Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human
Introduction
The development of therapeutic agents to treat central nervous system (CNS) conditions requires an understanding of the mechanisms of drug entry and efflux into the different brain compartments. Drug compounds can enter the brain by both active transport and passive permeability mechanisms; however, the effective concentration of any compound can be affected by the interaction of these compounds with efflux pumps in the brain. To address this, we investigated expression patterns of the ABC family of transporters in brain and brain microvessel endothelial cells (BMEC) of the blood brain barrier (BBB) in human and other species of pharmacological interest. By understanding expression of ABC transporters, we hope to enable a more educated drug design to achieve therapeutic concentrations of CNS agents at their site of action.
While both molecular physiochemical properties and a variety of enzymes and transporters contribute to BBB impermeability [1], [2], [3], members of the ATP-cassette-binding protein (ABC) family of efflux transporters have been identified as major contributors in the exclusion of drugs from the brain [3]. In particular, P-glycoprotein (PGP; ABCB1) is highly expressed in BMEC of the blood–brain barrier and is known to limit transport of many compounds into the central nervous system. However, other compounds show limited transport across the BBB even when PGP is blocked or absent, indicating that additional transporters limit xenobiotic penetration of the brain. Most antihistamines are PGP substrates, and compounds such as terfenadine and loratadine can achieve substantial brain accumulation when PGP is inhibited by cyclosporin A [4]. However, other second-generation antihistamines, such as fexofenadine and cetirizine, have very poor brain penetration even when co-administered with cyclosporin A, suggesting that they may be substrates of additional efflux pumps [4]. Studies of ABCG2- and PGP-knockout mice have shown that both of these efflux pumps contribute to limiting brain uptake of dehydroepiandrosterone and mitoxantrone; however, there are also additional unidentified efflux pumps that can block uptake of these compounds into the brain [5]. Another recent report [6] using PGP-deficient mice studied the brain distribution of 34 drugs. For 21 of these 34 drugs, the PGP efflux ratio and plasma/brain ratio were in concurrence, indicating that any impairment of CNS uptake would be consistent with PGP efflux. However, for 10 of the 34 drugs studied, the levels in plasma were relatively high while the levels in brain were relatively low despite only having weak to moderate PGP activity, suggesting that other non-PGP-mediated mechanisms limit the CNS distribution of these drugs. Some of these (digoxin, doxorubicin, ivermectin) appear to be substrates of PGP as well as additional efflux transporters. Others (cimetidine, ranitidine) show little if any PGP-mediated efflux, yet appear to be limited by unidentified efflux pumps [4].
Forty-eight ABC transporters have been identified [7], although only a few of these have been well characterized. To identify which of these transporters may be involved in drug efflux at the BBB, we measured mRNA expression levels in brain and BMEC of 41 of these genes in human, 39 in rat, and 38 in mouse using quantitative real-time PCR. We also measured mRNA expression levels of 21 of these genes in cow and pig brain and BMEC, because primary BMEC cultures from these species are commonly used BBB models.
The importance of active mechanisms for uptake and efflux of compounds from the CNS has received wide attention by the scientific community. However, most of the studies so far have conducted assessments in rodent species, with somewhat conflicting results. No measurement of the transporter expression in the human BBB has previously been published, primarily due to the difficulty in obtaining fresh samples of human brain capillaries for analysis. In this study, human brain and BMEC samples were obtained from patients undergoing neurosurgery as part of epilepsy surgery. This is the first report of a comprehensive profile of ABC transporters in human BBB and brain, as well as four additional species commonly used to study BBB drug permeability in vivo (mouse and rat) and in vitro (pig and cow).
Section snippets
Materials
Heparin and bovine serum albumen (BSA) were from Sigma (Saint Louis, MO, USA). PBS, Hepes solution, and antibiotic/antimycotic solution were from Invitrogen (Carlsbad, CA, USA). Low glucose DMEM and fetal bovine serum (FBS) were from Hyclone (Logan, UT, USA). Collagenase/dispase was from Roche (Indianapolis, IN, USA).
Isolation of brain microvessel endothelial cells (BMEC)
All protocols involving animals were approved by the XenoPort Animal Use Committee in accordance with the recommendations outlined in the NIH Guide for the Care and Use of
Results
The mRNA expression levels of a variety of ABC transporters were measured in human and rodent brain and BMEC. The expression levels of a subset of transporters also were examined in pig and cow brain and BMEC. The results, shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4, are grouped by ABC subfamilies. Transporter mRNA levels were normalized to the BMEC expression level of GLUT1 (SLC2A1) mRNA for each species. GLUT1 is expressed at high levels in the BMEC of all species examined and is important for
Discussion
Several reports in the literature indicate that efflux pumps other than PGP may be important in drug exclusion at the BBB [11], [12], [16], [17], [18]. In this study, we have examined the expression in both the brain and BMEC of a broad selection of ABC transporters across five species. Our qPCR results are consistent with previous reports of ABC transporter expression at the BBB for a number of transporters [11], and provide novel data on many poorly characterized family members. Although
Conclusions
We have evaluated expression of ABC transporters in BMEC of the BBB in multiple species. ABC expression profiles may aid in understanding the physiological basis of drug distribution in the brain, and thus for the development of therapeutic agents for CNS diseases. BMEC to brain enrichment ratios of ABC transporters that are expressed at a level that is at least 0.5% of GLUT1 in the BBB of one or more species are shown in Table 1. Many of these transporters show BMEC enrichment and could be
Acknowledgements
We would like to acknowledge Mei Zhou, Steve Cwirla, Toni Yan, and Kent Grindstaff for technical assistance and helpful discussions, as well as Dr. Hans Rollema for providing information.
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Present address: Amgen, Pharmacokinetics and Drug Metabolism, 1201 Amgen Court W., Seattle, WA 98119, United States.