A little over a year ago researchers began describing something that sounded more like science fiction than science. A sinister sounding mechanism in human cells was found to actually suck the revolutionary class of anti-HIV drugs, the protease inhibitors, out of the very cells where they were doing so much good. Not since the days of superfit multi-nucleoside resistant HIV had the therapeutic order seemed so convoluted. A cellular "protease pump?" How could this be? And might such a phenomenon explain the frequent failure of these drug regimens? If the effect turned out to be significant, would there be any way to reverse the process?
Now we're delving into serious grad school cell biology here, so the sciency talk quickly gets thick. Yvette has done her best to bring it down to earth. Her full report, complete with a deluge of references, is available on our website, www.treatmentactiongroup.org. But for now, take a deep breath and slog on.
What is P-Glycoprotein?
P-glycoprotein (P-gp) is a plasma membrane protein which acts as a localized drug transport mechanism, actively exporting drugs out of the cell. The effects of P-gp on the distribution, metabolism and excretion of drugs -- including protease inhibitors -- in the body is great. P-gp activity, for example, decreases the intracellular concentration of cancer drugs, enabling resistance to develop to them. The same may be true for protease inhibitors.
What is the function of P-gp?
The normal physiological function of P-gp in the absence of therapeutics or toxins is unclear. Studies of MDR-1 knock-out mice (mice bred in the lab specifically for the absence of the MDR-1 gene and, therefore, no P-gp activity) show that they have normal viability, fertility and a range of biochemical and immunological parameters. Predictably, they do have delayed kinetics and clearance of vinblastine, and they accumulate high levels of certain drugs (vinblastine, ivermectin, cyclosporin A, dexamethasone and digoxin) in their brains. The mice also demonstrated marked increases in the levels of these drugs in the tests, ovaries and adrenal gland compared with wild-type mice. It has been reported that some MDR-1a knock-out mice develop a severe, spontaneous intestinal inflammation similar to human inflammatory bowel disease; however, this has not been observed by other researchers.
Mechanism of Action
The majority of published data suggest that P-gp acts as a transmembrane pump which removes drugs from the cell membrane and cytoplasm. It has further been proposed that P-gp acts like a hydrophobic vacuum cleaner or "flippase," transporting drugs from the inner leaflet of the plasma membrane lipid bilayer to the outer leaflet or to the external medium.
P-gp Substrates and Inhibitors
There have been various attempts to classify compounds based on their effect on or interaction with P-gp. A number of chemicals, including anticancer drugs, have been categorized based on their effect on ATPase activity of human P-gp. Class I compounds in low concentrations stimulate ATPase activity and in high concentrations inhibit it. Kinetic analyses show they have high affinity for the active site and low affinity for the inhibitory site. They include vinblastine, verapamil and taxol. Class II compounds stimulate ATPase activity in a dose-dependent manner without any inhibition and interact only with the active site. They include bisantrene, valinomycin and diltiazem. Class III compounds, which bind to the inhibitory site with high affinity, inhibit both basal and verapamil-stimulated ATPase activity. They include cyclosporin A, rapamycin and gramicidin D.
Some studies support a model of P-gp in which there is a region or multiple regions of interaction rather than one or two simple binding sites. Molecules interacting with P-gp may be classified as "substrate" or "antagonist." It has also been demonstrated that one possible mechanism of action for P-gp-mediated resistance to chemotherapeutic agents is through gene rearrangement.
P-gp and HIV
All HIV protease inhibitors currently in use are transported by P-gp, with affinities in the order ritonavir>nelfinavir>indinavir>saquinavir. In MDR-1a knock-out mice, plasma levels of indinavir, saquinavir and nelfinavir were 2-5 times higher compared with control mice. This strongly suggests that P-gp transport at the intestinal and/or hepatic level limits the systemic bioavailability of these drugs. The effect of P-gp on limiting oral bioavailability and tissue distribution of protease inhibitors has obvious implications for the effectiveness of protease inhibitor-containing regimens. Poor penetration of protease inhibitors into the brain, testis and other "sanctuary sites" may result in de facto compartmental mono or dual antiretroviral therapy with ongoing HIV replication and development of resistance.
Blockage of P-gp may be useful in facilitating greater intestinal absorption, bioavailability and penetration of protease inhibitors into HIV sanctuary sites as well as reduced excretion. It may also simplify protease inhibitor-containing regimens by reducing the oral doses of protease inhibitors and the frequency at which they are taken. Higher protease inhibitor levels in these sites may result in greater suppression of viral replication in these sites, but they may also result in unwanted adverse effects. These effects may not be limited to protease inhibitors but may extend to other co-administered drugs. For example, the antidiarrheal agent loperamide is an opiate which acts peripherally and penetrates the brain poorly. However, in MDR-1a knock-out mice, loperamide exhibits strong morphine-like central nervous system (CNS) effects.
Transport of HIV protease inhibitors can be inhibited by P-gp inhibitors like cyclosporin A, verapamil, and PSC833. Ritonavir, saquinavir, nelfinavir and indinavir have also been shown to inhibit transport of some of the known P-gp substrates. But, with the exception of ritonavir and possibly saquinavir, the P-gp inhibiting effects of the protease inhibitors are weaker than those of the established inhibitors like verapamil or cyclosporin A.
The P-gp transport system clearly has major implications for HIV infection and its treatment. There is still much left to be understood. The effects of P-gp expression, or alterations of P-gp expression, on the immune systems of HIV-infected individuals need to be fully studied and evaluated. The impact of P-gp expression and its inhibition on protease inhibitor therapy needs to be assessed.