Boosted saquinavir hard gel formulation exposure in HIV-infected subjects: ritonavir 100 mg once daily versus twice daily

Marta Boffito1,*, Desmond Maitland1, Laura Dickinson2, David Back2, Andrew Hill2,3, Carl Fletcher1, Graeme Moyle1, Mark Nelson1, Brian Gazzard1 and Anton Pozniak1

1 PK Research Ltd, St Stephen's Centre, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK; 2 University of Liverpool, Liverpool, UK; 3 Roche, Welwyn, UK


* Corresponding author. Tel: +44-20-8846-6507; Fax: +44-20-8746-5628; Email: marta.boffito{at}chelwest.nhs.uk

Received 12 November 2004; returned 15 December 2004; revised 4 January 2005; accepted 5 January 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The amount of ritonavir needed to enhance saquinavir hard gel (hg) plasma concentrations is unclear. Reduced ritonavir dosing may help to reduce ritonavir-related side effects and costs. This study examined the pharmacokinetics of twice-daily saquinavir-hg (1000 mg) in the presence of ritonavir 100 mg, dosed twice-daily and once-daily on one single occasion.

Methods: Eighteen HIV-infected adults taking saquinavir/ritonavir 1000/100 mg twice-daily underwent pharmacokinetic (PK) assessment of saquinavir/ritonavir on day 1 following a morning saquinavir/ritonavir dose. On day 2, PK assessment was repeated when subjects took saquinavir without ritonavir. Drug intake (with a standard meal containing 20 g of fat) was timed on days –1, 1 and 2. Geometric mean ratios (GMR) and 95% confidence intervals (CI) were calculated to assess changes in saquinavir PK parameters.

Results: Geometric mean saquinavir AUC0–12, Ctrough, Cmax and elimination half-life on days 1 and 2 were 14 389 and 9590 ng·h/mL, 331 and 234 ng/mL, 2503 and 1893 ng/mL and 2.80 and 2.82 h, respectively. The GMR (95% CI) for these parameters were 0.67 (0.53–0.84), 0.71 (0.48–1.04), 0.76 (0.58–0.98) and 1.01 (0.86–1.18), respectively.

Conclusions: Withholding a ritonavir dose significantly reduces overall saquinavir exposure and Cmax, but had no impact on the elimination half-life. These data establish the need to administer saquinavir and ritonavir simultaneously.

Keywords: saquinavir , ritonavir , pharmacokinetics , boosted protease inhibitors


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of low-dose ritonavir as a pharmacokinetic (PK) enhancer of other protease inhibitors (PIs) has allowed for less frequent dosing and a lower pill burden for most PI-containing therapies. Although ritonavir is available as a liquid, ritonavir capsules are available only in a 100 mg formulation, and most (though not all) boosted PIs use a single 100 mg capsule per dose. However, the minimum effective dose of ritonavir for adequate inhibition of cytochrome P450 isoenzymes, in the gut and/or liver, is unknown.

Despite this, there is some evidence that 100 mg may be a more than adequate boosting dose for some PIs, particularly those administered on a twice-daily schedule. For example, Kurowski et al. have shown that twice-daily dosing of amprenavir (600 mg) with 50 mg ritonavir in healthy volunteers results in an amprenavir exposure profile that is not significantly different from that obtained with 100 mg ritonavir.1 Similarly, Boffito et al. showed that plasma amprenavir exposure was similar for twice-daily dosing of fosamprenavir 700 mg with saquinavir hard gel (hg) 1000 mg and either 100 mg or 200 mg of ritonavir,2 suggesting that 100 mg ritonavir lies on the plateau of the dose–response curve for fosamprenavir boosting. Kilby et al. have shown that saquinavir is boosted to a similar extent by various ritonavir doses in the range 100–400 mg,3 again implying that a plateau in boosting efficacy was reached by 100 mg ritonavir and that lower doses may efficiently boost saquinavir exposure. More indirectly, Stephan et al. observed that saquinavir exposure parameters in a group of 45 patients taking both lopinavir (400 mg) and saquinavir (1000 mg), jointly boosted with ritonavir (100 mg) on a twice-daily schedule, were similar to a control group of 32 patients taking saquinavir/ritonavir (1000/100 mg) without lopinavir.4 The similarity of the profiles occurred in spite of significantly lower plasma levels of ritonavir in the lopinavir/saquinavir/ritonavir group.

These data raise the possibility that, for at least some twice-daily boosted PIs, residual ritonavir levels from the previous dose may be sufficient to boost the next dose without additional ritonavir. In the case of boosted saquinavir, both serum triglyceride increases and the overall number of adverse events have been shown to be higher for increasing booster doses of ritonavir,5 a situation that can also be expected to apply to other boosted PIs. Thus, halving daily exposure to ritonavir by boosting a twice-daily PI with once-daily ritonavir may have implications for improved long-term safety and tolerability, as well as reducing treatment cost. A small proof-of-concept study in six HIV-infected patients recently demonstrated that there is sufficient residual ritonavir 12 h after a dose of saquinavir/ritonavir 1600/100 mg to boost a second dose of saquinavir (1600 mg).6 However, a formal PK analysis was not undertaken and this study had several limitations regarding small sample size with wide interpatient variability, lack of steady-state dosing and control for food intake, and the use of an investigational once-daily dose of saquinavir-hg rather than the standard 1000 mg twice-daily dose. We therefore undertook a study to investigate more fully whether a single daily dose of 100 mg ritonavir can boost two twice-daily doses of 1000 mg saquinavir-hg to a comparable extent.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design and procedures

This was an exploratory, prospective, open-label pilot study with a single arm, designed to compare the pharmacokinetics of saquinavir-hg when co-administered with ritonavir 100 mg twice-daily with the pharmacokinetics of saquinavir-hg after the omission of the ritonavir 100 mg morning dose on one occasion. HIV-1 antibody-seropositive male or female subjects aged 19–65 years who were receiving ongoing treatment with a saquinavir/ritonavir-containing regimen were eligible for enrolment. Subjects were excluded if they had any active clinically significant disease or any grade 3 or 4 toxicity according to the AIDS Clinical Trial Group (ACTG) grading severity list, other than grade 3 or 4 asymptomatic triglyceride/cholesterol elevations. Subjects were also ineligible for enrolment if they had taken corticosteroids systemically or drugs known to induce or inhibit hepatic enzymes within 14 days of study entry. Approval for the study was obtained from the local ethics committee (Riverside Research Ethics Committee) and all subjects gave written informed consent to participate in the study.

All patients were screened at day –14. From the evening dose on day –1 to the morning dose of day 2, inclusive, drug intake was timed and observed and all doses were taken after a standard meal containing 20 g of fat.

Steady-state PK assessment was carried out on blood samples drawn pre-dose (0 h) and at 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h after drug intake on the morning of day 1, when saquinavir-hg was dosed with ritonavir, and again after drug intake on the morning of day 2 when ritonavir was withheld. Total saquinavir and ritonavir plasma concentrations were determined at a Clinical Pathology Accredited laboratory within the University of Liverpool by a fully validated high-performance liquid chromatography–tandem mass spectrometry method adapted from Reynolds et al.7 Lower limit of quantification was 41 and 24 ng/mL for saquinavir and ritonavir, respectively and inter-assay and intra-assay variability did not exceed 11% and 7%, respectively. Assay performance is routinely monitored by assessment of internal quality controls and the laboratory participates in an external quality assurance programme twice yearly (International Interlaboratory Quality Control Program for Therapeutic Drug Monitoring in HIV Infection, Nijmegen, The Netherlands).

Data analysis

Parameters determined for saquinavir were maximum drug concentration (Cmax), trough concentration (Ctrough), elimination half-life (t1/2) and total drug exposure, expressed as the area under the concentration–time curve from 0 to 12 h (AUC0–12). Non-compartmental analyses (WinNonlin software; Pharsight Corporation, Mountain View, CA, USA) were used to derive the AUC for each patient over the dosing interval. Parameters determined for ritonavir were the AUC from 0 to 12 h on day 1 and from 12 to 24 h on day 2.

The value of each saquinavir PK parameter was expressed as the geometric mean (GM) and 95% confidence interval (95% CI) calculated across all the study participants for whom data were available. Changes in PK parameters between day 1 and day 2 were assessed by calculating the geometric mean ratio (GMRDay2/Day1) and 95% CI. A difference was significant at the 5% level where the 95% CI of the GMR did not cross 1.0.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighteen HIV-infected adult patients on a saquinavir/ritonavir-containing regimen were enrolled, whose baseline demographics are shown in Table 1. All patients completed the study with no adverse events observed.


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Table 1. Patient characteristics

 
The concentration–time profiles of saquinavir (0–12 h on days 1 and 2) and ritonavir (0–12 h on day 1 and 12–24 h on day 2) are shown in Figure 1. Concentrations of saquinavir on day 2 were numerically lower than the corresponding day 1 sample at all time points. Day 2 ritonavir exposure (AUC12–24: 2332 ng·h/mL) was only 28% that of day 1 (AUC0–12: 8271 ng·h/mL).



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Figure 1. (a) 12 h concentration–time profiles for saquinavir (SQV) 1000 mg, dosed on day 1 [with ritonavir (RTV) 100 mg] and day 2 (without RTV); (b) 0–12 h (day 1) and 12–24 h (day 2) concentration–time profiles for RTV 100 mg. Values are expressed as geometric means with the 95% confidence intervals as error bars. BID, twice-daily; w/o, without.

 
Saquinavir exposure parameters on days 1 and 2 and the associated GMRs are shown in Table 2. Total saquinavir exposure (AUC0–12) and Cmax were both significantly lower on day 2, whereas Ctrough and t1/2 were unchanged. Four patients (22%) on day 1, versus six patients (33%) on day 2 displayed saquinavir Ctrough levels below the minimum effective concentration threshold suggested for HIV wild-type (100 ng/mL).8


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Table 2. Geometric means (GM) and geometric mean ratios (GMR) with associated geometric 95% confidence intervals (95% CI) for saquinavir pharmacokinetic parameters on study day 1 (1000 mg with 100 mg ritonavir) and day 2 (1000 mg; ritonavir withheld)

 
Ritonavir PK parameters are illustrated in Table 3.


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Table 3. Geometric means (GM) with associated geometric 95% confidence intervals (95% CI) for ritonavir pharmacokinetic parameters on study day 1 (1000 mg with 100 mg ritonavir) and day 2 (1000 mg; ritonavir withheld)

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are no data available on the PK of unboosted saquinavir-hg at 1000 mg twice-daily, but, historically, plasma Cmax for an unboosted 600 mg thrice-daily dose of saquinavir-hg is ~200 ng/mL.9 This study's results show that there were residual levels of ritonavir present in plasma 12 h after administration of 100 mg that continued to boost a 1000 mg dose of saquinavir-hg. However, although Ctrough levels of saquinavir on day 2 were not significantly lower than on day 1, the significantly lower total exposure and Cmax values observed indicate that residual ritonavir does not boost saquinavir-hg to a comparable extent to concomitant administration. Thus, although evidence indicates that 100 mg of ritonavir is more than required for adequate boosting of saquinavir, a single daily dose of 100 mg will not adequately boost both doses of saquinavir-hg 1000 mg twice-daily.

Our observation that Cmax and total saquinavir exposure are lower on day 2 while the elimination rate is unchanged is consistent with a model of saquinavir boosting that occurs predominantly through ritonavir inhibition of gut wall cytochrome P450. Ritonavir inhibition of gut-wall P-glycoprotein (P-gp), which actively transports saquinavir and limits its oral availability,10,11 may also be implicated in the boosting process; however, the effect of ritonavir on P-gp transport is not as well established as its effect on P450. Some groups report strong inhibition in vitro12,13 while others report poor activity in vivo.10,14

Inhibition of first-pass metabolism and/or efflux transport results in significant elevations in plasma Cmax with a lesser effect on the elimination rate, which is mediated by hepatic metabolism. This type of boosting is common to poorly absorbed PIs, and Cmax elevation is the principal mechanism of boosting for saquinavir15 and lopinavir.16 This mechanism requires adequate levels of ritonavir to be present in the gut at the time of dosing of the concomitant PI in order to enhance absorption; but, since elimination is not as affected, adequate ritonavir will only need to be maintained while dissolution and absorption of the second PI is occurring. Thus, the timing of the ritonavir component with respect to the second PI can be considered more important to this type of boosting than the maintenance of sustained effective levels of ritonavir. Consistent with this hypothesis, therapeutic Ctrough concentrations of saquinavir are present with saquinavir-hg/ritonavir 2000/100 mg once-daily despite half the ritonavir being used relative to 1000/100 mg twice-daily administration and despite little or no plasma ritonavir remaining at the end of the 24 h dosing interval.17 Studies of once-daily lopinavir/ritonavir also show lower but still therapeutic Ctrough concentrations of lopinavir despite little or no ritonavir remaining at the end of the interval.18

In contrast, adequately absorbed PIs show little or no elevation in Cmax with ritonavir boosting but do demonstrate significantly extended elimination half-lives through ritonavir inhibition of hepatic cytochrome P450. For these PIs, which include indinavir19 and amprenavir,20 it can be speculated that the maintenance of adequate ritonavir over time is more important to ensure that systemic clearance is reduced than for PIs boosted primarily through Cmax elevation.

Our observations help explain the reduced boosting efficacy observed for 12 h residual ritonavir, and provide a theoretical framework for the importance of co-dosing saquinavir with ritonavir, whether administered on a twice-daily or once-daily schedule. The same rationale should apply to other PIs for which the major mechanism of boosting occurs at the level of first-pass metabolism as opposed to reduced systemic clearance. It can be hypothesized that once-daily ritonavir might effectively boost at least some PIs for which it significantly prolongs elimination rather than elevates absorption, but further data are needed to confirm or refute this. Moreover, it is unclear if lower concentrations of ritonavir (and saquinavir) would be observed following the administration of ritonavir once daily for a longer period of time. However, similar concentrations of ritonavir 24 h after 100 mg intake have been observed at steady-state.17

Finally, although once-daily ritonavir is not suitable for boosting twice-daily saquinavir-hg, it should be noted that the investigational once-daily combination saquinavir-hg/ritonavir 2000/100 mg has shown promising pharmacokinetics.17 This combination is currently under clinical evaluation as another potential way of minimizing daily ritonavir intake for boosted saquinavir regimens. Nevertheless, this study once more shows the need for a 50 mg ritonavir formulation.


    Acknowledgements
 
This study was financially supported by Roche, Welwyn, UK.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Kurowski, M., Staszewski, S., Arslan, A. et al. (2001). Influence of 50 mg, 100 mg and 200 mg ritonavir (RTV) on the pharmacokinetics (PK) of amprenavir (APV) after multiple doses in healthy volunteers for once daily (QD) and twice daily (BID) regimens. Presented at the First IAS Conference on HIV Pathogenesis and Treatment, Buenos Aires, Argentina, 2001. Abstract 351. Marathon Multimedia.

2 . Boffito, M., Dickinson, L., Hill, A. et al. (2004). Steady-state pharmacokinetics of saquinavir hard gel/ritonavir/fosamprenavir in HIV-1-infected patients. Journal of Acquired Immune Deficiency Syndrome 37, 1376–84.[Medline]

3 . Kilby, J. M., Hill, A. & Buss, N. (2002). The effect of ritonavir on saquinavir plasma concentration is independent of ritonavir dosage: combined analysis of pharmacokinetic data from 97 subjects. HIV Medicine 3, 97–104.[CrossRef][Medline]

4 . Stephan, C., Hentig, N., Kourbeti, I. et al. (2004). Saquinavir drug exposure is not impaired by the boosted double protease inhibitor combination of lopinavir/ritonavir. AIDS 18, 503–8.[CrossRef][ISI][Medline]

5 . Buss, N., Snell, P., Bock, J. et al. (2001). Saquinavir and ritonavir pharmacokinetics following combined ritonavir and saquinavir (soft gelatin capsules) administration. British Journal of Clinical Pharmacology 52, 255–64.[CrossRef][ISI][Medline]

6 . Luber, A., Anderson, D., Stryker, R. et al. (2004). Can ritonavir (RTV) once daily boost saquinavir (SQV) twice daily? A pilot study. Presented at the Fifth International Workshop on Clinical Pharmacology of HIV Therapy, Rome, Italy, 2004. Abstract 16, p. 13. Virology Education, Utrecht, The Netherlands.

7 . Reynolds, H. E., Tjia, J. F., Gibbons, S. E. et al. (2001). Simultaneous determination of four HIV protease inhibitors by HPLC-MS/MS for use in a therapeutic drug monitoring service [abstract]. British Journal of Clinical Pharmacology 52, 481P–2P.

8 . Back, D., Blaschke, T., Boucher, C. et al. (2003). Optimising TDM in HIV clinical care: a practical guide to performing therapeutic drug monitoring (TDM) for antiretroviral agents. Version 1.0. [Online.] www.hivpharmacology.com (1 November 2004, date last accessed).

9 . Roche Pharmaceuticals. Invirase® Summary of Product Characteristics. September 2002.

10 . Huisman, M. T., Smit, J. W., Wiltshire, H. R. et al. (2001). P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir. Molecular Pharmacology 59, 806–13.[Abstract/Free Full Text]

11 . Kim, A. E., Dintaman, J. M., Waddell, D. S. et al. (1998). Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein. Journal of Pharmacology and Experimental Therapeutics 286, 1439–45.[Abstract/Free Full Text]

12 . Drewe, J., Gutmann, H., Fricker, G. et al. (1999). HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833. Biochemical Pharmacology 57, 1147–52.[CrossRef][ISI][Medline]

13 . Gutmann, H., Fricker, G., Drewe, J. et al. (1999). Interactions of HIV protease inhibitors with ATP-dependent drug export proteins. Molecular Pharmacology 56, 383–9.[Abstract/Free Full Text]

14 . Huisman, M. T., Smit, J. W., Wiltshire, H. R. et al. (2003). Assessing safety and efficacy of directed P-glycoprotein inhibition to improve the pharmacokinetic properties of saquinavir coadministered with ritonavir. Journal of Pharmacology and Experimental Therapeutics 304, 596–602.[Abstract/Free Full Text]

15 . Kilby, J. M., Sfakianos, G., Gizzi, N. et al. (2000). Safety and pharmacokinetics of once-daily regimens of soft-gel capsule saquinavir plus minidose ritonavir in human immunodeficiency virus-negative adults. Antimicrobial Agents and Chemotherapy 44, 2672–8.[Abstract/Free Full Text]

16 . Sham, H. L., Kempf, D. J., Molla, A. et al. (1998). ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease. Antimicrobial Agents and Chemotherapy 42, 3218–24.[Abstract/Free Full Text]

17 . Boffito, M., Dickinson, L., Hill, A. et al. (2004). Pharmacokinetics of once-daily saquinavir/ritonavir in HIV-infected subjects: comparison with the standard twice-daily regimen. Antiviral Therapy 9, 423–9.[Medline]

18 . Eron, J. J., Feinberg, J., Kessler, H. A. et al. (2004). Once-daily versus twice-daily lopinavir/ritonavir in antiretroviral-naive HIV-positive patients: a 48-week randomized clinical trial. Journal of Infectious Diseases 189, 265–72.[CrossRef][ISI][Medline]

19 . Hugen, P. W., Burger, D. M., ter Hofstede, H. J. et al. (2000). Dose-finding study of a once-daily indinavir/ritonavir regimen. Journal of Acquired Immune Deficiency Syndrome 25, 236–45.[ISI][Medline]

20 . Sadler, B. M., Piliero, P. J., Preston, S. L. et al. (2001). Pharmacokinetics and safety of amprenavir and ritonavir following multiple-dose, co-administration to healthy volunteers. AIDS 15, 1009–18.[CrossRef][ISI][Medline]





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