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.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: [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.

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 (Aß) across the BBB. This study aimed to compare the abundance and activity of ABCG2 in a commonly used ß-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.

Generation and Characterization of an Abcc1 Humanized Mouse Model (hABCC1flx/flx ) 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.

Cross-Isotopic Bioorthogonal Tools as Molecular Twins for Radiotheranostic Applications.

Radiotheranostics are designed by labeling targeting (bio)molecules with radionuclides for diagnostic or therapeutic application. Because the pharmacokinetics of therapeutic compounds play a pivotal role, chemically closely related imaging agents are used to evaluate the overall feasibility of the therapeutic approach. "Theranostic relatives" that utilize different elements are frequently used in clinical practice. However, variations in pharmacokinetics, biodistribution, and target affinity due to different chemical properties of the radioisotopes remain as hurdles to the design of optimized clinical tools. Herein, the design and synthesis of structurally identical compounds, either for diagnostic (18 F and a stable metal isotope) or therapeutic application (radiometal and stable 19 F), are reported. Such "molecular twins" have been prepared by applying a modular strategy based on click chemistry that enables efficient radiolabeling of compounds containing a metal complex and a tetrazine moiety. This additional bioorthogonal functionality can be used for subsequent radiolabeling of (bio)molecules or pretargeting approaches, which is demonstrated in vitro.

Inhibition of ABCB1 and ABCG2 at the Mouse Blood-Brain Barrier with Marketed Drugs To Improve Brain Delivery of the Model ABCB1/ABCG2 Substrate [11C]erlotinib.

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters at the blood-brain barrier (BBB), which effectively restrict brain distribution of diverse drugs, such as tyrosine kinase inhibitors. There is a crucial need for pharmacological ABCB1 and ABCG2 inhibition protocols for a more effective treatment of brain diseases. In the present study, seven marketed drugs (osimertinib, erlotinib, nilotinib, imatinib, lapatinib, pazopanib, and cyclosporine A) and one nonmarketed drug (tariquidar), with known in vitro ABCB1/ABCG2 inhibitory properties, were screened for their inhibitory potency at the BBB in vivo. Positron emission tomography (PET) using the model ABCB1/ABCG2 substrate [11C]erlotinib was performed in mice. Tested inhibitors were administered as i.v. bolus injections at 30 min before the start of the PET scan, followed by a continuous i.v. infusion for the duration of the PET scan. Five of the tested drugs increased total distribution volume of [11C]erlotinib in the brain ( VT,brain) compared to vehicle-treated animals (tariquidar, + 69%; erlotinib, + 19% and +23% for the 21.5 mg/kg and the 43 mg/kg dose, respectively; imatinib, + 22%; lapatinib, + 25%; and cyclosporine A, + 49%). For all drugs, increases in [11C]erlotinib brain distribution were lower than in Abcb1a/b(-/-)Abcg2(-/-) mice (+149%), which suggested that only partial ABCB1/ABCG2 inhibition was reached at the mouse BBB. The plasma concentrations of the tested drugs at the time of the PET scan were higher than clinically achievable plasma concentrations. Some of the tested drugs led to significant increases in blood radioactivity concentrations measured at the end of the PET scan (erlotinib, + 103% and +113% for the 21.5 mg/kg and the 43 mg/kg dose, respectively; imatinib, + 125%; and cyclosporine A, + 101%), which was most likely caused by decreased hepatobiliary excretion of radioactivity. Taken together, our data suggest that some marketed tyrosine kinase inhibitors may be repurposed to inhibit ABCB1 and ABCG2 at the BBB. From a clinical perspective, moderate increases in brain delivery despite the administration of high i.v. doses as well as peripheral drug-drug interactions due to transporter inhibition in clearance organs question the translatability of this concept.

Effect of Rifampicin on the Distribution of [11C]Erlotinib to the Liver, a Translational PET Study in Humans and in Mice.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

Hepatocyte-Specific Deletion of EGFR in Mice Reduces Hepatic Abcg2 Transport Activity Measured by [11C]erlotinib and Positron Emission Tomography.

The epidermal growth factor receptor (EGFR) regulates cellular expression levels of breast cancer resistance protein (humans: ABCG2, rodents: Abcg2) via its downstream signaling pathways. Drugs that inhibit EGFR signaling (e.g., tyrosine kinase inhibitors, antibodies) may lead to ABCG2-mediated drug-drug interactions (DDIs) by changing the disposition of concomitantly administered ABCG2 substrate drugs. In this study, we used positron emission tomography and magnetic resonance imaging to compare disposition of the model Abcg2 substrate [11C]erlotinib in a mouse model of hepatocyte-specific deletion of EGFR (EGFR∆hep mice, n = 5) with EGFRfl/fl control mice (n = 6), which have normal EGFR expression levels in all tissues. Integration plot analysis was used to estimate the rate constants for transfer of radioactivity from the liver into bile (kbile) and from the kidney into urine (kurine). EGFR∆hep mice showed significantly lower radioactivity concentrations in the intestine (1.6-fold) and higher radioactivity concentrations in the urinary bladder (3.2-fold) compared with EGFRfl/fl mice. Kbile was significantly decreased (3.0-fold) in EGFR∆hep mice, whereas kurine was by 2.2-fold increased. Western blot analysis of liver tissue confirmed deletion of EGFR and showed significant decreases in Abcg2 and increases in P-glycoprotein (Abcb1a/b) expression levels in EGFR∆hep versus EGFRfl/fl mice. Our data show that EGFR deletion in hepatocytes leads to a reduction in Abcg2-mediated hepatobiliary clearance of a probe substrate accompanied by a shift to renal excretion of the drug, which raises the possibility that EGFR-inhibiting drugs may cause ABCG2-mediated DDIs.

[18F]Fluoroalkyl azides for rapid radiolabeling and (Re)investigation of their potential towards in vivo click chemistry.

In recent years, radiofluorinated alkyl azides have been reported for click radiolabeling and pretargeted PET imaging, but only little is known about the biodistribution and metabolism of these compounds. In this work, we present a significantly improved procedure for the synthesis of [18F]fluoroethyl azide and reinvestigated this radiolabeled probe in detail showing poor stability and very restricted suitability for in vivo application. Therefore, modified low-molecular-weight [18F]fluoroalkyl azides were developed. Propargyl-tagged endomorphin-1 (as model compound) was successfully radiolabeled in high yield and short reaction time making these probes useful and efficient bioorthogonal tools for rapid radiolabeling. Biodistribution, pharmacokinetics and in vivo stability were studied by preclinical PET/MR scanning and metabolite analysis. The results of this study revealed only limited applicability of [18F]fluoroalkyl azides for in vivo application.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

Design, Synthesis, and Evaluation of a Low-Molecular-Weight (11)C-Labeled Tetrazine for Pretargeted PET Imaging Applying Bioorthogonal in Vivo Click Chemistry.

A low-molecular-weight tetrazine labeled with the short-lived positron emitter carbon-11 was developed as a bioorthogonal PET probe for pretargeted imaging. A method for efficient and fast synthesis of this imaging agent is presented using radiolabeling of a readily available precursor. High reactivity with trans-cyclooctenes was observed and in vivo investigations including PET/MR scanning showed homogeneous biodistribution, good metabolic stability, and rapid excretion in naive mice. These properties are key to the success of bioorthogonal (11)C-PET imaging, which has been shown in a simple pretargeting experiment using TCO-modified mesoporous silica nanoparticles. Overall, this (11)C-labeled tetrazine represents a highly versatile and advantageous chemical tool for bioorthogonal PET imaging and enables pretargeting approaches using carbon-11 for the first time.

Synthesis and preclinical characterization of 1-(6'-deoxy-6'-[18F]fluoro-ß-d-allofuranosyl)-2-nitroimidazole (ß-6'-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia.

Positron emission tomography (PET) using fluorine-18 (18F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6'-deoxy-6'-[18F]fluoro-ß-d-allofuranosyl)-2-nitroimidazole (ß-[18F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [18F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (ß-6) in a final radiochemical yield of 12±8% (n=10, based on [18F]fluoride starting activity) in a total synthesis time of 60min with a specific activity at end of synthesis of 218±58GBq/µmol (n=10). Both radiolabeling precursor ß-6 and unlabeled reference compound ß-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of ß-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of ß-[18F]1 in tumor cell lines. In biodistribution studies in healthy mice ß-[18F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13±0.22 (n=4) at 2h after administration of ß-[18F]1. In ex vivo autoradiography experiments ß-[18F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, ß-[18F]1 shows potential as PET hypoxia radiotracer which merits further investigation.

[18F]FE@SUPPY: a suitable PET tracer for the adenosine A3 receptor? An in vivo study in rodents.

PURPOSE: The adenosine A3 receptor (A3R) is involved in cardiovascular, neurological and tumour-related pathologies and serves as an exceptional pharmaceutical target in the clinical setting. A3R antagonists are considered antiinflammatory, antiallergic and anticancer agents, and to have potential for the treatment of asthma, COPD, glaucoma and stroke. Hence, an appropriate A3R PET tracer would be highly beneficial for the diagnosis and therapy monitoring of these diseases. Therefore, in this preclinical in vivo study we evaluated the potential as a PET tracer of the A3R antagonist [(18)F]FE@SUPPY. METHODS: Rats were injected with [(18)F]FE@SUPPY for baseline scans and blocking scans (A3R with MRS1523 or FE@SUPPY, P-gp with tariquidar; three animals each). Additionally, metabolism was studied in plasma and brain. In a preliminary experiment in a mouse xenograft model (mice injected with cells expressing the human A3R; three animals), the animals received [(18)F]FE@SUPPY and [(18)F]FDG. Dynamic PET imaging was performed (60 min in rats, 90 min in xenografted mice). In vitro stability of [(18)F]FE@SUPPY in human and rat plasma was also evaluated. RESULTS: [(18)F]FE@SUPPY showed high uptake in fat-rich regions and low uptake in the brain. Pretreatment with MRS1523 led to a decrease in [(18)F]FE@SUPPY uptake (p = 0.03), and pretreatment with the P-gp inhibitor tariquidar led to a 1.24-fold increase in [(18)F]FE@SUPPY uptake (p = 0.09) in rat brain. There was no significant difference in metabolites in plasma and brain in the treatment groups. However, plasma concentrations of [(18)F]FE@SUPPY were reduced to levels similar to those in rat brain after blocking. In contrast to [(18)F]FDG uptake (p = 0.12), the xenograft model showed significantly increased uptake of [(18)F]FE@SUPPY in the tissue masses from CHO cells expressing the human A3R (p = 0.03). [(18)F]FE@SUPPY was stable in human plasma. CONCLUSION: Selective and significant tracer uptake of [(18)F]FE@SUPPY was found in xenografted mice injected with cells expressing human A3R. This finding supports the strategy of evaluating [(18)F]FE@SUPPY in "humanized animal models". In conclusion, preclinical evaluation points to the suitability of [(18)F]FE@SUPPY as an A3R PET tracer in humans.

Factors Governing P-Glycoprotein-Mediated Drug-Drug Interactions at the Blood-Brain Barrier Measured with Positron Emission Tomography.

The adenosine triphosphate-binding cassette transporter P-glycoprotein (ABCB1/Abcb1a) restricts at the blood-brain barrier (BBB) brain distribution of many drugs. ABCB1 may be involved in drug-drug interactions (DDIs) at the BBB, which may lead to changes in brain distribution and central nervous system side effects of drugs. Positron emission tomography (PET) with the ABCB1 substrates (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed direct comparison of ABCB1-mediated DDIs at the rodent and human BBB. In this work we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[(11)C]verapamil or [(11)C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously observed species differences between rodents and humans. We performed in vitro transport experiments with [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide in wild-type mice without and with tariquidar pretreatment and in homozygous Abcb1a/1b((-/-)) and heterozygous Abcb1a/1b((+/-)) mice. We found no differences for in vitro transport of [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [(3)H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [(3)H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [(11)C]-N-desmethyl-loperamide than for (R)-[(11)C]verapamil. Both radiotracers showed tariquidar dose dependent increases in brain distribution with tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930-1189) for (R)-[(11)C]verapamil and 1329 nM (95% CI: 980-1801) for [(11)C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b((-/-)) mice brain radioactivity distribution was increased by 3.9- and 2.8-fold and in heterozygous Abcb1a/1b((+/-)) mice by 1.5- and 1.1-fold, for (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide, respectively, as compared with wild-type mice. For both radiotracers radiolabeled metabolites were detected in plasma and brain. When brain and plasma radioactivity concentrations were corrected for radiolabeled metabolites, brain distribution of (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide was increased in tariquidar (15 mg/kg) treated animals by 14.1- and 18.3-fold, respectively, as compared with vehicle group. Isoflurane anesthesia altered [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]verapamil metabolism, and this had a direct effect on the magnitude of the increase in brain distribution following ABCB1 inhibition. Our data furthermore suggest that in the absence of ABCB1 function brain distribution of [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]verapamil may depend on cerebral blood flow. In conclusion, we have identified a number of important factors, i.e., substrate affinity to ABCB1, brain uptake of radiolabeled metabolites, anesthesia, and cerebral blood flow, which can directly influence the magnitude of ABCB1-mediated DDIs at the BBB and should therefore be taken into consideration when interpreting PET results.

Development of a (18) F-labeled tetrazine with favorable pharmacokinetics for bioorthogonal PET imaging.

A low-molecular-weight (18) F-labeled tetrazine derivative was developed as a highly versatile tool for bioorthogonal PET imaging. Prosthetic groups and undesired carrying of (18) F through additional steps were evaded by direct (18) F-fluorination of an appropriate tetrazine precursor. Reaction kinetics of the cycloaddition with trans-cyclooctenes were investigated by applying quantum chemical calculations and stopped-flow measurements in human plasma; the results indicated that the labeled tetrazine is suitable as a bioorthogonal probe for the imaging of dienophile-tagged (bio)molecules. In vitro and in vivo investigations revealed high stability and PET/MRI in mice showed fast homogeneous biodistribution of the (18) F-labeled tetrazine that also passes the blood-brain barrier. An in vivo click experiment confirmed the bioorthogonal behavior of this novel tetrazine probe. Due to favorable chemical and pharmacokinetic properties this bioorthogonal agent should find application in bioimaging and biomedical research.

Functionalization of 68Ga-Radiolabeled Nanodiamonds with Octreotide Does Not Improve Tumor-Targeting Capabilities.

Nanodiamonds (NDs) are emerging as a novel nanoparticle class with growing interest in medical applications. The surface coating of NDs can be modified by attaching binding ligands or imaging probes, turning them into multi-modal targeting agents. In this investigation, we assessed the targeting efficacy of octreotide-functionalized 68Ga-radiolabelled NDs for cancer imaging and compared it with the tumor uptake using [68Ga]Ga-DOTA-TOC. In vivo studies in mice bearing AR42J tumors demonstrated the highest accumulation of the radiolabeled functionalized NDs in the liver and spleen, with relatively low tumor uptake compared to [68Ga]Ga-DOTA-TOC. Our findings suggest that, within the scope of this study, functionalization did not enhance the tumor-targeting capabilities of NDs.