Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhibitor
Introduction
HIV protease inhibitors (PIs) have revolutionized the treatment of HIV infection (Roberts et al., 1990, Vacca et al., 1994, Danner et al., 1995, Kempf et al., 1995, Patick et al., 1996). Due to limited oral bioavailability and poor pharmacokinetics of many of the currently available PIs, additional efforts have been made to design more potent PIs with improved pharmacokinetic properties. Lopinavir (LVR), an analog of ritonavir (RVR) is a potent inhibitor of wild type and mutant HIV protease (Ki = 1.3 − 28 pM) (Kumar et al., 2004). Its structure is outlined in Fig. 1. The LVR:RVR combination (KALETRA) has been shown to be effective in the treatment of HIV infection and is approved for clinical use (Hurst and Faulds, 2000, Miller, 2000). LVR, as such, is extensively metabolized by CYP3A4 and produces low systemic availability when administered alone. RVR potently inhibits CYP3A4 and is used in combination with LVR to enhance the systemic exposure of LVR (Kumar et al., 1999). This combination results in LVR concentrations that greatly exceed those necessary in vitro to inhibit both wild-type and PI-resistant HIV isolates. (Kaletra® Prescribing Information, Abbott Laboratories, January 2002).
The low oral bioavailability of LVR was attributed to high first-pass metabolism (Kumar et al., 2004). In vitro investigations with human liver microsomes have shown that cytochrome P450 3A plays a predominant role in the metabolism of LVR. High first pass metabolism can also occur due to intestinal efflux which can lead to increased exposure time to metabolizing enzymes (Wacher et al., 1995, Wacher et al., 2001, Katragadda et al., 2005). We have hypothesized that the low oral bioavailability of LVR and possibly limited brain penetration could be in part due to efflux of LVR by several efflux pumps such as P-glycoprotein (P-gp), multidrug-resistance related proteins (MRPs) and breast cancer resistance protein (BCRP) present on intestinal epithelial and blood capillary endothelial cells. Potential interaction between efflux transporters in the GIT and CYP3A4 metabolizing enzymes may be a source of variation associated with LVR absorption and distribution (Williams and Sinko, 1999). In humans, CYP3A4 is the principal enzyme involved in the hepatic and intestinal drug metabolism, and there is a striking overlap of substrate specificites among CYP3A4, P-gp and MRPs. The coordinated function of both CYP3A and P-gp, MRPs can dramatically lower oral bioavailability of compounds which are substrates for both (Van Asperen et al., 1997, Wacher et al., 1998) and this may also be true for LVR.
P-gp-mediated efflux of LVR is known (Vishnuvardhan et al., 2003, Woodahl et al., 2005), but interactions of LVR with other adenosine triphosphate-binding cassette (ABC) efflux transporters such as MRPs and BCRP have not yet been investigated. Therefore, it is important to delineate quantitatively if these latter efflux transporters can restrict, at least in part, the permeation of LVR at both the intestinal and blood brain barrier (BBB) membranes.
ABC transporters comprise one of the largest membrane-bound protein families. These proteins transport substrates against a concentration gradient with ATP hydrolysis as a driving force across the membrane. P-gp, a multiple drug-resistant (MDR) gene product, transports a wide range of compounds, including anticancer drugs, steroids, calcium channel blockers and antihistamines (Endicott and Ling, 1989, Borst et al., 1993, Borst et al., 2000, Germann et al., 1993, Pal and Mitra, 2006). P-gp-mediated efflux reduces the intracellular accumulation of these compounds, thereby diminishing drug efficacy. P-gp is present on the apical membrane of many absorptive epithelial and endothelial cells. Because of its localization and distribution, P-gp limits the oral absorption and bioavailability of PIs across intestine, brain, testis and placenta (Kim et al., 1998, Polli et al., 1999, Smit et al., 1999, Choo et al., 2000, Huisman et al., 2001, Huisman et al., 2002).
Recent studies have demonstrated that the PIs are also substrates for the MRPs, belonging to the same ABC transporter family (Huisman et al., 2002, Bachmeier et al., 2005). So far, eight MRP homologs have been identified for ABC proteins, MRP1–8. MRP1 is a widely expressed transporter. When present in epithelial cells, this protein is found primarily in the basolateral membrane (Hipfner et al., 1999). However, it has been reported that MRP1 does not mediate substantial polarized transport of PIs in MDCKII-MRP1 cells (Huisman et al., 2002). In contrast to MRP1, MRP2 is localized on the apical membrane of several epithelia. Functionally, it is similar to P-gp-mediated elimination of toxic compounds in gut and placenta (Kruh and Belinsky, 2003). It has been fairly established that MRP2 effluxes PIs (Huisman et al., 2002, Williams et al., 2002).
Human BCRP/MXR is a relatively new ABC efflux transporter. Like P-gp, BCRP confers high levels of resistance to anthracyclines, mitoxantrone and the camptothecins by enhancing drug efflux from the cell to extracellular space (Litman et al., 2000, Bates et al., 2001, Ejendal and Hrycyna, 2002). BCRP is expressed in larger amounts than P-gp in the intestine (Taipalensuu et al., 2001). The expression of a BCRP homologue, known as brain multidrug resistance protein (BMRP), has also been reported in porcine brain capillary endothelial cells (Eisenblatter et al., 2003). Both BCRP and BMRP possess one half of the MDR1 P-gp structure with only six transmembrane domains and one ATP-binding domain (Doyle et al., 1998). In addition to this structural similarity, most known substrates for BCRP/BMRP are similar to P-gp (i.e., hydrophobic, amphiphilic xenobiotics), suggesting that PIs may also interact with BCRP/BMRP (Litman et al., 2001, Doyle and Ross, 2003). In fact, results from a recent study suggest that saquinavir (SQV), RVR and nelfinavir (NFV) may serve as inhibitors of BCRP (Gupta et al., 2004).
Thus, P-gp and MRPs, can play an important role in lowering intestinal absorption and brain penetration of LVR. Because these efflux transporters are oriented in the secretory (i.e., out of the organ or tissue) direction, high efflux will lead to lower net absorption for LVR. Sub-therapeutic concentrations of PIs in the sanctuary sites like brain, testes and bone marrow may cause persistence of viral infections leading to drug resistance (Williams and Sinko, 1999). Therefore, the purpose of this study is to assess the affinity of LVR for the efflux transporters using a well-defined system consisting of polarized non-human (canine) MDCKII cells, singly transfected with human MDR1, human MRP1/MRP2 complementary DNA (cDNA) or murine Bcrp1 cDNA and also to delineate quantitatively whether efflux limits permeation of LVR across intestinal and BBB absorptive cells.
Section snippets
Materials
Unlabeled (ulb) LVR, Fumitremorgin-C (FC) and GF120918 (GF) were generous gifts from Abbott Laboratories Inc., National Institutes of Health AIDS Research and Reference Reagent Program (National Institutes of Health, Bethesda, MD) and GlaxoSmithKline Ltd respectively. [3H] LVR (1 Ci/mmol) and [3H] Mitoxantrone (MX) (4 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA, USA). P-gp-4008 (P4) and MK-571 (MK) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Biomol (Plymouth
[3H] LVR transport across the MDCKII-WT cell line
[3H] LVR transport across the MDCKII-WT cells was determined in the absence and presence of various combinations of inhibitors. This study enabled us to compare the [3H] LVR permeabilities across the parental and the transfected cell lines in the absence and presence of specific efflux inhibitors. LVR permeabilities across MDCKII-WT cells are summarized in Table 1. Directional transport of [3H] LVR across MDCKII-WT cells in the absence of efflux inhibitors was very low (Fig. 2a). As a result,
Conclusion
Our results demonstrate for the first time that LVR is extruded from absorptive cells by MRP2 and confirm that P-gp/MDR1 is involved in its efflux. This efflux can be prevented by specific inhibitors such as P4, GF120918 for P-gp and MK for MRP2. LVR did not interact significantly with MRP1. Also, no substantial difference was noted in the ER values of LVR in MDCKII-Bcrp1 cells in the presence and absence of FC, a specific and potent BCRP efflux inhibitor. This study clearly indicates that LVR
Acknowledgment
This work was supported by National Institute of Health Grant GM 64320-03.
References (58)
- et al.
Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals
Pharmacol. Ther.
(1993) - et al.
Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR
Cancer Lett.
(1999) - et al.
Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood–brain barrier
Brain Res.
(2003) - et al.
Transport of glutathione prostaglandin A conjugates by the multidrug resistance protein 1
FEBS Lett.
(1997) - et al.
The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance
Biochem. Biophys. Res. Commun.
(1995) - et al.
P-glycoproteins: mediators of multidrug resistance
Semin. Cell Biol.
(1993) - et al.
Structural, mechanistic and clinical aspects of MRP1
Biochim. Biophys. Acta
(1999) - et al.
Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators
J. Biol. Chem.
(1993) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J. Immunol. Methods
(1983)- et al.
MDR- and CYP3A4-mediated drug–herbal interactions
Life Sci.
(2006)
Role of P-glycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics
J. Pharm. Sci.
Active secretion and enterocytic drug metabolism barriers to drug absorption
Adv. Drug Deliv. Rev.
Oral absorption of the HIV protease inhibitors: a current update
Adv. Drug Deliv. Rev.
Quantitative assessment of HIV-1 protease inhibitor interactions with drug efflux transporters in the blood–brain barrier
Pharm. Res.
The role of half-transporters in multidrug resistance
J. Bioenerg. Biomembr.
A family of drug transporters: the multidrug resistance-associated proteins
J. Natl. Cancer Inst.
Effect of multidrug resistance-reversing agents on transporting activity of human canalicular multispecific organic anion transporter
Mol. Pharmacol.
Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes
Drug Metab. Dispos.
A short-term study of the safety, pharmacokinetics, and efficacy of ritonavir, an inhibitor of HIV-1 protease
N. Engl. J. Med.
Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)
Oncogene
A multidrug resistance transporter from human MCF-7 breast cancer cells
Proc. Natl. Acad. Sci. U.S.A.
Multidrug resistance and cancer: the role of the human ABC transporter ABCG2
Curr. Protein. Pept. Sci.
The biochemistry of P-glycoprotein-mediated multidrug resistance
Annu. Rev. Biochem.
Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA
J. Clin. Invest.
Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export
Br. J. Cancer
Transport of HIV protease inhibitors through the blood–brain barrier and interactions with the efflux proteins, P-glycoprotein and multidrug resistance proteins
J. Acquir. Immune Defic. Syndr.
HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2)
J. Pharmacol. Exp. Ther.
P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir
Mol. Pharmacol.
Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs
AIDS
Cited by (96)
HIV and Associated TB: A Lethal Association for Kidney Health?
2023, Seminars in NephrologyFlammulina velutipes polysaccharide-iron(III) complex used to treat iron deficiency anemia after being absorbed via GLUT2 and SGLT1 transporters
2023, Food Science and Human WellnessPredicting disruptions to drug pharmacokinetics and the risk of adverse drug reactions in non-alcoholic steatohepatitis patients
2023, Acta Pharmaceutica Sinica BInsights into the binding properties of calf thymus DNA with lopinavir from spectroscopic and computational studies
2021, Journal of Molecular LiquidsConsiderations for Determining Direct Versus Indirect Functional Effects of Solubilizing Excipients on Drug Transporters for Enhancing Bioavailability
2020, Journal of Pharmaceutical Sciences