Assessment of species differences in P-glycoprotein function at the blood-brain barrier

The adenosine triphosphate (ATP) binding cassette (ABC) transporter P-glycoprotein (Pgp) acts as a gatekeeper at the level of the vascular endothelium of the blood-brain barrier (BBB) preventing brain uptake of a wide range of lipophilic drugs by active ATP-driven efflux transport, which may lead to therapeutic failure of drugs targeted to the central nervous system. The earliest possible prediction of the extent of Pgp efflux of new drug candidates at the human BBB can therefore be considered an important goal in drug development. This is frequently achieved by studying drug brain distribution in rodent models (mice, rats), in which Pgp is either genetically or chemically disrupted. However, previous data suggest that species-dependent differences in the substrate and inhibitor specificity as well as in expression levels may exist between human Pgp (MDR1) and rodent Pgp (mdr1a/b). The proposed project plans to investigate species differences in the function of human and murine Pgp at the BBB in vivo by using a novel humanized mouse model in which the murine mdr1a/b gene is replaced by the human MDR1 gene (C57BL/6 MDR1). Pgp function will be assessed by studying brain distribution of microdoses (<1 g) of the radiolabeled model Pgp substrates (R)-[ 11C]verapamil and [ 11C]-N-desmethyl-loperamide after administration of different doses of the potent Pgp inhibitor tariquidar by means of small-animal positron emission tomography (PET) imaging. Data obtained in C57BL/6 MDR1 mice will be compared with their wild-type counterparts (C57BL/6 mdr1), which provides the unique opportunity to directly compare transporter function under otherwise identical physiological and experimental conditions. Furthermore, dose-response relationships of tariquidar to enhance brain uptake of (R)-[ 11C]verapamil in C57BL/6 MDR1 and C57BL/6 mdr1 mice will be compared with data in healthy human subjects obtained previously in the applicant`s laboratory. To complement the in vivo data, inhibition of (R)-verapamil and N-desmethyl-loperamide transport by tariquidar will be studied in vitro using LLC- PK1 cells transfected with either MDR1 or mdr1a. The results of the proposed project will answer the basic question if species differences between human and murine Pgp exist and may therefore enable an improved prediction of drug distribution into the human brain in future drug research.

The adenosine triphosphate-binding cassette transporter P-glycoprotein (humans: ABCB1; rodents: 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) is a non-invasive nuclear imaging method which allows to measure concentrations of radiolabelled drugs in the brain. PET with the ABCB1 substrates (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed to directly compare ABCB1-mediated DDIs at the rodent and human BBB. In this project we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[11C]verapamil or [11C]-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 [3H]verapamil and [3H]-N-desmethyl- loperamide in ABCB1 and Abcb1a overexpressing cell lines. We found no species differences for in vitro transport of [3H]verapamil and [3H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. Moreover we conducted in vivo experiments with (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide in mice without and with tariquidar pretreatment. These experiments showed that radiotracer metabolism and brain uptake of radiolabelled metabolites may exert a pronounced impact on the magnitude of ABCB1- mediated DDIs. Therefore differences in metabolism of radiotracers between rodents and humans may have contributed to differences seen in ABCB1-mediated DDIs measured with PET. Moreover we found that isoflurane anesthesia, which is commonly used in animal experiments, alters [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil metabolism. This also had a direct effect on the magnitude of the increase in brain distribution following ABCB1 inhibition. We could show that isoflurane anesthesia increases cerebral blood flow, which led to an increase in brain distribution of [11C]-N-desmethyl-loperamide but not (R)- [11C]verapamil. In conclusion, we have identified a number of important factors which can directly influence the magnitude of ABCB1-mediated DDIs at the BBB and should therefore be taken into consideration when interpreting PET results. The results of this project may enable an improved prediction of drug distribution to the human brain based on results from preclinical studies.