Pavitra Kannan

The "specific" P-glycoprotein inhibitor Tariquidar is also a substrate and an inhibitor for breast cancer resistance protein (BCRP/ABCG2).

Tariquidar was developed as a specific inhibitor of the efflux transporter ABCB1. Recent positron emission tomographic brain imaging studies using [(11)C]tariquidar to measure ABCB1 (P-gp, P-glycoprotein) density in mice indicate that the inhibitor may not be as specific as previously thought. We examined its selectivity as an inhibitor and a substrate for the human transporters P-gp, breast cancer resistance protein (BCRP, ABCG2), and multidrug resistance protein 1 (MRP1, ABCC1). Our results show that at low concentrations, tariquidar acts selectively as an inhibitor of P-gp and also as a substrate of BCRP. At much higher concentrations (≥100 nM), tariquidar acts as an inhibitor of both P-gp and BCRP. Thus, the in vivo specificity of tariquidar depends on concentration and the relative density and capacity of P-gp vs BCRP.

Radiodefluorination of 3-Fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a Radioligand for Imaging Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography, Occurs by Glutathionylation in Rat Brain

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[18F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [18F]-fluoride ion from [18F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [18F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [18F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [18F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [18F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H]+ of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H]+ of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [18F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.

Lysosomal trapping of a radiolabeled substrate of P-glycoprotein as a mechanism for signal amplification in PET

The radiotracer [11C]N-desmethyl-loperamide (dLop) images the in vivo function of P-glycoprotein (P-gp), a transporter that blocks the entry of drugs that are substrates into brain. When P-gp is inhibited, [11C]dLop, a potent opiate agonist, enters and becomes trapped in the brain. This trapping is beneficial from an imaging perspective, because it amplifies the PET signal, essentially by accumulating radioactivity over time. As we previously demonstrated that this trapping was not caused by binding to opiate receptors, we examined whether [11C]dLop, a weak base, is ionically trapped in acidic lysosomes. To test this hypothesis, we measured [3H]dLop accumulation in human cells by using lysosomotropics. Because the in vivo trapping of dLop was seen after P-gp inhibition, we also measured [3H]dLop uptake in P-gp–expressing cells treated with the P-gp inhibitor tariquidar. All lysosomotropics decreased [3H]dLop accumulation by at least 50%. In P-gp–expressing cells, tariquidar (and another P-gp inhibitor) surprisingly decreased [3H]dLop uptake. Consequently, we measured [11C]dLop uptake before and after tariquidar preadministration in lysosome-rich organs of P-gp KO mice and humans. After tariquidar pretreatment in both species, radioactivity uptake in these organs decreased by 35% to 40%. Our results indicate that dLop is trapped in lysosomes and that tariquidar competes with dLop for lysosomal accumulation in vitro and in vivo. Although tariquidar and dLop compete for lysosomal trapping in the periphery, such competition does not occur in brain because tariquidar has negligible entry into brain. In summary, tariquidar and [11C]dLop can be used in combination to selectively measure the function of P-gp at the blood–brain barrier.

Collateral sensitivity of multidrug-resistant cells to the orphan drug tiopronin.

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues.

Tariquidar is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein

Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [11C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes.

Regulation of O2 consumption by the PI3K and mTOR pathways contributes to tumor hypoxia

Background Inhibitors of the phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathway are currently in clinical trials. In addition to antiproliferative and proapoptotic effects, these agents also diminish tumor hypoxia. Since hypoxia is a major cause of resistance to radiotherapy, we sought to understand how it is regulated by PI3K/mTOR inhibition. Methods Whole cell, mitochondrial, coupled and uncoupled oxygen consumption were measured in cancer cells after inhibition of PI3K (Class I) and mTOR by pharmacological means or by RNAi. Mitochondrial composition was assessed by immunoblotting. Hypoxia was measured in spheroids, in tumor xenografts and predicted with mathematical modeling. Results Inhibition of PI3K and mTOR reduced oxygen consumption by cancer cell lines is predominantly due to reduction of mitochondrial respiration coupled to ATP production. Hypoxia in tumor spheroids was reduced, but returned after removal of the drug. Murine tumors had increased oxygenation even in the absence of average perfusion changes or tumor necrosis. Conclusions Targeting the PI3K/mTOR pathway substantially reduces mitochondrial oxygen consumption thereby reducing tumor hypoxia. These alterations in tumor hypoxia should be considered in the design of clinical trials using PI3K/mTOR inhibitors, particularly in conjunction with radiotherapy.

Factors That Limit Positron Emission Tomography Imaging of P‑Glycoprotein Density at the Blood−Brain Barrier

Efflux transporters located at the blood–brain barrier, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), regulate the passage of many drugs in and out of the brain. Changes in the function and density of these proteins, in particular P-gp, may play a role in several neurological disorders. Several radioligands have been developed for measuring P-gp function at the blood–brain barrier of human subjects with positron emission tomography (PET). However, attempts to measure P-gp density with radiolabeled inhibitors that bind to these proteins in vivo have not thus far provided useful, quantifiable PET signals. Herein, we argue that not only the low density of transporters in the brain as a whole but also their very high density in brain capillaries act to lower the concentration of ligand in the plasma and thereby contribute to absent or low signals in PET studies of P-gp density. Our calculations, based on published data and theoretical approximations, estimate that whole brain densities of many efflux transporters at the blood–brain barrier range from 0.04 to 5.19 nM. We conclude that the moderate affinities (>5 nM) of currently labeled inhibitors may not allow measurement of efflux transporter density at the blood–brain barrier, and inhibitors with substantially higher affinity will be needed for density imaging of P-gp and other blood–brain barrier transporters.

The Role of Oxygen in Avascular Tumor Growth

The oxygen status of a tumor has significant clinical implications for treatment prognosis, with well-oxygenated subvolumes responding markedly better to radiotherapy than poorly supplied regions. Oxygen is essential for tumor growth, yet estimation of local oxygen distribution can be difficult to ascertain in situ, due to chaotic patterns of vasculature. It is possible to avoid this confounding influence by using avascular tumor models, such as tumor spheroids, a much better approximation of realistic tumor dynamics than monolayers, where oxygen supply can be described by diffusion alone. Similar to in situ tumours, spheroids exhibit an approximately sigmoidal growth curve, often approximated and fitted by logistic and Gompertzian sigmoid functions. These describe the basic rate of growth well, but do not offer an explicitly mechanistic explanation. This work examines the oxygen dynamics of spheroids and demonstrates that this growth can be derived mechanistically with cellular doubling time and oxygen consumption rate (OCR) being key parameters. The model is fitted to growth curves for a range of cell lines and derived values of OCR are validated using clinical measurement. Finally, we illustrate how changes in OCR due to gemcitabine treatment can be directly inferred using this model.

Estimating oxygen distribution from vasculature in three-dimensional tumour tissue.

Regions of tissue which are well oxygenated respond better to radiotherapy than hypoxic regions by up to a factor of three. If these volumes could be accurately estimated, then it might be possible to selectively boost dose to radio-resistant regions, a concept known as dose-painting. While imaging modalities such as 18F-fluoromisonidazole positron emission tomography (PET) allow identification of hypoxic regions, they are intrinsically limited by the physics of such systems to the millimetre domain, whereas tumour oxygenation is known to vary over a micrometre scale. Mathematical modelling of microscopic tumour oxygen distribution therefore has the potential to complement and enhance macroscopic information derived from PET. In this work, we develop a general method of estimating oxygen distribution in three dimensions from a source vessel map. The method is applied analytically to line sources and quasi-linear idealized line source maps, and also applied to full three-dimensional vessel distributions through a kernel method and compared with oxygen distribution in tumour sections. The model outlined is flexible and stable, and can readily be applied to estimating likely microscopic oxygen distribution from any source geometry. We also investigate the problem of reconstructing three-dimensional oxygen maps from histological and confocal two-dimensional sections, concluding that two-dimensional histological sections are generally inadequate representations of the three-dimensional oxygen distribution.

Kinetic brain analysis and whole-body imaging in monkey of [11C]MNPA: a dopamine agonist radioligand.

With a view to future extension of the use of the agonist radioligand [11C]MNPA ([O‐methyl‐11C]2‐methoxy‐N‐propylnorapomorphine) from animals to humans, we performed two positron emission tomography (PET) studies in monkeys. First, we assessed the ability to quantify the brain uptake of [11C]MNPA with compartmental modeling. Second, we estimated the radiation exposure of [11C]MNPA to human subjects based on whole‐body imaging in monkeys. Brain PET scans were acquired for 90 min and included concurrent measurements of the plasma concentration of unchanged radioligand. Time‐activity data from striatum and cerebellum were quantified with two methods, a reference tissue model and distribution volume. Whole‐body PET scans were acquired for 120 min using four bed positions from head to mid thigh. Regions of interest were drawn on compressed planar whole‐body images to identify organs with the highest radiation exposures. After injection of [11C]MNPA, the highest concentration of radioactivity in brain was in striatum, with lowest levels in cerebellum. Distribution volume was well identified with a two‐tissue compartmental model and was quite stable from 60 to 90 min. Whole‐body PET scans showed the organ with the highest radiation burden (μSv/MBq) was the urinary bladder wall (26.0), followed by lungs (22.5), gallbladder wall (21.9), and heart wall (16.1). With a 2.4‐h voiding interval, the effective dose was 6.4 μSv/MBq (23.5 mrem/mCi). In conclusion, brain uptake of [11C]MNPA reflected the density of D2/3 receptors, quantified relative to serial arterial measurements, and caused moderate to low radiation exposure. Synapse 62:700–708, 2008. Published 2008 Wiley‐Liss, Inc.