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Saquinavir pharmacokinetics alone and in combination with ritonavir in HIV-infected patients

Merry, Concepta1,2; Barry, Michael G.1,3; Mulcahy, Fiona2; Ryan, Mairin2; Heavey, Jane2; Tjia, John F.1; Gibbons, Sara E.1; Breckenridge, Alasdair M.1; Back, David J.1

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In 1996, treatment with zidovudine (ZDV) plus didanosine (ddI) or ZDV plus zalcitabine (ddC) was demonstrated to be more effective than ZDV monotherapy in preventing disease progression and reducing mortality in patients with HIV disease [1,2]. Further progress followed recent studies with three reverse transcriptase (RT) inhibitors or two RT inhibitors and a protease inhibitor producing greater antiviral activity with prolonged reductions of 2.0–3.0 log in plasma HIV RNA [3,4]. The HIV protease enzyme is responsible for post-translational processing of Gag and Gag–Pol polyprotein precursors into their functional units and inhibition of the enzyme results in the production of immature, non-infectious virions. Initial studies suggest that the protease inhibitors are very potent anti-HIV drugs with ritonavir (RIT) producing a 1.7-log reduction in plasma viraemia, which is similar to the combination of lamivudine (3TC) plus ZDV. As with the nucleoside analogues, viral resistance may develop with the protease inhibitors; for example, incidence is approximately 45% after 1 year of saquinavir (SQV) monotherapy [5]. Furthermore, cross-resistance to other protease inhibitors has been demonstrated following administration of indinavir suggesting initial therapy with indinavir may limit the benefit of subsequent treatment with others [6]. In vitro work demonstrates that isolates from patients treated with RIT may show decreased sensitivity to RIT but remain susceptible to SQV suggesting that combination of these two protease inhibitors may be more effective than monotherapy [7]. However, the administration of protease inhibitors in combination does raise important pharmacokinetic issues in particular clinically relevant drug interactions.

In vitro studies using human liver microsomes have demonstrated that RIT is a potent inhibitor of cytochrome P450 3A4 (CYP3A4) and to a lesser extent other cytochrome P450 isozymes [8]. SQV is exten-sively metabolised by CYP3A4, and in rats coadminis-tration of RIT increases the area under the concentration–time curve (AUC) of SQV by 36-fold [9]. Single and multiple dose studies in healthy volunteers have also shown that RIT greatly increases the AUC and maximum concentration (Cmax) of coadmin-istered SQV [10]. We have now determined the magnitude of the RIT–SQV interaction in the clinical setting and report the pharmacokinetics of SQV when administered alone and in combination with RIT in HIV-infected patients.



Seven male patients, aged 28–45 years, with sexually acquired HIV infection participated in this study. All patients had advanced HIV disease (Centers for Disease Control and Prevention 1993 criteria, stage IV) with a mean CD4 count of 25 × 10 6/l (range, 10–60 × 10 6/l). Current medications included co-trimoxazole 960 mg daily as prophylaxis for Pneumocystis carinii pneumonia. All patients were receiving triple therapy with ZDV 250 mg twice daily, 3TC 150 mg twice daily and SQV 600 mg three times daily. The mean duration of nucleoside therapy was 2.5 years. All patients were consid-ered for quadruple therapy with the addition of RIT to their current regimen. There was no evidence of significant hepatic (greater than twofold elevation of hepatic transaminases) or renal dysfunction and no medication known to interfere with SQV metabolism (except RIT) was prescribed during the study period. Approval for the study was obtained from the St James's Hospital Ethics Committee and each patient provided written informed consent.

On the first study day, patients attended at 0830 h after an overnight fast. An indwelling intravenous cannula was inserted in the cubital fossa to facilitate blood sampling. At 0900 h a fasting blood sample was taken (time 0 h). Patients ingested ZDV 250 mg, 3TC 150 mg and SQV 600 mg. Blood samples were then taken at 1, 2, 3, 4, 6 and 8 h post-dosing. Prior to the second study day 1 week later, patients had been treated with their usual medications (ZDV, 3TC, SQV) plus RIT 300 mg twice daily for 4 days. The procedure on study day 2 was identical to that of the first day, except for the addition of RIT 300 mg to ZDV, 3TC and SQV prior to sampling. All samples were centrifuged without delay and the separated plasma was heated to 58°C for at least 30 min to inactivate HIV. Plasma was then stored at −80°C until SQV analysis using high performance liquid chromatography (HPLC).

Drug analysis

SQV concentrations in plasma were determined as follows. Plasma samples in duplicate (0.2–0.5 ml, depending on the phase of the study) were pipetted into glass tubes, and sodium hydroxide (0.5 M; 0.3 ml) and internal standard (31–9564; Roche Products, Welwyn Garden City, Hertfordshire, UK) added. Tube contents were mixed thoroughly. Standard curves were prepared containing blank plasma and SQV at a concentration range of 20–400 or 500–8000 ng/ml. Quality control samples for the assessment of precision and accuracy of the assay were prepared by adding known quantities of SQV to blank plasma samples. Test samples, standards and quality control samples were extracted with diethyl ether (5 ml) for 10 min. After centrifuging for 5 min at 3000 rpm the organic phase was transferred to clean tubes and evaporated to dryness. Extracts were reconstituted in the HPLC mobile phase (0.15 ml) and washed with hexane (1 ml). Hexane was subsequently discarded and washed extracts transferred to vials for injection into the HPLC.

SQV and the internal standard were resolved on a Megellen 5C8 column (5 μm; 250 X 4.6 mm; Phenomenex, Macclesfield, Cheshire, UK) with a mobile phase of acetontrile : water (63 : 37) at a flow rate of 1 ml/min. Absorbance was monitored at 238 nm. Peaks of interest, SQV (retention time, 10.5 min) and internal standard (retention time, 25 min) were quantified using a Kontron MT2 data acquisition system (Kontron Instruments, Watford, Hertfordshire, UK).

The limit of quantification was 20 ng/ml. Interassay variability was determined with two different control samples containing nominal concentrations of 100 and 1000 ng/ml. The coefficients of variation were 3.6 and 4.1%, respectively. Intra-assay precision was determined with samples containing 100 and 1000 ng/ml. The coefficients of variation were 8.2 and 3.6%, respectively.

Pharmacokinetic and statistical analysis

SQV concentrations were evaluated for Cmax, time to Cmax and AUC to 8 h (AUC0–8h). AUC values were determined by non-compartmental analysis using the TOPFIT computer software (Gustav Fischer Verlag, Stuttgart, Germany).

Differences in SQV pharmacokinetic parameters were compared using the Mann–Whitney U test. A P value < 0.05 was considered statistically significant.


The results are summarized in Table 1. Patients administered SQV 600 mg three times daily demonstrated a 12-fold variability in SQV AUC0–8h ranging from 293 to 3446 ng•h/ml (Fig. 1). When combined with RIT there was a marked increase in the maximum plasma concentration of SQV: median (range), 146 (57–702) versus 4795 (1420–15 810) ng/ml, ∼95% confidence interval (CI), 2988–6819; P = 0.0006, Mann–Whitney U test (Fig. 2). Because SQV plasma concentrations are shown on an arithmetic scale, the profile of the SQV alone phase appears virtually flat. However, there was an approximate threefold increase in SQV concentration from trough (0 h) to peak (Cmax). The AUC0–8h for SQV was also significantly increased in the presence of RIT: median (range), 470 (293–3446) versus 27 458 (7357–108 001) ng•h/ml; ∼95% CI, 16 628–35 111; P = 0.0006 (Fig. 3).

Table 1
Table 1:
. Pharmacokinetic parameters for saquinavir (SQV) 600 mg three times daily alone and in combination with ritonavir (RIT) 300 mg twice daily.
Figure 1
Figure 1:
. Area under the concentration–time curve (AUC0–8h) for saquinavir (SQV) in seven patients treated with SQV 600 mg three times daily.
Figure 2
Figure 2:
. Mean (±SEM) plasma saquinavir (SQV) steady state concentrations following treatment with SQV 600 mg three times daily alone and in combination with ritonavir (RIT) 300 mg twice daily.
Figure 3
Figure 3:
. Area under the concentration–time curve (AUC0–8h) for saquinavir (SQV) in seven patients treated with SQV 600 mg three times daily alone and in combination with ritonavir (RIT) 300 mg twice daily.


It is estimated that there are at least 15 human liver cytochrome P450s involved in drug metabolism with the isozymes CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 responsible for the metabolism of the majority of drugs [11]. The single most important drug metabolising cytochrome P450 is CYP3A and in vitro evidence suggests that this is the most influential isoen-zyme involved in the metabolism of the protease inhibitors with the isoforms CYP2C9 and CYP2D6 also contributing [9]. If drugs are substrates for a com-mon cytochrome P450 enzyme it may be anticipated that the elimination of one or both may be delayed if the drugs are coadministered. If, as is likely, two pro-tease inhibitors are administered in combination as therapy for HIV we would predict a drug interaction, which is indeed supported by recent in vitro and in vivo studies. RIT potently inhibited the metabolism of SQV in rat and human liver microsomes with median inhibitory concentration values of 0.42 and 0.25 μM, respectively [9]. Recent single and multiple dose inter-action studies between SQV and RIT in healthy volunteers confirm the increased SQV concentration when coadministered with RIT, whereas RIT pharma-cokinetics remained unaltered [10].

In this study we investigated the potential clinical relevance of such an interaction by determining SQV kinetics when administered alone and in combination with RIT in HIV-infected patients attending our clinic. Steady-state SQV profiles were obtained on two occasions following treatment with SQV 600 mg three times daily and when administered with RIT 300 mg twice daily. Our results demonstrate a significant drug interaction between RIT and SQV probably mediated by enzyme inhibition as demonstrated by in vitro studies [9]. In the presence of RIT the maximum plasma concentration of SQV was increased 30-fold with a 58-fold increase in the median AUC0–8h compared with SQV administered alone. This represents a very potent inhibitory effect of RIT on SQV metabolism exposing patients to very high SQV concentrations. It has been proposed that an interaction between RIT and SQV may be beneficial and overcome problems associated with the poor bioavailability of SQV [12]. Doses of SQV higher than the standard 600 mg three times daily produce a greater and more sustained suppression of HIV viral load and elevation of CD4 counts. Treatment with SQV 3600 mg daily resulted in a maxi-mum mean decrease in plasma HIV RNA levels of 1.06 log RNA copies/ml and a maximum increase in CD4 cell counts of 72 × 10 6/l. Increasing the SQV dose to 7200 mg daily resulted in a mean decrease in HIV RNA of 1.34 log RNA copies/ml with a maximum increase in CD4 counts of 121 × 10 6/l [13]. Therefore, the ability of RIT to increase SQV levels together with the non-overlapping resistance patterns of SQV and RIT would appear to offer an attractive therapeutic option for the treatment of HIV disease.

Our data show that levels of SQV achieved with SQV 600 mg three times daily plus RIT 300 mg twice daily would be approximately six times greater than those after SQV 7200 mg daily. Significant toxicity occurred in patients treated with SQV 7200 mg daily with 5% of patients developing severe (grade 3) elevations in liver function tests, 5% developed severe (grade 3) neutrope-nia, and 5% developed life-threatening (grade 4) elevations in creatinine phosphokinase levels [13]. Therefore, it is not surprising that dosing regimens such as RIT 600 mg twice daily plus SQV 600 mg twice daily, which have been proposed as a therapeutic option and produce SQV levels much higher than monotherapy with SQV 7200 mg daily, result in the appearance of severe (grade 3) or life-threatening (grade 4) elevations in liver function tests in approximately 20% of patients [14]. Therefore, the choice of dosing regimen will be crucial in order to optimize the antiviral efficacy and minimize toxicity with this combination. Preliminary results (unpublished data) from our clinic suggest that the SQV AUC0–14h achieved by the combination of SQV 200 mg once daily plus RIT 600 mg twice daily may exceed that following SQV 7200 mg daily, giving some indication of the SQV dose that may suffice when combined with RIT.

As SQV is predominantly metabolised by CYP3A4 present in the gastrointestinal tract and the liver, and as significant intersubject variability in CYP3A4 exists it explains, in part, the 12-fold variability in SQV AUC0–8h following SQV 600 mg three times daily. This variability is also seen in the presence of RIT with the SQV AUC0–8h ranging from 7357 to 108 001 ng•h/ml. As a consequence of this observed variability, for some patients treatment with SQV 600 mg three times daily will result in very low SQV plasma levels and possibly little antiviral effect. When combined with RIT the variability of SQV levels will result in some patients being exposed to very high SQV levels and subsequent toxicity.

As RIT is a potent inhibitor of CYP3A it would be expected to inhibit the metabolism of other CYP3A substrates including protease inhibitors (indinavir, nelfi-navir), azole antifungal drugs (ketoconazole, itracona-zole), hypnotics (midazolam) and macrolide antibiotics (erythromycin, clarithromycin). Coadministration of RIT and clarithromycin in healthy volunteers produced a 77% increase in clarithromycin AUC and a threefold increase in the elimination half-life [15]. Treatment with RIT 500 mg twice daily plus rifabutin 150 mg daily resulted in a fourfold increase in rifabutin AUC [16]. The ability of RIT to inhibit cytochrome P450 enzymes other than CYP3A is demonstrated by the inhibition of desipramine (CYP2D6) metabolism [17]. As a consequence of the potent inhibition of cytochrome P450 enzymes, RIT is contraindicated in patients receiving some non-sedating antihistamines (astemizole, terfenadine), sedative hypnotics (midazolam) and antiarrhythmics (amiodarone, encainide, flecainide, propafenone, quinidine). However, in addition to these inhibitory effects, RIT may also induce CYP1A2 [18] and glucuronyl transferase activity [19] thereby increasing the metabolism of drugs such as theophylline (CYP1A2), zidovudine, opiates and ethinyloestradiol (all metabolised by glucuronyl trans-ferase). As the mean AUC of ethinyloestradiol may be reduced by 40% during treatment with RIT 500 mg twice daily, use of alternative contraceptive measures should be considered [19]. The potential for multiple drug interactions mediated predominantly by enzyme inhibition but also by induction, together with the poor tolerability of RIT that we have described in patients with advanced disease (30% discontinuation of therapy [20]), may well limit the use of this potent anti-HIV drug in some patients [21].

This study clearly demonstrates the ability of RIT to inhibit SQV metabolism in the clinical setting. The very high SQV levels as a consequence of this interaction will expose patients to SQV toxicity (increased liver function tests, increased creatinine phosphokinase, neutropenia). If combination therapy with RIT plus SQV is considered, then the dose of SQV should be greatly reduced. Further studies are in progress to determine the optimum dosage.


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Saquinavir; ritonavir; pharmacokinetics; HIV; AIDS

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