Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates

Abstract

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K and K of both P-gp substrates were indicative of drug efflux. Also, K for the BCRP substrates was low. In contrast, K for both BCRP substrates was close to unity, resulting in K/K ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Age-Related Functional and Expressional Changes in Efflux Pathways at the Blood-Brain Barrier

“…The human expression of ABCG2 and ABCB1 was about 40% greater in the occipital lobe than in the parietal lobe (Billington et al, 2019). In the canine brain, regional differences in Abcg2 protein expression levels were reported for the cerebellum and the brain stem (Braun et al, 2017).…”

Blood-Brain Barrier and Delivery of Protein and Gene Therapeutics to Brain

“…The relative expression of CMT, RMT, or AET systems at the brain capillary endothelium, which forms the BBB, as compared to the choroid plexus, which forms the blood-CSF barrier, has also been analyzed by QTAP. Whereas P-glycoprotein, or BCRP, are expressed at the BBB, the expression of these AET systems at the choroid plexus is only 0%-2% of the comparative expression at the BBB (Braun et al, 2017). With regard to RMT systems, the expression of the TfR1 at the choroid plexus, in the dog, is 16-fold higher than the expression of the IR at the choroid plexus.…”

SGLT6 - A pharmacological target for the treatment of obesity?

“…Plasma protein binding was determined by equilibrium dialysis as described. 15 Briefly, compound was spiked into plasma to get a final plasma concentration of 10 µM. Equilibrium dialysis against 100 mM potassium phosphate buffer, pH 7.4 was carried out for 2 hours at 37°C under rotation.…”

“…Animals were sacrificed at the time points indicated in the results section, plasma, a piece of leg muscle and brain were taken. Tissue was homogenized as described 15 and concentrations were determined by means of HPLC-MS/MS. Tissue concentration ratios were calculated per animal and descriptive statistics calculated.…”