In vivo characterization of [18F]AVT-011 as a radiotracer for PET imaging of multidrug resistance

Multidrug resistance (MDR) impedes cancer treatment. Two efflux transporters from the ATP-binding cassette (ABC) family, ABCB1 and ABCG2, may contribute to MDR by restricting the entry of therapeutic drugs into tumor cells. Although a higher expression of these transporters has been correlated with an unfavorable response to chemotherapy, transporter expression does not necessarily correlate with function. In this study, we characterized the pharmacological properties of [18F]AVT-011, a new PET radiotracer for imaging transporter-mediated MDR in tumors.AVT-011 was radiolabeled with 18F and evaluated with PET imaging in preclinical models. Transport of [18F]AVT-011 by ABCB1 and/or ABCG2 was assessed by measuring its uptake in the brains of wild-type, Abcb1a/b-/-, and Abcg2-/- mice at baseline and after administration of the ABCB1 inhibitor tariquidar (n = 5/group). Metabolism and biodistribution of [18F]AVT-011 were also measured. To measure ABCB1 function in tumors, we performed PET experiments using both [18F]AVT-011 and [18F]FDG in mice bearing orthotopic breast tumors (n = 7-10/group) expressing clinically relevant levels of ABCB1.At baseline, brain uptake was highest in Abcb1a/b-/- mice. After tariquidar administration, brain uptake increased 3-fold and 8-fold in wild-type and Abcg2-/- mice, respectively, but did not increase further in Abcb1a/b-/- mice. At 30 min after injection, the radiotracer was > 90% in its parent form and had highest uptake in organs of the hepatobiliary system. Compared with that in drug-sensitive tumors, uptake of [18F]AVT-011 was 32% lower in doxorubicin-resistant tumors with highest ABCB1 expression and increased by 40% with tariquidar administration. Tumor uptake of [18F]FDG did not significantly differ among groups.[18F]AVT-011 is a dual ABCB1/ABCG2 substrate radiotracer that can quantify transporter function at the blood-brain barrier and in ABCB1-expressing tumors, making it potentially suitable for clinical imaging of ABCB1-mediated MDR in tumors

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

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 24-Nor-Ursodeoxycholic Acid on Hepatic Disposition of [(18)F]Ciprofloxacin, a Positron Emission Tomography Study in Mice

24-nor-ursodeoxycholic acid (norUDCA) is a novel therapeutic approach to cholestatic liver diseases. In mouse models of cholestasis, norUDCA induces basolateral multidrug resistance-associated proteins 4 (Mrp4) and 3 in hepatocytes, which provide alternative escape routes for bile acids accumulating during cholestasis but could also result in altered hepatic disposition of concomitantly administered substrate drugs. We used positron emission tomography imaging to study the influence of norUDCA on hepatic disposition of the model Mrp4 substrate [(18)F]ciprofloxacin in wild-type and Mdr2((-/-)) mice, a model of cholestasis. Animals underwent [(18)F]ciprofloxacin positron emission tomography at baseline and after norUDCA treatment. After norUDCA treatment, liver-to-blood area under the curve ratio of [(18)F]ciprofloxacin was significantly decreased compared to baseline, both in wild-type (-34.0 ± 2.1%) and Mdr2((-/-)) mice (-20.5 ± 6.0%). [(18)F]Ciprofloxacin uptake clearance from blood into liver was reduced by -17.1 ± 9.0% in wild-type and by -20.1 ± 7.3% in Mdr2((-/-)) mice. Real-time PCR analysis showed significant increases in hepatic Mrp4 and multidrug resistance-associated protein 3 mRNA after norUDCA. Transport experiments in organic anion transporting polypeptide (OATP)1B1-, OATP1B3-, and OATP2B1-transfected cells revealed weak transport of [(14)C]ciprofloxacin by OATP1B3 and OATP2B1 and no inhibition by norUDCA. In conclusion, our data suggest that changes in hepatic [(18)F]ciprofloxacin disposition in mice after norUDCA treatment were caused by induction of basolateral Mrp4 in hepatocytes

Measurement of cerebral ABCC1 transport activity in wild-type and APP/PS1-21 mice with positron emission tomography

Previous data suggest a possible link between multidrug resistance-associated protein 1 (ABCC1) and brain clearance of beta-amyloid (Aβ). We used PET with 6-bromo-7-[11C]methylpurine ([11C]BMP) to measure cerebral ABCC1 transport activity in a beta-amyloidosis mouse model (APP/PS1-21) and in wild-type mice aged 50 and 170 days, without and with pretreatment with the ABCC1 inhibitor MK571. One hundred seventy days-old-animals additionally underwent [11C]PiB PET scans to measure Aβ load. While baseline [11C]BMP PET scans detected no differences in the elimination slope of radioactivity washout from the brain (kelim) between APP/PS1-21 and wild-type mice of both age groups, PET scans after MK571 pretreatment revealed significantly higher kelim values in APP/PS1-21 mice than in wild-type mice aged 170 days, suggesting increased ABCC1 activity. The observed increase in kelim occurred across all investigated brain regions and was independent of the presence of Aβ plaques measured with [11C]PiB. Western blot analysis revealed a trend towards increased whole brain ABCC1 levels in 170 days-old-APP/PS1-21 mice versus wild-type mice and a significant positive correlation between ABCC1 levels and kelim. Our data point to an upregulation of ABCC1 in APP/PS1-21 mice, which may be related to an induction of ABCC1 in astrocytes as a protective mechanism against oxidative stress

Comparison of fully-automated radiosyntheses of [11C]erlotinib for preclinical and clinical use starting from in target produced [11C]CO2 or [11C]CH4

Abstract Background [11C]erlotinib has been proposed as a PET tracer to visualize the mutational status of the epidermal growth factor receptor (EGFR) in cancer patients. For clinical use, a stable, reproducible and high-yielding radiosynthesis method is a prerequisite. In this work, two production schemes for [11C]erlotinib applied in a set of preclinical and clinical studies, starting from either [11C]CH4 or [11C]CO2, are presented and compared in terms of radiochemical yields, molar activities and overall synthesis time. In addition, a time-efficient RP-HPLC method for quality control is presented, which requires not more than 1 min. Results [11C]erlotinib was reliably produced applying both methods with decay-corrected radiochemical yields of 13.4 ± 6.2% and 16.1 ± 4.9% starting from in-target produced [11C]CO2 and [11C]CH4, respectively. Irradiation time for the production of [11C]CO2 was higher in order to afford final product amounts sufficient for patient application. Overall synthesis time was comparable, mostly attributable to adaptions in the semi-preparative HPLC protocol. Molar activities were 1.8-fold higher for the method starting from [11C]CH4 (157 ± 68 versus 88 ± 57 GBq/μmol at the end of synthesis). Conclusions This study compared two synthetic protocols for the production of [11C]erlotinib with in-target produced [11C]CO2 or [11C]CH4. Both methods reliably yielded sufficiently high product amounts for preclinical and clinical use

Generation and characterization of an ABCC1 humanized mouse model (HABCC1<sup>FLX/FLX</sup>) with knockout capability

ATP-binding cassette (ABC) transporters such as ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP) are well known for their role in rendering cancer cells resistant to chemotherapy. Additionally, recent research provided evidence that, along with other ABC transporters (ABCA1 and ABCA7), they might be cornerstones to tackle neurodegenerative diseases. Overcoming chemoresistance in cancer, understanding drug-drug interactions, and developing efficient and specific drugs that alter ABC transporter function are hindered by a lack of in vivo research models, which are fully predictive for humans. Hence, the humanization of ABC transporters in mice has become a major focus in pharmaceutical and neurodegenerative research. Here, we present a characterization of the first Abcc1 humanized mouse line. To preserve endogenous expression profiles, we chose to generate a knockin mouse model that leads to the expression of a chimeric protein that is fully human except for one amino acid. We found robust mRNA and protein expression within all major organs analyzed (brain, lung, spleen, and kidney). Furthermore, we demonstrate the functionality of the expressed human ABCC1 protein in brain and lungs using functional positron emission tomography imaging in vivo. Through the introduction of loxP sites, we additionally enabled this humanized mouse model for highly sophisticated studies involving cell type–specific transporter ablation. Based on our data, the presented mouse model appears to be a promising tool for the investigation of cell-specific ABCC1 function. It can provide a new basis for better translation of preclinical research

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 μg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n= 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg,n= 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p≤ 0.05) and -41 ± 16% (p≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinibVTin the human brain (+20 ± 16%,p≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution

Brain Distribution of Dual ABCB1/ABCG2 Substrates Is Unaltered in a Beta-Amyloidosis Mouse Model

Background: ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein) are co-localized at the blood-brain barrier (BBB), where they restrict the brain distribution of many different drugs. Moreover, ABCB1 and possibly ABCG2 play a role in Alzheimer’s disease (AD) by mediating the brain clearance of beta-amyloid (Aβ) across the BBB. This study aimed to compare the abundance and activity of ABCG2 in a commonly used β-amyloidosis mouse model (APP/PS1-21) with age-matched wild-type mice. Methods: The abundance of ABCG2 was assessed by semi-quantitative immunohistochemical analysis of brain slices of APP/PS1-21 and wild-type mice aged 6 months. Moreover, the brain distribution of two dual ABCB1/ABCG2 substrate radiotracers ([11C]tariquidar and [11C]erlotinib) was assessed in APP/PS1-21 and wild-type mice with positron emission tomography (PET). [11C]Tariquidar PET scans were performed without and with partial inhibition of ABCG2 with Ko143, while [11C]erlotinib PET scans were only performed under baseline conditions. Results: Immunohistochemical analysis revealed a significant reduction (by 29–37%) in the number of ABCG2-stained microvessels in the brains of APP/PS1-21 mice. Partial ABCG2 inhibition significantly increased the brain distribution of [11C]tariquidar in APP/PS1-21 and wild-type mice, but the brain distribution of [11C]tariquidar did not differ under both conditions between the two mouse strains. Similar results were obtained with [11C]erlotinib. Conclusions: Despite a reduction in the abundance of cerebral ABCG2 and ABCB1 in APP/PS1-21 mice, the brain distribution of two dual ABCB1/ABCG2 substrates was unaltered. Our results suggest that the brain distribution of clinically used ABCB1/ABCG2 substrate drugs may not differ between AD patients and healthy people