounts should be determined in order to calculate the contribution of each metabolite to the total amount of radioactivity measured by the PET scan in the intestine. In this case, the intrinsic biliary clearance of each metabolite can be determined from eq 3. If the metabolites pattern is unknown, the same equation can be used to calculate the canalicular clearance of the total radioactivity associated with the PET tracer and its metabolites. As a reflection of the uptake from the systemic circulation and the efflux from the canalicular membrane, the biliary secretion clearance, CLbile, can be calculated using the following eq 4: CL bile =

Xbile(0 − t ) AUC blood(0 − t )

(4)

where Xbile(0−t) is the amount of radioactivity secreted in the bile between time 0 and t.

3. PET TRACERS TO STUDY HEPATIC TRANSPORTERS The ideal PET tracer to study hepatic transporters should exhibit the following characteristics: • Actively and selectively transported by specific uptake and/or efflux transporters • Show low/no passive diffusion through cellular membranes • Metabolically stable B

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Molecular Pharmaceutics • Safe to administer in vivo • Efficiently and easily labeled with a PET isotope having half-life long enough to allow the imaging of the clearance process • Easily formulated for intravenous dosing In reality it might be difficult to find a PET tracer with all the aforementioned attributes. If the tracer is employed to study the uptake process in hepatocytes, the absence of hepatic metabolism is not strictly required, but the transport should not be blood-flow limited in order to observe the effect of transporter inhibitors on the tracer uptake. On the other hand, if observing the biliary efflux process is the objective of the study, high metabolic stability of the tracer is desirable, and the biological half-life of the radiolabeled tracer should be comparable with the physical half-life time of the labeled radioisotope. Clinically used drugs that undergo hepatic excretion are common starting points for the design of a PET tracer for the study of hepatic transporters, because their pharmacokinetics and toxicology have already been extensively studied throughout the drug development process. Also, drug metabolites17,19 and endogenous compound derivatives20,21 have been successfully labeled with 11C and employed as PET tracers. Table 1 lists the PET tracers that have been used to study hepatic transporters.

3.1. [11C]-Dehydropravastatin as a PET Tracer To Study OATPs and MRP2 in Rats. Dehydropravastatin 1 (DPV) is an analogue of the lipoprotein-lowering drug pravastatin 2, which was developed to evaluate the functions of OATP1B1 and MRP2 in vivo.22 In comparison to pravastatin, dehydropravastatin lacks the stereogenic center at the C2′ carbon and the presence of the double bond between the C2′ and C3′ allowed the 11C labeling via cross coupling chemistry (Figure 1). The slight difference of

Figure 1. Chemical structures of dehydropravastatin 1 and pravastatin 2.

molecular structure between DPV 1 and pravastatin 2 is postulated to have a negligible effect on the potential of DPV to be an OATPs and MRP2 substrate. The synthesis of [11C]-1 was accomplished via a palladium mediated Suzuki coupling of the organoboron precursor 3 and [11C]-CH3I, followed by deprotection of the hydroxyl groups concomitant with lactone ring opening and formation of the sodium salt (Scheme 1).23

Table 1. PET Tracers That Had Been Used To Study Hepatic Transporters PET tracer 11

[ C]Dehydropravastatin [11C]-Rosuvastatin

hepatic transporters studied

used preclinically or clinically (species)

refs

OATPs, MRP2

Preclinical (rat)

22, 23

Preclinical (rat)

24

Preclinical (rat) and clinical

18, 25

[11C]-Glyburide

OATPs, NTCP, BCRP OATP1B1, OATP1B3, MRP2 OATPs

(baboon,

26

[11C]-Telmisartan

OATP1B3

(rat) and

27−29

[11C]-Metformin

OCT1, MATE1

(mouse,

30, 31

[11C]-Rhodamine-123

OCT1, P-gp

(rat,

32

[11C]SC-62807 [11C]-Cholylsarcosine

OATPs, BCRP Bile acid transporters OATPs, MRP2

Preclinical mouse) Preclinical clinical Preclinical rat, pig) Preclinical mouse) Preclinical Preclinical

(mouse) (pig)

17, 19 20

[11C]-TIC-Me

[11C]-N-acetylcysteinylleukotriene E4

Preclinical (rat, monkey)

Scheme 1a

a Reagents and conditions: (i) [11C]-CH3I, Pd2(dba)3, P(o-tolyl)3, K2CO3, THF, 65 °C, 5 min; (ii) NBu4F, 60 °C, 2 min; (iii) 0.1 M NaOH, r.t., 1 min.

The uptake of DPV was examined in vitro using freshly prepared rat hepatocytes and rodent MRP2-expressing membrane vesicles. The kinetic transport parameters for DPV were found to be similar to those obtained for pravastatin 2. In the presence of rifampicin (an OATPs inhibitor), the uptake of DPV by hepatocytes was completely inhibited, supporting the hypothesis that DPV, like pravastatin, is a substrate of uptake transporters such as OATPs. DPV and pravastatin were also found to inhibit the MRP2 transport of estradiol 17βglucuronide (E217βG), a probe substrate of MRP2. The metabolism of [11C]-1 was studied in rats and human cryopreserved hepatocytes. While no metabolism was observed within 90 min of incubation with human hepatocytes, at least two major metabolites were found in the blood, liver, and bile of rats after the intravenous administration of [11C]-1. Time−plasma concentration profile, hepatic uptake, and canalicular efflux clearance of [11C]-1 were studied in control, rifampicin-treated, and MRP2-deficient rats (Eiasi hyperbilirubemic mutant rat). Within 5 min of the intravenous

21

The following sections describe the synthesis and in vitro characterization of the reported tracers employed to study hepatic transporters, along with a discussion of the in vivo imaging results. In most cases, in vitro studies have been performed in cells expressing human transporters, while in vivo experiments were conducted with rodents or other animal models (pigs, baboons). This practice is generally acceptable, considering the high functional homology between human and rodent transporters. However, during the interpretation of the results, one should take into account possible specific differences in the substrate recognition of human and murine transporters. C

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Molecular Pharmaceutics injection of [11C]-1, the radioactivity had cleared from the blood in all the animal models. After the same period of time, the elimination in rifampicin-treated and MRP2-deficient rats was significantly reduced. [11C]-1 extensively accumulated in the liver, in which the uptake was blood-flow limited, with a maximum peak at about 2 min after administration. In rifampicin-treated and MRP2deficient rats, the AUCs of radioactivity in the liver were 1.8and 3-fold larger than those in the control rats. The maximum amount of radioactivity in the intestine was found to be 2.5and 4-fold less in rifampicin-treated and MRP2-deficient rats than in control rats, respectively. The hepatic uptake clearance and the canalicular efflux clearance were also calculated for [11C]-1 using the integration plot method. As expected, the hepatic uptake clearance in MRP2-deficient rats was similar to that of the control rats, while the canalicular efflux clearance was about 9-fold smaller. In rifampicin-treated rats, the hepatic uptake clearance of [11C]-1 was only slightly smaller than the value found in control rats. This was probably due to the bloodflow-limited hepatic uptake of [11C]-1. The canalicular efflux clearance was 2-fold smaller in rifampicin-treated rats than in control rats, demonstrating the inhibitory effect of this molecule on the activity of MRP2.22 3.2. [11C]-Rosuvastatin as a PET Tracer To Study OATPs and MRP2 in Rats. Rosuvastatin 5 is a 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitor, clinically used for the treatment of dyslipidemia. Because rosuvastatin is a known substrate of the transporters OATPs, NTCP, MRP2, and BCRP,33−36 the [11C]-isotopomer [11C]-5 was developed to study the hepatobiliary transport of the drug by means of quantitative PET imaging.24 [11C]-5 was prepared by N-methylation of the desmethyl precursor 4 with [11C]-CH3I in the presence of tetrabutylphosphonium hydroxide (Scheme 2). After HPLC purification, [11C]-5 was obtained in 54% decay-corrected yield in about 35 min.24

that [11C]-5 is a sensitive PET tracer for studying the inhibition of hepatic transporters by rifamipicin in rats, and the tracer may be a useful PET probe for human hepatic transporters. By coadministration of inhibitor drugs with [11C]-5 by i.v. and oral routes separately, it might be possible to understand the impact of gut transporters in rosuvastatin DDIs. The use of pitavastatin as a PET probe might be a useful strategy, as this molecule has recently been shown to be a more specific and sensitive clinical probe for OATP1B1 inhibition when coadministered with rifampicin in humans.39 3.3. [11C]-TIC-Me as a PET Tracer To Study OATP1B1, OATP1B3, and MRP2 in Rats and Humans. (15R)-TICMe, (15R)-16-m-tolyl-17,18,19,20-tetranoisocarbacyclin methyl ester (6), was originally developed as a radioligand for the imaging of prostacyclin receptor PGI2 in the thalamus.40 15-(R)-TIC-Me (6) is a prodrug of the pharmacologically active acid 15-(R)-TIC (7) (Figure 2) that is subjected to

Figure 2. Chemical structures of (15R)-TIC 7 and its prodrug (15R)TIC-Me 6.

hepatic clearance.18 Due to the structural similarity between (15R)-TIC and prostaglandin E2, which is a well-known OATP substrate, it was postulated that [11C]-6 could be a good tracer to study hepatobiliary transport in vivo. The 11C labeling was achieved by using a Stille coupling of stannane 8 and [11C]-CH3I (Scheme 3).41 In vitro assays were performed in order to study the uptake of [3H]-(15R)-TIC in OATP1B1, OATP1B3, OATP2B1, NTCP-expressing HEK239 cells, control cells, and human

Scheme 2a

Scheme 3a

a

Reagents and conditions: (i) [11C]-CH3I, PBu4OH, MeCN/DMSO, 105−120 °C, 5 min.

In a preclinical study, [11C]-5 was administered in rats intravenously (i.v. bolus dose of 1.41 μg/kg, 1 mCi) over 30 s with and without rifampicin (40 mg/kg i.v. bolus plus 1.85 mg/ min/kg infusion).24 It was observed that [11C]-5 selectively accumulated in the liver and kidneys. In the rat, rosuvastatin is transported into the liver by uptake transporters OATPs (1A1, 1A4, 1A5, and 1B2), but not by NTCP.34 In the liver, it is biotransformed by metabolizing enzymes and/or excreted by MRP2 and BCRP.36,37 In the kidneys, rosuvastatin is actively taken up by the renal transporter OAT3.38 The coadministration of rifampicin increased systemic AUC by 3-fold, while it decreased accumulation (AUC0−15 min) of [11C]-5 in the liver and kidneys by 54% and 73%, respectively. The study showed

a

Reagents and conditions: (i) [11C]-CH3I, Pd2(dba)3/P(o-tolyl)3 (1:4), CuCl, K2CO3 DMF, 65 °C, 5 min. D

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Molecular Pharmaceutics Scheme 4a

a

Reagents and conditions: (i) [11C]-CH3OTf, 3 N NaOH, acetone, 110 °C, 2 min.

hepatocytes.18 The OATP1B1 and OATP1B3 contributions to the hepatic uptake of [3H]-(15R)-TIC were calculated by fractional functional contribution study using reference substrates for OATP1B1 and OATP1B3. In the hepatic uptake of (15R)-TIC, the contributions of OATP1B1 and OATP1B3 were 69.3% and 30.7%, respectively. No differences between the transport of [3H]-(15R)-TIC in OATP2B1 and control cells were found, suggesting that the molecule is not a substrate of that transporter. Although the authors did not find a sodium dependence in the transport of [3H]-(15R)-TIC in human hepatocytes, indicating that NTCP is not involved in the transport, it was observed to be transported in NTCP expressing cells. The contribution of NTCP in the transport of [3H]-(15R)-TIC was considered negligible if compared to the contribution of OATPs. The metabolism of the PET tracer [11C]-6 was thoroughly investigated in normal and MRP2-deficient rats. In neither of them could the methyl ester prodrug be detected in the blood 1 min after the administration. The carboxylic acid [11C]-7 was detectable for up to 5 min after injection. At least three metabolites were detected in the blood, liver, and bile of normal and MRP2-deficient rats. The three major metabolites were identified as a beta oxidation (de-ethylation) product of [11C]-7 (M1), the acylglucoronide of [11C]-7 (M2), and the acylglucoronide of M1 (M3). Most of the radioactivity found in the liver of MRP2-deficient and control rats was derived from the metabolite M2, while, in the bile, the most abundant metabolite was M3 in both animal models. The hepatobiliary transport of [11C]-7 and its metabolites was extensively investigated in normal and MRP2-deficient rats. After 2 min from the injection of [11C]-(15R)-TIC-Me, the radioactivity was mainly located in the liver and kidneys. After 60 min, most of the radioactivity was localized in the intestine of normal rats while no signal was detected in the intestine of MRP2-deficient rats. The AUCliver0−90 was 2.7-fold higher in MRP2-deficient rats than in control rats. The hepatic uptake clearance of [11C]-6 was calculated by means of the integration plot method, using eq 1. The value found for control rats (45 mL/min/kg) was comparable with the rat’s hepatic blood flow rate (55 mL/min/kg), suggesting a very effective hepatic uptake of [11C]-(15R)-TIC. In MRP2deficient rats, the hepatic uptake clearance of [11C]-6 was slightly reduced, probably due to the lower OATP expression42 and to the high bilirubin glucuronide concentration, which may inhibit the uptake of OATP ligands.43 The intrinsic biliary clearance values of the two metabolites found in the bile, M2 and M3, were calculated using eq 2. For the major metabolite found in the bile, M3, the canalicular efflux clearance CLint,bile,M3 was decreased to 14% in MRP2deficient rats, while, for the metabolite M2, the reduction of the CLint,bile,M2 was not statistically significant, probably because of the involvement of other efflux transporters in the excretion of M2 from the hepatocyte. This was supported by in vitro assays

employing MRP2 and BCRP expressing vesicles: M2 and M3 were good substrates of MRP2, while only M2 was significantly transported by BCRP. In humans, a similar metabolic profile pattern was found,25 with the conversion of [11C]-6 to the free carboxylic acid [11C]7 in less than 2 min and the formation of M1, M2, and M3. A new metabolite was also found, but its structure was not clarified in the study. The hepatobiliary transport of [11C]-6 was then studied in healthy human volunteers with and without rifampicin treatment. After 17 min from the intravenous administration of [11C]-6, the maximum levels of radioactivity were found in the liver in both control and rifampicin-treated subjects. In rifampicintreated subjects the AUCblood (0−30) was 1.5-fold higher than in control subjects. Consequently, the amount of radioactivity in the bile excreted into the intestine of rifampicin-treated subjects was reduced to 50% of the control. The hepatic uptake clearance of [11C]-7 was calculated as described before by employing eq 3. The value found in control subjects ranged from 46% to 79% of the blood flow rate. At the dose employed in the study, rifampicin was able to reduce the hepatic uptake clearance by 45%, while no effects were observed on the renal uptake clearance. This was in agreement with the Ki values of rifampicin found for OATP1B1 and OATP1B3 (0.62 μM and 0.39 μM, respectively) and the concentration of unbound rifampicin present in the systemic circulation (1.3 μM) after 1 h of administration of a 600 mg dose. The systemic concentration of rifampicin was higher than the Ki values; therefore, it could produce a detectable inhibition of transporters activity. Even though the effects of rifampicin on the efflux clearance were variable among the subjects, a significant reduction of the canalicular efflux of the radioactivity (by 62%) was observed in rifampicin-treated subjects, probably due to the inhibition of the MRP2 transporter. [11C]-6 was successfully employed as a PET tracer to quantitatively study the activity of the rat transporter MRP2, and due to its history of clinical use, it was safely employed in humans to assess the effect of rifampicin on the hepatobiliary functionality. In this valuable work, the authors proved the use of PET imaging to analyze the kinetics of the hepatobiliary transport in humans and demonstrated that, at the clinical dose, rifampicin is able to inhibit the hepatic uptake via OATP1B1 and OATP1B3. 3.4. [11C]-Glyburide as a PET Tracer To Study OATP1B1 in Baboons. Glyburide 10, a sulphonylurea receptor 1 inhibitor, is a widely prescribed antidiabetic agent, used in the treatment of type 2 diabetes. It is a substrate of several ABC transporters (ATP Binding Cassette proteins) (BCRP, MRP1, and MRP3)44,45 but is also a substrate of OATP1B1, OATP2B1, and OATP1A2.46,47 E

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Molecular Pharmaceutics [11C]-Glyburide [11C]-10 was obtained via methylation of the desmethyl precursor 9 with [11C]-CH3OTf at 110 °C for 2 min (Scheme 4).26 In vitro studies were performed to measure the uptake of [3H]-glyburide in P-gp and BCRP-expressing MDCKII cells, with or without the presence of FTC (a BCRP inhibitor),48,49 PSC833 (a P-gp inhibitor),50,51 and GF 120918 (a dual P-gp/ BCRP inhibitor).52 The uptake of [3H]-glyburide in P-gp expressing MDCKII cells was significantly increased by the presence of PSC833 and GF120918, while the effect of the FTC was negligible. Similarly, the uptake of [3H]-glyburide in BCRP-expressing MDCKII cells was significantly increased by the presence of FTC and GF120918, while the specific P-gp inhibitor PSC833 did not affect the uptake. PET imaging and metabolism experiments were performed in anesthetized baboons with or without the coadministration of pantoprazole (CYP3A4 inhibitor), rifampicin (OATPs inhibitor), and cyclosporine A (OATPs/ABC transporters and CYP3A4 inhibitor). No metabolites of [11C]-10 were found in blood when rifampicin or cyclosporine A were coadministered, suggesting that the transport of glyburide from the bloodstream to the liver is the first key step in glyburide metabolism. [11C]-Glyburide accumulated in the liver of control baboons, showing the hepatobiliary extraction of the labeled drug. The rifampicin and cyclosporine A treatments significantly reduced the liver-to-blood AUC ratio, Kp,liver, by 14- and 18-fold, respectively. As a consequence of the decreased hepatic uptake, the [11C]-glyburide exposure to the renal cortex, myocardium, pancreas, and lungs increased. Regarding the biliary excretion, no significant results were obtained due to great variability in the PET imaging experiments. This might be due to the relatively short time period investigated (60 min) compared to the glyburide half-life (4.7 h in humans). The results confirmed that OATP transporters control the glyburide biodistribution and have an important impact on its metabolism. As no difference was found between the rifampicin and cyclosporine A treatments, the impact of P-gp inhibition in the biodistribution of glyburide was believed to be minor, at least when OATP was already inhibited. 3.5. [11C]-Telmisartan as a PET Tracer To Study OATP1B3 in Rats and Humans. Telmisartan (12) is an angiotensin receptor antagonist used in the management of hypertension. It is a lipophilic compound mainly transported into the liver by the OATP1B3 transporter.53 [11C]-Telmisartan ([11C]-12) was selected as a PET tracer to study the activity of OATP1B3 and was prepared by the coupling of desmethyl precursor 11 with [11C]-CH3I, followed by methyl ester hydrolysis (Scheme 5). An automated synthesis was also developed for the clinical production of [11C]-12.28 The metabolism of [11C]-12 was investigated in normal rats. No metabolites were found in the blood after 20 min of the

administration, while, in the liver, telmisartan was transformed into the glucuronide metabolite by UDP-glucuronyltransferases.54 The biliary excretion of telmisartan-glucuronide (the sole metabolite of the drug) was observed. The hepatobiliary transport of [11C]-12 was at first studied in normal and rifampicin-treated rats and then in healthy human volunteers.27,29,55 In control rats, a maximum of the radioactivity was observed at 14 min postinjection, and from that point, the radioactivity was observed to accumulate gradually in the intestine. In rifampicin-treated rats, the radioactivity observed in the liver decreased in a dose dependent manner while the radioactivity in the intestine was unaffected. Compared to control rats, the AUC0−90blood was 2.4- and 3.9fold higher in rats treated with a constant infusion of rifampicin at 0.5 and 1.5 μmol/min/kg, respectively. The hepatic uptake clearance CLuptake calculated by the integration plot method was found to be very close to the hepatic blood flow in control rats, but it was possible to observe a significant reduction of CLuptake to 65% in rats treated with rifampicin at 1.5 μmol/min/kg. No significant difference was found in the biliary efflux clearance (CLint,bile) between control and rifampicin-treated rats. The effect of the administration of 1, 4, and 10 mg/kg of unlabeled telmisartan on the biodistribution of [11C]-12 was also investigated. The elimination of the radioactivity from the systemic circulation was reduced in a dose dependent manner. The hepatic uptake clearance of [11C]-12 was significantly reduced to 52% at the dose of 10 mg/kg of unlabeled telmisartan. On the other hand, at the dose of 4 and 10 mg/kg, unlabeled telmisartan increased the biliary efflux of total radioactivity probably due to up-regulation of efflux transporters. A clinical study was performed on healthy human volunteers in order to determine the biodistribution and radiation dosimetry of [11C]-12 in humans.27 A few minutes after the [11C]-12 injection, the kidneys, intestine, and other organs were weakly visible. Subsequent imaging confirmed the hepatobiliary clearance of the tracer by accumulation at first in the liver and gradual elimination into the gallbladder and the intestine. The liver was the organ in which the highest level of radioactivity was observed, with a peak of 55.7 ± 3.3% of the injected dose at 0.80 h. The effective dose for [11C]-12 was found comparable to the doses of other 11C tracers published in literature, and no adverse effects, changes in physical conditions, or abnormal findings were observed after the termination of the scan. The tracer was thus found to be safe and promising in order to study OATP1B3 in humans. 3.6. [11C]-Metformin as a PET Tracer To Study OCT1 and MATE1 in Mice, Rats, and Pigs. Metformin 15 is a biguanide drug widely employed in the treatment of type II diabetes.56 Its site of action is the hepatocyte, in which it inhibits two fundamental enzymes involved in the gluconeogenesis: glucose-6-phosphatase and phosphoenolpyruvate carboxykinase.57 Metformin is transported into the hepatocyte via the OCT1 transporter58 and is then excreted unchanged into the bile via the efflux transporters MATE1 and MATE2K.59 While [14C]metformin has been generally employed as a probe substrate for in vitro testing of OCT1 activity, the 11C labeled form of metformin for PET imaging was first published in the literature in 2013.30 The synthesis was initiated by reacting [11C]-CH3I with methylamine to give [11C]-dimethylamine ([11C]-13), which was subsequently treated with

Scheme 5a

Reagents and conditions: (i) 1 M KOH, [11C]-CH3I DMSO, 120 °C, 5 min, (ii) 1 M NaOH, 100 °C, 3 min. a

F

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Molecular Pharmaceutics cyanogen bromide to afford [11C]-cyanamide ([11C]-14). Reaction of [11C]-14 with guanidine hydrochloride at high temperature in the presence of sodium hydroxide gave [11C]15 in an overall 17% radiochemical yield (Scheme 6).

The tracer was stable for at least 2.5 h in the plasma of rats, but some metabolites, whose structures were not resolved, were found during in vivo experiments. The biodistribution of [11C]17 was investigated with PET in different groups of mice: wild type, P-gp knock out, OCT1/2 knockout, and cimetidine pretreated P-gp knockout mice. The higher accumulation of [11C]-17 in the kidneys of wild type mice compared to that of OCT1/2 knock out mice confirmed that [11C]-17 is a good in vivo substrate for OCT1/2 transporters, as observed in vitro.66 There was no difference in the time−activity profile of [11C]-17 in the kidneys of wild type and P-gp knockout mice, indicating that there is no involvement of P-gp in the kidneys distribution of [11C]-17. Pretreatment of wild type rats with DCPQ, i.e., ((2R)-anti-5-{3-[4-9,10,11-dichloromethanobibenzosuber-5yl)piperazin-1-yl]-2-hyroxypropyl}quinoline trihydrochloride a P-gp inhibitor, resulted in greater peak radioactivity in the liver, confirming the role of P-gp in the biliary excretion of [11C]-17 in rats. Pretreatment of rats with cimetidine increased the systemic concentrations of [11C]-17, which may be due to inhibition uptake in the kidneys by renal transporters, as described previously, and/or due to inhibition of metabolic pathways by cimetidine in the liver.67,68 Since no data is available to compare accumulation of [11C]-17 between cimetidine-treated and -untreated rat liver, it is difficult to conclude what is the exact role of OCT1 in the liver uptake of [11C]-17. 3.8. [11C]SC-62807 as a PET Tracer To Study OATP1B1, OATP1B3, and BCRP in Mice. SC-62807 (20) is the principal metabolite of the selective cyclooxygenase inhibitor celecoxib. It is produced in the liver by two-step oxidation of celecoxib and is then excreted in the bile by the BCRP.69 [11C]SC-62807 was prepared via a Suzuki reaction of [11C]CH3I and boronate ester 18 to give [11C]-celecoxib [11C]-19, which was oxidized to [11C]-SC-62807 [11C]-20 by means of basic potassium permanganate under microwave irradiation (Scheme 8).17

Scheme 6a

a Reagents and conditions: (i) 2 M MeNH2 in THF, dimethylacetamide, 45 °C, 5 min; (ii) BrCN, IPr2NEt, dimethylacetamide, 45 °C, 5 min; (iii) guanidine hydrochloride, NaOH, H2O, 175 °C, 5 min.

The biodistribution of [11C]-15 was studied in pyrimethamine (an inhibitor of MATE1 and OCT1)-treated and control mice by injecting the compounds into the tail vein.30 In both the animal groups, the accumulation of radioactivity was observed in the kidneys and the urinary bladder. The radioactivity in the liver of control mice rapidly decreased after 2 min. In pyrimethamine-treated mice, an enhanced accumulation of the radioactivity was observed in the liver, which reached a peak between 6 and 7 min after the administration. This effect was due to the effective inhibition of the extrusion transporter MATE1 by the presence of pyrimethamine, reducing the canalicular efflux of the radioactivity. Negligible effects on OCT1 inhibition were observed in the imaging experiment because, at the concentrations employed in the study (5 mg/kg, corresponding to unbound concentration 0.3 μM), pyrimethamine has no effects on OCT1 (Ki = 3.6 μM), while it is able to inhibit MATE1 (Ki = 0.14 nM). Although the work did not describe the impact on human renal transporters (OCT2 and MATE1/2K), it is proposed that it may also be possible to use [11C]-15 to study the activities of OCT2 and MATEs in the kidney. In a different study, the biodistribution of [11C]-15 was also investigated in rats and pigs.31 In both the animal models, the activity accumulated in liver and kidneys. Administration of cold metformin delayed the hepatic and renal clearance.31 3.7. [11C]-Rhodamine-123 as a PET Tracer To Study OCT1 and P-gp in Rats. Rhodamine 123 17 is a weakly basic fluorone dye, known to accumulate in the mitochondria. It has also been employed in oncology research in order to measure the P-gp expression in certain drug resistant cancer cells60−63 and at the blood−brain barrier.64,65 In vitro experiments using OCT1-expressing HEK293 cells demonstrated that rhodamine123 is a high-affinity substrate for both the organic cation transporters.66 [11C]-Rhodamine-123([11C]-17) was synthesized by esterifying rhodamine-110 (16) with [11C]-CH3I (Scheme 7).32

Scheme 8a

a

Reagents and conditions: (i) (a) [11C]-CH3I, Pd2(dba)3, P(o-tolyl)3, DMF, 65 °C, 4 min. (ii) KMnO4, 0.2 M NaOH, 140 °C (microwave), 5 min.

In vitro studies were performed to evaluate the uptake of [11C]-20 in OATP1B1- and OATP1B3-expressing HEK 239 cells, human and rodents BCRP vesicles, and human hepatocytes. [11C]-20 proved to be a good substrate for rodent and human BCRP, OATP1B1, and OATP1B3 transporters. The relative contributions of OATP1B1 and OATP1B3 transporters in the human hepatocyte uptake were found to be similar. Metabolism and imaging studies were performed in wild-type and BCRP knockout mice.19 No metabolites of [11C]-20 were found in the blood, bile, liver, and urine of normal and BCRPdeficient mice. After 2 min from the injection of [11C]-20, the radioactivity was mainly located in the liver and kidneys of both

Scheme 7a

a

Reagents and conditions: (i) [11C]-CH3I, NBu4OH, DMF. G

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Reagents and conditions: (i) (a) [11C]-CH3I, 1,2,2,6,6-pentamethylpiperidine, DMSO, 50 °C, 5 min. (ii) diethyl cyanophosphonate, cholic acid, DMSO, 50 °C, 5 min. (iii) 0.25 M NaOH, room temperature, 2 min.

a

rate of the radioactivity into the intestine. The binding of [11C]23 to plasma proteins was found to be very similar to that of endogenous cholylderivatives (60−80%). It was also demonstrated that cholyltaurine displaced [11C]-23 from plasma proteins, meaning that [11C]-23 binds to the same proteins as endogenous bile acids. The hepatobiliary transport of a bile acid analogue and the effects of alterations of bile acid transporters were analyzed in a noninvasive way, showing how the imaging of hepatic transporters could be employed in the characterization of normal and pathological liver functionality. 3.10. [11C]-N-Aacetyl-cysteinyl-leukotriene E4 for the Study of OATP1B1 and MRP1/2 in Rats and Monkeys. NAcetyl-cysteinyl-leukotriene E4 (LTE4NAc) (25) is the major metabolite of endogenous cysteinyl leukotrienes in rodents.71 Leukotrienes, produced from arachidoic acid in the lipoxygenase pathway, are important mediators of inflammatory response72 and are generally substrates of OATP1B1 and MRP1/2.73 In order to assess the contribution of different organs to the clearance of leukotrienes, a 11C-labeled version of LTE4Nac, [11C]-25, was prepared and its biodistribution was studied in normal rats, transporter-deficient mutant rats (with a hereditary defect in the leucotriene elimination),74 and monkeys.21 [11C]-Acetyl chloride ([11C]-24) was obtained by reaction of 11 [ C]-carbon dioxide with methylmagnesium chloride and subsequent treatment with phthaloyl chloride. The acetylation of LTE4 was then performed in the presence of 2,6dimethylpyridine to afford [11C]-LTE4NAc ([11C]-25) after HPLC purification (Scheme 10).75

animal groups. By 30 min, most of the radioactivity was localized in the intestine and in the urinary bladder of normal mice whereas the AUCblood(0−30) was 2.2-fold higher in BCRPdeficient mice, indicating that the transportation of the radioactivity in BCRP-deficient mice had been slowed. Biliary secretion clearance (eq 4) and canalicular clearance (eq 3) were significantly lower in BCRP-deficient mice than in control mice (10- and 4-fold, respectively). Similar results were found for the renal secretion clearance and the kidney brush border efflux clearance, whose values were found to be 25- and 100-fold lower in BCRP-deficient mice, proving that BCRP is essential in the biliary and renal excretion of anionic drug metabolites such as SC-62807. The authors also conducted a study to assess the capability of [11C]-20 to study BCRP function at the blood−brain barrier (BBB). Unfortunately, low uptake of the tracer into the brain was observed in both BCRP-deficient and control mice probably because of the low membrane permeability of [11C]-20. 3.9. [11C]-Cholylsarcosine as a PET Tracer To Study Bile Acid Transporters in Pigs. Cholylsarcosine (23) is an analogue of the bile acid conjugated cholyl-glycine. It is a metabolically stable derivative of cholic acid and the amino acid sarcosine (N-methylglycine) and is subjected to enterohepatic circulation in humans.20,70 [11C]-Cholylsarcosine ([11C]-23) was prepared by Nmethylation of the methylester of glycine with [11C]-CH3I and subsequent coupling with cholic acid. The [11C]cholylsarcosine methylester so obtained was purified by HPLC and then hydrolyzed with aqueous NaOH to give [11C]-cholylsarcosine ([11C]-23) (Scheme 9).20 Because of the high similarity between cholylsarcosine and cholylglycine, it has been postulated that cholylsarcosine is a substrate of NTCP and BSEP transporters. However, no in vitro transport studies have been performed as yet and the contribution of other transporters such as OATPs and BCRP in the hepatobiliary transport of this bile acid analogue cannot be excluded. Imaging experiments were performed in pigs to investigate the hepatocellular transport and biodistribution of [11C]-23.20 The tracer rapidly accumulated in the liver (peak within 3 min postinjection) and was then excreted into the intrahepatic and common bile duct and eventually accumulated in the gallbladder or intestines. As a consequence of enterohepatic circulation, the radioactivity in the liver and bile ducts increased again 75 min after the injection. The imaging experiment was repeated in one of the animals after administration of cholyltaurine, an endogenous bile acid conjugate (286 mg/ kg), to investigate the inhibition of the hepatobiliary transport of [11C]-23. Inhibition of both hepatic uptake and biliary excretion was observed, with a substantial reduction of the liver AUC and choledochus AUC, and a much slower transportation

Scheme 10a

Reagents and conditions: (i) CH3MgCl, THF, −78 °C. (ii) phthaloyl chloride, THF, 80 °C, 5 min. (iii) leukotriene E4, 2,6-dimethylpyridine, THF, 45 °C, 10 min.

a

To the best of our knowledge, no in vitro transport studies have been performed to investigate the transport kinetics of LTE4NAc 25 on specific transporters. Metabolism of 25 was studied in normal rats, transporter-deficient mutant rats (impaired bilirubin secretion),76 and cholestatic rats, employing the tritium-labeled [3H]-LTE4NAc. Major metabolites were polar, shorter-chain, oxidation products. The biological half-life of [3H]-LTE4NAc was about 40 s in normal rats, and about 85% of the administrated radioactivity was excreted into the H

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Figure 3. Schematic representation of the development process of a PET tracer to study hepatic transporters.

bile within 1 h.21 In the same animal model, the amount of radioactivity found in urine was about 4%. In transporterdeficient mutant rats, 6% of the administrated radioactivity was excreted in the bile and 21% in the urine within 1 h, whereas in cholestatic rats a delayed renal elimination of the radioactivity was observed.21 PET imaging of [11C]-25 showed that the radioactivity accumulated in the liver (with a maximum activity concentration in the liver at 4 min for control rats, and 9−10 min for transporter mutant and cholestatic rats) and then in the intestine of normal rats or in the urinary bladder of mutant and cholestatic rats. The mean liver transit time was found to be 17 min for normal rats and 54 min for transporter mutant rats. In monkeys, as in normal rats, the hepatobiliary elimination exceeded the renal clearance of [11C]-25, with a maximum liver activity at 12 min postinjection and mean liver transit time of 34 min.21 This pioneering PET study showed how, in the case of impaired hepatic functionality (due to hereditary transporter deficiencies or bile duct obstruction), the extraction of leukotriene metabolites can be switched from hepatobiliary to renal clearance.

development of the 11C carbonylation methodology, for example, allows the introduction of the radioisotope in metabolically stable carbonyl, carboxyl functional, and heterocyclic compounds.77 Fluorine-18 radiochemistry has expanded considerably as well,78 leading to the development of new strategies that, in principle, could allow the late stage radiofluorination of almost all the fluorinated clinically used drugs. Ex novo design of completely new hepatic PET probes would involve a multidisciplinary approach consisting of specific syntheses based on structure activity relationships, radiolabeling, in vitro and in vivo evaluation, and kinetic modeling of the radiolabeled compound before it is applied to preclinical or clinical research (Figure 3). The key medicinal chemistry principles for designing hepatoselective PET tracers can be generated based on in silico and computational modeling of substrates and/or inhibitors of major uptake (OATP1B1/1B3, NTCP, OCT1) and efflux (P-gp, BCRP, MATE1, MRP2) drug transporters. In order to design hepatoselective PET probes for uptake transporter(s), the following attributes are desirable: • Structural features: Several studies have shown that incorporation of an acidic moiety (anionic) is a useful strategy to enable recognition of compounds by uptake transporters such as OATP1B1/1B3.79,80 Inclusion of a cationic tertiary amine could favor uptake by the OCT1 transporter. For NTCP inhibitors, pharmacophores consisting of hydrophobic regions and one hydrogen bond acceptor have been proposed.81,82 • Molecular weight (MW) and relative polar surface area (RPSA): MW of ≥400 Da and RPSA ≥20% have been observed with known OATP substrates.80 • Low passive permeability: Poor permeability of compounds prevents diffusion of the probe from hepatocytes back into sinusoidal blood capillaries and is helpful in terms of intracellular accumulation of the PET probe. Generally, a membrane permeability value of 10−6 cm/s

4. DESIGN OF PET TRACERS FOR HEPATIC TRANSPORTERS Although PET has been extensively used in various areas of drug discovery and development in the past decades, its use to study the involvement of hepatic transporters in drug pharmacokinetics is a relatively new but burgeoning area of research. In principle, incorporation of a PET isotope into the structure of most drug molecules to study hepatic transport should be feasible; however, there are significant technical challenges to overcome, mainly due to the short half-lives of PET tracers. While, for decades, the very short half-life times of 11 C and 18F have limited the chemical space of PET tracers to a few entities, now the evolution of novel synthetic methodologies virtually allows the labeling of any existing drug. The I

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PET tracers in in vitro models and determination of apparent Michaelis−Menten kinetics (Km), maximal transport velocity (Vmax), active clearance (CLactive), and passive clearance (CLdiff). If multiple transporters are involved, then the kinetics for each transporter should be evaluated. These constants, along with physicochemical properties (log P, pKa, B/P ratio, PPB), would be valuable if full physiologically based pharmacokinetic (PBPK) modeling of data generated from the PET study is desired. In vitro transporter inhibition assays using a PET tracer as probe substrate will help to gauge the sensitivity of the PET tracer for in vivo DDI studies. The determination of metabolic clearance (CLin) in human liver microsomes or similar systems and identification of generated metabolite(s) is necessary to aid the interpretation of in vivo findings in PET tracer studies. A preclinical PK study of the PET tracer will help to establish its distribution and elimination pathways. A DDI study in preclinical species using the PET tracer as the probe and a known potential inhibitor can provide results on the efficacy of use of the PET tracer as suitable probe. In vitro transporter inhibition results can be correlated to in vivo findings to validate the model. Although there are no direct orthologs between human and rodent hepatic OATP isoforms, despite these fundamental differences, they are functionally comparable at a collective level.90 Thus, preliminary DDI studies in preclinical species using PET probes still offer an important preparatory step before taking the probe into clinical studies. Most PET probes for hepatic transporters studied so far lack specificity in transport mechanism and are also eliminated by renal transporters (Table 1). For example, [11C]-rosuvastatin is not only eliminated by hepatic (OATPs, MRP2, and BCRP) but also by renal transporters (OAT3). Since it is possible to obtain concentration versus time profiles of specific organ systems in a PET study, “integration plots” from these concentration profiles can provide data on changes in concentrations/distribution at specific organs in the presence of a “perpetrator” candidate drug. This will help not only to identify the level of interactions at organ levels but also to provide a mechanistic interpretations of the interaction. For example, after intravenous administration of [11C]-rhodamine123 to wild-type rodents, PET and ex vivo measurements showed that radioactivity uptake was very low in brain, but relatively high in some other organs, such as heart and especially liver and kidneys. Inhibition of P-gp increased uptake in brain, heart, kidney, and liver, but only by up to 2-fold. Secretion of radioactivity from kidneys was markedly reduced by OCT knockout or pretreatment with cimetidine. [11C]Rhodamine-123 was unsuitable as a PET probe for P-gp function and appears to be a strong substrate of OCT1/2 in kidneys. Cimetidine appears to be effective for blocking OCT1/ 2 in kidneys in vivo. This is especially advantageous in light of the fact that most in vitro models cannot simultaneously study the effect of all transporters involved in the PK of a “victim” drug and cannot provide information on whole body PK when coadministered with a “perpetrator” drug. However, PET cannot distinguish the individual contribution of specific transporters colocated in the same organ. For example, [11C](15R)-TIC is actively transported into the liver, PET demonstrated CLuptake,liver of 21 mL/min/kg in humans, and the tracer-related radioactivity reached 37% of dose in the liver within 17 min of intravenous administration.25 However, it could not be confirmed from the PET study how much of this uptake clearance is contributed by OATP1B1 and/or

or lower, as determined from in vitro transcellular assays, is classified as low permeability of a compound.83 • Low lipophilicity (log D at pH7.4 ≤ 2): Most of the known good substrates of OATP1B1/1B3 have low log D values.84 Although low log D is the preferred property to impart hepatoselectivity to compounds, very low log D may have adverse ADME issues. Tu et al.79 had analyzed hepatoselective properties of a large set of diverse compounds from varied chemical spaces against their physiochemical and pharmacokinetic properties. The authors observed that the optimal lipophilicity for orally administered hepatoselective drugs lies between log D of 0.5 and 2.0. • Optimum solubility: High solubility of PET probes is desirable in order to enable higher fractions of the dose to be absorbed after oral administration. However, if the PET tracer is developed for intravenous administration, then this attribute may not be critical. There is considerable overlap of substrates between uptake and efflux transporters,80 and because of this, they share similar sets of important molecular descriptors for design of optimal PET tracers. However, there might be subtle differences in the desirable values of these molecular descriptors required for good substrates of efflux transporters, when compared to uptake transporters. For example, the structural features log D and the membrane permeability value requirements for substrates of efflux transporters require them to be more lipophilic85 than substrates of uptake transporters. The molecular descriptors proposed for P-gp substrates are a log P value of ≥2.92, ≥18-atom long molecular axis, high energy of the highest occupied orbital, and at least one tertiary basic nitrogen atom.85 Similarly, quantitative structure activity relationships (QSARs) had been reported and extensively reviewed for BCRP,86 MATE1/2K,87 MRP2,88 and BSEP89 transporters. Although a detailed analysis of QSAR for each hepatic transporter lies outside the scope of this review, the key findings concerning these aspects can be helpful stepping stones for the design of completely novel hepatic PET tracers. As evident from this section, most hepatic PET probes reported in the literature so far are well established drugs, drug metabolites, or their synthetic derivatives. This obviates the need to establish safety margins for them if they are used at microdose or subtherapeutic dose levels in PET studies. Their transporter kinetics are well characterized in in vitro and/or in vivo models and readily available from the literature. In principle, instead of sticking to the prototypical probe drugs already reported in the literature, it might be possible to study any drug of interest as a “victim” by labeling it with a PET isotope. Labeling a drug and coadministering it with the investigational “perpetrator” drug would enable not only the study pairs of drugs but also the study in the patient population of interest, instead of normal healthy volunteers. Also, because only trace doses of a PET probe are needed, drugs with narrow therapeutic index (for example, paclitaxel) could be used as PET tracers.

5. VALIDATION, DESIGN, AND INTERPRETATION OF PET STUDIES The validation of PET tracers as tools for studying hepatic transport may be done prior to their recommendation as in vivo probes for routine drug−drug interaction studies (Figure 3). This validation should involve full transporter kinetics of the J

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tissues in which they are abnormally expressed would also be useful for the diagnosis of liver diseases and cancers.

OATP1B3. Therefore, the relative activity factor (RAF) or fractional functional contribution was determined in vitro using HEK293 expressing OATP1B1 or OATP1B3 transporters along with human hepatocytes.25 The complete knowledge of elimination mechanisms and their relative contribution to the overall elimination of the proposed hepatic PET probe can provide useful information about the principal component involved in an observed DDI with the probe.91 For example, [11C]-rosuvastatin absorption is affected by BCRP and the systemic concentrations are controlled by active hepatic (OATP1B1, OATP1B3, NTCP) and renal uptake (OAT3). When administered by an oral or intravenous route, rosuvastatin can be used as probe for hepatic uptake transporters (OATP1B1 and OATP1B3), intestinal efflux transporters (BCRP), and/or renal uptake transporter (OAT3). The visualization of the PET probe and changes in local accumulation pattern will help identify the actual transporter(s) implicated in any observed DDI. While PET studies are easy to perform in conscious humans, they are difficult to perform in moving preclinical species. Often this requires sedation of animals with suitable anesthetics. This might interfere with the normal physiology of the body and introduce artifacts in PK observations by PET in preclinical species. Another major challenge in the interpretation of data from hepatic PET studies is the biotransformation of probes by action of metabolizing enzymes to generate various fractions of metabolites containing PET isotopes. This makes the determination of the mass balance of the parent PET tracer relative to the metabolites difficult, unless additional metabolite profiling studies are performed (which might also involve withdrawing blood samples at time points to know the exact concentrations of radiometabolites if formed at appreciable levels). The interest in the imaging of hepatic transporters, especially OATP1B1 and OATP1B3,92 is not limited to the field of DMPK: recently OATPs have been recognized as potential biomarkers for gastrointestinal,93 breast,94 prostate,95 pancreas,96 and lung97 cancers. Moreover, OATPs, for which expression is often altered in abnormal cells, play an important role in hormone98,99 and drug distribution in cancer cells.97 A selective PET probe for OATP expression would allow for a better understanding of cancer cell metabolism.100 Moreover, the use of PET tracers such as [ 11 C]cholylsarcosine [11C]-21 transported by bile acid transporters, NTCP and BSEP, could potentially provide valuable insight as diagnostic agents for pathophysiological hepatic conditions and also for drug induced hepatotoxicities which produce a perturbation of these transporters.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +44-(0) 1625 234281. Notes

The authors declare the following competing financial interest(s): The work for this review article is supported by grants from AstraZeneca Ltd. UK and SINAPSE (Scottish Imaging Network), Scotland, towards PhD studentship of AT. PS and CE are employees of AstraZeneca Ltd., UK.

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ACKNOWLEDGMENTS The critical review of this manuscript by Katherine Fenner, DMPK DSM, AstraZeneca Ltd. UK, is appreciated. ABBREVIATIONS AUC, area under the curve; DDI, drug−drug interaction; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPV, dehydropravastatin; HPLC, high-performance liquid chromatography; MRI, magnetic resonance imaging; PET, positron emission tomography; PK, pharmacokinetic; RAF, relative activity factor; ROI, region of interest; RPSA, relative polar surface area; THF, tetrahydrofuran

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REFERENCES

(1) Elliott, J. I.; Raguz, S.; Higgins, C. F. Multidrug transporter activity in lymphocytes. Br. J. Pharmacol. 2004, 143, 899−907. (2) Koepsell, H.; Endou, H. The SLC22 drug transporter family. Pflugers Arch. 2004, 447, 666−676. (3) Niessen, J.; Jedlitschky, G.; Grube, M.; Bien, S.; Schwertz, H.; Ohtsuki, S.; Kawakami, H.; Kamiie, J.; Oswald, S.; Starke, K.; Strobel, U.; Siegmund, W.; Rosskopf, D.; Greinacher, A.; Terasaki, T.; Kroemer, H. K. Human platelets express organic anion-transporting peptide 2B1, an uptake transporter for atorvastatin. Drug Metab. Dispos. 2009, 37, 1129−1137. (4) Chan, L. M.; Lowes, S.; Hirst, B. H. The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur. J. Pharm. Sci. 2004, 21, 25−51. (5) Tachibana-Iimori, R.; Tabara, Y.; Kusuhara, H.; Kohara, K.; Kawamoto, R.; Nakura, J.; Tokunaga, K.; Kondo, I.; Sugiyama, Y.; Miki, T. Effect of genetic polymorphism of OATP-C (SLCO1B1) on lipid-lowering response to HMG-CoA reductase inhibitors. Drug Metab. Pharmacokinet. 2004, 19, 375−380. (6) Sharma, P.; Butters, C. J.; Smith, V.; Elsby, R.; Surry, D. Prediction of the in vivo OATP1B1-mediated drug-drug interaction potential of an investigational drug against a range of statins. Eur. J. Pharm. Sci. 2012, 47, 244−255. (7) Sai, Y.; Tsuji, A. Transporter-mediated drug delivery: recent progress and experimental approaches. Drug Discovery Today 2004, 9, 712−720. (8) Kato, Y.; Suzuki, H.; Sugiyama, Y. Toxicological implications of hepatobiliary transporters. Toxicology 2002, 181−182, 287−290. (9) The International Transporter Consortium Membrane transporters in drug development. Nat. Rev. Drug Discovery 2010, 9, 2015− 236. (10) VanWert, A. L.; Gionfriddo, M. R.; Sweet, D. H. Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology. Biopharm. Drug Dispos. 2010, 31, 1−71. (11) Abu-Qare, A. W.; Elmasry, E.; Abou-Donia, M. B. A role for Pglycoprotein in environmental toxicology. J. Toxicol. Environ. Health B Crit. Rev. 2003, 6, 279−288. (12) Geldenhuys, W. J.; Manda, V. K.; Mittapalli, R. K.; Van der Schyf, C. J.; Crooks, P. A.; Dwoskin, L. P.; Allen, D. D.; Lockman, P.

6. CONCLUSIONS The in vivo PET imaging of hepatic transporters is still an emerging technology that allows the quantification of critical kinetic parameters useful to describe the activity of such transporters. The effects of chemical inhibition and reduced activity of specific hepatic transporters on the pharmacokinetic profile of reference drugs have been successfully studied, prompting the development of standard imaging protocols that can be adopted in drug safety studies. The mechanism of some DDIs has been clarified and unequivocally attributed to inhibition of transporters. A deeper understanding of the function of transporters in the liver and in K

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Molecular Pharmaceutics R. Predictive screening model for potential vector-mediated transport of cationic substrates at the blood-brain barrier choline transporter. Bioorg. Med. Chem. Lett. 2010, 20, 870−877. (13) Abuasal, B.; Sylvester, P. W.; Kaddoumi, A. Intestinal absorption of gamma-tocotrienol is mediated by Niemann-Pick C1-like 1: in situ rat intestinal perfusion studies. Drug Metab. Dispos. 2010, 38, 939− 945. (14) Sharma, P.; Holmes, V. E.; Elsby, R.; Lambert, C.; Surry, D. Validation of cell-based OATP1B1 assays to assess drug transport and the potential for drug−drug interaction to support regulatory submissions. Xenobiotica 2010, 40, 24−37. (15) DeGorter, M. K.; Kim, R. B. Use of transgenic and knockout mouse models to assess solute carrier transporter function. Clin. Pharmacol. Ther. 2011, 89, 612−616. (16) Kim, D. C.; Sugiyama, Y.; Satoh, H.; Fuwa, T.; Iga, T.; Hanano, M. Kinetic analysis of in vivo receptor-dependent binding of human epidermal growth factor by rat tissues. J. Pharm. Sci. 1988, 77, 200− 207. (17) Takashima-Hirano, M.; Takashima, T.; Katayama, Y.; Wada, Y.; Sugiyama, Y.; Watanabe, Y.; Doi, H.; Suzuki, M. Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation. Bioorg. Med. Chem. 2011, 19, 2997−3004. (18) Takashima, T.; Nagata, H.; Nakae, T.; Cui, Y.; Wada, Y.; Kitamura, S.; Doi, H.; Suzuki, M.; Maeda, K.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. Positron emission tomography studies using (15R)16-m-[11C]tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester for the evaluation of hepatobiliary transport. J. Pharmacol. Exp. Ther. 2010, 335, 314−323. (19) Takashima, T.; Wu, C.; Takashima-Hirano, M.; Katayama, Y.; Wada, Y.; Suzuki, M.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with [11C]-SC-62807. J. Nucl. Med. 2013, 54, 267−276. (20) Frisch, K.; Jakobsen, S.; Sorensen, M.; Munk, O. L.; Alstrup, A. K.; Ott, P.; Hofmann, A. F.; Keiding, S. N-methyl-[11C]cholylsarcosine, a novel bile acid tracer for PET/CT of hepatic excretory function: radiosynthesis and proof-of-concept studies in pigs. J. Nucl. Med. 2012, 53, 772−778. (21) Guhlmann, A.; Krauss, K.; Oberdorfer, F.; Siegel, T.; Scheuber, P. H.; Muller, J.; Csuk-Glanzer, B.; Ziegler, S.; Ostertag, H.; Keppler, D. Noninvasive assessment of hepatobiliary and renal elimination of cysteinyl leukotrienes by positron emission tomography. Hepatology 1995, 21, 1568−1575. (22) Shingaki, T.; Takashima, T.; Ijuin, R.; Zhang, X.; Onoue, T.; Katayama, Y.; Okauchi, T.; Hayashinaka, E.; Cui, Y.; Wada, Y.; Suzuki, M.; Maeda, K.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. Evaluation of Oatp and Mrp2 activities in hepatobiliary excretion using newly developed positron emission tomography tracer [11C]dehydropravastatin in rats. J. Pharmacol. Exp. Ther. 2013, 347, 193− 202. (23) Ijuin, R.; Takashima, T.; Watanabe, Y.; Sugiyama, Y.; Suzuki, M. Synthesis of [11C]dehydropravastatin, a PET probe potentially useful for studying OATP1B1 and MRP2 transporters in the liver. Bioorg. Med. Chem. 2012, 20, 3703−3709. (24) He, J.; Yu, Y.; Prasad, B.; Link, J.; Miyaoka, R. S.; Chen, X.; Unadkat, J. D. PET Imaging of Oatp-Mediated Hepatobiliary Transport of [11C] Rosuvastatin in the Rat. Mol. Pharmaceutics 2014, 11, 2745−2754. (25) Takashima, T.; Kitamura, S.; Wada, Y.; Tanaka, M.; Shigihara, Y.; Ishii, H.; Ijuin, R.; Shiomi, S.; Nakae, T.; Watanabe, Y.; Cui, Y.; Doi, H.; Suzuki, M.; Maeda, K.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me. J. Nucl. Med. 2012, 53, 741−748. (26) Tournier, N.; Saba, W.; Cisternino, S.; Peyronneau, M. A.; Damont, A.; Goutal, S.; Dubois, A.; Dolle, F.; Scherrmann, J. M.; Valette, H.; Kuhnast, B.; Bottlaender, M. Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide. AAPS J. 2013, 15, 1082−1090.

(27) Shimizu, K.; Takashima, T.; Yamane, T.; Sasaki, M.; Kageyama, H.; Hashizume, Y.; Maeda, K.; Sugiyama, Y.; Watanabe, Y.; Senda, M. Whole-body distribution and radiation dosimetry of [11C]telmisartan as a biomarker for hepatic organic anion transporting polypeptide (OATP) 1B3. Nucl. Med. Biol. 2012, 39, 847−853. (28) Iimori, H.; Hashizume, Y.; Sasaki, M.; Kajiwara, Y.; Sugimoto, Y.; Sugiyama, Y.; Watanabe, Y.; Senda, M. First automatic radiosynthesis of 11C labeled Telmisartan using a multipurpose synthesizer for clinical research use. Ann. Nucl. Med. 2011, 25, 333−337. (29) Takashima, T.; Hashizume, Y.; Katayama, Y.; Murai, M.; Wada, Y.; Maeda, K.; Sugiyama, Y.; Watanabe, Y. The involvement of organic anion transporting polypeptide in the hepatic uptake of telmisartan in rats: PET studies with [11C]telmisartan. Mol. Pharmaceutics 2011, 8, 1789−1798. (30) Hume, W. E.; Shingaki, T.; Takashima, T.; Hashizume, Y.; Okauchi, T.; Katayama, Y.; Hayashinaka, E.; Wada, Y.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. The synthesis and biodistribution of [11C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo. Bioorg. Med. Chem. 2013, 21, 7584−7590. (31) Jakobsen, S.; Busk, M.; Munk, O.; Aalstrup, A.; Gormsen, L.; Jessen, N.; Frokiaer, J. Preclinical validation of [11]C-Metformin: A novel PET tracer targeting organic cation transporters. J. Nucl. Med. 2014, 55, 385−385. (32) Bao, X.; Lu, S.; Liow, J. S.; Morse, C. L.; Anderson, K. B.; Zoghbi, S. S.; Innis, R. B.; Pike, V. W. [11C]Rhodamine-123: synthesis and biodistribution in rodents. Nucl. Med. Biol. 2012, 39, 1128−1136. (33) Kitamura, S.; Maeda, K.; Wang, Y.; Sugiyama, Y. Involvement of multiple transporters in the hepatobiliary transport of rosuvastatin. Drug Metab. Dispos. 2008, 36, 2014−2023. (34) Ho, R. H.; Tirona, R. G.; Leake, B. F.; Glaeser, H.; Lee, W.; Lemke, C. J.; Wang, Y.; Kim, R. B. Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression, and pharmacogenetics. Gastroenterology 2006, 130, 1793−1806. (35) Huang, L.; Wang, Y.; Grimm, S. ATP-dependent transport of rosuvastatin in membrane vesicles expressing breast cancer resistance protein. Drug Metab. Dispos. 2006, 34, 738−742. (36) Hobbs, M.; Parker, C.; Birch, H.; Kenworthy, K. Understanding the interplay of drug transporters involved in the disposition of rosuvastatin in the isolated perfused rat liver using a physiologicallybased pharmacokinetic model. Xenobiotica 2012, 42, 327−338. (37) Jemnitz, K.; Veres, Z.; Tugyi, R.; Vereczkey, L. Biliary efflux transporters involved in the clearance of rosuvastatin in sandwich culture of primary rat hepatocytes. Toxicol. In Vitro 2010, 24, 605− 610. (38) Windass, A. S.; Lowes, S.; Wang, Y.; Brown, C. D. The contribution of organic anion transporters OAT1 and OAT3 to the renal uptake of rosuvastatin. J. Pharmacol. Exp. Ther. 2007, 322, 1221− 1227. (39) Prueksaritanont, T.; Chu, X.; Evers, R.; Klopfer, S. O.; Caro, L.; Kothare, P. A.; Dempsey, C.; Rasmussen, S.; Houle, R.; Chan, G.; Cai, X.; Valesky, R.; Fraser, I. P.; Stoch, S. A. Pitavastatin is a more sensitive and selective OATP1B clinical probe than rosuvastatin. Br. J. Clin. Pharmacol. 2014, 78, 587. (40) Suzuki, M.; Kato, K.; Noyori, R.; Watanabe, Y.; Takechi, H.; Matsumura, K.; Långström, B.; Watanabe, Y. (15 R)-16-m-Tolyl17,18,19,20-tetranorisocarbacyclin: A Stable Ligand with High Binding Affinity and Selectivity for a Prostacyclin Receptor in the Central Nervous System. Angew. Chem., Int. Ed. Engl. 1996, 35, 334−336. (41) Bjorkman, M.; Andersson, Y.; Doi, H.; Kato, K.; Suzuki, M.; Noyori, R.; Watanabe, Y.; Langstrom, B. Synthesis of 11C/13Clabelled prostacyclins. Acta Chem. Scand. 1998, 52, 635−640. (42) Kuroda, M.; Kobayashi, Y.; Tanaka, Y.; Itani, T.; Mifuji, R.; Araki, J.; Kaito, M.; Adachi, Y. Increased hepatic and renal expressions of multidrug resistance-associated protein 3 in Eisai hyperbilirubinuria rats. J. Gastroenterol. Hepatol. 2004, 19, 146−153. (43) Kurisu, H.; Kamisaka, K.; Koyo, T.; Yamasuge, S.; Igarashi, H.; Maezawa, H.; Uesugi, T.; Tagaya, O. Organic anion transport study in L

DOI: 10.1021/acs.molpharmaceut.5b00059 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Review

Molecular Pharmaceutics mutant rats with autosomal recessive conjugated hyperbilirubinemia. Life Sci. 1991, 49, 1003−1011. (44) Gedeon, C.; Behravan, J.; Koren, G.; Piquette-Miller, M. Transport of glyburide by placental ABC transporters: implications in fetal drug exposure. Placenta 2006, 27, 1096−1102. (45) Cygalova, L. H.; Hofman, J.; Ceckova, M.; Staud, F. Transplacental pharmacokinetics of glyburide, rhodamine 123, and BODIPY FL prazosin: effect of drug efflux transporters and lipid solubility. J. Pharmacol. Exp. Ther. 2009, 331, 1118−1125. (46) Koenen, A.; Kock, K.; Keiser, M.; Siegmund, W.; Kroemer, H. K.; Grube, M. Steroid hormones specifically modify the activity of organic anion transporting polypeptides. Eur. J. Pharm. Sci. 2012, 47, 774−780. (47) van de Steeg, E.; Greupink, R.; Schreurs, M.; Nooijen, I. H.; Verhoeckx, K. C.; Hanemaaijer, R.; Ripken, D.; Monshouwer, M.; Vlaming, M. L.; DeGroot, J.; Verwei, M.; Russel, F. G.; Huisman, M. T.; Wortelboer, H. M. Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1. Drug Metab. Dispos. 2013, 41, 592−601. (48) Rabindran, S. K.; He, H.; Singh, M.; Brown, E.; Collins, K. I.; Annable, T.; Greenberger, L. M. Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C. Cancer Res. 1998, 58, 5850−5858. (49) Rabindran, S. K.; Ross, D. D.; Doyle, L. A.; Yang, W.; Greenberger, L. M. Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein. Cancer Res. 2000, 60, 47−50. (50) Loor, F.; Boesch, D.; Gaveriaux, C.; Jachez, B.; PourtierManzanedo, A.; Emmer, G. SDZ 280−446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the Pglycoprotein-mediated tumour cell multidrug resistance. Br. J. Cancer 1992, 65, 11−18. (51) Boesch, D.; Gaveriaux, C.; Jachez, B.; Pourtier-Manzanedo, A.; Bollinger, P.; Loor, F. In vivo circumvention of P-glycoproteinmediated multidrug resistance of tumor cells with SDZ PSC 833. Cancer Res. 1991, 51, 4226−4233. (52) Hyafil, F.; Vergely, C.; Du Vignaud, P.; Grand-Perret, T. In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Res. 1993, 53, 4595−4602. (53) Ishiguro, N.; Maeda, K.; Kishimoto, W.; Saito, A.; Harada, A.; Ebner, T.; Roth, W.; Igarashi, T.; Sugiyama, Y. Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin II receptor antagonist, in humans. Drug Metab. Dispos. 2006, 34, 1109−1115. (54) Stangier, J.; Schmid, J.; Turck, D.; Switek, H.; Verhagen, A.; Peeters, P. A.; van Marle, S. P.; Tamminga, W. J.; Sollie, F. A.; Jonkman, J. H. Absorption, metabolism, and excretion of intravenously and orally administered [14C]telmisartan in healthy volunteers. J. Clin. Pharmacol. 2000, 40, 1312−1322. (55) Kataoka, M.; Takashima, T.; Shingaki, T.; Hashidzume, Y.; Katayama, Y.; Wada, Y.; Oh, H.; Masaoka, Y.; Sakuma, S.; Sugiyama, Y.; Yamashita, S.; Watanabe, Y. Dynamic analysis of GI absorption and hepatic distribution processes of telmisartan in rats using positron emission tomography. Pharm. Res. 2012, 29, 2419−2431. (56) Kirpichnikov, D.; McFarlane, S. I.; Sowers, J. R. Metformin: an update. Ann. Int. Med. 2002, 137, 25−33. (57) Viollet, B.; Guigas, B.; Sanz Garcia, N.; Leclerc, J.; Foretz, M.; Andreelli, F. Cellular and molecular mechanisms of metformin: an overview. Clin. Sci. (London) 2012, 122, 253−270. (58) Wang, D. S.; Jonker, J. W.; Kato, Y.; Kusuhara, H.; Schinkel, A. H.; Sugiyama, Y. Involvement of organic cation transporter 1 in hepatic and intestinal distribution of metformin. J. Pharmacol. Exp. Ther. 2002, 302, 510−515. (59) Tanihara, Y.; Masuda, S.; Sato, T.; Katsura, T.; Ogawa, O.; Inui, K. Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H(+)-organic cation antiporters. Biochem. Pharmacol. 2007, 74, 359−371. (60) Canitrot, Y.; Lautier, D. Use of rhodamine 123 for the detection of multidrug resistance. Bull. Cancer 1995, 82, 687−697.

(61) Wagner-Souza, K.; Diamond, H. R.; Ornellas, M. H.; Gomes, B. E.; Almeida-Oliveira, A.; Abdelhay, E.; Bouzas, L. F.; Rumjanek, V. M. Rhodamine 123 efflux in human subpopulations of hematopoietic stem cells: comparison between bone marrow, umbilical cord blood and mobilized peripheral blood CD34+ cells. Int. J. Mol. Med. 2008, 22, 237−242. (62) Perloff, M. D.; Stormer, E.; von Moltke, L. L.; Greenblatt, D. J. Rapid assessment of P-glycoprotein inhibition and induction in vitro. Pharm. Res. 2003, 20, 1177−1183. (63) Lee, J. S.; Paull, K.; Alvarez, M.; Hose, C.; Monks, A.; Grever, M.; Fojo, A. T.; Bates, S. E. Rhodamine efflux patterns predict Pglycoprotein substrates in the National Cancer Institute drug screen. Mol. Pharmacol. 1994, 46, 627−638. (64) de Lange, E. C.; de Bock, G.; Schinkel, A. H.; de Boer, A. G.; Breimer, D. D. BBB transport and P-glycoprotein functionality using MDR1A (−/−) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123. Pharm. Res. 1998, 15, 1657− 1665. (65) Wang, Q.; Yang, H.; Miller, D. W.; Elmquist, W. F. Effect of the p-glycoprotein inhibitor, cyclosporin A, on the distribution of rhodamine-123 to the brain: an in vivo microdialysis study in freely moving rats. Biochem. Biophys. Res. Commun. 1995, 211, 719−726. (66) Jouan, E.; Le Vee, M.; Denizot, C.; Da Violante, G.; Fardel, O. The mitochondrial fluorescent dye rhodamine 123 is a high-affinity substrate for organic cation transporters (OCTs) 1 and 2. Fundam. Clin. Pharmacol. 2014, 28, 65−77. (67) Martinez, C.; Albet, C.; Agundez, J. A.; Herrero, E.; Carrillo, J. A.; Marquez, M.; Benitez, J.; Ortiz, J. A. Comparative in vitro and in vivo inhibition of cytochrome P450 CYP1A2, CYP2D6, and CYP3A by H2-receptor antagonists. Clin. Pharmacol. Ther. 1999, 65, 369−376. (68) Emery, S.; Oldham, H. G.; Norman, S. J.; Chenery, R. J. The effect of cimetidine and ranitidine on paracetamol glucuronidation and sulphation in cultured rat hepatocytes. Biochem. Pharmacol. 1985, 34, 1415−1421. (69) Paulson, S. K.; Hribar, J. D.; Liu, N. W.; Hajdu, E.; Bible, R. H., Jr; Piergies, A.; Karim, A. Metabolism and excretion of [(14)C]celecoxib in healthy male volunteers. Drug Metab. Dispos. 2000, 28, 308−314. (70) Schmassmann, A.; Fehr, H. F.; Locher, J.; Lillienau, J.; Schteingart, C. D.; Rossi, S. S.; Hofmann, A. F. Cholylsarcosine, a new bile acid analogue: metabolism and effect on biliary secretion in humans. Gastroenterology 1993, 104, 1171−1181. (71) Hagmann, W.; Denzlinger, C.; Rapp, S.; Weckbecker, G.; Keppler, D. Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyl leukotriene E4. Prostaglandins 1986, 31, 239−251. (72) Samuelsson, B.; Dahlen, S. E.; Lindgren, J. A.; Rouzer, C. A.; Serhan, C. N. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 1987, 237, 1171−1176. (73) Abe, T.; Kakyo, M.; Tokui, T.; Nakagomi, R.; Nishio, T.; Nakai, D.; Nomura, H.; Unno, M.; Suzuki, M.; Naitoh, T.; Matsuno, S.; Yawo, H. Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1. J. Biol. Chem. 1999, 274, 17159− 17163. (74) Huber, M.; Guhlmann, A.; Jansen, P. L.; Keppler, D. Hereditary defect of hepatobiliary cysteinyl leukotriene elimination in mutant rats with defective hepatic anion excretion. Hepatology 1987, 7, 224−228. (75) Oberdorfer, F.; Siegel, T.; Guhlmann, A.; Keppler, D.; MaierBorst, W. The preparation of a 11C-labelled 5-lipoxygenase product. 5(S)-hydroxy-6(R)-(N-[1−11C]acetyl)cysteinyl-7,9-trans-11,14-ciseicosatetraenoic acid. J. Labelled Compd. Radiopharmaceut. 1992, 31, 903−913. (76) Jansen, P. L.; Peters, W. H.; Lamers, W. H. Hereditary chronic conjugated hyperbilirubinemia in mutant rats caused by defective hepatic anion transport. Hepatology 1985, 5, 573−579. (77) Rotstein, B. H.; Liang, S. H.; Holland, J. P.; Collier, T. L.; Hooker, J. M.; Wilson, A. A.; Vasdev, N. 11CO2 fixation: a renaissance in PET radiochemistry. Chem. Commun. (Cambridge) 2013, 49, 5621− 5629. M

DOI: 10.1021/acs.molpharmaceut.5b00059 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Review

Molecular Pharmaceutics (78) Brooks, A. F.; Topczewski, J. J.; Ichiishi, N.; Sanford, M. S.; Scott, P. J. H. Late-stage [18F]fluorination: new solutions to old problems. Chem. Sci. 2014, 5, 4545. (79) Tu, M.; Mathiowetz, A. M.; Pfefferkorn, J. A.; Cameron, K. O.; Dow, R. L.; Litchfield, J.; Di, L.; Feng, B.; Liras, S. Medicinal chemistry design principles for liver targeting through OATP transporters. Curr. Top. Med. Chem. 2013, 13, 857−866. (80) Varma, M. V.; Chang, G.; Lai, Y.; Feng, B.; El-Kattan, A. F.; Litchfield, J.; Goosen, T. C. Physicochemical property space of hepatobiliary transport and computational models for predicting rat biliary excretion. Drug Metab. Dispos. 2012, 40, 1527−1537. (81) Dong, Z.; Ekins, S.; Polli, J. E. Structure-activity relationship for FDA approved drugs as inhibitors of the human sodium taurocholate cotransporting polypeptide (NTCP). Mol. Pharmaceutics 2013, 10, 1008−1019. (82) Greupink, R.; Nabuurs, S. B.; Zarzycka, B.; Verweij, V.; Monshouwer, M.; Huisman, M. T.; Russel, F. G. In silico identification of potential cholestasis-inducing agents via modeling of Na(+)dependent taurocholate cotransporting polypeptide substrate specificity. Toxicol. Sci. 2012, 129, 35−48. (83) US FDA Draft guidance. Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations. Website: http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf. 2006. (84) Karlgren, M.; Ahlin, G.; Bergstrom, C. A.; Svensson, R.; Palm, J.; Artursson, P. In vitro and in silico strategies to identify OATP1B1 inhibitors and predict clinical drug-drug interactions. Pharm. Res. 2012, 29, 411−426. (85) Wang, R. B.; Kuo, C. L.; Lien, L. L.; Lien, E. J. Structure-activity relationship: analyses of p-glycoprotein substrates and inhibitors. J. Clin. Pharm. Ther. 2003, 28, 203−228. (86) Pan, Y.; Chothe, P. P.; Swaan, P. W. Identification of novel breast cancer resistance protein (BCRP) inhibitors by virtual screening. Mol. Pharmaceutics 2013, 10, 1236−1248. (87) Schlessinger, A.; Khuri, N.; Giacomini, K. M.; Sali, A. Molecular modeling and ligand docking for solute carrier (SLC) transporters. Curr. Top. Med. Chem. 2013, 13, 843−856. (88) Xing, L.; Hu, Y.; Lai, Y. Advancement of structure-activity relationship of multidrug resistance-associated protein 2 interactions. AAPS J. 2009, 11, 406−413. (89) Warner, D. J.; Chen, H.; Cantin, L. D.; Kenna, J. G.; Stahl, S.; Walker, C. L.; Noeske, T. Mitigating the inhibition of human bile salt export pump by drugs: opportunities provided by physicochemical property modulation, in silico modeling, and structural modification. Drug Metab. Dispos. 2012, 40, 2332−2341. (90) Higgins, J. W.; Bao, J. Q.; Ke, A. B.; Manro, J. R.; Fallon, J. K.; Smith, P. C.; Zamek-Gliszczynski, M. J. 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. 2014, 42, 182−192. (91) Elsby, R.; Hilgendorf, C.; Fenner, K. Understanding the critical disposition pathways of statins to assess drug-drug interaction risk during drug development: it’s not just about OATP1B1. Clin. Pharmacol. Ther. 2012, 92, 584−598. (92) Wlcek, K.; Svoboda, M.; Riha, J.; Zakaria, S.; Olszewski, U.; Dvorak, Z.; Sellner, F.; Ellinger, I.; Jager, W.; Thalhammer, T. The analysis of organic anion transporting polypeptide (OATP) mRNA and protein patterns in primary and metastatic liver cancer. Cancer Biol. Ther. 2011, 11, 801−811. (93) Lee, W.; Belkhiri, A.; Lockhart, A. C.; Merchant, N.; Glaeser, H.; Harris, E. I.; Washington, M. K.; Brunt, E. M.; Zaika, A.; Kim, R. B.; ElRifai, W. Overexpression of OATP1B3 confers apoptotic resistance in colon cancer. Cancer Res. 2008, 68, 10315−10323. (94) Wlcek, K.; Svoboda, M.; Thalhammer, T.; Sellner, F.; Krupitza, G.; Jaeger, W. Altered expression of organic anion transporter polypeptide (OATP) genes in human breast carcinoma. Cancer Biol. Ther. 2008, 7, 1450−1455.

(95) Wright, J. L.; Kwon, E. M.; Ostrander, E. A.; Montgomery, R. B.; Lin, D. W.; Vessella, R.; Stanford, J. L.; Mostaghel, E. A. Expression of SLCO transport genes in castration-resistant prostate cancer and impact of genetic variation in SLCO1B3 and SLCO2B1 on prostate cancer outcomes. Cancer Epidemiol. Biomarkers Prev. 2011, 20, 619− 627. (96) Thakkar, N.; Kim, K.; Jang, E. R.; Han, S.; Kim, K.; Kim, D.; Merchant, N.; Lockhart, A. C.; Lee, W. A cancer-specific variant of the SLCO1B3 gene encodes a novel human organic anion transporting polypeptide 1B3 (OATP1B3) localized mainly in the cytoplasm of colon and pancreatic cancer cells. Mol. Pharmaceutics 2013, 10, 406− 416. (97) Olszewski-Hamilton, U.; Svoboda, M.; Thalhammer, T.; Buxhofer-Ausch, V.; Geissler, K.; Hamilton, G. Organic Anion Transporting Polypeptide 5A1 (OATP5A1) in Small Cell Lung Cancer (SCLC) Cells: Possible Involvement in Chemoresistance to Satraplatin. Biomark. Cancer 2011, 3, 31−40. (98) Pressler, H.; Sissung, T. M.; Venzon, D.; Price, D. K.; Figg, W. D. Expression of OATP family members in hormone-related cancers: potential markers of progression. PLoS One 2011, 6, e20372. (99) Hamada, A.; Sissung, T.; Price, D. K.; Danesi, R.; Chau, C. H.; Sharifi, N.; Venzon, D.; Maeda, K.; Nagao, K.; Sparreboom, A.; Mitsuya, H.; Dahut, W. L.; Figg, W. D. Effect of SLCO1B3 haplotype on testosterone transport and clinical outcome in caucasian patients with androgen-independent prostatic cancer. Clin. Cancer Res. 2008, 14, 3312−3318. (100) Buxhofer-Ausch, V.; Secky, L.; Wlcek, K.; Svoboda, M.; Kounnis, V.; Briasoulis, E.; Tzakos, A. G.; Jaeger, W.; Thalhammer, T. Tumor-specific expression of organic anion-transporting polypeptides: transporters as novel targets for cancer therapy. J. Drug Delivery 2013, 2013, 863539.

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