Ejf Franssen

Complete in vivo reversal of P-glycoprotein pump function in the blood-brain barrier visualized with positron emission tomography

1 Homozygously mdr1a gene disrupted mice (mdr1a(−/−) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P‐glycoprotein (P‐gp) function imaging non‐invasively and to study the effect of a P‐gp reversal agent on its function in vivo. 2 [11C]verapamil (0.1 mg/kg) was administered and the changes in tissue concentrations were determined ex vivo by organ extirpation and in vivo with PET. To block P‐gp function, cyclosporin A was administered. 3 Biodistribution studies revealed 9.5‐fold (P<0.001) and 3.4‐fold (P<0.001) higher [11C]verapamil in the brain and testes of mdr1a(−/−) mice than in mdr1a(+/+) mice. Cyclosporin A (25 mg/kg) increased [11C]verapamil levels in the brain and testes of mdr1a(+/+) mice in both cases 3.3‐fold (P<0.01 (brain); P<0.001 (testes)). Fifty mg/kg cyclosporin A increased [11C]verapamil in the brain 10.6‐fold (P<0.01) and in the testes 4.1‐fold (P<0.001). No increases were found in the mdr1a(−/−) mice. This indicates complete inhibition of P‐gp mediated [11C]verapamil efflux. 4 Positron camera data showed lower [11C]verapamil levels in the brain of mdr1a(+/+) mice compared to those in mdr1a(−/−) mice. [11C]verapamil accumulation in the brain of mdr1a(+/+) mice was increased by cyclosporin A to levels comparable with those in mdr1a(−/−) mice, indicating that reversal of P‐gp mediated efflux can be monitored by PET. 5 We conclude that cyclosporin A can fully block the P‐gp function in the blood brain barrier and the testes and that PET enables the in vivo measurement of P‐gp function and reversal of its function non‐invasively.

Visualization of multidrug resistance in vivo

Abstract. Various mechanisms are involved in multidrug resistance (MDR) for chemotherapeutic drugs, such as the drug efflux pumps, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP). In this review the mechanisms involved in MDR are described and results are reviewed with particular attention to the in vivo imaging of Pgp and MRP. Various detection assays provide information about the presence of drug efflux pumps at the mRNA and protein levels. However, these methods do not yield information about the dynamic function of Pgp and MRP in vivo. For the study of Pgp- and MRP-mediated transport, single-photon emission tomography (SPET) and positron emission tomography (PET) are available. Technetium-99m sestamibi is a substrate for Pgp and MRP, and has been used in clinical studies for tumour imaging, and to visualize blockade of Pgp-mediated transport after modulation of the Pgp pump. Other 99mTc radiopharmaceuticals, such as 99mTc-tetrofosmin and several 99Tc-Q complexes, are also substrates for Pgp, but to date only results from in vitro and animal studies are available for these compounds. Several agents, including [11C]colchicine, [11C]verapamil and [11C]daunorubicin, have been evaluated for the quantification of Pgp-mediated transport with PET in vivo. The results suggest that radiolabelled colchicine, verapamil and daunorubicin are feasible substrates with which to image Pgp function in tumours. Uptake of [11C]colchicine and [11C]verapamil is relatively high in the chest area, reducing the value of both tracers for monitoring Pgp-mediated drug transport in tumours located in this region. In addition, it has to be borne in mind that only comparison of Pgp-mediated transport of radioalabelled substrates in the absence and in the presence of Pgp blockade gives quantitative information on Pgp-mediated pharmacokinetics. Leukotrienes are specific substrates for MRP. Therefore, N-[11C]acetyl-leukotriene E4 provides an opportunity to study MRP function non-invasively. Results obtained in MRP2 mutated GY/TR rats have demonstrated visualization of MRP-mediated transport. This tracer permits the study of MRP transport function abnormalities in vivo, e.g. in Dubin-Johnson patients, who are MRP2 gene deficient. Results obtained show the feasibility of using SPET and PET to study the functionality of MDR transporters in vivo.

99mTc-sestamibi is a substrate for P-glycoprotein and the multidrug resistance-associated protein.

99mTc-sestamibi (99mTc-MIBI) is a substrate for the P-glycoprotein (P-gp) pump but it is not known whether it is a substrate for the multidrug resistance-associated protein (MRP) pump. Therefore, 99mTc-MIBI was evaluated in the GLC4 cell line and its doxorubicin-resistant MRP-, but not P-gp-, overexpressing GLC4/ADR sublines as well as in the S1 cell line and its MRP-transfected subline S1-MRP. 99mTc-MIBI concentration decreased in the GLC4/ADR sublines with increasing MRP overexpression and was lower in S1-MRP than in S1. 99mTc-MIBI plus vincristine increased 99mTc-MIBI concentration in GLC4 lines compared with 99mTc-MIBI alone. 99mTc-MIBI efflux raised with increasing MRP expression in the GLC4 lines. Glutathione depletion elevated 99mTc-MIBI concentration in GLC4/ADR150x. Cross resistance for 99Tc-MIBI, used to test cytotoxicity of the Tc compound, was observed in GLC4/ADR150x vs GLC4. 99Tc-MIBI induced a synergistic effect on vincristine cytotoxicity in GLC4/ADR150x. These results show that 99mTc-MIBI is involved in MRP-mediated efflux. The fact that 99mTc-MIBI efflux is influenced by MDR1 and MRP expression must be taken into account when this gamma-rays-emitting complex is tested for tumour efflux measurements.

Low Molecular Weight Proteins as Carriers for Renal Drug Targeting: Naproxen–Lysozyme

AbstractLow molecular weight proteins (LMWPs), such as lysozyme, may be suitable carriers to target drugs to the kidney. In this study the antiinflammatory drug naproxen was covalently bound to lysozyme (1:1). Pharmacokinetics of the conjugate, naproxen–lysozyme (nap-LYSO), were compared to that of an equimolar mixture of uncoupled naproxen with lysozyme in freely moving rats. Similar plasma kinetics and organ distribution for native lysozyme and the drug conjugate were observed (Clp = 1.2 and 1.1 ml/min;

Enzymatic synthesis of [4-methoxy-11C]daunorubicin for functional imaging of P-glycoprotein with PET

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Fluorine-18-fluorodeoxyglucose PET imaging of soft-tissue sarcoma

= 85 and 75 min, respectively). In case of the uncoupled naproxen–lysozyme mixture, a monoexponential plasma disappearance of naproxen with a

Carbon-11-Labeled Daunorubicin and Verapamil for Probing P-Glycoprotein in Tumors with PET

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