Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes

Toxicology in Vitro 23 (2009) 185–193

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Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes N. Treijtel a,*, J.C.H. van Eijkeren b, S. Nijmeijer a, I.C.J. de Greef-van der Sandt c, A.P. Freidig d a

Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands National Institute for Public Health and the Environment, RIVM, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands c Kinesis Pharma, Lage Mosten 29, 4822 NK Breda, The Netherlands d BU Biosciences, TNO Quality of Life , P.O. Box 360, 3700 AJ Zeist, The Netherlands b

a r t i c l e

i n f o

Article history: Received 27 December 2007 Accepted 6 November 2008 Available online 13 November 2008 Keywords: HIV-protease inhibitors Sandwich-culture Rat hepatocytes, rat liver microsomes P-Glycoprotein Metabolism

a b s t r a c t The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich-culture and 14 different metabolites in microsomes. Ketoconazole did not affect the metabolism of ritonavir in sandwich-culture or microsomes and slightly inhibited the metabolism of saquinavir in sandwich-culture. This inhibition resulted in a different metabolite profile for saquinavir in microsomes. Ritonavir had a pronounced inhibiting effect on the metabolism of saquinavir and affected the hydroxylation of 6b-testosterone negatively. In the active transport studies, cyclosporin A and PSC833 enhanced the metabolism of ritonavir, suggesting that ritonavir is normally excreted into the bile canaliculi. Verapamil, showed no effect on the metabolism of ritonavir. The intrinsic clearance was estimated at 1.65 and 67.5 ll/min/1  106 cells and the hepatic metabolism clearance at 0.017 and 6.83 ml/min/SRW for ritonavir and saquinavir respectively. In conclusion, for saquinavir the metabolism rate and the amount of metabolites produced was higher than for ritonavir. Ritonavir had a strong inhibitory effect on the metabolism of saquinavir and seemed to be excreted into the bile. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Ritonavir and saquinavir are frequently used HIV-protease inhibitors (HIV-PI’s) in the treatment of AIDS. HIV-protease is an enzyme required for the proteolytic cleavage of viral polyprotein precursors into individual functional proteins found in infectious HIV. Inhibition of HIV protease prevents the assembly and maturation of infectious virions, resulting in the formation of non-infectious viral progeny and effectively blocking HIV replication (Markowitz et al., 1995). Abbreviations: BSA, bovine serum albumine; CLbile, biliary clearance (ml/min/kg bodyweight); CLhep, hepatic clearance (ml/min/kg bodyweight); CLint, intrinsic clearance (ml/min); CsA, cyclosporin A; DMEM, Dulbecco’s modified eagle’s medium; FCS, foetal calf serum; HPLC, high performance liquid chromatography; Km, Michaelis-Menten constant lM; MRP, multi drug resistance related protein; NADPH, nicotinamide adenine dinucleotide phosphate; PBS, phosphate-buffered saline; Pgp, P-glycoprotein; Qh, hepatic blood flow (L/h); S.D., standard deviation; SRW, standard rat bodyweight (250 g); Vmax, maximum rat of metabolism (lM/ min). * Corresponding author. Address: IRAS, Yalelaan 2, 3584 CM Utrecht, The Netherlands. Tel.: +31 30 2535400. E-mail address: [email protected] (N. Treijtel). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.11.001

High plasma levels of HIV-protease inhibitors are necessary to prevent the formation of resistant virus particles (Molla et al., 1996). However, most HIV-PI’s and especially saquinavir have a limited oral bioavailability, low area under the plasma concentration-time curve (AUC) values, and short plasma half-lives (Williams and Sinko, 1999). The unfavourable pharmacokinetics is mostly due to poor absorption, fast metabolism (except for ritonavir) and hepatobiliary elimination. Most HIV-PI’s are extensively metabolised by CYP3A (Kumar et al., 1996; Fitzsimmons and Collins, 1997; Koudriakova et al., 1998; Eagling et al., 2002). In addition, the drug transporter protein P-glycoprotein also plays a role in the limited oral absorption and biliary excretion (Alsenz et al., 1998; Kim et al., 1998b; Williams and Sinko, 1999). Therefore, HIV-PI’s needed to be taken in frequent and high doses in the past (Perry and Noble, 1998; Cameron et al., 1999). Nowadays, HIV-PI’s are usually given in combination with a low dose of ritonavir. Ritonavir is a potent inhibitor of CYP3A (von Moltke et al., 2000). When ritonavir is co-administered with other HIV-PI’s such as saquinavir, it serves to enhance their plasma concentration and prolongs drug residence in the circulation (Hsu et al., 1998a; Barry et al., 1999). Thus, potent antiviral effects can be achieved with

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lower doses of each protease inhibitor and with a less frequent dosing regimen. As the pharmacokinetics of these compounds play a major role in the management of HIV, it is important to study their metabolism as well as interaction with other HIV-PI’s or drugs on a cellular basis. An appropriate system to study this is by using sandwichcultures of rat hepatocytes. In sandwich-cultured rat hepatocytes, the hepatocytes are plated between two layers of collagen thereby maintaining enzyme activities and the expression of drug transporter proteins (Koebe et al., 1994; Beken et al., 1997b; Liu et al., 1999). Furthermore, the hepatocytes form bile canaliculi in which the substrates of the transporters are excreted and accumulated. The sandwich-culture thus contains the most important pathways involved in the elimination of HIV-PI’s and should therefore be investigated for its suitability in studying the hepatic first-pass effects of these compounds in vitro. In addition, it is possible to estimate an in vitro clearance in the sandwich-culture when using substrate depletion data. This in vitro clearance can be extrapolated to the in vivo clearance (Treijtel et al., 2004). The results of such thorough in vitro studies could make a considerable contribution to the design of new therapies. The aim of this study was to show the suitability of the sandwich system to study complicated processes underlying the clearance of these compounds. For this purpose, metabolic patterns of the HIV-PI’s in sandwich-cultured rat hepatocytes were compared to metabolic patterns in rat microsomes and to in vivo data. The parent compound depletion data of the sandwich-culture were used to estimate the in vitro intrinsic clearance and were extrapolated to the in vivo hepatic clearance. In addition, the pharmacokinetic interaction of ketoconazole, which is a reference CYP3A inhibitor (Eagling et al., 1998; von Moltke et al., 2000) with saquinavir and ritonavir was studied. The inhibitory effect of ritonavir on the clearance of saquinavir was studied as well. Finally, the influence of drug transporter inhibitors on the metabolism of ritonavir was investigated. 2. Materials and Methods 2.1. Chemicals Fetal calf serum (FCS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco BRL (Breda, The Netherlands). Insulin, glucagon, hydrocortison, gentamycin, bovine serum albumin (BSA), collagenase type 1A, testosterone and its metabolites, ketoconazole, cyclosporin A and verapamil were obtained from Sigma Chemical Co. (St. Louis MO, USA). Collagenase type B was purchased from Boehringer (Mannheim, Germany). Ritonavir, 3HRitonavir, and 3H-Saquinavir with a specific activity of 1.6 Ci/mmol and 1.0 Ci/mmol respectively were obtained from Moravek Biochemicals (Brea CA, USA). Saquinavir mesylate was kindly provided by F. Hoffmann-La Roche Ltd (Basel, Switzerland) and PSC833 was a kind gift from Novartis Pharma AG (Basel, Switzerland). All other chemicals and reagents were of analytical grade.

were used for hepatocyte isolation. Acidified water and food was provided ad libitum before the liver perfusion. The rats were anesthetised by injecting pentobarbital i.p. (NembutalÒ, 120 mg/kg bodyweight). Rat hepatocytes were isolated using the two-step collagenase perfusion technique as described by Seglen (1976) and modified according to Paine (1979). 2.4. Hepatocyte culture The cells were seeded on Ø 60 mm tissue culture dishes, precoated with 0.25 ml of collagen, at a density of 5  106 cells/ dish in 4 ml of DMEM. DMEM was supplemented with 10% fetal calf serum (FCS), 0.5 U/ml insulin, 0.007 lg/ml glucagon, 7.5 lg/ml of hydrocortisone, 50 lg/ml gentamycin and 1 lM of dexamethasone. After 4 h of incubation, cultures were washed twice with DMEM (without FCS) and 0.25 ml of collagen was added as a top layer on the dishes. Cultures were placed in the incubator (37 °C, 5% CO2) for 1 h to allow gellation. Subsequently, 4 ml of DMEM (without FCS) was added. Medium was refreshed every day. 2.5. Cell viability Morphology of hepatocytes was checked daily under the microscope and cell viability was determined by trypan blue exclusion. 2.6. Enzyme activities Testosterone hydroxylation activity was determined directly and 2, 4, 5, and 6 days after isolation according to the method of Wortelboer et al. (1990) in triple. Testosterone hydroxylation was also determined on day 5 and 6 after incubation with 1 lM ritonavir or saquinavir on day 4. After 30 min of incubation with testosterone but without the ritonavir or saquinavir, both medium and sandwich were analysed separately for parent compound and metabolites. Cytosolic protein was determined separately in three separate culture dishes measured according to Lowry et al. (1951) using BSA as a standard. Mean hydroxylation values were divided by mean protein values. 2.7. Kinetic profiles in the sandwich rat hepatocyte culture 4 days after hepatocyte isolation, sandwich-cultures were incubated with 4 ml of 1 lM 3H-labeled-ritonavir or 3H-labeled-saquinavir in DMEM with supplements (without FCS). 3H-labeled ritonavir was mixed with non-labelled substrate in a ratio of 2:1 to yield a concentration of 1 lM and 1.07 Ci/mmol. After 0, 2, 5, 15, 30, 60 min and 4, 12, 24, and 48 h of incubation, a culture was sacrificed. The medium was transferred to a glass centrifuge tube. One millilitre of ice-cold PBS was added to the sandwich-culture and the total of PBS, collagen and hepatocytes was transferred to a glass centrifuge tube as well. All the tubes were ice-cold and stored at 20 °C immediately after harvesting. The experiments were carried out in duplicate.

2.2. Preparation of rat tail tendon collagen Type I collagen was prepared from rat tail tendons as described by Koebe et al. (1994) by a modified procedure of Elsdale and Bard (1972). This preparation yields type I collagen, mostly in its native, not cross-linked, triple-helical form (Beken et al., 1997a). 2.3. Isolation of hepatocytes Male Wistar rats (CPB:uWU) from the Central Animal Laboratory (Utrecht University, The Netherlands), weighing 190–250 g

2.8. Inhibition studies Sandwich-cultures were pre-incubated with 2 ml of 10 lM of ketoconazole in DMEM with supplements for 30 min in an incubator. Hereafter, 2 ml medium containing 2 lM of ritonavir (in a mixture of 3H-labeled and non-labelled as described above) or 2 lM of 3H-labeled saquinavir, was added to yield concentrations of 5 lM of ketoconazole and 1 lM ritonavir or saquinavir. After 12 and 24 h medium and sandwich were sampled as described above.

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2.9. Ritonavir metabolism in combination with drug transporter inhibitors Sandwich-cultures were incubated with 1 lM 3H-labeled ritonavir (in a mixture as described above) in combination with 20, 10 and 1 lM of verapamil, cyclosporin A and PSC833 respectively. After 24 h medium and sandwich were sampled as described above. 2.10. Interaction study with ritonavir and saquinavir To determine the inhibitory effect of ritonavir on saquinavir metabolism, sandwich-cultures were pre-incubated with 2 ml of medium containing 2 lM ritonavir for 30 min in an incubator. Hereafter, 2 ml medium (DMEM) containing 2 lM of 3H-labeled saquinavir was added to the sandwich-cultures. Medium and sandwich were sampled as described above after 4, 12, and 24 h. 2.11. Microsomal incubation Metabolism of ritonavir and saquinavir was studied in rat microsomes. A rat liver was perfused with ice-cold saline and homogenised in 0.15 M KCl containing 0.1 mM EDTA using a Potter-Elvhejem glass-Teflon homogeniser. Microsomes were prepared by centrifugation (2  20 min, 9000g; supernatant 60 min, 105,000g). The microsomal pellet was resuspended in sodiumphosphate buffer (0.1 M, pH 7.8) containing 0.1 mM EDTA and frozen quickly in liquid N2 stored at 70 °C. Microsomal protein content was determined by the method of Lowry et al. (1951). Determinations were carried out at 37 °C in 5 ml incubation mixture consisting of potassium-phosphate buffer (0.1 M, pH 7.4), containing 500 ll microsomes (2.0 mg/ml final concentration), 5 ll ritonavir (1 lM, in a mixture of 3H-labeled and nonlabelled as described above) or 1 lM of 3H-labeled saquinavir. The reaction was started with the addition of 50 ll NADPH (100 mM). The reactions were terminated after 60 min by the addition of 8 ml of a mixture consisting of dieethylether/dichloromethane/iso-propanol in a 60:40:1 ratio (v/v). Incubations without NADPH served as the controls. For inhibition studies, microsomes were pre-incubated for ten minutes with 10 lM of ketoconazole or 2 lM of ritonavir. The reaction was started after addition of 2 lM of substrate (ritonavir or saquinavir) and 50 ll NADPH (100 mM) to yield a concentration of 5 lM ketoconazole or 1 lM ritonavir and a substrate concentration of 1 lM. 2.12. Sample analysis Samples were analysed for parent compound (ritonavir or saquinavir) and metabolites by means of a slightly modified HPLC method according to Denissen et al. (1997). In short, the sandwich samples (1.5 ml) were thawed and 1.5 ml of a collagenase-Krebs solution (100 U/ml) was added. The samples were incubated at 37 °C for 60 min after which all collagen was digested. Subsequently, the sandwich samples and the medium samples were extracted with 8 ml of a mixture consisting of dieethylether/ dichloromethane/iso-propanol in a 60:40:1 ratio (v/v). 7 ml of the organic layer was transferred to a clean centrifuge tube and evaporated. The residue was dissolved in 100 ll of 50% acetonitrile in 0.1% trifluoroacetic acid (adjusted to pH 4.8 with ammonium acetate). Samples (20 ll) were separated using an Alltima C18 5 lm (250  3.2 mm) column at 40 °C and profiled on a HPLC system (Agilent, Palo Alto, CA, USA) with a UV and online RA-detector (A515 flow scintillation detector). A linear gradient of 25–60% acetonitrile in 0.1% trifluoroacetic acid (adjusted to pH 4.8 with

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ammonium acetate) over 50 min was used as column eluent at a flow rate of 0.16 ml/min. UV-absorption was measured at 230 nm. Total radioactivity was measured on a Minaxi Tri-carb 4000 liquid scintillation counter (Packard Bioscience Co., Meriden, CT, USA) using Ultima GOLD scintillation liquid (Packard Bioscience Co., Meridin, CT, USA). 2.13. Mathematical model To calculate the intrinsic clearance of ritonavir and saquinavir from the sandwich-culture data a mathematical model was used. For a more extensive description of this mathematical model we refer to Treijtel et al (2004, 2005). In short, three processes determine the time course of a parent compound in the medium-sandwich experimental model: transport by molecular diffusion partitioning and biotransformation. The initial decrease from medium is mostly due to diffusion from medium to sandwich, resulting in a corresponding initial increase in sandwich. This initial phase is followed by a terminal phase of decrease in both medium and sandwich due to metabolism. The model calculations for all three processes were performed using the result of fitting the model to the data of depletion both in medium and sandwich. In the model, the clearance per unit volume of hepatocytes is parameterised, which allows for a straightforward in vitro–in vivo extrapolation to the liver intrinsic clearance once the relative volume of hepatocytes in the liver is known. To account for the decrease in cell number, cell viability and biotransformation capacity in vitro, the true clearance parameter is corrected by multiplying with the corresponding fractions. 2.14. In vitro–in vivo extrapolation For ritonavir and saquinavir, the intrinsic hepatocyte clearance was extrapolated to the intrinsic liver clearance by accounting for the volume fraction of hepatocytes in the rat liver, which was assumed to be about 90%. Next, this estimate of the intrinsic liver clearance was used for an estimation of the hepatic clearance of the compounds, using the well-stirred liver model (Wilkinson, 1987). The well-stirred liver model assumes instantaneous and complete mixing and reads

CLh ¼

Q h fu CLint Q h þ fu CLint

Here, CLh is the hepatic clearance from blood, Qh is the hepatic blood flow and fu is the free fraction in blood.

3. Results 3.1. Culture characteristics Cell viability was determined using trypan blue exclusion. After 4, 5 and 6 days of isolation viability yielded 80%. Protein content decreased to 55% at day 4, 5 and 6. Testosterone hydroxylation on several positions, representing a variety of cytochrome P450 enzyme activity, was measured in freshly isolated hepatocytes and after 2, 4, 5, and 6 days in culture. Enzyme activity of cytochrome P4503A, which is the main enzyme involved in the metabolism of ritonavir and saquinavir, decreased to 34% of its value on day 4, 5, and 6, compared to freshly isolated hepatoyctes. Other enzyme activities decreased in a similar manner as was described earlier (Treijtel et al., 2004). Over this time period sandwich-cultured rat hepatocytes were used for studies on ritonavir and saquinavir metabolism.

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3.2. Inhibition of CYP3A enzyme activity CYP3A4 enzyme activity in sandwich-cultures was also measured after incubation with ritonavir or saquinavir on day 4 exactly as in the metabolism studies. Cultures that were not incubated with these drugs were used as controls. Formation rate of 6b-hydroxytestosterone, representing the activity of the cytochrome P4503A enzyme, is shown in Fig. 1. There is a significant decrease of enzyme activity after incubation with ritonavir on day 5 and 6 after isolation. For saquinavir no difference in 6b-hydroxytestosterone formation was observed compared to the control situation. 3.3. Metabolite profiles in sandwich-cultured rat hepatoyctes

formation (pmol/min/mg protein)

After incubation of 1 lM ritonavir for 48 h in sandwich-cultured rat hepatocytes, 4 metabolites could be distinguished. In Fig. 2A a

control

400

ritonavir

300

saquinavir

200 100 0

0

2

4 time (days)

5

6

Fig. 1. Formation of 6b-testosterone metabolite in sandwich-cultured rat hepatoyctes after incubation with ritonavir or saquinavir on day 4 after isolation. Controls were not incubated with ritonavir or saquinavir (mean ± S.D., n = 3).

A

representative RA-chromatogram of ritonavir in medium is shown. Metabolites are indicated as R1–R4 in order of their retention time. In sandwich-cultured rat hepatocytes that were incubated with 1 lM of saquinavir for 4 h, eight metabolites could be distinguished. These metabolites are indicated as S1–S8 in order of their retention time. Fig. 2B shows a representative RA-chromatogram of saquinavir and its metabolites. 3.4. Inhibition of metabolism in sandwich-cultured rat hepatocytes After 24 h incubation with ritonavir in combination with 5 lM of ketoconazole almost no inhibiting effect was observed in sandwich-cultured rat hepatocytes. Results are shown in Fig. 3A in which the amount of ritonavir and metabolites present in the culture system are expressed as percentage of the total amount present (100%). As the R2 metabolite could not be detected after incubation with ketoconazole, its formation seems to be inhibited. The amount of ritonavir in the culture was higher compared to the control situation. The amount of the R3 metabolite formed increased slightly. Results of saquinavir metabolism inhibition are shown in Fig. 3B. The amount of saquinavir and metabolites present in the culture system are expressed as percentage of the total amount present. After 12 h of incubation with saquinavir in combination with 5 lM of ketoconazole the amount of saquinavir present was higher in sandwich-cultured rat hepatoyctes. This increase was more pronounced when saquinavir was incubated for 12 h in combination with 1 lM of ritonavir. A slight decrease in the amount of metabolites formed could be observed after incubation with ketoconazole, except for S7 which showed a strong increase and S8 where no difference could be observed. After inhibition with ritonavir this decrease was more pro-

B

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4000.0

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Fig. 2. Representative RA-chromatograms presenting metabolite profiles of ritonavir (A) and saquinavir (B) in sandwich-cultured rat hepatoyctes. Cultures were incubated with 1 lM of ritonavir for 48 h and 1 lM saquinavir for 4 h.

A

B 80%

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Saq

Fig. 3. Inhibition of ritonavir (A) and saquinavir (B) metabolism by ketoconazole or ritonavir after 12 h of incubation in sandwich-cultured rat hepatocytes. Data are expressed as a percentage of the total amount present (n = 2). Metabolites are referred to as in order of their retention time (see Fig. 2). Metabolites R1 and R4 were not detected at 12 h.

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nounced. Except for the S7 metabolite for which a relevant increase was observed. 3.5. Ritonavir metabolism in combination with drug transporter inhibitors To study the influence of the drug transporter inhibitors verapamil, cyclosporin A and PSC833 on the metabolism of ritonavir, sandwich-cultures were incubated with ritonavir in combination with these inhibitors for 24 h. The percentage of ritonavir that was still present in the culture after 24 h was measured. In the control situation without inhibitor, 66% of ritonavir was still present in the sandwich-culture. After co-incubation with verapamil the percentage of ritonavir present was 70%. After co-incubation of ritonavir with cyclosporin A and PSC833 this percentage decreased to 39% and 33% respectively. 3.6. Metabolite profiles in rat microsomes When rat liver microsomes were incubated with 1 lM ritonavir for 1 h, the metabolite profile obtained was similar to the metabo-

A

lite profile found in sandwich-cultured rat hepatocytes after a 48 h incubation period. When rat liver microsomes were incubated with 1 lM ritonavir in combination with 5 lM ketoconazole, no difference could be observed compared to the incubation without ketoconazole. Metabolites are indicated as R1–R4 in order of their retention time. Representative RA-chromatograms are shown in Fig. 4A for ritonavir alone and in Fig. 4B for ritonavir in combination with ketoconazole. When rat liver microsomes were incubated with 1 lM saquinavir for 1 h, the metabolite profile obtained was different compared to the metabolite profile found in sandwich-cultured rat hepatocytes after a 4 h incubation period. In Fig. 5A a representative RA-chromatogram is shown in which more than 14 different metabolites could be distinguished instead of eight for the sandwich-cultured rat hepatocytes. The metabolites formed in microsomes could be observed at different retention times than in sandwich-cultured hepatocytes. Metabolites in microsomes are therefore numbered from Sm1 to Sm14 in order of their retention times. Only the metabolite Sm13 had the same retention time as the metabolite S1 that was found in sandwich-cultured rat hepatocytes.

B 120000.0

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80000.0 60000.0 40000.0 20000.0 0.0 0:00

R1R2

20:00

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40:00

time (min)

60:00

Fig. 4. Representative RA-chromatograms presenting metabolite profiles of ritonavir (A) and ritonavir in combination with ketoconazole (B) in rat microsomes. Microsomes were incubated for 1 h with 1 lM of ritonavir alone or in combination with 5 lM ketoconazole.

A

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C

Fig. 5. Representative RA-chromatograms of saquinavir (A), saquinavir in combination with ketoconazole (B) and saquinavir in combination with ritonavir (C) and their metabolite patterns. Rat microsomes were incubated for 1 h with 1 lM of each compound.

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When rat liver microsomes were incubated with 1 lM saquinavir in combination with 5 lM ketoconazole (Fig. 5B), there was a clear inhibiting effect on the metabolism of saquinavir. 20% of saquinavir was still present after the incubation with ketoconazole in contrast to 5% for the control situation. Furthermore, the metabolite profile was completely different after incubation with ketoconazole. Only the metabolites Sm11–Sm14 were recovered. When rat liver microsomes were incubated with 1 lM saquinavir in combination with 1 lM ritonavir (Fig. 5C), there was an overall inhibition of saquinavir metabolism. 92% of saquinavir was still present after the incubation with ritonavir in contrast with 5% for the control situation.

ume (3.4%) were taken from Brown et al. (1997). Standard rat weight is 250 g and the tissues specific density was taken to be 1. The free fraction of 0.01 for ritonavir was taken from Denissen et al. (1997) and the free fraction 0.15 for saquinavir was taken from Shibata et al. (2002). Thus, for ritonavir the hepatic clearance was estimated to be 0.017 ml/min/SRW. For saquinavir the hepatic clearance was estimated to be 6.8 ml/min/SRW. 4. Discussion In the present study, several aspects of the pharmacokinetics of the HIV-protease inhibitors ritonavir and saquinavir were studied. Metabolic profiles, clearance rates, metabolic inhibition and the influence of drug transporter inhibitors were investigated and the results obtained show that the sandwich system is suitable for studying complicated processes underlying the clearance of these compounds in vitro. Ritonavir and saquinavir were used in a concentration of 1 lM. As this concentration was below the Michaelis-Menten constant Km in rat liver microsomes (Yamaji et al., 1999), metabolism was assumed to be linear. This concentration was also still in the range of clinical relevance since maximal plasma concentrations are about 15 lM and 3.7 lM for ritonavir and saquinavir when taking 600 mg twice a day and 1200 mg three times a day, respectively (Hsu et al., 1998b). After 48 h of incubation, results from the metabolite profile of ritonavir could be compared to profiles obtained in rat bile by Denissen et al. (1997). As we used exactly the same HPLC method, we were able to compare the retention times of the metabolites formed. The R1 metabolite was assumed to be the M-11/12 metabolites, the retention time of the R2 metabolite from our study was comparable to the M-2 metabolite, the R3 metabolite corresponded with the M-9 metabolite and the R4 metabolite was assumed to be the M-1 metabolite. The structures are shown in Fig. 7. As these metabolites were the major metabolites detected by Denissen et al. (1997), the metabolite profile obtained from our study seems to have a close resemblance to the metabolite profile in the in vivo situation. Based on retention times, the same metabolites could be observed after a microsomal incubation with ritonavir. This indicates that there was no difference between hepatocyte and microsomal metabolism. After incubation of saquinavir in sandwich-cultures, all the metabolites were detected after 60 min, indicating very fast metabolism. Eight metabolites could be distinguished. After a microsomal incubation with saquinavir, more than 14 metabolites

3.7. In vitro intrinsic clearance The experimental data on depletion of ritonavir and saquinavir in medium and sandwich are shown in Fig. 6A and B, respectively, together with the corresponding concentration-time course model calculations (log scale was used for the time axis for a better resolution of the dense sample scheme during the initial phase of the experiment). The initial decrease from medium is mostly due to diffusion from medium to sandwich, resulting in a corresponding initial increase in sandwich. This initial phase is followed by a terminal phase of decrease in both medium and sandwich due to metabolism. The model calculations were performed using the result of fitting the model to the data of depletion both in medium and sandwich. For ritonavir this resulted in a value for the intrinsic clearance per unit of hepatocyte volume of 0.22 [–/min], a value for the hepatocyte-collagen partition coefficient of 30, and a value for the diffusion coefficient of 7.9  102 cm2/min. For saquinavir this resulted in a value for the intrinsic clearance per unit of hepatocyte volume of 9.0 [–/min], a value for the hepatocyte-collagen partition coefficient of 62, and a value for the diffusion coefficient of 7.3  102 cm2/min. 3.8. In vitro–in vivo extrapolation The in vitro intrinsic hepatocyte clearance of ritonavir and saquinavir, that was estimated using sandwich-cultured rat hepatocytes, was extrapolated to the intrinsic liver clearance by accounting for the volume fraction of 90% of hepatoyctes in the rat liver (10% other cell types). Subsequently, this intrinsic clearance was extrapolated to the corresponding hepatic clearance from blood, using the well-stirred liver model. Rat cardiac output (110 ml/min), relative hepatic blood flow (18.3%), relative liver vol-

B

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Fig. 6. Concentration of ritonavir (A) and saquinavir (B) in the culture medium (solid line, closed circles) and sandwich (dashed line, open circles) over time. Model calculations are based on fitting the specific hepatic clearance, the hepatocyte to medium partition and the molecular diffusion coefficient to the corresponding data in medium and sandwich.

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Ritonavir

M-1

O N

S

N

CH3

O

H N

N H

N H

OH

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O

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S

N

S

N

N

CH3

N H

O

H N O

OH

N H

O

S N

OH OH

Denissen et al. 1997 Fig. 7. The molecular structures of ritonavir and its metabolites that were identified by Denissen et al. (1997). The retention times of these structures matched with the retention times of the molecules detected after a 48 h incubation period in sandwich-cultured rat hepatoyctes. M-1 corresponds to R4, M-2 corresponds to R2, M-9 corresponds to R3, and M-11/12 corresponds to R4 from Figs. 2A and 4.

could be observed, suggesting a difference in metabolism after incubation in hepatoyctes compared to microsomes. These results show that there can be a major difference in metabolism between different in vitro systems. Therefore, such a system should be chosen carefully and preferably resemble the in vivo situation as close as possible. To our knowledge, there were no literature data on saquinavir rat metabolites to compare our results. However, after incubation using human liver microsomes 11 metabolites could be distinguished (Eagling et al., 2002). None of the metabolites could be identified using literature data because of different retention times. Ritonavir is mainly metabolised by the cytochrome P4503A4 enzyme and to a lesser extent by cytochrome P4502D6 in humans (Kumar et al., 1996). According to Eagling et al (Eagling et al., 1999; Eagling et al., 2002), the cytochrome P4503A4 is the only metabolising enzyme for saquinavir in human liver microsomes. Kempf et al. (1997) suggested a major role of CYP3A2 in male rat liver microsomes. Ketoconazole is a potent inhibitor of cytochrome P4503A at low concentrations in human, based on reversible binding (Eagling et al., 1998; Dresser et al., 2000; von Moltke et al., 2000). In rat microsomes, ketoconazole has been shown an inhibitor of CYP3A as well as other enzymes. As ritonavir and saquinavir are mainly metabolised by CYP3A, a co-incubation with ketoconazole should have pronounced effects on the metabolism of both drugs. Nevertheless, for ritonavir no influence of ketoconazole could be observed, not in sandwich-culture or in microsomes. This finding was supported by Hsu et al., 1998a,b, who stated that ritonavir is minimally affected by CYP3A inhibitors because of its high affinity for the enzyme. Ketoconazole did show an effect on the metabolism of saquinavir. However, after microsomal incubation with

ketoconazole the metabolism changed quantitatively as well as qualitatively. Although several metabolites were detected, only 3 of the 14 microsomal metabolites from the control situation were recovered after inhibition with ketoconazole. This might suggest the involvement of other enzymes in the metabolism of saquinavir when CYP3A is inhibited. Besides a substrate for CYP3A, ritonavir also proved to be a potent inhibitor of this enzyme and to a lesser extent of the human CYP2D6 enzyme (Eagling et al., 1997; von Moltke et al., 1998). The exact mechanism of inhibition of ritonavir on these enzymes is not completely elucidated yet. It is mentioned that the inhibiting mechanism is purely by reversible binding of ritonavir to the enzyme (Hsu et al., 1998a), but others have suggested a mechanism-based inhibition of the enzyme (Koudriakova et al., 1998; von Moltke et al., 2000). When ritonavir was used as an inhibitor for the metabolism of saquinavir, a very pronounced inhibiting effect was observed. Almost no saquinavir was metabolised in the presence of ritonavir. Hardly any metabolites were formed except for the S7 metabolite that changed from a minor metabolite in the normal situation to major metabolite after inhibition with ritonavir. The fact that this inhibitory effect of ritonavir is so much stronger than the inhibitory effect of ketoconazole in both sandwichculture and microsomes, suggest that ritonavir can also inhibit other enzymes besides CYP3A. The inhibitory effect of ritonavir was also studied measuring testosterone hydroxylation after 1 and 2 days of incubation in sandwich-culture. A significant effect of ritonavir on the 6bhydroxylation of testosterone could be observed compared to the control situation, indicating the involvement of an inhibiting effect on CYP3A. Saquinavir did not show an inhibitory effect on CYP3A;

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no difference in testosterone hydroxylation could be observed compared to the control situation. These studies confirm the auto-inhibitory properties of ritonavir towards its own metabolism (Hsu et al., 1998b; McConn and Zhao, 2004). Besides biotransformation, ritonavir is also eliminated by biliary excretion. Denissen et al. (1997), showed a biliary elimination of 85.5% in the male rat. Ritonavir is excreted into the bile by the drug transporter protein P-glycoprotein and to a lesser extent by mrp2 (Lee et al., 1998; Meaden et al., 2002). In the sandwich-culture, these drug transporters are present and substrates are excreted into the bile canaliculi which are formed after 4 days in culture. When ritonavir was incubated with the P-glycoprotein inhibitors cyclosporin A and PSC833 metabolism was increased whereas verapamil had no effect on the metabolism of ritonavir. It was hypothesised that this increase was due to an inhibition of the P-glycoprotein efflux pump, thus increasing the amount of ritonavir available for metabolism. It should be noted here that cyclosporin A can also act as a CYP3A inhibitor (Wu et al., 1995; Dresser et al., 2000) but apparently to a lesser extent than for P-glycoprotein. Although this experiment was not performed for saquinavir, similar results are expected as saquinavir is a mrp2 substrate as well as a P-glycoprotein substrate (Kim et al., 1998a; Lee et al., 1998; Meaden et al., 2002; Su et al., 2004). Intrinsic clearance was estimated for both ritonavir and saquinavir using a substrate depletion approach over 48 h. A mathematical model was developed to estimate a true intrinsic clearance from these data that is devoid of system dependent parameters such as protein binding, hepatocyte-medium partition and molecular diffusion of compounds. Thus the estimated in vitro intrinsic clearance can easily be extrapolated to the in vivo situation. This approach already proved to be useful in estimating the intrinsic clearance of tolbutamide, warfarin and 7-ethoxycoumarin (Treijtel et al., 2004; Treijtel et al., 2005). An intrinsic clearance of 1.65 ll/min/1  106 cells and 67.5 ll/ min/1  106 cells leading to an in vivo hepatic clearance of 0.0168 and 6.83 ml/min/SRW was estimated for ritonavir and saquinavir, respectively. For saquinavir, the intrinsic clearance value was similar to the intrinsic clearance determined by Yamaji et al. (1999) of 170.9 ll/min/mg microsomal protein considering a CYP content in hepatocytes of 0.14 nmol CYP/106 cells and 0.32 nmol CYP/mg microsomal protein in humans (Iwatsubo et al., 1997). The hepatic clearance was comparable to the in vivo value obtained by Shibata et al. (Shibata et al., 2002) of 8.63 ml/ min/300 g which equals 7.19 ml/min/SRW. For ritonavir, there were no intrinsic clearance data in rat available to compare with. However, the metabolic clearance of saquinavir is known to be much higher than for ritonavir (Yamaji et al., 1999; Shibata et al., 2002). Finally, as we have suggested that biliary excretion also plays a significant role in the elimination of these compounds in the sandwich-culture, the values of the metabolic clearance that were estimated here, could be an under prediction of the overall hepatic in vivo clearance. The clearance of ritonavir by the hepatobiliary path reported by Denissen et al. (1997) seems almost as efficient as the clearance of saquinavir reported in Shibata et al. (2002). In this study, the complex pharmacokinetics and drug–drug interactions of HIV-PI’s were confirmed. This complexity emphasises the necessity to further investigate the processes that play a role both in the metabolism and the biliary excretion of these compounds and the possibility of regulation of these processes. As shown in this study sandwich-cultures of rat hepatocytes possess properties that play an important role in the clearance of these drugs (i.e. cell viability, enzyme activity and biliary excretion), and thus proved to be a very suitable system for these kinds of studies in vitro.

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