4.1.1  P-gp
P-gp, first discovered in 1976, is a 150–180 kDa membrane protein encoded by the multi-drug resistance gene 1 (MDR-1). It is widely expressed in tissues such as the liver, kidney and intestine, as well as the luminal membrane of the BBB (Cordon-Cardo et al., 1989; Jette et al., 1993, 1995; Soontornmalai et al., 2006) and the choroid plexus epithelium (Rao et al., 1999; Gazzin et al., 2008). At the BBB it appears to be important in protecting the brain from hydrophobic molecules and drugs. However, its function at the blood–CSF barrier is ambiguous and it may not have a major neuroprotective role at this site (Gazzin et al., 2008). Uncharged or weakly basic molecules are most efficiently transported by P-gp, but acidic compounds can also be transported. A large body of evidence suggests that PIs are substrates of P-gp and as a result the limited ability of these drugs to transverse the blood–brain barrier is attributed to the activity of this efflux transporter (Kim et al., 1998; Polli et al., 1999; Choo et al., 2000; Park and Sinko, 2005; Bachmeier et al., 2005; Eilers et al., 2008).
In comparison to PIs, the interaction of P-gp and NRTIs has been less extensively studied. Recently a study provided in vivo and in vitro evidence that the nucleoside reverse transcriptase inhibitor, abacavir ([(−)-(1S, 4R)-4-[2-amino-6 (cyclopropylamino)-H-purin-9-yl]-2-cyclopentene-1-methanol; 1592U89]) is a P-gp substrate (Shaik et al., 2007; Giri et al., 2008). In fact it is likely that P-gp is the dominant transporter limiting the CNS penetration of abacavir (Giri et al., 2008). Similarly, earlier studies showed that both HIV-infected T cell and monocytic cell lines had increased P-gp expression which accumulated significantly less zidovudine (azidodeoxythymidine (AZT)) in comparison to uninfected cells (Gollapudi and Gupta, 1990). Likewise, a decrease in AZT accumulation in P-gp-over-expressing CEM VBL100 cells with a corresponding decline in antiviral efficacy of the drug has been observed (Antonelli et al., 1992).
Using P-gp in Caco-2 cell lines, the NNRTIs nevirapine, efavirenz, and delavirdine were found not to be substrates of this transporter. However, all of these drugs were found to induce the expression and function of P-gp, with nevirapine being the more potent inducer compared with the other two NNRTIs (Stormer et al., 2002). Alternatively, a recent study demonstrated that the effect of P-gp on intracellular HIV-1 replication may be more clinically relevant than the efflux function of P-gp on PIs. The data suggested that high-cellular P-gp activity corresponds with a lower intracellular HIV-1 load in vivo (Sankatsing et al., 2007). Interestingly, Langford et al. (2004) showed that AIDS patients with HIV encephalitis (HIVE) have higher brain P-gp levels than HIVE-negative patients. However, despite studies showing an upregulation of P-gp in HIV-1 infected macrophages, CD4+ T lymphocytes and glial cells (Langford et al., 2004), the pump function of P-gp in HIV-1 infected patients is thought to be decreased (Sankatsing et al., 2004).
Recent experiments using primary culture of rat astrocytes have demonstrated that both the expression and the transport function of P-gp are downregulated following exposure to HIV viral envelope protein, gp120. Collectively, these crucial glial cells that harbour the virus within the CNS are thought to form a dynamic barrier behind the BBB to further impede the access of anti-HIV drugs to sites of infection within the CNS (Ronaldson and Bendayan, 2006). Furthermore, using intact, isolated rat brain capillaries, Hartz et al. (2004) revealed that subnanomolar to nanomolar concentrations of the hormone endothelin-1 (ET-1) rapidly and reversibly attenuated P-gp-mediated transport function over the short term (minutes). This effect was found to be due to the stimulation of the ETB receptor with subsequent activation of nitric oxide synthase and protein kinase C. The release of ET-1 has been apparent in a number of CNS disorders including HIVE (Hartz et al., 2004) and AIDS dementia complex however the effect of ET-1 on brain capillary permeability remains controversial, with some studies claiming that ET-1 significantly increases brain permeability and others suggesting no effect. This discrepancy can be attributed to the different durations of the experiments. An increase in permeability was observed over hours to days, raising the possibility that capillary permeability may remain unchanged during early ET-1 exposure (Hartz et al., 2004).
Inflammation is a central pathophysiological mechanism in the majority of CNS diseases and is reproduced experimentally by the injection of the bacterial endotoxin—lipopolysaccharide (LPS). Altered P-gp expression and corresponding changes in the disposition of several xenobiotics have been observed in the LPS model (Miller et al., 2008). Recent studies have demonstrated evidence in line with these findings. P-gp was downregulated via an unknown mechanism following the administration of LPS into rat intracranial ventricles. This subsequently caused an accumulation of the P-gp substrate, digoxin, within the brain (Goralski et al., 2003). Other studies have shown that the proinflammatory cytokine TNF-α causes a rapid and reversible loss of P-gp activity in rat brain capillaries.
The proposed mechanism suggested that short-term exposure to the cytokine caused TNF receptor 1 stimulation resulting in ET-1 release and consequent ETB receptor, nitric oxide synthase and protein kinase C activation. This pathway was thought to be activated by LPS to reduce P-gp transporter activity (Hartz et al., 2006). More recently, the same research group found that this initial rapid decrease in transport preceded a 2–3-h plateau at this reduced level of transporter activity, and was then followed by a rapid increase in both transporter activity and protein expression. Collectively, these findings demonstrate that chronic inflammation can tighten the BBB to CNS drugs which are P-gp substrates by upregulating P-gp expression (Bauer et al., 2007). An upregulation of P-gp in rat brain endothelium was also observed in an inflammatory pain model causing a decrease in the penetration of the P-gp substrate, morphine and consequent antinociception (Seelbach et al., 2007).
HIV-Tat, a protein thought to be responsible for the vascular abnormalities and neurotoxicity in HIV, also induces the expression of P-gp in brain endothelial cells which correlated with a functional upregulation of the transporter function of P-gp (Hayashi et al., 2005). A similar change in P-gp expression has been observed following chronic exposure of bovine brain microvessel endothelial cells to ritonavir. In fact, the HIV PI increased P-gp activity and expression in a concentration-dependent manner in this in vitro model of the BBB, raising the possibility that HAART could itself contribute to the brain as a HIV sanctuary site by the induction of drug transporters (Perloff et al., 2007). Collectively, these studies suggest that the selective inhibition of P-gp may facilitate the entry of PIs and certain NRTIs into viral sanctuaries and enhance the concentration of anti-HIV drugs in these sites to therapeutic levels.