The role of drug transporters at the blood brain barrier

Posted: 29 February 2016 | | No comments yet

The human brain is the most highly perfused organ in the body, being composed of over 100 billion capillaries with an average inter-capillary distance of 50μm and a length greater than 600km. This extensive network of blood vessels facilitates the delivery of nutrients and oxygen to the brain, while providing a physical, metabolic and immunologic barrier, the blood brain barrier (BBB), to protect it. The BBB comprises a very tight layer of capillary endothelial cells with elaborate tight junctions between adjacent cells that prevent most soluble materials from crossing, while allowing gases such as oxygen and carbon dioxide to diffuse through.

The role of drug transporters at the blood brain barrier

Passage into the brain from the circulatory system is limited by a number of mechanisms. Endothelial cells of the central nervous system (CNS) have extremely low rates of transcytosis, thereby limiting passage of endogenous substrates and larger molecules. When small molecules diffuse through the cell membrane, the highly expressed efflux transport proteins pump these out, further limiting access to the brain. This efficient and essential barrier is a major hurdle for researchers developing drugs for CNS disorders, since overcoming these protective mechanisms, while reaching the target at an efficacious and safe concentration, is a challenge. Developing drugs becomes increasingly complicated in disease states caused by Alzheimer’s and multiple sclerosis, since the barrier function changes, potentially resulting in alterations in CNS drug exposure.

Membrane transport proteins at the BBB are binned into two categories – uptake and efflux transporters. At the BBB, multiple uptake transporters are responsible for bringing solutes from circulation into the endothelial cells (apical/luminal membrane) and then into the brain across the basolateral membrane. Meanwhile, the efflux transporters pump compounds back into the blood as they traverse the apical cell membrane (i.e., the blood side) and also pump compounds out of the cell into the brain on the basolateral side. These transporters are present at very high concentrations on the apical membrane, and have been studied extensively.

Efflux transporters

Multiple efflux transporters are involved in drug transport at the BBB. These include P-glycoprotein (P-gp), breast cancer resistant protein (BCRP) and the multidrug resistance-associated proteins MRP1, MRP3, MRP4 and MRP6. The activities of the efflux transporters at the luminal BBB are dominated by P-gp, followed by BCRP and finally the MRP transporters. P-gp and BCRP work in cooperation as ‘gatekeepers’ of the BBB. The consequence of this cooperation was first demonstrated with the chemotherapeutic agent topotecan, a topoisomerase inhibitor that is transported by both P-gp and BCRP. In a study using transgenic mice, it was shown that brain uptake did not increase much in mice that were deficient in Bcrp (Bcrp1−/−) or P-gp (Mdr1a/1b−/−) and increased dramatically when both transporters were knocked out (Mdr1a/1b−/−Bcrp1−/−). 

More extensive studies were conducted by Polli et al2 using the tyrosine kinase inhibitor, lapatinib (Tykerb/Tyverb, GlaxoSmithKline), which is used to treat advanced or metastatic breast cancers overexpressing human epidermal receptor 2. As compared with wild-type mice, lapatinib brain-to-plasma ratios were no different in the Bcrp knockout mice, were three-fold to four-fold higher in P-gp knockout mice and were an astounding 40-fold higher in mice lacking both transporters, establishing that P-gp and Bcrp work in concert. Similar studies with the P-gp and BCRP dual substrate drugs dasatinib (Syrycel, Bristol-Myers Squibb), gefitinib (Iressa, AstraZeneca and Teva) and sorafenib (Nexavar, Bayer and Onyx Pharmaceuticals) revealed that absence of either one of the transporters was significantly less effective at increasing brain concentrations, relative to when both were knocked down. This finding has important potential consequences for the treatment of CNS disorders with small molecule drugs, since it is not advisable to develop drugs that are dual substrates of both these transporters.

The dominant thinking at this time is that P-gp plays a greater role than BCRP in pumping out drugs from the CNS, for many dual substrates3. Due to this, P-gp has remained the transporter of focus in neuroscience drug discovery and development. Multiple clinical studies have corroborated the role of P-gp in limiting brain penetration, e.g., co-administration of the cancer drug etoposide (Etopophos, Bristol-Myers Squibb) with the P-gp inhibitor drug cyclosporine results in more severe nausea4 and co-administration with the P-gp inhibitor valspodar, an experimental cancer treatment drug, causes severe ataxia5. In both cases, the co-administered P-gp inhibitor allowed for higher concentrations of etoposide to cross the BBB, inducing neurotoxicity.

Similarly, when the hypertension drug verapamil (a P-gp substrate and inhibitor) is co-administered with tamoxifen, neurologic side effects result. To understand these mechanisms further, a clinical experimental study was conducted with the experimental oncology drug tariquidar, which is a potent P-gp inhibitor. Positron emission tomography scans with radiolabelled verapamil were conducted in five healthy volunteers. Total distribution volume of verapamil in whole-brain gray matter increased about 300% in the presence of continuous intravenous infusion of tariquidar, indicating almost complete inhibition of P-gp6. While there is unlikely to be this extent of P-gp inhibition at therapeutic concentrations, the study demonstrates that it is possible to achieve this and potentially use it in limited cases, in a controlled clinical setting.

BBB function in health and disease

Expression of efflux transporters at the BBB can vary with gender, age and health state.   Exposure to certain medicines and environmental toxins, over longer periods of time (typically over a week) and at relatively high concentrations (usually more than 100mg per day), can activate nuclear receptors’ transporter expression in the endothelial cells of the BBB7. Multiple nuclear receptors, including the pregnane X (PXR), constitutive androstane (CAR), aryl hydrocarbon (AhR) and glucocorticoid (GR) receptors are involved in this regulation. An actual difference in BBB transporter activity, resulting from up-regulation under physiological conditions, is difficult to assess in humans. A range of neurological diseases are associated with disruption of the BBB at the tight junctions and due to alterations in expression of transporters, resulting in a decrease in barrier integrity and enhanced BBB permeability.

Alzheimer’s disease

The Alzheimer’s brain progressively accumulates amyloid-beta, a protein that is also found at higher concentrations in the geriatric population. Alzheimer’s is marked by impaired barrier function, including a decrease in expression and activity of P-gp at the BBB. Since P-gp plays a key role in CNS extrusion of amyloid-beta from the brain, the decrease in this transporter potentially contributes to disease progression, particularly in the aging population8. More women than men are diagnosed with Alzheimer’s, which is likely because women live longer and P-gp expression is lower in the female brain9.  Interestingly, BCRP expression is significantly up-regulated in the Alzheimer’s brain, when compared with age-matched controls10. This balancing of transporters makes it difficult to predict drug exposure with progressing disease and much more work needs to be conducted in this area.

Multiple Sclerosis

Changes in the expression of BBB efflux transporters occurs with neuroinflammation, for example, in MS, trauma, infection and stroke. Inflammatory responses are mediated by cytokines such as tumor necrosis factor-α (TNF-α) and the interleukins IL-1and IL-6, which bind to extracellular receptors on endothelial cells of the BBB. Decreased expression of endothelial cell P-gp but not BCRP, MRP1 and MRP2 are reported in MS11. A different pattern is reported in brain lesions of these patients, with increased P-gp, MRP1 and MRP2 expression in reactive astrocytes in lesions but not in healthy regions. Data from preclinical studies further implicate P-gp in immunomodulation. A significant reduction in clinical symptoms of experimental autoimmune encephalomyelitis (MS model) was observed in P-gp knockout mice.

Amyotrophic lateral sclerosis

Patients with amyotrophic lateral sclerosis (ALS) have a significant increase in P-gp and BCRP but not MRP 1, 2, 4 or 5 expression in the cerebral cortex and spinal cord, with no change in the cerebellum. Riluzole (Rilutek, Sanofi Pharmaceuticals), the only United States Food and Drug Administration-approved drug for ALS is a dual P-gp and BCRP substrate that provides limited CNS exposure and efficacy12 and is most effective in the first 12 months of treatment. Reduction in mortality decreases from 38% to 19% at 21 months of treatment13. In an ALS mouse model, the dual P-gp/BCRP inhibitor elacridar significantly increased riluzole concentrations in the brain and spinal cord and also increased efficacy12, demonstrating that these transporters contribute to the low CNS exposure of the drug.

This data presents a conundrum for researchers developing CNS therapeutics, as while under experimental conditions, it is possible to inhibit transporter activity at the BBB, achieving this at pharmacological and safe concentrations is another matter. It is still not clear whether the mild disruption of the BBB and reduced expression and activity of efflux transporters such as P-gp and BCRP at the BBB with age and in certain diseases, results in higher or lower efficacy of therapeutics. Biologic drugs do not have the liability of being pumped out by efflux transporters and instead have limited access to the brain due to reduced transcytosis in the endothelial cells. The best way forward for CNS therapeutics is to develop potent and specific drugs that are not substrates of efflux transporters. A way around this problem may be targeting uptake transporters at the BBB (Figure 2; page 00).

Uptake transporters at the BBB

Uptake systems are expressed at the BBB to bring in essential nutrients, such as glucose and amino acids, which cannot diffuse through due to their polarity. Some of the membrane proteins in brain capillary endothelial cells implicated in drug transport include the organic anion transporters OATP1A2, OATP1C1 and OATP2B1, the organic cation transporters OCT1, OCT2, OCT3 and OCTN2, and the multidrug and toxin extrusion protein, MATE1 (reviewed in 14). There is mRNA and protein expression data for all these at the BBB but no clinical activity data. Moreover, the apical vs. basolateral location of some of these, e.g., OCT3 and MATE1, is yet to be confirmed (Figure 2; page 00). Irrespective of membrane location, the uptake transporters are entry gates for xenobiotics into the BBB, working in concert to allow specific polar substrates to cross into the endothelial cells and enter the brain.

The OATP1C1 transporter facilitates uptake of thyroid hormones that are required for normal brain development, and in adults, for metabolic adaptation. The role of this transporter in drug transport across the BBB has not been reported. OATP1A2 has emerged as potentially important for drug uptake across the BBB and has a wide substrate spectrum that includes drugs that are neuroactive (e.g., opioid analgesic peptides), have detrimental CNS side effects (e.g., levofloxacin) and toxicities (e.g., methotrexate). Statins that are OATP substrates demonstrate neuroprotection in hypoxia and inflammatory disorders including multiple sclerosis. Several of the triptan drugs are also substrates of OATP1A2.

Zolmitriptan (Zomig), a selective serotonin receptor agonist marketed by AstraZeneca, is transported by both P-gp and OATP1A2, demonstrates efficacy for migraines and has CNS access15,16. Preclinical data indicates that OATP1A2 may be a valid target for CNS- active drugs, since it has broad substrate specificity, high mRNA expression (protein levels are not yet confirmed in human brain microcapillaries) and data indicates that this transporter could be leveraged to counteract P-gp efflux17. Designing molecules that are substrates for OATP1A2 is worth exploring for CNS indications. A word of caution is warranted here, since targeting brain uptake transporters could attenuate neurotoxicity. The most extreme case study of this was seen when a bacterial infection leading to microcystin intoxication spread in a hemodialysis unit in Brazil. Sixty of the 126 patients died from acute neurotoxicity and subacute hepatotoxicity18. Microcystin is a substrate of OATP1A2 and uptake across the BBB and into neurons likely contributed to the impaired neuronal function.


The unique structure of the BBB provides very effective protection from circulating xenobiotics, due to the strong tight junctions between endothelial cells, reduced endocytosis at the cell membrane and a combination of transporters that allow for uptake of nutrients, while minimising uptake of xenobiotics into the brain. The transporter most relevant to this neuroprotection is P-gp, although BCRP, MRP1, MRP2 and MRP4 also contribute to limiting CNS access of polar solvents. Uptake transporters such as OATP1A2 work in the opposite direction, bringing some drugs into the brain. Theoretically, the intricate interplay between the uptake and efflux transporters can be leveraged to design new chemical entities that would by-pass the efflux transporters by using uptake transporters to traverse through both membranes. This gets more complex in disease states and in the older population, since there may be alterations in BBB permeability and transporter expression, potentially resulting in altered CNS exposure. Research over the next few years will further delineate these processes and improve our ability to safely access the CNS for targeted therapeutics.


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Jasminder Sahi is Senior Director and Head of Disposition, Safety and Animal Research for the Asia Pacific region at Sanofi R&D. Previously, she led the DMPK group at GlaxoSmithKline Shanghai, where the focus was on CNS drug discovery and the blood brain barrier. She has also worked at Parke Davis/Pfizer, Ann Arbor USA, where she developed models of induction and drug transporters. She is a member of the ISSX council and a reviewer for five journals. Her research contributions include over 40 peer-reviewed research articles, book chapters and invited reviews. Jasminder’s contact details are: Jasminder Sahi, Drug Metabolism, Safety and Animal Research (DSAR), Sanofi R&D, Tower III Kerry Center, 1228 Middle Yan’an Road, Jing’an District Shanghai 200040, China. Phone: +86 182 0172 5919. E-mail: [email protected]

Dr. Yi (Shelley) Li received her PhD degree in Analytical Chemistry from Shanghai Institute of Organic Chemistry (SIOC) in 2007. Subsequently, she worked for GSK R&D Shanghai in the department of drug metabolism and pharmacokinetics (DMPK), where she supported neuroscience drug discovery projects for seven years. She is an expert in bioanalysis for quantitation of both small molecules and biomolecules by LC-MS/MS. She currently works at Sanofi R&D in Shanghai, China, supporting both research and clinical platforms.