Introduction

Top Summary Introduction Materials and Methods Results Discussion References

A major strategy used to fight infection with HIV type 1 (HIV-1) is treatment with protease inhibitors, which are small polypeptide derivatives (Fig. 1). Several of these demonstrate high antiretroviral potency to HIV-1 in vitro. However, the low and variable oral bioavailability of protease inhibitors is a significant impediment to their use (e.g., saquinavir has the lowest oral bioavailability of the protease inhibitors, only about 4%). It is becoming clear that several types of mechanism contribute to this problem. Initially, saquinavir's low bioavailability was attributed to metabolism by the cytochrome P-450 3A4 isoform (CYP3A4; Eagling et al., 1997), but recent studies have implicated another process: active extrusion of saquinavir out of enterocytes back into the gut lumen, mediated by the ATP-driven, drug efflux pump, P-glycoprotein (Alsenz et al., 1998; Kim et al., 1998). P-glycoprotein-mediated transport at the blood-brain barrier has also been implicated in the near-exclusion of saquinavir and other protease inhibitors from the central nervous system, an important target of protease inhibitor therapy (Drewe et al., 1999; Glynn and Yazdanian, 1998). The relevance of these mechanisms to the difficulties encountered during saquinavir treatment is further emphasized by recent studies in which the HIV protease inhibitor ritonavir increased saquinavir plasma concentrations in rats and humans by up to 50-fold (Kempf et al., 1997, Merry et al., 1997). Ritonavir has been shown to be a potent inhibitor of both CYP3A4 activity and P-glycoprotein-mediated transport (Eagling et al., 1997; Alsenz et al., 1998; Koudriakova et al., 1998; Lee et al., 1998).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structures of HIV-1 protease inhibitors and the FL-Saq derivative used in the present study.

The present report is concerned with the interactions between HIV-1 protease inhibitors and drug-transporting ATPases. These plasma membrane transport proteins play a major role in determining drug uptake, distribution, and excretion. P-glycoprotein and the recently characterized multidrug resistance-associated proteins (Mrps) were originally discovered as overexpressed proteins in tumor cells with a multidrug-resistant phenotype. Subsequently, P-glycoprotein and the Mrp2 isoform were also found to be at high levels in certain excretory or barrier tissues (e.g., gut, liver, and renal proximal tubule), where their polar distributions within the plasma membrane puts them in the correct orientation to: 1) limit xenobiotic uptake into blood and 2) drive xenobiotic excretion into bile and urine (see, for example, Thiebault et al., 1987; Lieberman et al., 1989; Hsing et al., 1992; Muller and Jansen, 1997; Schaub et al., 1997; Makhey et al., 1998). Although P-glycoprotein and Mrp2 are both members of the ATP-binding cassette family of transporters and share a common distribution in epithelial tissues, they have different specificities. In general, P-glycoprotein transports uncharged and cationic drugs and Mrp2 transports primarily anionic compounds, although there is some overlap of specificities (Elbling et al., 1998; Kusuhara et al., 1998). Along with drug-metabolizing enzymes, these transporters are important determinants of drug effectiveness on the one hand and of drug toxicity on the other hand. In addition, because of their wide specificity limits, these transporters also provide a mechanism, namely competition for transport, by which drugs with very different structures may interact.

In the present study, we used isolated killifish renal proximal tubules, fluorescent substrates, confocal microscopy, and image analysis to assess the interactions between the HIV-1 protease inhibitors saquinavir and ritonavir and drug-transporting ATPases, P-glycoprotein, and Mrp2. Renal tissue from teleost fish offers several important advantages for the study of excretory transport mechanisms in the proximal tubule (Miller, 1987). Proximal tubules are easily isolated with broken ends resealed to form a closed, fluid-filled luminal compartment that communicates with the medium only through the tubular epithelium. Thus, the preparation has the correct geometry to facilitate the study of excretory transport. The use of imaging techniques allows us to probe mechanisms responsible for transport at both the basolateral and luminal membranes of the tubular epithelial cells. Moreover, the xenobiotic transport mechanisms found in teleost tubules appear to be identical to those found in mammalian renal proximal tubules. Finally, recent studies have identified fluorescent substrates and specific inhibitors of P-glycoprotein and Mrp2 (Miller, 1995; Schramm et al., 1995; Masereeuw et al., 1996; Miller and Pritchard, 1997; R. Masereeuw and D.S.M., unpublished data), so that the physiology and pharmacology of these transporters may be studied in an intact native epithelium.

Here we report that the HIV-1 protease inhibitors saquinavir and especially ritonavir are potent inhibitors of drug secretion mediated by P-glycoprotein and Mrp2. These are the first data indicating that these compounds can interact with the Mrp family of major drug-transporting ATPases.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion References

Chemicals. The HIV protease inhibitors saquinavir, mesylate, and ritonavir, as well as fluorescein saquinavir (FL-Saq), were a kind gift from Dr. H. Wiltshire (Roche Ltd., Lewes, UK). The fluorescent labeled cyclosporin analog [N-(4-nitrobenzofurazan-7-yl)-D-Lys8]cyclosporin (NBDL-CSA) was obtained from Novartis Ltd. (Basel, Switzerland), and the fluorescent methotrexate derivative fluorescein-methotrexate (FL-MTX) was obtained from Molecular Probes (Madison, WI). All other chemicals were of analytical grade and were obtained from commercial sources.

Animals and Tissue Preparation. Killifish (fundulus heteroclitus) were purchased from local fishermen in the vicinity of Mount Desert Island, Maine, and maintained at the Mount Desert Island Biological Laboratory in tanks with aerated, natural flowing sea water. Renal proximal tubules were isolated in marine teleost saline based on the buffer system of Forster and Taggart (1950), containing 140 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl₂, 1.0 mM MgCl₂, and 20 mM Tris, pH 8.0. Under a dissecting microscope, each kidney was teased with fine forceps to remove adherent hematopoietic tissue. Individual killifish proximal tubules were dissected and transferred to an aluminum foil-covered Teflon incubation chamber containing 1.5 ml of marine teleost saline with fluorescent compound and added effectors. The chamber floor was a 4 × 4-cm glass coverslip to which the tubules adhered lightly and through which the tissue could be viewed by means of an inverted confocal laser microscope. Fluorescent compounds and inhibitors were added to the incubation medium as stock solutions in dimethyl sulfoxide. Previous experiments have shown that the concentrations of dimethyl sulfoxide used (1%) had no significant effects on the uptake and distribution of the fluorescent labeled test compounds as measured by confocal microscopy (Schramm et al., 1995; Masereeuw et al., 1996; D.S.M., unpublished data). Analysis of tubule extracts by HPLC showed no degradation of NBDL-CSA, FL-MTX, or FL-Saq during 60-min transport experiments (Schramm et al., 1995; Masereeuw et al., 1996; H.G., G.F., J.D. and D.S.M., unpublished data).

Confocal Microscopy. The chamber containing tubules was mounted onto the stage of an Olympus FluoView inverted confocal laser scanning microscope and viewed through a 40× water immersion objective (NA = 1.15). The 488-nm laser line, a 510-nm dichroic filter, and a 515-nm long-pass emission filter were used. Low laser intensity (6% of maximum) was used to avoid photobleaching of the dyes. With the photomultiplier gain set to give an average luminal fluorescence intensity of 1500 to 3000 (full scale, 4096), tissue autofluorescence was undetectable.

Statistics. Data are given as mean ± S.E.. Mean values were considered to be statistically different at a value of P < .05 by use of the appropriate paired or unpaired t test or by one-way ANOVA.

	Results

Top Summary Introduction Materials and Methods Results Discussion References

NBDL-CSA and FL-MTX Transport. The confocal micrograph shown in Fig. 2 demonstrates the basic characteristics of NBDL-CSA and FL-MTX transport in killifish renal proximal tubules. In control tubules, the steady-state distributions of both fluorescent compounds were similar: the lumens were much brighter than the cells, which in turn were brighter than the medium (Fig. 2). Although both compounds exhibited similar steady-state distribution patterns in control tubules, they were differentially affected by compounds that interact specifically with each of the ATP-driven transporters (Fig. 3). For example, verapamil and leukotriene (LT)C₄ are potent competitive inhibitors of P-glycoprotein and Mrp2, respectively (Leier et al., 1996; Ling, 1997; Kusuhara et al., 1998). In agreement with previous experiments using killifish tubules (Schramm et al., 1995; Masereeuw et al., 1996), NBDL-CSA transport from cell to tubular lumen was reduced by 10 µM verapamil but not by 500 nM LTC₄; conversely, LTC₄ inhibited cell-to-lumen transport of FL-MTX, but 10 µM verapamil was without effect (Fig. 3). Neither inhibitor altered the cellular accumulation of NBDL-CSA or FL-MTX, a result consistent with previous studies (Schramm et al., 1995; Masereeuw et al., 1996) and taken to indicate that events at the luminal membrane do not significantly affect the steady-state cellular accumulation of these fluorescent substrates.

View larger version (107K): [in this window] [in a new window]   Fig. 2.   Confocal micrograph showing steady-state distribution of NBDL-CSA fluorescence in a killifish renal proximal tubule. Tubules were incubated for 30 min in medium with 1 µM NBDL-CSA, and confocal images were acquired as described in Materials and Methods. Notice the intensely fluorescent lumimal space and the modest level of fluorescence within the cells. A similar distribution of fluorescence was found for tubules incubated in medium containing FL-MTX.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of 10 µM verapamil (VERAP) and 0.5 µM LTC4 on the transport of NBDL-CSA and FL-MTX. Killiifish tubules were incubated for 30 min in medium containing 1 µM NBDL-CSA or FL-MTX and the indicated additions. Confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for 15 to 22 tubules. **P < .01, significantly lower than control.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Inhibition of NBDL-CSA transport by saquinavir and ritonavir. Tubules were incubated for 30 min in medium containing 1 µM NBDL-CSA and the indicated concentration of protease inhibitor. Confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for 6 to 21 tubules. *P < .05, **P < .01, significantly lower than control.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Inhibition of FL-MTX transport by saquinavir and ritonavir. Tubules were incubated for 30 min in medium containing 1 µM FL-MTX with the indicated concentration of protease inhibitor. Confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for 8 to 18 tubules. *P < .05, **P < .01, significantly lower than control.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Lack of effects of saquinavir and ritonavir on fluorescein (FL) transport in killifish tubules. Tubules were incubated for 30 min in medium containing 1 µM FL and the indicated concentration of protease inhibitor. Confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for 9 to 23 tubules.

FL-Saq Transport. To determine whether protease inhibitors are substrates for renal P-glycoprotein and Mrp2, we measured the transport of a FL-Saq derivative. Figure 7 shows the time course of FL-Saq accumulation in killifish tubules. FL-Saq was rapidly taken up by the tissue, with a steady-state distribution of the drug attained within 20 min of incubation. At steady state, the lumen-to-cell fluorescence ratio averaged 3 to 5, which is similar to that found for NBDL-CSA and FL-MTX (above). When verapamil or LTC₄ was added to the medium bathing the tubules, steady-state luminal fluorescence was partially reduced (Fig. 8). The effect of verapamil and LTC₄ in combination on luminal fluorescence was significantly greater than that of LTC₄ alone (one-way ANOVA; Fig. 8). Both verapamil and LTC₄ also caused a significant reduction in cellular fluorescence. No additional reduction in cellular fluorescence was found when the two inhibitors were used in combination. These data indicate that the fluorescent saquinavir derivative is secreted into the tubular lumen by a process that is uphill and specific and that may involve both P-glycoprotein and Mrp2.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Time course of NBD-Saq accumulation in killifish renal tubules. Tubules were incubated for the time indicated in medium containing 1 µM NBD-Saq, and confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for five tubules.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effects of 10 µM verapamil (VERAP) and 0.3 µM LTC4 on the transport of NBD-Saq in killifish tubules. Tubules were incubated for 30 min in medium containing 1 µM NBD-Saq and the indicated additions. Confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for 19 to 31 tubules. **P < .01, significantly lower than control.

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Inhibition of NBD-Saq transport by saquinavir and ritonavir. Tubules were incubated for 30 min in medium containing 1 µM NBD-Saq and the indicated concentration of protease inhibitor. Confocal images were acquired as described in Materials and Methods. Data given as mean ± S.E. for 14 to 28 tubules. *P < .05, **P < .01, significantly lower than control.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

The success of protease inhibitor-based therapies against acquired immunodeficiency syndrome have been limited by a variety of problems; these include not only strict and rigid drug regimens with the potential for noncompliance but also the presence of pharmacodynamic barriers, which limit entry of orally administered drugs from the gastrointestinal tract and access of absorbed drugs to sites of HIV-1 infection in the central nervous system. Although first-pass metabolism by the 3A4 isoform (CYP3A4) of the cytochrome P-450 system may partially account for low bioavailability after oral administration, another important factor affecting drug bioavailability, specific transport, is just beginning to receive attention. Indeed, recent studies implicate P-glycoprotein in the excretory transport of saquinavir and ritonavir in an intestinal cell line in vitro and in the blood-brain barrier in vitro and in vivo (Alsenz et al., 1998; Drewe et al., 1999; Glynn and Yazdanian, 1998; Kim et al., 1998).

Interactions with P-Glycoprotein and Mrp2. Previous studies have established killifish renal proximal tubules as a model system in which to study excretory drug transport mediated by P-glycoprotein and Mrp2 in an intact, native epithelium (Schramm et al., 1995; Masereeuw et al., 1996). This system provides information of two types about interactions of drugs with these transporters: 1) a quantitative measure of the drug's ability to specifically inhibit the transport of model substrates and, thus, interact at the transport level with other drugs handled by that carrier, and 2) an indication of whether the drug itself or a fluorescent analog is transported by P-glycoprotein or Mrp2. The results of the present study show that two protease inhibitors extensively used in anti-HIV-1 therapy were potent inhibitors of the luminal accumulation of fluorescent substrates for both P-glycoprotein (NBDL-CSA) and Mrp2 (FL-MTX). Except for the highest concentration of saquinavir used, this decrease in luminal substrate accumulation was generally seen in the absence of reduced cellular accumulation, indicating that uptake at the basolateral membrane and intracellular sequestration of the two substrates were not affected but that cell-to-lumen transport was reduced. Inhibition of transport was not due to toxicity or inhibition of cellular metabolism because neither saquinavir nor ritonavir, at concentrations that substantially reduced NBDL-CSA and FL-MTX transport, had any effect on the transport of FL, a substrate for the classic renal organic anion system. The organic anion transport system is particularly sensitive to disruption of metabolism or ion gradients (Miller, 1981; Sullivan and Grantham, 1990; Miller and Pritchard, 1991; Pritchard and Miller, 1993). Together, these inhibition data indicate that in renal proximal tubule, both saquinavir and ritonavir inhibit drug transport mediated by both P-glycoprotein and Mrp2. Although it can be inferred from recent transport studies with Caco-2 cell monolayers that saquinavir and ritonavir can be transported by P-glycoprotein and can inhibit P-glycoprotein-mediated transport (Alsenz et al., 1998; Kim et al., 1998), the present data are the first published evidence that these protease inhibitors interact with the second major epithelial drug transporter, Mrp2.

View this table: [in this window] [in a new window]   TABLE 1 Effectiveness of several compounds as inhibitors of P-glycoprotein- and Mrp2-mediated transport in killifish renal proximal tubules Values were estimated from dose-response curves in Figs. 3 and 4 and from data of Schramm et al., 1995; Masereeuw et al., 1996; and Miller et al., unpublished observations.

FL-Saq Transport. Although the NBDL-CSA and FL-MTX inhibition studies provided clear evidence of interactions between the HIV-1 protease inhibitors and P-glycoprotein and Mrp2, the experiments designed to characterize the mechanisms driving transport of the fluorescent saquinavir derivative were more difficult to interpret. Confocal micrographs showed both cellular accumulation and uphill transport of FL-Saq from cell to tubular lumen. Little is known about the renal handling of protease inhibitors. Renal excretion can account for as little as a few percent of administered dose to as much as 20% (Hilgeroth, 1998). Hsu et al. (1998) reported that the renal clearance of saquinavir in patients is 0.5 liter/h, a value well below the glomerular filtration rate. However, interpretation of this low value is confounded by high (98%) binding of saquinavir to plasma proteins (Hilgeroth, 1998) and the drug's nonpolar nature. Extensive binding in plasma and reabsorption in more distal nephron segments could mask active secretion in proximal tubule.