P-glycoproteins belong to a family of plasma membrane proteins encoded by the MDR (multi-drug resistance) gene(s) that are well-conserved in nature. P-glycoprotein (P-gp) functions as a membrane-localized drug transport mechanism that has the ability to actively pump out all currently prescribed HIV-protease inhibitors (PIs) from the intracellular cytoplasm. This effect may result in limited oral bioavailability of PIs as well as a decreased ability of the drugs to cross blood-tissue barriers such as the blood-brain barrier (BBB), the blood-testis barrier (BTB) and the materno-fetal barrier (MFB). MDR1 encoded P-glycoprotein has also been implicated in the cytotoxicity process and with the induction of immune responses during HIV infection. It also plays a role in oxidative and inflammatory processes and it may be involved in lipid transport and metabolism.

What Is P-Glycoprotein?

P-gp is a phosphorylated and glycosylated plasma membrane protein belonging to the ATP-binding cassette superfamily of transport proteins. MDR1 P-gp (referred to simply as P-gp in this article) is a transmembrane protein that is 1,280 amino acids long and consists of two homologous halves of 610 amino acids joined by a flexible linker region of 60 amino acids.

When viewed from above the plasma membrane, P-gp is donut shaped with 6-fold symmetry, a diameter of about 10 nm and a large central pore of about 5 nm in diameter. It has a thickness in the plane of the plasma membrane of about 8 nm. Since the depth of the plasma membrane lipid bilayer is about 4 nm, about half of the molecule is within the plasma membrane.

Mechanism of Action, P-Gp Substrates and Inhibitors

The majority of published data suggest that P-gp acts as a transmembrane pump which removes drugs from the cell membrane and cytoplasm. ATP hydrolysis provides the energy for active drug transport, which can occur against steep concentration gradients. It was initially hypothesized that P-gp forms a hydrophilic pathway and that drugs are transported from the cytoplasm to the extracellular medium through the central pore, thereby shielding the substrate from the hydrophobic lipid phase. It has more recently been proposed that P-gp acts like a hydrophobic vacuum cleaner or flippase. In this model P-gp intercepts the drug as it moves through the lipid membrane and flips the drug from the inner leaflet of the plasma membrane lipid bilayer to the outer leaflet and into the extracellular medium.

Molecules interacting with P-gp may be classified as substrate or antagonist (inhibitor). Cancer drugs in the substrate group are characterized by a >4-fold increase in cytotoxicity in MDR cells. Compounds in the antagonist group increase the intracellular accumulation of the P-gp substrates and display reversal of drug cytotoxicity. Inhibitors may bind P-gp more tightly and, failing to be transported, prevent the transport of other compounds, while substrates, in being transported, do not block the transport of other substrates.

A partial list of P-gp substrates would include cancer drugs, such as doxorubicin, daunorubicin, vinblastine, and vincristine; immunosuppressive drugs, including cyclosporin A; steroids like aldosterone, hydrocortisone, and cortisol; HIV PIs, such as amprenavir (APV), indinavir (IDV), nelfinavir (NFV), ritonavir (RTV) and saquinavir (SQV); the antihistamine terfenadine; cardiac drugs, such as digoxin and quinidine; the lipid lowering agent lovastatin; the antibiotic erythromycin; and the anti-tuberculous agent rifampicin. P-gp activity decreases the intracellular concentration of cancer drugs, thus enabling resistance to develop; the same may be true for PIs.

P-gp inhibitors include the immunosuppressant cyclosporin A; the calcium channel blocker verapamil; the PIs RTV, SQV, NFV and possibly IDV; the progesterone antagonist mifepristone (RU486); the sedative midazolam; the anti-estrogen tamoxifen; the antibiotic erythromycin; and the antifungal ketoconazole.

P-gp-dependent drug transport activity depends on the level of expression of the MDR1 gene as well as on the functionality of the MDR1-encoded P-gp. Induction of intestinal P-gp by rifampicin has been shown to be the major mechanism responsible for reduced digoxin levels during concomitant rifampicin therapy.

Gene amplifications, rifampicin induction and probably other factors cause MDR1 over-expression. Polymorphism in exon 26 (C3435T) of MDR1 is significantly correlated with levels of expression and function of MDR1. Individuals homozygous for this polymorphism (TT allele) showed significantly lower duodenal MDR1 expression and higher digoxin plasma levels than volunteers with the CC genotype. Evaluation of maximum plasma concentrations (Cmax) during steady state conditions of digoxin administration showed a mean difference of 38% in digoxin Cmax in the homozygous TT genotype compared with the CC genotype.

Where Is P-Gp Found?

Monoclonal antibody MRK16 was used to localize P-gp in normal human tissues. Most tissues examined revealed very little P-gp. However, P-gp was found in the liver, pancreas, kidney, colon and jejunum and the adrenal gland. P-gp is also found in the epithelium of the choroid plexus of the brain (which forms the blood-cerebrospinal fluid (CSF) barrier) as well as on the luminal surface of blood capillaries of the brain (the blood-brain barrier). In mice, MDR mRNA expression levels increase dramatically during pregnancy and are expressed at extremely high levels in the gravid compared with the non-gravid uterus. P-gp is also expressed in the testes and ovaries of mice and in the steroid-producing endometrial glands of the pregnant uterus.

P-gp has been found in normal bone marrow in hematopoietic stem cells and in peripheral blood mononuclear cells (PBMCs), mature macrophages, natural killer (NK) cells, antigen-presenting dendritic cells (DCs) and T- and B-lymphocytes. P-gp and MDR1 are expressed to different levels in normal leukocytes. The presence of P-gp has been demonstrated at relatively high levels in CD56+ cells (NK cells), high to moderate levels in CD4+, CD8+ and CD15+ cells (T-helper cells, T-suppressor cells and granulocytes, respectively) and lower levels in CD19+ and CD14+ cells (B-lymphocytes and monocytes, respectively).

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 MDR1 knock out (KO) mice (mice lacking the MDR1 genes) 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, cyclosporin A, dexamethasone, loperamide and digoxin) in their brains. These mice also demonstrated marked increases in the levels of these drugs in the testis, ovary and adrenal gland compared with wild-type mice.

1. Protection From Drugs and Toxins
   Expression of P-gp on the luminal surfaces of the epithelial cells of the small and large intestine, biliary ductules, and proximal tubules of the kidney suggest a role in decreasing the absorption from the gut and/or the excretion of endogenous and exogenous hydrophobic amphipathic toxins. P-gp plays a role in the intestinal excretion and, hence, in the reduced bioavailability after oral ingestion of several drugs, including digoxin, paclitaxel, and HIV PIs. The plasma concentrations after oral administration of IDV, NFV and SQV were 2- to 5-fold higher in MDR1a KO mice compared with wild-type (WT) or normal mice.

   Expression in the capillary endothelial cells of the brain, nerves, testes and placenta suggest a barrier function to keep toxins out of the nervous system, gonads and fetus. Many relatively hydrophobic drugs that were expected to diffuse easily across lipid membranes did not readily enter the brain. P-gp has been found in hematopoietic stem cells and probably contributes significantly to the removal of drugs and toxins from the bone marrow. Studies have also noted that P-gp can be detected in human placental trophoblasts from the first trimester of pregnancy to full term, making it very likely that placental P-gp protects the developing embryo and fetus from toxic insult in humans as well.

2. Steroid Metabolism
   The presence of P-gp in the adrenal and in steroid-producing cells of the endometrium suggest it may also have a role in the handling of steroids, possibly providing a protective function for the plasma membranes of steroid-producing cells. Furthermore, it has been found that P-gp-expressing epithelial monolayers of cells are able to transport steroids and that some lymphoid cells expressing P-gp are resistant to the cytotoxic effects of steroids.

3. Cholesterol Metabolism
   P-gp appears to have a role in cholesterol metabolism. Cholesterol esterification is one of the mechanisms that cells use to control the amount of toxic free cholesterol. Under conditions of excess cholesterol, cholesterol is transported from the plasma membrane to the endoplasmic reticulum (ER) where it is esterified. P-gp functions to increase esterification of cholesterol derived from plasma membrane by facilitating the movement of cholesterol from the plasma membrane to the ER.

4. Immune System
   Immune responses in a peripheral organ like skin are initiated when antigen-presenting cells, especially dendritic cells (DCs), capture antigens locally. The DCs then migrate via lymphatic vessels to draining lymph nodes where they select T lymphocytes that bear receptors for the presented antigen. In vitro models have demonstrated that P-gp facilitates this migration of DCs and that in the presence of P-gp antagonists, DCs are retained in the epidermis.

   There is also evidence that P-gp may be involved in the transport of some cytokines (CKs), particularly interleukin-1 (IL-1), IL-2, IL-4 and interferon-gamma (IFN-y) out of activated normal lymphocytes. However, P-gp does not seem to transport IL-6. The biological importance of P-gp to CK secretion during an immune response is still to be clarified.

5. Cell Death and Cell Differentiation
   The high level of P-gp expression on NK and CD8+ T cells has raised the question of the role of P-gp in the function of these cells. Inhibition of P-gp results in a reduction in NK and CD8+ T cell cytolytic activity. The majority of physiological cell death pathways appear to involve cystein-aspases (caspases). Cell death due to membrane and cytosolic perturbations by cytotoxic granules occurs in the absence of activation of the caspase pathway, whereas nuclear damage requires caspase activation. NK and cytotoxic CD8+ T cells bind their target cells and induce death either via the Fas/Fas ligand system or, usually, by release of cytotoxic granules, such as perforin and granzymes, into the target cell. In tumor cells, P-gp confers resistance to Fas-mediated apoptosis. It has been demonstrated that tumor cells expressing P-gp are resistant to a wide range of stimuli that activate the caspase apoptotic cascade, but are not resistant to caspase-independent cell death mediated by pore-forming proteins and granzyme B. Inhibition of P-gp completely reverses this resistance to caspase-dependent cell death.

6. Chloride Channels
   P-gp does not seem to have intrinsic channel activity, but may regulate an endogenous chloride channel that is yet to be identified. The physiological importance of this indirect modulation of chloride channels is controversial.

7. Cytochromes
   Many of the same drugs that are transported by P-gp are metabolized by some of the cytochromes (CYPs), especially CYP450 3A. CYP3A substrates include HIV PIs, the antibiotic erythromycin, and rifampicin. CYP3A can be induced by many agents, including glucorticoids, barbiturates and rifampicin. Human variation in CYP3A expression is believed to influence drug response for up to one-third of all drugs.

   It is likely that because P-gp can influence the intracellular concentration of many CYP3A substrates, it may also affect the availability of those substrates to CYP3A and therefore the extent of CYP3A metabolism of those substrates. P-gp thus plays an important role in modulating expression of CYP3A and this is likely to complicate the prediction of drug interactions among drugs that are substrates for both P-gp and CYP3A systems.

P-Gp and HIV

All HIV PIs currently in use (IDV, SQV, NFV, RTV and APV) are transported by P-gp, which can actively expel these PIs from cells. Transport of HIV PIs can be inhibited by P-gp inhibitors like cyclosporin A, quinidine, verapamil, and PSC833. PIs interact with P-gp with affinities in the order RTV>NFV>IDV>SQV. RTV, SQV, NFV and possibly IDV have also been shown to inhibit transport of some of the known P-gp substrates. Except for RTV and maybe SQV, their inhibitory effects are weaker than established inhibitors like verapamil or cyclosporin A.

It has been demonstrated that plasma levels of oral IDV, SQV and NFV were 2 to 5 times higher in MDR1a KO mice compared with WT mice. This strongly suggests that P-gp transport at the intestinal and/or hepatic level limits the systemic bioavailability of these drugs. Studies in Caco-2 cells, which exhibit many of the morphological and biochemical characteristics of human small intestine, suggest that P-gp transports absorbed PIs (APV, RTV, IDV, NFV and SQV) back into the intestinal lumen, thus limiting oral bioavailability.

Studies have reported that the rank order of in vitro intracellular accumulation of PIs was SQV>RTV>IDV. P-gp, MRP (multidrug resistance protein, another drug transporter), protein binding and HIV infection all decreased the intracellular accumulation of PIs. Compared with human erythroleukemia cells that don't express P-gp, cells over-expressing P-gp demonstrated a 10-fold reduction in APV and IDV intracellular concentrations, 3- and 6-fold reductions for RTV and SQV, respectively, while there was no difference in NFV intracellular concentrations.

P-gp may also limit the penetration of PIs into several tissue compartments in the body, thereby possibly creating sanctuary sites, such as the brain and gonads. Brain penetration of IDV, SQV and NFV were increased 7-, 10- and 36-fold respectively in MDR1a KO mice compared with WT mice. A study using an in vitro BBB model demonstrated that APV, RTV and IDV are actively transported by P-gp across the BBB. Another study demonstrated that brain and testis levels of NFV, APV, IDV and SQV were significantly increased in mice when the potent P-gp inhibitor LY335797 was administered intravenously and that this increase was not due to increased PI plasma levels. While IDV achieves good penetration into the semen (seminal plasma (SP): blood plasma (BP) ratio = 0.9), RTV and SQV penetrated very poorly (SP:BP were 0.02 and 0.03, respectively).

The effect of P-gp on limiting oral bioavailability and tissue distribution of PIs has obvious implications for the effectiveness of PI-containing regimens. Poor penetration of PIs into the brain, testes and other sanctuary sites may result in de facto compartmental mono- or dual antiretroviral therapy with ongoing HIV replication and development of resistance.

HIV PIs do not cross the placental barrier appreciably and placental P-gp may be an important factor in this low penetration. PIs are therefore generally considered unsuitable for prevention of mother-to-infant transmission. After intravenous administration of SQV to pregnant mice, the ratios of SQV concentration in fetal tissue to that in maternal plasma were 5-7 fold higher in MDR1a/1b KO mice than in WT mice. P-gp fetal and blood-brain barriers are not abolished by co-administration of high doses of RTV.

The effects of P-gp on the distribution, metabolism and excretion of drugs, including PIs, in the body is great. Blockage of P-gp may prove useful in facilitating greater intestinal absorption, bioavailability and penetration of PIs into HIV sanctuary sites as well as in reducing PI excretion. It may also simplify PI containing regimens by reducing the oral doses of PIs and the frequency at which they are taken. Higher PI levels in these sites may result in greater suppression of viral replication in these sanctuary sites, but they may also result in unwanted adverse effects. The effects of P-gp inhibition may not be limited to PIs but may extend to other co-administered drugs. For example, the antidiarrheal agent loperamide is a peripherally acting opiate which penetrates the brain poorly. However, in MDR1a KO mice, loperamide exhibits strong morphine-like effects on the central nervous system.

In HIV infected cells with high P-gp expression, both accumulation and antiviral efficacy of IDV, SQV and RTV are diminished. One study found that 90% of all peripheral blood lymphocyte subsets (CD4+, CD8+, CD56+ and CD10+ cells) expressed surface P-gp in both HIV-infected patients and controls. However, P-gp function was significantly reduced in CD16+ NK cells and CD19+ B-cells from HIV+ patients compared with controls. This reduced function significantly correlated with decreased NK cytotoxicity observed in HIV+ patients. P-gp can also be detected on an intracellular level in different peripheral blood monocyte subpopulations, mainly CD8+ T cells, CD16+ NK cells and CD14+ monocytes. This intracellular expression was decreased in CD8+ T cells and CD16+ NK cells from HIV-infected patients.

In addition, a significantly increased proportion of CD4+ T-cells from HIV-infected patients expressed P-gp compared with controls. This resulted in a significantly increased ratio of the proportions of CD4+/P-gp+ to CD8+/P-gp+ cells. This ratio was significantly higher in patients with CD4+ cell counts of <200/mL than in those with CD4+ cell counts >200/mL. However, both CD4+ and CD8+ T-cells from HIV-infected patients accumulated more of the P-gp substrate rhodamine 123 compared with controls. P-gp inhibitors failed to increase further this intracellular accumulation in HIV+ patients. This suggests that in HIV infection there is increased expression of a functionally defective P-gp in CD4+ and CD8+ T-cells that appears to increase with disease progression.

P-gp expression may affect HIV-infectivity. Studies have demonstrated a reduction in virus production when P-gp was over-expressed at the surface of 12D7, a continuous CD4+ human T cell leukemia cell line, infected with a laboratory strain of HIV-1. Reduction in infectivity occurred both during the fusion of viral and plasma membranes and at subsequent steps in the HIV life cycle. P-gp over-expression did not significantly alter the surface expression or distribution of either the CD4 receptor or the CXCR4 coreceptor.

PIs can cause hypercholesterolemia and, as explained earlier, P-pg plays a role in cholesterol metabolism and possibly in atherogenesis. Whether P-gp plays any role in PI-mediated dyslipidemia is not known.

Further Research on P-Gp and HIV

The P-gp transport system clearly has major implications for HIV infection and its treatment. There is still much left to be understood. P-gp expression and function in HIV-infection needs to be studied -- both in different tissues as well as in various stages of disease. The possibility of using P-gp function and expression as another surrogate marker for HIV disease progression should be explored. The effects of P-gp expression -- and alterations in P-gp expression on HIV infectivity, on the immune and other systems of HIV-infected individuals and on HIV therapy should be fully evaluated. Does P-gp play a role in the failure of antiretroviral therapy and the development of resistance to PIs?

The safety and efficacy of P-gp modulation in the management of HIV disease, especially in the use of PI-containing regimens, require further study. This includes the use of recognized P-gp inhibitors like PSC833, LY335979, and PIs like RTV, as well as the possibility of using P-gp maturation inhibitors (proteasome inhibitors). The optimal therapeutic dose of RTV required to inhibit P-gp; its effects on intracellular concentrations of PIs in HIV infected cells and on tissue penetration of PIs; its effects on concomitantly administered drugs; and the clinical value of using RTV as a P-gp inhibitor in the treatment of HIV disease remain to be evaluated. Are the different P-gp modulators site-specific; do they inhibit P-gp to different degrees depending on location?

The interactions and interdependence of the P-gp transport and the cytochrome metabolic systems need further elucidation. Both are important causes of drug-drug interactions and HIV PIs interact with both systems. It may be necessary in the future to determine the interactions of HIV drugs not only with the cytochrome system, but also with the P-gp transport system.

It is necessary to investigate properly whether the co-administration of P-gp inhibitors with PIs is safe and effective for prophylaxis of mother-to-child transmission. Administration of P-gp inhibitors may be best done in later pregnancy to minimize the adverse effects of drugs and toxins on the developing fetus.

Studies also need to be undertaken to discern the mechanism of action of PI-induced dyslipidemias and what role, if any, P-gp plays. Does HIV disease itself affect the role of P-gp in cholesterol metabolism?

The P-gp transport system is complex and poorly understood. It is even less well understood in HIV disease, in which it may play a significant role. The role of P-gp in HIV disease pathogenesis and its effect on HIV drugs are undoubtedly deserving of greater study. It may become routine in the future to determine the interactions of HIV drugs not only with the cytochrome system, but also with the P-gp transport system.

An extended, fully referenced version of this article entitled, "P-Glycoprotein: A Tangled Web Waiting to Be Unraveled" is available at the TAG Web site: www.treatmentactiongroup.org.

Areas for Further Research

Further research on P-gp is needed in the following areas:

1. Related to HIV Disease and Progression

   • P-gp expression and function in HIV infection, both in different tissues of the body as well as in various stages of disease.

   • The possibility of using P-gp function and expression as one of the surrogate markers for HIV disease progression.

   • The effects of P-gp expression -- and alterations in P-gp expression:

     • on HIV infectivity,

     • on the immune systems of HIV-infected individuals, and

     • on HIV therapy.

   • The role of P-gp in viral failure and the development of resistance to PIs.

2. Related to Treatment for HIV

   • The safety and efficacy of P-gp modulation in the management of HIV disease, especially during the use of PI-containing regimens.

     • Do the modulators affect P-gp differently in different tissues?

   • Inhibition of P-gp transport by RTV (and possibly other PIs).

   • Clinical value of using RTV (and other PIs) as a P-gp inhibitor.

     • Safety and efficacy.

   • Optimal therapeutic dose(s) of RTV (and of other PIs).

   • Effects of P-gp on intracellular concentrations of PIs in HIV infected cells and on tissue penetration of PIs.

   • Effects of P-gp on concomitantly administered drugs.

3. Related to the Cytochrome System

   • Pharmokinetic interactions and interdependence of the P-gp transport and the cytochrome metabolic systems.

4. Mother-to-Child Transmission

   • Safety and efficacy of co-administration of P-gp inhibitors with PIs for prophylaxis of vertical transmission.

   • The optimal time to administer P-gp inhibitors to minimize the adverse effects of drugs and toxins on the developing fetus.

5. Cholesterol Metabolism and Dyslipidemias

   • The mechanism of action of PI-induced dyslipidemias and what role, if any, P-gp plays.
   • Whether HIV disease itself affects the role of P-gp in cholesterol metabolism.