[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.

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.

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.

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.