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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Assessing Safety and Efficacy of Directed P-Glycoprotein Inhibition to Improve the Pharmacokinetic Properties of Saquinavir Coadministered with Ritonavir

Division of Experimental Therapy, The Netherlands Cancer Institute, Amsterdam, The Netherlands (M.T.H., A.H.S.); Cobra Therapeutics Limited, Keele, United Kingdom (J.W.S.); Pharma Development, Roche Products Limited, Welwyn Garden City, United Kingdom (H.R.W.); and Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam, The Netherlands (J.H.B.)

Received September 11, 2002; accepted October 22, 2002.

	Abstract

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Using a mouse model, we tested the effects of in vivo P-glycoprotein inhibition to enhance the oral uptake and penetration into pharmacological sanctuary sites of the human immunodeficiency virus protease inhibitor (HPI) saquinavir. The HPI ritonavir is frequently coadministered with saquinavir to improve saquinavir plasma levels since it strongly reduces the cytochrome P450 3A4-mediated metabolism of saquinavir. Previously, we demonstrated that ritonavir is not an efficient P-glycoprotein inhibitor in vivo, evidenced by the limited oral uptake of saquinavir and its penetration into brain and fetus. Increasing drug concentrations in these sites using more effective P-gp inhibitors might improve therapy but could also lead to toxicity. We orally coadministered ritonavir and saquinavir to mice, with or without the potent P-glycoprotein inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Upon GF120918 coadministration, two of seven P-glycoprotein-deficient animals died. Using a decreased ritonavir dose, GF120918 coadministration led to a 4.4-fold increase in the saquinavir plasma area under the curve in wild-type mice, whereas no such effect was observed in P-glycoprotein-deficient mice. Despite the decreased ritonavir dose, all mice did suffer from impaired gastric emptying. Including GF120918 in a multiple (bidaily) dosing regimen, we found continued accumulation of saquinavir in brain over several days, resulting in 10-fold higher levels compared with vehicle-treated mice. Transient ritonavir-related neurotoxicity, however, was observed after the fourth and final drug dosing. Clinical attempts to efficiently inhibit P-glycoprotein function for improved HPI disposition may therefore be feasible, but they should be performed without ritonavir and monitored carefully for unexpected toxicities.

The most important cause for the low oral bioavailability of saquinavir is an extensive first-pass effect, which is mainly due to cytochrome P450 3A4 (CYP3A4)-mediated metabolism (Cameron et al., 1999; Huisman et al., 2001; Kaufmann et al., 1998; Koudriakova et al., 1998). Cyp3A4, strategically located in the enterocytes and hepatocytes (Guengerich, 1999), metabolizes saquinavir very efficiently (Fitzsimmons and Collins, 1997). It has been demonstrated that the HPI ritonavir is a potent inhibitor of CYP3A4 and other cytochrome P450 isoforms and that coadministration of ritonavir with saquinavir leads to highly elevated saquinavir plasma levels both in animals and humans (Eagling et al., 1997; Fitzsimmons and Collins, 1997; Cameron et al., 1999; Dresser et al., 2000).

Another factor limiting the oral uptake of saquinavir is P-glycoprotein (P-gp) function (Alsenz et al., 1998; Kim et al., 1998a,b; Williams and Sinko, 1999). Most HPIs, including saquinavir and ritonavir, are P-gp substrates. P-gp is an active drug transporter, which extrudes a wide range of substrates from cells, and belongs to the ATP binding cassette transporter family (Juliano and Ling, 1976; Higgins, 1992; Gottesman and Pastan, 1993). One can distinguish several pharmacological functions for P-gp. It limits the oral bioavailability of its substrates, as it is present in the apical membrane of the intestinal epithelium and can mediate direct intestinal excretion of its substrates (Sparreboom et al., 1997). It further mediates secretion of its substrates into bile by its presence in the bile canalicular membrane of hepatocytes (Thiebaut et al., 1987). Finally, it contributes to the protective function of important blood-tissue barriers, as it is expressed in the blood-brain, blood-nerve, and blood-testis barrier and in the materno-fetal barrier formed by placental trophoblasts (Holash et al., 1993; Choo et al., 2000).

It is known that saquinavir and other HPI concentrations in pharmacological sanctuary sites are limited by P-gp function in blood-tissue barriers (Kim et al., 1998a,b; Huisman et al., 2001), and it is assumed that these sites could be a breeding ground for therapy resistant viruses due to the low drug concentrations. The resistant viruses may then migrate back into the circulation, leading to failure of therapy (Chun and Fauci, 1999). Thus, inhibiting P-gp function to increase the drug concentration in these sites might have beneficial effects. On the other hand, increased drug penetration could also lead to increased toxicity since for instance the CNS will be exposed to higher drug concentrations.

Some groups have suggested that ritonavir is an efficient P-gp inhibitor (Drewe et al., 1999; Gutmann et al., 1999). We previously demonstrated, however, that high-dose ritonavir does not abrogate P-gp function in mice and, therefore, even when saquinavir is coadministered with ritonavir, P-gp function still contributes to low oral bioavailability of drugs and to poor penetration into pharmacological sanctuary sites. Thus, ritonavir is a poor P-gp inhibitor in vivo (Huisman et al., 2001). Similar results in vitro and in vivo have been reported by others (Van der Sandt et al., 2001). The purpose of this study, therefore, was to test whether a highly potent P-gp inhibitor, GF120918 (Hyafil et al., 1993), could be safely and effectively used in mice to enhance the oral bioavailability and sanctuary penetration of saquinavir.

	Materials and Methods

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Chemicals. [¹⁴C]Saquinavir (41.3 µCi mg^-1) and saquinavir were provided by Roche Discovery Welwyn (Welwyn Garden City, UK). Ritonavir was purchased from Abbott Laboratories, Inc. (Abbott Park, IL) as Norvir (80 mg ml^-1). GF120918 was generously provided by GlaxoSmithKline (Uxbridge, Middlesex, UK). Metofane (methoxyflurane) was from Medical Developments Australia, Pty. Ltd. (Melbourne, Australia). Deionized water was obtained using the Milli-Q Plus system (Millipore Corp., Bedford, Massachusetts). Bovine serum albumin was from Boehringer Mannheim GmbH (Mannheim, Germany). TaqDNA polymerase and deoxynucleoside-triphosphates were purchased from Invitrogen (Breda, The Netherlands).

Drug Distribution Studies. Mice used in all experiments were males, except for the mice that were used in the fetal distribution studies, and between 10 and 14 weeks of age. Animals were housed and handled according to institutional guidelines complying with Dutch legislation under a 12-h light/dark cycle at a temperature of 22°C. Wild-type, Mdr1a^+/-/1b^+/-, and Mdr1a^-/-/1b^-/- mice were of a >99% FVB genetic background (Schinkel et al., 1994, 1997). The mice received a standard chow (AM-II; Hope Farms, Woerden, The Netherlands) and acidified water ad libitum. At t =-30 min, the mice received ritonavir and/or GF120918 (100 mg kg^-1), and at t = 0 min, the mice received 5 mg kg^-1[¹⁴C]saquinavir. All drugs were dosed orally in a volume of 2.5 µl/gram b.wt.

Norvir was diluted with a control vehicle for Norvir containing 43% v/v ethanol. This vehicle resembles the matrix of liquid Norvir and contains Cremophor EL (105 mg ml^-1) (Sigma-Aldrich, St. Louis, Missouri), propylene glycol (0.25 ml ml^-1), peppermint oil (3.5 mg ml^-1), and water-free citric acid (2.8 mg ml^-1). Vehicle pH was 4.3. Animals received 1 to 2 µCi (37–74 kBq) of the radiolabeled drugs, and 40-µl blood samples were taken from the tail vein at the appropriate time points, or mice were sacrificed by orbital bleeding or cardiac puncture under Metofane anesthesia, followed by cervical dislocation.

Tissues were collected and processed as described previously by Smit et al. (1999). In short, scintillation counting was applied to Ultima Gold scintillation cocktail (Packard Bioscience B.V., Groningen, The Netherlands) mixed with a fraction of in 4% bovine serum albumin (w/v) homogenized tissue. Tissue concentrations of [¹⁴C]saquinavir were thus assessed by total radioactivity measurements. Unchanged saquinavir and ritonavir were determined in plasma by HPLC according to Van Heeswijk et al. (1998). The within-day precision ranged from 1.8 to 6.7%, and between-day precision ranged from 0.7 to 7.6%. The lower limits of quantification for saquinavir and ritonavir were 25 and 50 ng/ml, respectively. Unchanged fetal saquinavir concentrations were too low to be determined by HPLC and were for that reason determined as the radioactive drug equivalent per weight. Genotype analysis was done by polymerase chain reaction, according to Smit et al. (1999).

Statistical Analysis. Statistical analysis was performed using the Student's t test (unpaired and two-tailed). The modified Bonferroni procedure was applied to the data presented in Fig. 2. The regression coefficient R² was calculated using Pearson's standard linear regression. Differences between two sets of data were considered statistically significant if P < 0.05. Unless indicated otherwise, errors are represented as the standard deviation.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of ritonavir and GF120918 on the percentage of the [14C]saquinavir dose remaining in the stomach. Wild-type and P-gp-deficient (knockout) mice were analyzed 8 h after oral administration of 5 mg kg-1 [14C]saquinavir and pretreatment with or without oral GF120918 (100 mg kg-1) and different doses of oral ritonavir (0 to 50 mg kg-1) as indicated. Data are expressed as average ± S.D. (n = 3–6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

	Results

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Increased Ritonavir Toxicity Caused by Coadministration of GF120918. We first set out to determine whether 100 mg kg^-1 of the potent P-gp inhibitor GF120918 was able to efficiently inhibit P-gp function at various tissue barriers. GF120918 was coadministered with ritonavir (50 mg kg^-1)at t =-30 min and [¹⁴C]saquinavir (5 mg kg^-1) at t = 0 min to wild-type and P-gp-deficient mice. This high dose of ritonavir, close to the maximum tolerated dose, was used to inhibit saquinavir metabolism to the highest possible extent. The HPI doses were equal to those used in our previous study (Huisman et al., 2001) in which we demonstrated that at this dose of ritonavir P-glycoprotein function was not abrogated. With the P-gp inhibitor GF120918 included, however, two of seven P-gp-deficient mice died within 1 h after drug administration showing signs of ataxia and tremor. The wild-type mice showed no signs of toxicity. Since we observed similar toxicity signs previously when determining the maximum tolerated dose of ritonavir in mice (Huisman et al., 2001), the toxicity was most likely due to ritonavir overexposure. When the ritonavir dose was lowered to 25 mg kg^-1, toxicity effects were not observed in any of the animals.

The Effect of GF120918 on the [¹⁴C]Saquinavir Plasma Concentration. We first determined whether 100 mg kg^-1 of the potent P-gp inhibitor GF120918 could increase the [¹⁴C]saquinavir plasma concentration after oral saquinavir administration. Wild-type and P-gp knockout mice received oral ritonavir (12.5 mg kg^-1) and [¹⁴C]saquinavir (5 mg kg^-1) with either GF120918 (100 mg kg^-1) or vehicle (Fig. 1). Subsequently, we determined the [¹⁴C]saquinavir plasma concentration every hour up to 11 h. Note that we demonstrated previously that even a ritonavir dose of 50 mg kg^-1 does not abrogate P-glycoprotein function (Huisman et al., 2001).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Effect of P-gp inhibitor GF120918 on plasma levels of [14C]saquinavir. Wild-type (a) and P-gp-deficient mice (b) received oral ritonavir (12.5 mg kg-1), with or without the potent P-gp inhibitor GF120918 (100 mg kg-1), followed 30 min later by oral [14C]saquinavir (5 mg kg-1). The plasma [14C]saquinavir concentration was determined in each animal every hour by total radioactivity (micrograms per milliliter). Error bars indicate the S.D. (n = 5).

As can be seen in Fig. 1a, the [¹⁴C]saquinavir plasma concentration and AUC were considerably increased (4.4-fold) in GF120918-treated wild-type animals compared with the vehicle-treated wild-types (P < 0.0001). Next, we determined the effect of GF120918 on the [¹⁴C]saquinavir plasma concentration in P-gp knockout animals under otherwise identical conditions, although one would of course not expect to find an effect (Fig. 1b). The [¹⁴C]saquinavir plasma AUCs for these groups of animals were identical (P = 0.67), indicating that the effect of GF120918 in wild-type mice was primarily mediated by P-gp inhibition.

The overall [¹⁴C]saquinavir plasma concentration seemed to be slightly higher in GF120918-treated wild-type animals than in GF120918-treated knockouts, but the AUCs were not significantly different (P > 0.1), indicating that GF120918 inhibited P-gp efficiently. Noteworthy, GF120918-treated knockout mice showed considerable fluctuation in the [¹⁴C]saquinavir plasma concentration in time (Fig. 1b). Based on the data for individual mice, we were under the impression that, in this specific group of five animals, emptying of the stomach was somewhat delayed in a few mice (data not shown).

In this experiment, impaired gastric emptying might be due to several causes, but both saquinavir (Washington et al., 2000) and GF120918 (Polli et al., 1999) have been given at very high dosages to rodents without obvious adverse side effects. We hypothesized therefore that it was most likely that ritonavir was responsible for this toxic effect and that GF120918 possibly exacerbated this.

To test whether ritonavir can indeed cause delayed gastric emptying and to determine a drug-dose combination that does not cause this possible toxic effect, wild-type and P-gp-deficient mice received various dosages of ritonavir, ranging from 0 to 50 mg kg^-1, with or without 100 mg kg^-1 GF120918, followed 30 min later by oral [¹⁴C]saquinavir (5 mg kg^-1). The mice were sacrificed 8 h later, after which the percentage of the initially applied [¹⁴C]saquinavir dose remaining in the stomach was determined (Fig. 2).

Focusing on the wild-type data in Fig. 2, it is obvious that 25 mg kg^-1 of ritonavir or 100 mg kg^-1 GF120918 alone does not lead to markedly impaired gastric emptying. In wild-type mice treated with GF120918, however, increasing dosages of ritonavir up to 25 mg kg^-1 caused increasingly delayed gastric emptying, demonstrating the role of ritonavir and the involvement of GF120918 in the toxicity (R² = 0.893). Similar ritonavir dose-dependent toxicity was observed in knockout mice without (P < 0.05) or with (R² = 0.889) GF120918 treatment (Fig. 2), although knockout mice without GF120918 treatment appeared to be somewhat less sensitive than wild-type or knockout mice treated with GF120918.

Accumulation of [¹⁴C]Saquinavir in Pharmacological Sanctuary Sites upon Multiple Drug Treatment. Since HIV/AIDS patients on highly active antiretroviral therapy take drugs life-long and at least once daily, we wanted to determine whether GF120918 could be included in a long term ritonavir-saquinavir coadministration regimen. The aim was to achieve improved HPI penetration in the pharmacological sanctuary sites brain, testis, and glandula vesicularis but without causing unacceptable toxicity due to possible long-term accumulation of drugs in the CNS or other organs.

To test this, wild-type mice were treated every 12 h with 5 mg kg^-1 ritonavir, with or without 100 mg kg^-1 GF120918, followed 30 min later by 5 mg kg^-1 [¹⁴C]saquinavir. The ritonavir dose was decreased to 5 mg kg^-1 to minimize effects on the gastric release of drugs, although this dose may be too low to inhibit metabolism of [¹⁴C]saquinavir fully. One group of animals was sacrificed 12 h after the first [¹⁴C]saquinavir administration, and the remaining group received the drug combination four times (at 0, 12, 24, and 36 h) and was sacrificed 12 h after the final drug administration. Animals in the latter group were thus exposed to the drugs for 48 h. All animals were visually checked for toxicity signs for at least 1.5 h after each drug dosing.

Following the final drug administration at t = 36 h, all GF120918-treated mice showed transient signs of passivity and unresponsiveness, reminiscent of a mild form of previously observed ritonavir toxicity (data not shown). The animals fully recovered within 90 min. There were no pronounced abnormalities observed in the percentages of the dose in any part of the gastrointestinal tract, indicating that the ritonavir dose was below the level at which it causes considerable delayed gastric emptying (data not shown). The applied ritonavir dose was not sufficient to inhibit [¹⁴C]saquinavir metabolism fully since the plasma levels of [¹⁴C]saquinavir, saquinavir, and ritonavir were below the detection limit at the 12 and 48 h time points.

Since in our experience the relative drug accumulation in the spleen is not significantly affected by P-gp function, we used the [¹⁴C]saquinavir spleen concentration as a reference reflecting possible differences in plasma exposure between the treatment groups (Fig. 3).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of P-gp inhibition on the distribution of saquinavir in a repeated administration regimen. Concentration of [14C]saquinavir in wild-type mice is expressed as nanograms per gram. Mice received 5 mg kg-1 ritonavir, with or without GF120918 (100 mg kg-1), followed 30 min later by [14C]saquinavir. One half of the animals were sacrificed after 12 h; the other half received the appropriate drug combination every 12 h for four times and was sacrificed 12 h after the final drug dosing. This latter group is referred to as "48 h". Data are expressed as average ± S.D. (n = 5).

The [¹⁴C]saquinavir brain concentration was almost 4-fold higher in the GF120918-treated group than in the vehicle-treated group after 12 h. At t = 48 h, there was a 10-fold difference in brain penetration, whereas brain concentration in the vehicle-treated group was not significantly different from that at 12 h. Testis concentrations were 1.5- and 3.9-fold increased due to GF120918 at t = 12 and 48 h, respectively. In contrast, reference spleen concentrations were only 1.7- and 2.6-fold increased due to GF120918 treatment at 12 and 48 h. Effects of GF120918 treatment on glandula vesicularis [¹⁴C]saquinavir levels were similar to those for spleen. Of note, the absolute brain and testis levels of [¹⁴C]saquinavir had clearly increased between 12 and 48 h in the GF120918-treated mice but not in vehicle-treated mice. In contrast, continued [¹⁴C]saquinavir accumulation was not observed for spleen or glandula vesicularis either with or without GF120918.

Increased [¹⁴C]Saquinavir Fetal Penetration upon GF120918 Pretreatment. We previously demonstrated that despite high ritonavir plasma concentrations in mice P-gp function in the materno-fetal barrier still limits penetration of orally administered [¹⁴C]saquinavir into fetuses, resulting in an 18-fold higher [¹⁴C]saquinavir penetration in P-gp-deficient fetuses compared with wild-type fetuses (Huisman et al., 2001). We now investigated whether in a similar setup P-gp function in the materno-fetal barrier could be blocked efficiently by oral coadministration of GF120918.

Since P-gp is expressed in trophoblasts, which are of fetal origin, the genotype of the fetus determines the expression of placental P-gp. Heterozygous dams (Mdr1a^+/-/1b^+/-) were mated to heterozygous males (Mdr1a^+/-/1b^+/-), resulting in a fetal offspring of all three genotypes (Mdr1a^+/+/1b^+/+, Mdr1a^+/-/1b^+/-, and Mdr1a^-/-/1b^-/-). Intrinsic to this setup, all fetuses in one dam are exposed to the same maternal plasma concentrations of drugs. Pregnant dams at gestation day 15 received 100 mg kg^-1 GF120918 and 25 mg kg^-1 ritonavir orally at t =-30 min, followed by 5 mg kg^-1 oral [¹⁴C]saquinavir at t = 0 min. The maternal plasma and fetal [¹⁴C]saquinavir concentrations were determined 4 h after the [¹⁴C]saquinavir administration. The maternal [¹⁴C]saquinavir plasma concentrations at t = 4 h varied widely [i.e., 0.11–3.37 µg ml^-1 (Table 1)]. The same was true for unchanged saquinavir and ritonavir concentrations (data not shown). In retrospect, this effect was presumably caused by various degrees of impaired gastric emptying due to ritonavir (see above). Fortuitously, however, this wide variation in maternal plasma levels allowed us to assess the efficacy of placental P-gp inhibition at a wide range of plasma exposure levels, as fetuses of all P-gp genotypes are usually represented within each mother and can thus be directly compared with each other. Table 1 shows the [¹⁴C]saquinavir data collected for all individual dams and for the corresponding fetuses of each genotype. It is clear that relative to wild-type fetuses on average only 1.08- and 1.25-fold more [¹⁴C]saquinavir accumulated in heterozygous and knockout fetuses. In our previous study, without GF120918 (Huisman et al., 2001), in which 50 mg kg^-1 ritonavir was used but under otherwise identical conditions, these ratios were 1.33 and 18.1 for heterozygous and knockout fetuses, respectively. Taken together, our data show that P-gp function at the maternal-fetal barrier in mice can be inhibited almost fully over a wide range of saquinavir plasma concentrations, following coadministration of oral GF120918, ritonavir, and saquinavir.

	Discussion

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Several pharmacological factors may limit the therapeutic efficacy of HPIs, including low oral bioavailability and poor penetration into pharmacological sanctuary sites. In this study, we have tested in mice the safety and efficacy of a regimen to optimize the pharmacokinetic behavior of saquinavir, using ritonavir as an effective inhibitor of metabolism and GF120918 as an effective P-gp inhibitor. All drugs were administered orally to emulate the clinical setup as much as possible.

A number of relevant conclusions can be drawn. By extensively inhibiting P-gp function in clinically important barriers, we were able to increase the saquinavir plasma AUC by 4.4-fold and the relative penetration into fetus as well. This regimen, however, also revealed a number of ritonavir dose-related adverse events resulting from the absence or pharmacological inhibition of P-gp activity. In extreme cases a lethal, probably CNS-related toxicity of ritonavir, necessitating a reduction in dose. And, even at a reduced dose, ritonavir can cause a marked delay in gastric emptying.

From our data, it appears that when ritonavir is present GF120918 also contributes to some toxicity events, independent of its inhibition of P-gp. It is unlikely that the lethal toxicity we observed was due to GF120918 or saquinavir by itself. GF120918 has been orally administered in high doses to both mice (Polli et al., 1999; Bardelmeijer et al., 2000) and patients (Malingré et al., 2001) without obvious toxicity, and saquinavir has also been given at very high oral dosages to P-gp-deficient mice (500 mg kg^-1; Washington et al., 2000) without adverse toxicity events. Furthermore, it is unlikely that GF120918 inhibits the CYP3A-mediated metabolism of ritonavir or saquinavir, as it is not a significant CYP3A4 inhibitor (Cummins et al., 2002). The exact mechanism behind the GF120918-related toxicity therefore remains to be investigated, but we note that the effects are limited compared with those caused by ritonavir.

We have further demonstrated that, in a multiple dosing regimen with a greatly reduced dose of ritonavir, GF120918 treatment resulted in a progressive accumulation of saquinavir in brain and testis, but again toxicity was encountered that was most likely caused by ritonavir. Previously, several groups including our own have demonstrated improved brain, testis, or fetal penetration of various HPIs (saquinavir, amprenavir, and nelfinavir) by coadministration of effective P-gp inhibitors (Polli et al., 1999; Smit et al., 1999; Choo et al., 2000). None of these studies, however, employed a clinically realistic administration schedule, involving (repeated) oral administration of all relevant drugs, nor did they reveal the toxicity risks that we encountered.

We note that the importance of active transporters in HIV therapy could well extend beyond oral bioavailability and tissue penetration, as illustrated for instance by Meaden et al. (2002) who provided data suggesting that active transporters may play a role in limiting accumulation of ritonavir and saquinavir in lymphocytes of HIV-infected patients. Inhibition of transport proteins may therefore improve this aspect of HPI-based therapy as well.

To our knowledge, we are the first to describe a severely delayed gastric emptying caused by ritonavir. It is well known that in humans ritonavir frequently causes severe nausea and vomiting, which can be a dose-limiting toxicity (Gatti et al., 1999). Perhaps the same pharmacodynamic toxic effect of ritonavir plays a role in the stomach of mice and humans, but as mice are physically incapable of vomiting, the toxicity reveals itself as a greatly delayed gastric emptying. As this toxicity was only observed in P-gp-deficient or GF120918-treated wild-type animals, it is tempting to speculate that it resulted from increased penetration of ritonavir in the CNS or some other sanctuary site. In our experiments and also in HIV/AIDS patients treated with highly active antiretroviral therapy, ritonavir is primarily coadministered to inhibit rapid metabolism of other HPIs like saquinavir. Obviously, from our data, we would advise to avoid ritonavir from any clinical regimen including highly effective P-gp inhibitors. For rapidly metabolized HPIs such as saquinavir, this would mean that alternative CYP3A inhibitors should be included that do not cause severe toxicity in the absence of P-gp activity. Perhaps, ketoconazole would be a good candidate (Jordan, 1998). Alternatively, one could consider the use of compounds that are both effective P-gp and CYP3A inhibitors (Wandel et al., 1999), but their in vivo toxicity profiles should first be carefully investigated.

In conclusion, our data show that pharmacological P-gp inhibition with the aim of enhancing the oral uptake and penetration of the HPI saquinavir into pharmacological sanctuary sites is feasible. This could mean that viral replication in sanctuary sites is more efficiently suppressed. The observed toxicities, most likely caused by ritonavir, however, demonstrate that great caution must be exercised when trying to inhibit P-gp in a clinical setting. Even when omitting ritonavir from such HPI treatment regimens, one should always be aware that other (co-)administered drugs may reveal unexpected side effects due to the effective inhibition of P-gp.

	Acknowledgements

 
We thank GlaxoSmithKline and Dr. R. C. Jewell for providing GF120918 and further support and Prof. Dr. J. H. M. Schellens for arranging availability of the compound. We thank R. van der Put, I. Bedeker, and M. Kemper for the HPLC analyses, J. W. Jonker and Dr O. van Tellingen for critically reading the manuscript, and Dr. A. A. M. Hart for statistical analysis.

	Footnotes

 
This study was funded in part by the AIDS Fonds (The Netherlands) Project 4011.

DOI: 10.1124/jpet.102.044388.

ABBREVIATIONS: HIV, human immunodeficiency virus; HPI, HIV protease inhibitor; AIDS, acquired immunodeficiency syndrome; P-gp P-glycoprotein; CNS, central nervous system; GF120918, 1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; HPLC, high-performance liquid chromatography; AUC, area under the curve.

Address correspondence to: Dr. A. H. Schinkel, Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail: a.schinkel{at}nki.nl

	References

Top Abstract Materials and Methods Results Discussion References

 

Alsenz J, Steffen H, and Alex R (1998) Active apical secretory efflux of the HIV protease inhibitors saquinavir and ritonavir in Caco-2 cell monolayers. Pharm Res (NY) 15: 423–428.[CrossRef][Medline]

Bardelmeijer HA, Beijnen JH, Brouwer KR, Rosing H, Nooijen WJ, Schellens JHM, and van Tellingen O (2000) Increased oral bioavailability of paclitaxel by GF120918 in mice through selective modulation of P-glycoprotein. Clin Cancer Res 6: 4416–4421.[Abstract/Free Full Text]

Cameron DW, Japour AJ, Xu Y, Hsu A, Mellors J, Farthing C, Cohen C, Poretz D, Markowitz M, Follansbee S, et al. (1999) Ritonavir and saquinavir combination therapy for the treatment of HIV infection. AIDS 13: 213–224.[CrossRef][Medline]

Choo EF, Leake B, Wandel C, Imamura H, Wood AJ, Wilkinson GR, and Kim RB (2000) Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes. Drug Metab Dispos 28: 655–660.[Abstract/Free Full Text]

Chun TW and Fauci AS (1999) Latent reservoirs of HIV: obstacles to the eradication of virus. Proc Natl Acad Sci USA 96: 10958–10961.[Free Full Text]

Cummins CL, Jacobsen W, and Benet LZ (2002) Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4. J Pharmacol Exp Ther 300: 1036–1045.[Abstract/Free Full Text]

Dresser GK, Spence JD, and Bailey DG (2000) Pharmacokinetic-pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clin Pharmacokinet 38: 41–57.[CrossRef][Medline]

Drewe J, Gutmann H, Fricker G, Torok M, Beglinger C, and Huwyler J (1999) HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833. Biochem Pharmacol 57: 1147–1152.[CrossRef][Medline]

Eagling VA, Back DJ, and Barry MG (1997) Differential inhibition of cytochrome P450 isoforms by the protease inhibitors, ritonavir, saquinavir and indinavir. Br J Clin Pharmacol 44: 190–194.[CrossRef][Medline]

Eisen SA, Miller DK, Woodward RS, Spitznagel E, and Przybeck TR (1990) The effect of prescribed daily dose frequency on patient medication compliance. Arch Intern Med 150: 1881–1884.[Abstract/Free Full Text]

Fitzsimmons ME and Collins JM (1997) Selective biotransformation of the human immunodeficiency virus protease inhibitor saquinavir by human small-intestinal cytochrome P4503A4: potential contribution to high first-pass metabolism. Drug Metab Dispos 25:25 6–266.

Gatti G, Di Biagio A, Casazza R, De Pascalis C, Bassetti M, Cruciani M, Vella S, and Bassetti D (1999) The relationship between ritonavir plasma levels and side-effects: implications for therapeutic drug monitoring. AIDS 13:20 83–2089.

Gottesman MM and Pastan I (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:38 5–427.

Guengerich FP (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39: 1–17.[CrossRef][Medline]

Gutmann H, Fricker G, Drewe J, Toeroek M, and Miller DS (1999) Interactions of HIV protease inhibitors with ATP-dependent drug export proteins. Mol Pharmacol 56:38 3–389.

Higgins CF (1992) ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8: 67–113.

Holash JA, Harik SI, Perry G, and Stewart PA (1993) Barrier properties of testis microvessels. Proc Natl Acad Sci USA 90: 11069–11073.[Abstract/Free Full Text]

Huisman MT, Smit JW, Wiltshire HR, Hoetelmans RMW, Beijnen JH, and Schinkel AH (2001) P-glycoprotein limits oral availability, brain and fetal penetration of saquinavir even with high doses of ritonavir. Mol Pharmacol 59: 806–813.[Abstract/Free Full Text]

Hyafil F, Vergely C, Du Vignaud P, and Grand-Perret T (1993) In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Res 53: 4595–4602.[Abstract/Free Full Text]

Jordan WC (1998) The effectiveness of combined saquinavir and ketoconazole treatment in reducing HIV viral load. J Natl Med Assoc 90: 622–624.[Medline]

Juliano RL and Ling V (1976) A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455: 152–162.[Medline]

Kaufmann GR, Duncombe C, Cunningham P, Beveridge A, Carr A, Sayer D, French M, and Cooper DA (1998) Treatment response and durability of a double protease inhibitor therapy with saquinavir and ritonavir in an observational cohort of HIV-1-infected individuals. AIDS 12: 1625–1630.[CrossRef][Medline]

Kim AE, Dintaman JM, Waddell DS, and Silverman JA (1998a) Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein. J Pharmacol Exp Ther 286: 1439–1445.[Abstract/Free Full Text]

Kim RB, Fromm MF, Wandel C, Leake B, Wood AJ, Roden DM, and Wilkinson GR (1998b) The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Investig 101: 289–294.[Medline]

Koudriakova T, Iatsimirskaia E, Utkin I, Gangl E, Vouros P, Storozhuk E, Orza D, Marinina J, and Gerber N (1998) Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human intestinal microsomes and expressed cytochrome P4503A4/3A5: mechanism-based inactivation of cytochrome P4503A by ritonavir. Drug Metab Dispos 26: 552–561.[Abstract/Free Full Text]

Malingré MM, Beijnen JH, Rosing H, Koopman FJ, Jewell RC, Paul EM, Huinink WW, and Schellens JH (2001) Coadministration of GF120918 significantly increases the systemic exposure to oral paclitaxel in cancer patients. Br J Cancer 84: 42–47.

Meaden ER, Hoggard PG, Newton P, Tjia JF, Aldam D, Cornforth D, Lloyd J, Williams I, Back DJ, and Khoo SH (2002) P-Glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals. J Antimicrob Chemother 50: 583–588.[Abstract/Free Full Text]

Perry CM and Noble S (1998) Saquinavir soft-gel capsule formulation. A review of its use in patients with HIV infection. Drugs 55: 461–486.[CrossRef][Medline]

Polli JW, Jarrett JL, Studenberg SD, Humphreys JE, Dennis SW, Brouwer KR, and Woolley JL (1999) Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor. Pharm Res (NY) 16: 1206–1212.[CrossRef][Medline]

Schinkel AH, Mayer U, Wagenaar E, Mol CAAM, van Deemter L, Smit JJ, van der Valk MA, Voordouw AC, Spits H, van Tellingen O, et al. (1997) Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci USA 94: 4028–4033.[Abstract/Free Full Text]

Schinkel AH, Smit JJ, van Tellingen O, Beijnen JH, Wagenaar E, van Deemter L, Mol CAAM, van der Valk MA, Robanus-Maandag EC, and te Riele HP (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77: 491–502.[CrossRef][Medline]

Smit JW, Huisman MT, van Tellingen O, Wiltshire HR, and Schinkel AH (1999) Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure. J Clin Investig 104: 1441–1447.[Medline]

Sparreboom A, van Asperen J, Mayer U, Schinkel AH, Smit JW, Meijer DKF, Borst P, Nooijen WJ, Beijnen JH, and van Tellingen O (1997) Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc Natl Acad Sci USA 94: 2031–2035.[Abstract/Free Full Text]

Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, and Willingham MC (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 84: 7735–7738.[Abstract/Free Full Text]

Van der Sandt I, Vos CM, Nabulsi L, Blom-Roosemalen MC, Voorwinden HH, de Boer AG, and Breimer DD (2001) Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood-brain barrier. AIDS 15: 483–491.[CrossRef][Medline]

Van Heeswijk RPG, Hoetelmans RMW, Harms R, Meenhorst PL, Mulder JW, Lange JM, and Beijnen JH (1998) Simultaneous quantitative determination of the HIV protease inhibitors amprenavir, indinavir, nelfinavir, ritonavir and saquinavir in human plasma by ion-pair high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed Sci Appl 719: 159–168.[CrossRef][Medline]

Wandel C, Kim RB, Kajiji S, Guengerich P, Wilkinson GR, and Wood AJ (1999) P-Glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies. Cancer Res 59: 3944–3948.[Abstract/Free Full Text]

Washington CB, Wiltshire HR, Man M, Moy T, Harris SR, Worth E, Weigl P, Liang Z, Hall D, Marriott L, and Blaschke TF (2000) The disposition of saquinavir in normal and P-glycoprotein-deficient mice, rats and in cultured cells. Drug Metab Dispos 28: 1058–1062.[Abstract/Free Full Text]

Williams GC and Sinko PJ (1999) Oral absorption of the HIV protease inhibitors: a current update. Adv Drug Deliv Rev 39: 211–238.[CrossRef][Medline]

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