4.1 Two Gatekeepers Team Up at the Blood-Brain Barrier
The discovery of BCRP in brain endothelial cells changed the long standing opinion that P-gp is the sole important transporter responsible for efflux of drugs at the BBB. However, BCRP expression at the BBB has not been unequivocally correlated to low brain penetration of all BCRP substrates. For example, Lee et al.
conducted in situ
brain perfusion studies using the BCRP substrates dehydroepiandrosterone sulfate and mitoxantrone and reported that brain penetration of the two compounds was not increased in Bcrp1(−/−)
(BCRP knockout) mice [82
]. Si milarly, Giri and coworkers showed that BCRP mediated efflux of the antiretroviral drugs abacavir and zidovudine in vitro
]. However, despite the absence of BCRP, brain uptake of these two compounds was not elevated in Bcrp1(−/−)
]. One conclusion drawn from these studies was that BCRP played a minor role in drug efflux at the BBB and another study showed that interaction of BCRP with substrates in vitro
rarely translates to visible effects at the BBB in vivo
In contrast, other studies demonstrated BCRP transport activity at the BBB. Cisternino and colleagues showed that BCRP limits prazosin and mitoxantrone, two prototypical BCRP substrates, from penetrating into the brain [86
]. Likewise, Enokizono et al.
and Breedveld et al.
reported that brain distribution of drugs increased significantly in Bcrp1(−/−)
]. Moreover, we recently reported that sorafenib transport into the brain was significantly increased in Bcrp1(−/−)
Taken together, conflicting results on BCRP-mediated drug efflux from the brain initiated a controversy on the role of this transporter at the BBB that lead to further studies. With the development of the P-gp/BCRP knockout mouse (Mdr1a/1b(−/−) Bcrp1(−/−)
]), researchers have been provided with the opportunity to study the combined impact of these two efflux transporters on the delivery of drugs across the BBB. de Vries et al.
showed that brain uptake of topotecan, a substrate for both P-gp and BCRP, was not increased in mice lacking BCRP (Bcrp1(−/−)
]. In P-gp knockout mice (Mdr1a/1b(−/−)
) topotecan brain levels increased slightly by 1.5-fold. In contrast, in mice lacking both P-gp and BCRP (Mdr1a/1b(−/−)Bcrp1(−/−)
), topotecan brain uptake was increased by more than 12-fold. Thus, absence of both P-gp and BCRP resulted in an effect that was significantly larger than the combined effects from the single transporter knockout mice. This finding was confirmed by Polli et al
. using lapatinib in P-gp/BCRP knockout mice [16
]. We have shown the same with dasatinib [14
], gefitinib [12
] and sorafenib [13
]. Even though these drugs are substrates for both P-gp and BCRP, absence of only one of the transporters did not significantly increase delivery of either drug to the brain, but the greatest enhancement in brain penetration was seen when both transporters were absent or inhibited at the BBB. Several studies now show that this is true for other dual P-gp and BCRP substrates as well (, [69
]). summarizes recent data by Kawamura et al
] that demonstrate this phenomenon. These findings suggest that inhibition of either P-gp or BCRP can be compensated by the respective other transporter, and that both transporters “cooperate” with each other in preventing chemotherapeutic drugs from entering the brain.
Brain Distribution of Dual P-gp and BCRP Substrates
Figure 1 Figure 1a. Transaxial PET images showing [11C] GF120918 in the brain of a (A) wild-type, (B) P-gp knockout, (C) Bcrp knockout and (D) P-gp/Bcrp knockout mouse. GF120918 is a substrate for and inhibits both P-gp and BCRP. The radioactivity level is low (more ...)
P-gp and BCRP cooperation implies that absence of either P-gp or BCRP alone does not result in an appreciable increase in brain penetration of dual substrates. In BCRP knockout mice (where P-gp is present), P-gp alone is sufficient to prevent drugs from penetrating into the brain. Likewise, in P-gp knockout mice (where BCRP is present) BCRP alone is sufficient to limit drug uptake into the brain. The greatest enhancement in brain penetration of dual substrates is always seen when both P-gp and BCRP are absent in the combined P-gp/BCRP knockout mice ( and 2).
An insight into the mechanism of P-gp/BCRP cooperation can be gained by looking at relative transporter affinities of substrate drugs, and relative transporter expression levels at the BBB (assuming protein expression correlates with transport capacity for both transporters). In this regard, Kamiie et al
. used LC-MS to quantify membrane transporter expression at the mouse BBB and found approximately 5-fold higher P-gp protein levels compared to those of BCRP [92
]. Significantly higher protein expression levels at the BBB make P-gp appear to be the dominant efflux transporter for many dual substrates that have similar affinities to both P-gp and BCRP. In comparison, due to lower protein expression levels, BCRP-mediated efflux appears to be minor and becomes apparent only when P-gp or both transporters are absent. For example, for a compound with moderate P-gp affinity, higher P-gp expression levels (higher P-gp transport capacity) will compensate for lower transporter affinity, resulting in a pronounced P-gp effect on the efflux of this compound at the BBB. This is true for almost all anti-cancer drugs mentioned above (), with the exception of sorafenib and dantrolene. Both these compounds have a significantly higher affinity for BCRP than for P-gp [13
]. Therefore, BCRP is the dominant transporter in keeping these drugs out of the brain and an effect of P-gp on drug penetration is only noticeable in BCRP and P-gp/BCRP knockout mice. Kodaira et al
. explained P-gp/BCRP cooperation by determining the net contribution of each transporter to the overall efflux of various drugs at the BBB [90
]. The authors showed that for many dual substrates, P-gp-mediated efflux out of the brain was greater than that by BCRP. On the other hand, P-gp-mediated efflux of dantrolene (high affinity BCRP substrate) was 10-fold lower than BCRP-mediated dantrolene efflux.
While the above studies have been conducted in animal models, it is now clear that BBB P-gp and BCRP expression is species-dependent. In this regard, Uchida et al
. recently reported that at the human BBB, BCRP protein levels are higher compared to P-gp protein levels [93
]. Using LC-MS, the authors determined 8 fmol/μg total protein for BCRP vs. 6 fmol/μg total protein for P-gp in human brain capillaries. However, to draw a clear conclusion from these absolute transporter protein levels on the importance of each transporter for brain drug delivery is difficult. LC-MS measures total transporter protein and does not distinguish between transporter protein that is functionally active in the luminal membrane of the brain capillary endothelium and transporter protein that is inactive in intracellular vesicle membranes. For example, LC-MS measures both BCRP monomer and dimer, but only BCRP dimer is the functionally active form [94
]. From what we know today only functionally active transporter protein in the luminal membrane of the brain capillary endothelium affects drug delivery across the BBB. Thus, although total BCRP protein expression at the human BBB is higher compared to P-gp, it is impossible to say at this point in time which transporter is more important for brain drug delivery in patients. To make such a statement we will need information on the functional expression of each transporter at the BBB, the local drug concentration, and the drug-transporter affinity.
P-gp/BCRP cooperation at the BBB suggests two fundamental realities. First, these two transporters can significantly affect drug delivery to the brain, thereby influencing drug efficacy. Second, combined inhibition of both P-gp and BCRP is potentially an attractive therapeutic strategy to improve delivery and thus efficacy of substrate drugs in the CNS. Many of the chemotherapeutic drugs mentioned above have been clinically unsuccessful in treating brain cancers. Even though P-gp- and BCRP-mediated cooperative efflux transport is not limited to anti-cancer agents, combined inhibition of both transporters might have the biggest impact in the treatment of brain cancers, where a small increase in drug brain uptake might dramatically improve anti-cancer efficacy.
In summary, absence of either P-gp or BCRP alone does not enhance brain distribution of dual substrates, but genetic or chemical knockout of both transporters is required to significantly increase brain uptake of dual P-gp/BCRP substrate anti-cancer drugs. Thus, current research indicates that P-gp and BCRP team up at the BBB and “cooperate” in preventing dual substrates from entering the brain. This finding has lead to a paradigm shift in the field of BBB transporter research.
4.2 Dual Inhibition of P-gp and BCRP at the BBB
Given the cooperation of P-gp and BCRP at the BBB, developing compounds that are potent inhibitors of both transporters may prove beneficial. Elacridar (GF120918) is a dual P-gp/BCRP inhibitor that has undergone extensive preclinical and clinical evaluation [95
]. Elacridar has been used in several preclinical studies to inhibit P-gp and BCRP at the BBB with the purpose of enhancing brain distribution of simultaneously administered compounds [40
]. These studies demonstrated that the greater than additive increase in brain penetration is not restricted to P-gp/BCRP knockout animals, but can also be observed with dual P-gp/BCRP inhibitors. For example, Chen et al
. showed that brain penetration of dasatinib increased dramatically with co-administration of elacridar [14
]. Likewise, we showed that elacridar significantly enhanced gefitinib and sorafenib brain uptake [12
], and de Vries et al
. published similar findings for topotecan [15
]. Thus, in preclinical studies, elacridar significantly increased brain penetration of drugs that are dual P-gp/BCRP substrates.
Apart from these compounds that were developed for use as multi-drug resistance reversal agents, several studies examined drugs that are dual P-gp/BCRP substrates to competitively inhibit both transporters. These include several anti-cancer tyrosine kinase inhibitors that have been shown to be substrates for both P-gp and BCRP. In vitro
studies show that tyrosine kinase inhibitors like erlotinib [100
], gefitinib [101
], lapatinib [102
] and sunitinib [103
] inhibit ABC transporters, mainly P-gp and BCRP, and suggest the potential use of these agents as combination therapy to improve drug pharmacokinetics. In 2006, Zhuang et al
. showed that concurrent administration of gefitinib results in a significant increase in brain penetration of topotecan [104
]. The same group showed that gefitinib also increased intracellular tumor exposure to topotecan in a mouse model of glioma [105
]. In a recent clinical trial, Furman and coworkers used gefitinib to inhibit intestinal P-gp and BCRP and showed that it increased oral bioavailability of irinotecan [106
]. An interesting study by Nakanishi et al
. in 2006 showed that imatinib attenuated its BCRP-mediated resistance by suppressing BCRP expression [107
]. The underlying mechanism for these differential responses involved downstream effects of imatinib leading to decreased phosphorylation of Akt, subsequently leading to reduced BCRP expression [107
]. Many tyrosine kinase inhibitors have an inhibitory effect on the PTEN/PI3K/Akt signaling pathway. These drugs can thus reduce functional activity and protein expression of ABC transporters, especially BCRP, by blockade of PI3K/Akt signaling. Combination of tyrosine kinase inhibitors with other anti-cancer drugs can therefore have a bimodal effect on ABC transporters, wherein decreased transporter expression/function coupled with competitive inhibition can result in significantly increased drug penetration across the BBB and potentially substantially increased drug levels in brain tumors.
In summary, concurrent treatment with dual P-gp/BCRP inhibitors can improve delivery and thus efficacy of substrate drugs in the CNS. Recent data imply that the use of tyrosine kinase inhibitors to inhibit P-gp/BCRP could have multiple benefits, especially if the anti-cancer agent enhances its own delivery to the brain.