Performance and Sensitivity of [99mTc]Tc-sestamibi Compared with Positron Emission Tomography Radiotracers to Measure P-glycoprotein Function in the Kidneys and Liver.

P-glycoprotein (P-gp, encoded in humans by the ABCB1 gene and in rodents by the Abcb1a/b genes) is a membrane transporter that can restrict the intestinal absorption and tissue distribution of many drugs and may also contribute to renal and hepatobiliary drug excretion. The aim of this study was to compare the performance and sensitivity of currently available radiolabeled P-gp substrates for positron emission tomography (PET) with the single-photon emission computed tomography (SPECT) radiotracer [99mTc]Tc-sestamibi for measuring the P-gp function in the kidneys and liver. Wild-type, heterozygous (Abcb1a/b(+/-)), and homozygous (Abcb1a/b(-/-)) Abcb1a/b knockout mice were used as models of different P-gp abundance in excretory organs. Animals underwent either dynamic PET scans after intravenous injection of [11C]N-desmethyl-loperamide, (R)-[11C]verapamil, or [11C]metoclopramide or consecutive static SPECT scans after intravenous injection of [99mTc]Tc-sestamibi. P-gp in the kidneys and liver of the mouse models was analyzed with immunofluorescence labeling and Western blotting. In the kidneys, Abcb1a/b() mice had intermediate P-gp abundance compared with wild-type and Abcb1a/b(-/-) mice. Among the four tested radiotracers, renal clearance of radioactivity (CLurine,kidney) was significantly reduced (-83%) in Abcb1a/b(-/-) mice only for [99mTc]Tc-sestamibi. Biliary clearance of radioactivity (CLbile,liver) was significantly reduced in Abcb1a/b(-/-) mice for [11C]N-desmethyl-loperamide (-47%), [11C]metoclopramide (-25%), and [99mTc]Tc-sestamibi (-79%). However, in Abcb1a/b(+/-) mice, CLbile,liver was significantly reduced (-47%) only for [99mTc]Tc-sestamibi. Among the tested radiotracers, [99mTc]Tc-sestamibi performed best in measuring the P-gp function in the kidneys and liver. Owing to its widespread clinical availability, [99mTc]Tc-sestamibi represents a promising probe substrate to assess systemic P-gp-mediated drug-drug interactions and to measure renal and hepatic P-gp function under different (patho-)physiological conditions.

Positron Emission Tomography-Based Pharmacokinetic Analysis To Assess Renal Transporter-Mediated Drug-Drug Interactions of Antimicrobial Drugs.

Transporter-mediated drug-drug interactions (DDIs) are of concern in antimicrobial drug development, as they can have serious safety consequences. We used positron emission tomography (PET) imaging-based pharmacokinetic (PK) analysis to assess the effect of different drugs, which may cause transporter-mediated DDIs, on the tissue distribution and excretion of [18F]ciprofloxacin as a radiolabeled model antimicrobial drug. Mice underwent PET scans after intravenous injection of [18F]ciprofloxacin, without and with pretreatment with either probenecid (150 mg/kg), cimetidine (50 mg/kg), or pyrimethamine (5 mg/kg). A 3-compartment kidney PK model was used to assess the involvement of renal transporters in the examined DDIs. Pretreatment with probenecid and cimetidine significantly decreased the renal clearance (CLrenal) of [18F]ciprofloxacin. The effect of cimetidine (-86%) was greater than that of probenecid (-63%), which contrasted with previously published clinical data. The kidney PK model revealed that the decrease in CLrenal was caused by inhibition of basal uptake transporters and apical efflux transporters in kidney proximal tubule cells. Changes in the urinary excretion of [18F]ciprofloxacin after pretreatment with probenecid and cimetidine resulted in increased blood and organ exposure to [18F]ciprofloxacin. Our results suggest that multiple membrane transporters mediate the tubular secretion of ciprofloxacin, with possible species differences between mice and humans. Concomitant medication inhibiting renal transporters may precipitate DDIs, leading to decreased urinary excretion and increased blood and organ exposure to ciprofloxacin, potentially exacerbating adverse effects. Our study highlights the strength of PET imaging-based PK analysis to assess transporter-mediated DDIs at a whole-body level.

Use of PET Imaging to Assess the Efficacy of Thiethylperazine to Stimulate Cerebral MRP1 Transport Activity in Wild-Type and APP/PS1-21 Mice.

Multidrug resistance-associated protein 1 (MRP1, encoded by the ABCC1 gene) may contribute to the clearance of amyloid-beta (Aß) peptides from the brain into the blood and stimulation of MRP1 transport activity may be a therapeutic approach to enhance brain Aß clearance. In this study, we assessed the effect of thiethylperazine, an antiemetic drug which was shown to stimulate MRP1 activity in vitro and to decrease Aß load in a rapid ß-amyloidosis mouse model (APP/PS1-21), on MRP1 transport activity by means of positron emission tomography (PET) imaging with the MRP1 tracer 6-bromo-7-[11C]methylpurine. Groups of wild-type, APP/PS1-21 and Abcc1(-/-) mice underwent PET scans before and after a 5-day oral treatment period with thiethylperazine (15 mg/kg, once daily). The elimination rate constant of radioactivity (kelim) was calculated from time-activity curves in the brain and the lungs as a measure of tissue MRP1 activity. Treatment with thiethylperazine had no significant effect on MRP1 activity in the brain and the lungs of wild-type and APP/PS1-21 mice. This may either be related to a lack of an MRP1-stimulating effect of thiethylperazine in vivo or to other factors, such as substrate-dependent MRP1 stimulation, insufficient target tissue exposure to thiethylperazine or limited sensitivity of the PET tracer to measure MRP1 stimulation.

Influence of Cation Transporters (OCTs and MATEs) on the Renal and Hepatobiliary Disposition of [11C]Metoclopramide in Mice.

PURPOSE: To investigate the role of cation transporters (OCTs, MATEs) in the renal and hepatic disposition of the radiolabeled antiemetic drug [11C]metoclopramide in mice with PET. METHODS: PET was performed in wild-type mice after administration of an intravenous microdose (<1 µg) of [11C]metoclopramide without and with co-administration of either unlabeled metoclopramide (5 or 10 mg/kg) or the prototypical cation transporter inhibitors cimetidine (150 mg/kg) or sulpiride (25 mg/kg). [11C]Metoclopramide PET was also performed in wild-type and Slc22a1/2(-/-) mice. Radiolabeled metabolites were measured at 15 min after radiotracer injection and PET data were corrected for radiolabeled metabolites. RESULTS: [11C]Metoclopramide was highly metabolized and [11C]metoclopramide-derived radioactivity was excreted into the urine. The different investigated treatments decreased (~2.5-fold) the uptake of [11C]metoclopramide from plasma into the kidney and liver, inhibited metabolism and decreased (up to 3.8-fold) urinary excretion, which resulted in increased plasma concentrations of [11C]metoclopramide. Kidney and liver uptake were moderately (~1.3-fold) reduced in Slc22a1/2(-/-) mice. CONCLUSIONS: Our results suggest a contribution of OCT1/2 to the kidney and liver uptake and of MATEs to the urinary excretion of [11C]metoclopramide in mice. Cation transporters may contribute, next to variability in the activity of metabolizing enzymes, to variability in metoclopramide pharmacokinetics and side effects.

Measurement of Hepatic ABCB1 and ABCG2 Transport Activity with [11C]Tariquidar and PET in Humans and Mice.

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in the canalicular membrane of hepatocytes mediate the biliary excretion of drugs and drug metabolites. To measure hepatic ABCB1 and ABCG2 activity, we performed positron emission tomography (PET) scans with the ABCB1/ABCG2 substrate [11C]tariquidar in healthy volunteers and wild-type, Abcb1a/b(-/-), Abcg2(-/-), and Abcb1a/b(-/-)Abcg2(-/-) mice without and with coadministration of unlabeled tariquidar. PET data were analyzed with a three-compartment pharmacokinetic model. [11C]Tariquidar underwent hepatobiliary excretion in both humans and mice, and tariquidar coadministration caused a significant reduction in the rate constant for the transfer of radioactivity from the liver into bile (by -74% in humans and by -62% in wild-type mice), suggesting inhibition of canalicular efflux transporter activity. Radio-thin-layer chromatography analysis revealed that the majority of radioactivity (>87%) in the mouse liver and bile was composed of unmetabolized [11C]tariquidar. PET data in transporter knockout mice revealed that both ABCB1 and ABCG2 mediated biliary excretion of [11C]tariquidar. In vitro experiments indicated that tariquidar is not a substrate of major hepatic basolateral uptake transporters (SLCO1B1, SLCO1B3, SLCO2B1, SLC22A1, and SLC22A3). Our data suggest that [11C]tariquidar can be used to measure hepatic canalicular ABCB1/ABCG2 transport activity without a confounding effect of uptake transporters.

[11C]metoclopramide is a sensitive radiotracer to measure moderate decreases in P-glycoprotein function at the blood-brain barrier.

The efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier limits the cerebral uptake of various xenobiotics. To assess the sensitivity of [11C]metoclopramide to measure decreased cerebral P-gp function, we performed [11C]metoclopramide PET scans without (baseline) and with partial P-gp inhibition by tariquidar in wild-type, heterozygous Abcb1a/b(+/-) and homozygous Abcb1a/b(-/-) mice as models with controlled levels of cerebral P-gp expression. Brains were collected to quantify P-gp expression with immunohistochemistry. Brain uptake of [11C]metoclopramide was expressed as the area under the brain time-activity curve (AUCbrain) and compared with data previously obtained with (R)-[11C]verapamil and [11C]N-desmethyl-loperamide. Abcb1a/b(+/-) mice had intermediate P-gp expression compared to wild-type and Abcb1a/b(-/-) mice. In baseline scans, all three radiotracers were able to discriminate Abcb1a/b(-/-) from wild-type mice (2.5- to 4.6-fold increased AUCbrain, p ≤ 0.0001). However, only [11C]metoclopramide could discriminate Abcb1a/b(+/-) from wild-type mice (1.46-fold increased AUCbrain, p ≤ 0.001). After partial P-gp inhibition, differences in [11C]metoclopramide AUCbrain between Abcb1a/b(+/-) and wild-type mice (1.39-fold, p ≤ 0.001) remained comparable to baseline. There was a negative correlation between baseline [11C]metoclopramide AUCbrain and ex-vivo-measured P-gp immunofluorescence (r = -0.9875, p ≤ 0.0001). Our data suggest that [11C]metoclopramide is a sensitive radiotracer to measure moderate, but (patho-)physiologically relevant decreases in cerebral P-gp function without the need to co-administer a P-gp inhibitor.

Effect of budesonide on pulmonary activity of multidrug resistance-associated protein 1 assessed with PET imaging in rats.

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is a highly abundant efflux transporter in the lungs, which protects cells from toxins and oxidative stress and has been implicated in the pathophysiology of chronic obstructive pulmonary disease and cystic fibrosis. There is evidence from in vitro studies that the inhaled glucocorticoid budesonide can inhibit MRP1 activity. We used positron emission tomography (PET) imaging with 6-bromo-7-[11C]methylpurine ([11C]BMP), which is transformed in vivo into a radiolabeled MRP1 substrate, to assess whether intratracheally (i.t.) aerosolized budesonide affects pulmonary MRP1 activity in rats. Three groups of rats (n = 5-6 each) underwent dynamic PET scans of the lungs after i.t. aerosolization of either [11C]BMP alone, or [11C]BMP mixed with either budesonide (0.04 mg, corresponding to the maximum soluble dose) or the model MRP1 inhibitor MK571 (2 mg). From PET-measured radioactivity concentration-time curves, the rate constant describing radioactivity elimination from the right lung (kE,lung) and the area under the curve (AUClung) were calculated from 0 to 5 min after start of the PET scan as measures of pulmonary MRP1 activity. Co-administration of MK571 resulted in a pronounced decrease in kE,lung (25-fold, p < 0.0001) and an increase in AUClung (5.3-fold, p < 0.0001) when compared with vehicle-treated animals. In contrast, in budesonide-treated animals kE,lung and AUClung were not significantly different from the vehicle group. Our results show that i.t. aerosolized budesonide at an approximately 5 times higher dose than the maximum clinical dose leads to no change in pulmonary MRP1 activity, suggesting a lack of an effect of inhaled budesonide treatment on the MRP1-mediated cellular detoxifying capacity of the lungs. However, the strong effect observed for MK571 raises the possibility for the occurrence of transporter-mediated drug-drug interactions at the pulmonary epithelium with inhaled medicines.

Influence of P-glycoprotein on pulmonary disposition of the model substrate [11C]metoclopramide assessed by PET imaging in rats.

In the lungs, the membrane transporter P-glycoprotein (P-gp) is expressed in the apical (i.e. lumen-facing) membrane of airway epithelial cells and in the luminal (blood-facing) membrane of pulmonary capillary endothelial cells. To better understand the influence of P-gp on the pulmonary disposition of inhaled P-gp substrate drugs, we measured the intrapulmonary pharmacokinetics of the intratracheally (i.t.) aerosolized model P-gp substrate [11C]metoclopramide in presence and absence of P-gp activity by means of positron emission tomography (PET) imaging in rats. Data were compared to data previously acquired with the model P-gp substrates (R)-[11C]verapamil and [11C]N-desmethyl-loperamide, using the same experimental set-up. Groups of wild-type rats, either untreated or treated with the P-gp inhibitor tariquidar, and Abcb1a/b(-/-) rats underwent 90-min dynamic PET scans after i.t. aerosolization of [11C]metoclopramide. Lung exposure to [11C]metoclopramide was expressed as the area under the right lung concentration-time curve (AUClung). AUClung values were significantly higher in Abcb1a/b(-/-) rats (1.8-fold, p ≤ 0.0001) and in tariquidar-treated wild-type rats (1.6-fold, p ≤ 0.01) than in untreated wild-type rats. This differed from previously obtained results with (R)-[11C]verapamil and [11C]N-desmethyl-loperamide, which showed decreased exposure in the rat lung in absence of P-gp activity. Our results suggest that transepithelial transfer of [11C]metoclopramide was not or only to a small extent affected by P-gp activity, presumably due to the compound's high passive permeability. The increased lung retention of [11C]metoclopramide may be due to decreased P-gp-mediated clearance into the blood in absence of P-gp activity in capillary endothelial cells. The overall effect of P-gp on the lung exposure to inhaled P-gp substrate drugs may, thus, be determined by a balance of opposing effects at the pulmonary epithelium and endothelium.

Nuclear medicine imaging methods as novel tools in the assessment of pulmonary drug disposition.

INTRODUCTION: Drugs for the treatment of respiratory diseases are commonly administered by oral inhalation. Yet surprisingly little is known about the pulmonary pharmacokinetics of inhaled molecules. Nuclear medicine imaging techniques (i.e. planar gamma scintigraphy, single-photon emission computed tomography [SPECT] and positron emission tomography [PET]) enable the noninvasive dynamic measurement of the lung concentrations of radiolabeled drugs or drug formulations. This review discusses the potential of nuclear medicine imaging techniques in inhalation biopharmaceutical research. AREAS COVERED: (i) Planar gamma scintigraphy studies with radiolabeled inhalation formulations to assess initial pulmonary drug deposition; (ii) imaging studies with radiolabeled drugs to assess their intrapulmonary pharmacokinetics; (iii) receptor occupancy studies to quantify the pharmacodynamic effect of inhaled drugs. EXPERT OPINION: Imaging techniques hold potential to bridge the knowledge gap between animal models and humans with respect to the pulmonary disposition of inhaled drugs. However, beyond the mere assessment of the initial lung deposition of inhaled formulations with planar gamma scintigraphy, imaging techniques have rarely been employed in pulmonary drug development. This may be related to several technical challenges encountered with such studies. Considering the wealth of information that can be obtained with imaging studies their use in inhalation biopharmaceutics should be further investigated.

PET imaging to assess the impact of P-glycoprotein on pulmonary drug delivery in rats.

Several drugs approved for inhalation for the treatment of pulmonary diseases are substrates of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (P-gp). P-gp is expressed in the apical membrane of pulmonary epithelial cells and could play a role in modulating the pulmonary absorption and distribution of inhaled drugs, thereby potentially contributing to variability in therapeutic response and/or systemic side effects. We developed a new in vivo experimental approach to assess the functional impact of P-gp on the pulmonary delivery of inhaled drugs in rats. By using positron emission tomography (PET) imaging, we measured the intrapulmonary pharmacokinetics of the model P-gp substrates (R)-[11C]verapamil ([11C]VPM) and [11C]-N-desmethyl-loperamide ([11C]dLOP) administered by intratracheal aerosolization in three rat groups: wild-type, Abcb1a/b(-/-) and wild-type treated with the P-gp inhibitor tariquidar. Lung exposure (AUClung_right) to [11C]VPM was 64% and 50% lower (p < 0.05) in tariquidar-treated and in Abcb1a/b(-/-) rats, respectively, compared to untreated wild-type rats. For [11C]dLOP, AUClung_right was 59% and 34% lower (p < 0.05) in tariquidar-treated and in Abcb1a/b(-/-) rats, respectively. Our results show that P-gp can affect the pulmonary disposition of inhaled P-gp substrates, whereby a decrease in P-gp activity may lead to lower lung exposure and potentially to a decrease in therapeutic efficacy. Our study highlights the potential of PET imaging with intratracheally aerosolized radiotracers to assess the impact of membrane transporters on pulmonary drug delivery, in rodents and potentially also in humans.

Pharmacokinetic Imaging Using 99mTc-Mebrofenin to Untangle the Pattern of Hepatocyte Transporter Disruptions Induced by Endotoxemia in Rats.

Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99mTc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99mTc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug-drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99mTc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99mTc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99mTc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99mTc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99mTc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.

Impact of P-gp and BCRP on pulmonary drug disposition assessed by PET imaging in rats.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two efflux transporters which are expressed in the apical (i.e. airway lumen-facing) membranes of lung epithelial cells. To assess the influence of P-gp and BCRP on the pulmonary disposition of inhaled drugs, we performed positron emission tomography (PET) imaging in rats after intratracheal aerosolization of two model P-gp/BCRP substrate radiotracers (i.e. [11C]erlotinib and [11C]tariquidar). We studied rat groups in which both transporters were active (i.e. wild-type rats), either of the two transporters was inactive (Abcb1a/b(-/-) and Abcg2(-/-) rats) or both transporters were inactive (Abcg2(-/-) rats in which pulmonary P-gp activity was inhibited by treatment with unlabeled tariquidar). PET-measured lung distribution data were compared with brain-to-plasma radioactivity concentration ratios measured in a gamma counter at the end of the PET scan. For [11C]erlotinib, lung exposure (AUClungs) was moderately but not significantly increased in Abcb1a/b(-/-) rats (1.6-fold) and Abcg2(-/-) rats (1.5-fold), and markedly (3.6-fold, p < 0.0001) increased in tariquidar-treated Abcg2(-/-) rats, compared to wild-type rats. Similarly, the brain uptake of [11C]erlotinib was substantially (4.5-fold, p < 0.0001) increased when both P-gp and BCRP activities were impaired. For [11C]tariquidar, differences in AUClungs between groups pointed into a similar direction as for [11C]erlotinib, but were less pronounced and lacked statistical significance. Our study demonstrates functional P-gp and BCRP activity in vivo in the lungs and further suggests functional redundancy between P-gp and BCRP in limiting the pulmonary uptake of a model P-gp/BCRP substrate, analogous to the blood-brain barrier. Our results suggest that pulmonary efflux transporters are important for the efficacy and safety of inhaled drugs and that their modulation may be exploited in order to improve the pharmacokinetic and pharmacodynamic performance of pulmonary delivered drugs.

Repurposing 99mTc-Mebrofenin as a Probe for Molecular Imaging of Hepatocyte Transporters.

Hepatocyte transporters control the hepatobiliary elimination of many drugs, metabolites, and endogenous substances. Hepatocyte transporter function is altered in several pathophysiologic situations and can be modulated by certain drugs, with a potential impact for pharmacokinetics and drug-induced liver injury. The development of substrate probes with optimal properties for selective and quantitative imaging of hepatic transporters remains a challenge. 99mTc-mebrofenin has been used for decades for hepatobiliary scintigraphy, but the specific transporters controlling its liver kinetics have not been characterized until recently. These include sinusoidal influx transporters (organic anion-transporting polypeptides) responsible for hepatic uptake of 99mTc-mebrofenin, and efflux transporters (multidrug resistance-associated proteins) mediating its canalicular (liver-to-bile) and sinusoidal (liver-to-blood) excretion. Pharmacokinetic modeling enables molecular interpretation of 99mTc-mebrofenin scintigraphy data, thus offering a widely available translational method to investigate transporter-mediated drug-drug interactions in vivo. 99mTc-mebrofenin allows for phenotyping transporter function at the different poles of hepatocytes as a biomarker of liver function.

Assessing the Functional Redundancy between P-gp and BCRP in Controlling the Brain Distribution and Biliary Excretion of Dual Substrates with PET Imaging in Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are co-localized at the blood-brain barrier, where they display functional redundancy to restrict the brain distribution of dual P-gp/BCRP substrate drugs. We used positron emission tomography (PET) with the metabolically stable P-gp/BCRP substrates [11C]tariquidar, [11C]erlotinib, and [11C]elacridar to assess whether a similar functional redundancy as at the BBB exists in the liver, where both transporters mediate the biliary excretion of drugs. Wild-type, Abcb1a/b(-/-), Abcg2(-/-), and Abcb1a/b(-/-)Abcg2(-/-) mice underwent dynamic whole-body PET scans after i.v. injection of either [11C]tariquidar, [11C]erlotinib, or [11C]elacridar. Brain uptake of all three radiotracers was markedly higher in Abcb1a/b(-/-)Abcg2(-/-) mice than in wild-type mice, while only moderately changed in Abcb1a/b(-/-) and Abcg2(-/-) mice. The transfer of radioactivity from liver to excreted bile was significantly lower in Abcb1a/b(-/-)Abcg2(-/-) mice and almost unchanged in Abcb1a/b(-/-) and Abcg2(-/-) mice (with the exception of [11C]erlotinib, for which biliary excretion was also significantly reduced in Abcg2(-/-) mice). Our data provide evidence for redundancy between P-gp and BCRP in controlling both the brain distribution and biliary excretion of dual P-gp/BCRP substrates and highlight the utility of PET as an upcoming tool to assess the effect of transporters on drug disposition at a whole-body level.

Influence of ABC transporters on the excretion of ciprofloxacin assessed with PET imaging in mice.

Ciprofloxacin is a commonly prescribed fluoroquinolone antibiotic which is cleared by active tubular secretion and intestinal excretion. Ciprofloxacin is a known substrate of the ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). In this work, we used positron emission tomography (PET) imaging to investigate the influence of BCRP, MRP4, MRP2 and P-glycoprotein (P-gp) on the excretion of [18F]ciprofloxacin in mice. Dynamic 90-min PET scans were performed after intravenous injection of [18F]ciprofloxacin in wild-type mice without and with pre-treatment with the broad-spectrum MRP inhibitor MK571. Moreover, [18F]ciprofloxacin PET scans were performed in Abcc4(-/-), Abcc2(-/-), Abcc4(-/-)Abcg2(-/-) and Abcb1a/b(-/-)Abcg2(-/-) mice. In addition to non-compartmental pharmacokinetic (PK) analysis, a novel three-compartment PK model was developed for a detailed assessment of the renal disposition of [18F]ciprofloxacin. In MK571 pre-treated mice, a significant increase in the blood exposure to [18F]ciprofloxacin was observed along with a significant reduction in the renal and intestinal clearances. PK modelling revealed a significant reduction in renal radioactivity uptake (CL1) and in the rate constants for transfer of radioactivity from the corticomedullary renal region into blood (k2) and urine (k3), respectively, after MK571 administration. No changes in the renal clearance or in the estimated kidney PK model parameters were observed in any of the studied knockout models, while a significant reduction in the intestinal clearance was observed in Abcc2(-/-) and Abcc4(-/-)Abcg2(-/-) mice. Our data failed to reveal a role of any of the studied ABC transporters in the tubular secretion of ciprofloxacin. This may indicate that ciprofloxacin is handled in the kidneys by more than one transporter family, most likely with a great degree of mutual functional redundancy. Our study highlights the potential of PET imaging for an assessment of transporter-mediated renal excretion of radiolabelled drugs.

Imaging-Based Characterization of a Slco2b1(-/-) Mouse Model Using [11C]Erlotinib and [99mTc]Mebrofenin as Probe Substrates.

Organic anion-transporting polypeptide 2B1 (OATP2B1) is co-localized with OATP1B1 and OATP1B3 in the basolateral hepatocyte membrane, where it is thought to contribute to the hepatic uptake of drugs. We characterized a novel Slco2b1(-/-) mouse model using positron emission tomography (PET) imaging with [11C]erlotinib (a putative OATP2B1-selective substrate) and planar scintigraphic imaging with [99mTc]mebrofenin (an OATP1B1/1B3 substrate, which is not transported by OATP2B1). Dynamic 40-min scans were performed after intravenous injection of either [11C]erlotinib or [99mTc]mebrofenin in wild-type and Slco2b1(-/-) mice. A pharmacokinetic model was used to estimate the hepatic uptake clearance (CL1) and the rate constants for transfer of radioactivity from the liver to the blood (k2) and excreted bile (k3). CL1 was significantly reduced in Slco2b1(-/-) mice for both radiotracers (p < 0.05), and k2 was significantly lower (p < 0.01) in Slco2b1(-/-) mice for [11C]erlotinib, but not for [99mTc]mebrofenin. Our data support previous evidence that OATP transporters may contribute to the hepatic uptake of [11C]erlotinib. However, the decreased hepatic uptake of the OATP1B1/1B3 substrate [99mTc]mebrofenin in Slco2b1(-/-) mice questions the utility of this mouse model to assess the relative contribution of OATP2B1 to the liver uptake of drugs which are substrates of multiple OATPs.

Complete inhibition of ABCB1 and ABCG2 at the blood-brain barrier by co-infusion of erlotinib and tariquidar to improve brain delivery of the model ABCB1/ABCG2 substrate [11C]erlotinib.

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict at the blood-brain barrier (BBB) the brain distribution of the majority of currently known molecularly targeted anticancer drugs. To improve brain delivery of dual ABCB1/ABCG2 substrates, both ABCB1 and ABCG2 need to be inhibited simultaneously at the BBB. We examined the feasibility of simultaneous ABCB1/ABCG2 inhibition with i.v. co-infusion of erlotinib and tariquidar by studying brain distribution of the model ABCB1/ABCG2 substrate [11C]erlotinib in mice and rhesus macaques with PET. Tolerability of the erlotinib/tariquidar combination was assessed in human embryonic stem cell-derived cerebral organoids. In mice and macaques, baseline brain distribution of [11C]erlotinib was low (brain distribution volume, VT,brain < 0.3 mL/cm3). Co-infusion of erlotinib and tariquidar increased VT,brain in mice by 3.0-fold and in macaques by 3.4- to 5.0-fold, while infusion of erlotinib alone or tariquidar alone led to less pronounced VT,brain increases in both species. Treatment of cerebral organoids with erlotinib/tariquidar led to an induction of Caspase-3-dependent apoptosis. Co-infusion of erlotinib/tariquidar may potentially allow for complete ABCB1/ABCG2 inhibition at the BBB, while simultaneously achieving brain-targeted EGFR inhibition. Our protocol may be applicable to enhance brain delivery of molecularly targeted anticancer drugs for a more effective treatment of brain tumors.

Use of imaging to assess the activity of hepatic transporters.

Introduction: Membrane transporters of the SLC and ABC families are abundantly expressed in the liver, where they control the transfer of drugs/drug metabolites across the sinusoidal and canalicular hepatocyte membranes and play a pivotal role in hepatic drug clearance. Noninvasive imaging methods, such as PET, SPECT or MRI, allow for measuring the activity of hepatic transporters in vivo, provided that suitable transporter imaging probes are available.Areas covered: We give an overview of the working principles of imaging-based assessment of hepatic transporter activity. We discuss different currently available PET/SPECT radiotracers and MRI contrast agents and their applications to measure hepatic transporter activity in health and disease. We cover mathematical modeling approaches to obtain quantitative parameters of transporter activity and provide a critical assessment of methodological limitations and challenges associated with this approach.Expert opinion: PET in combination with pharmacokinetic modeling can be potentially applied in drug development to study the distribution of new drug candidates to the liver and their clearance mechanisms. This approach bears potential to mechanistically assess transporter-mediated drug-drug interactions, to assess the influence of disease on hepatic drug disposition and to validate and refine currently available in vitro-in vivo extrapolation methods to predict hepatic clearance of drugs.

Assessing the Activity of Multidrug Resistance-Associated Protein 1 at the Lung Epithelial Barrier.

Multidrug resistance-associated protein 1 (adenosine triphosphate-binding cassette subfamily C member 1 [ABCC1]) is abundantly expressed at the lung epithelial barrier, where it may influence the pulmonary disposition of inhaled drugs and contribute to variability in therapeutic response. The aim of this study was to assess the impact of ABCC1 on the pulmonary disposition of 6-bromo-7-11C-methylpurine (11C-BMP), a prodrug radiotracer that is intracellularly conjugated with glutathione to form the ABCC1 substrate S-(6-(7-11C-methylpurinyl))glutathione (11C-MPG). Methods: Groups of Abcc1(-/-) rats, wild-type rats pretreated with the ABCC1 inhibitor MK571, and wild-type control rats underwent dynamic PET scans after administration of 11C-BMP intravenously or by intratracheal aerosolization. In vitro transport experiments were performed with unlabeled BMP on the human distal lung epithelial cell line NCI-H441. Results: The pulmonary kinetics of radioactivity significantly differed between wild-type and Abcc1(-/-) rats, but differences were more pronounced after intratracheal than after intravenous administration. After intravenous administration, lung exposure (area under the lung time-activity curve from 0 to 80 min after radiotracer administration [AUClung]) was 77% higher and the elimination slope of radioactivity washout from the lungs (kE,lung) was 70% lower in Abcc1(-/-) rats, whereas after intratracheal administration, AUClung was 352% higher and kE,lung was 86% lower in Abcc1(-/-) rats. Pretreatment with MK571 decreased kE,lung by 20% after intratracheal radiotracer administration. Intracellular accumulation of MPG in NCI-H441 cells was significantly higher and extracellular efflux was lower in the presence than in the absence of MK571. Conclusion: PET with pulmonary administered 11C-BMP can measure ABCC1 activity at the lung epithelial barrier and may be applicable in humans to assess the effects of disease, genetic polymorphisms, or concomitant drug intake on pulmonary ABCC1 activity.

Validation of Pharmacological Protocols for Targeted Inhibition of Canalicular MRP2 Activity in Hepatocytes Using [99mTc]mebrofenin Imaging in Rats.

The multidrug resistance-associated protein 2 (MRP2) mediates the biliary excretion of drugs and metabolites. [99mTc]mebrofenin may be employed as a probe for hepatic MRP2 activity because its biliary excretion is predominantly mediated by this transporter. As the liver uptake of [99mTc]mebrofenin depends on organic anion-transporting polypeptide (OATP) activity, a safe protocol for targeted inhibition of hepatic MRP2 is needed to study the intrinsic role of each transporter system. Diltiazem (DTZ) and cyclosporin A (CsA) were first confirmed to be potent MRP2 inhibitors in vitro. Dynamic acquisitions were performed in rats (n = 5-6 per group) to assess the kinetics of [99mTc]mebrofenin in the liver, intestine and heart-blood pool after increasing doses of inhibitors. Their impact on hepatic blood flow was assessed using Doppler ultrasound (n = 4). DTZ (s.c., 10 mg/kg) and low-dose CsA (i.v., 0.01 mg/kg) selectively decreased the transfer of [99mTc]mebrofenin from the liver to the bile (k3). Higher doses of DTZ and CsA did not further decrease k3 but dose-dependently decreased the uptake (k1) and backflux (k2) rate constants between blood and liver. High dose of DTZ (i.v., 3 mg/kg) but not CsA (i.v., 5 mg/kg) significantly decreased the blood flow in the portal vein and hepatic artery. Targeted pharmacological inhibition of hepatic MRP2 activity can be achieved in vivo without impacting OATP activity and liver blood flow. Clinical studies are warranted to validate [99mTc]mebrofenin in combination with low-dose CsA as a novel substrate/inhibitor pair to untangle the role of OATP and MRP2 activity in liver diseases.