Highly active antiretroviral therapy (HAART) including protease inhibitors is now standard care for HIV-infected patients. However, a considerable number of patients may not adequately respond to HAART, and rates of virological failure of up to 50% have been reported in the literature [1,2]. Until now, no standard therapy has been established for these patients. Changing at least two drugs and considering double protease inhibitor therapy has been recommended by consensus conferences [3,4]. However, extensive pretreatment with antiretroviral drugs in many patients failing HAART makes it difficult to apply these recommendations in clinical practice, and studies which have assessed this concept are scarce.
We studied the virological and immunological efficacy and the toxicity of double protease inhibitor therapy in addition to reverse transcriptase inhibitors in patients with virological treatment failure under HAART. Additionally, the influence of plasma concentrations of protease inhibitors on virological response was examined.
Adult patients with documented HIV infection treated at an outpatient clinic (University of Cologne) were included into this prospective, non-controlled study. For inclusion, patients had to be treated with HAART containing a potent protease inhibitor for at least 3 months. HIV-RNA had to be >4log10 on two occasions during a screening process. There was no cutoff value for CD4 cells. Written informed consent was necessary. Exclusion criteria were: life expectancy of less than 3 months, concomitant treatment with drugs known to interfere with ritonavir, liver disease (transaminases five-fold upon upper limit of normal range), pregnancy and known intolerability to ritonavir or saquinavir.
The study was approved by the institutional review board of the Faculty of Medicine, University of Cologne.
All patients received ritonavir 400mg twice daily and saquinavir (Invirase) 600mg twice daily. Saquinavir was dosed higher than in other studies using 400mg twice daily because high plasma concentrations were considered mandatory for virological success in pretreated patients. At least one reverse transcriptase inhibitor was prescribed concomitantly. The reverse transcriptase inhibitors were selected by the treating physician according to the patient‚s history of antiretroviral treatment. As many new rerverse transcriptase inhibitors as possible should be used. In patients receiving a non-nucleoside reverse transcriptase inhibitor (NNRTI), no dose adjustments were performed.
Patients were seen in our outpatient clinic once each month and a complete clinical examination was performed at each visit. Laboratory tests were carried out at monthly intervals and included a complete blood count, chemistry [electrolytes, creatinine, glucose, uric acid, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma-glutamyl transpeptidase (gamma-GT), amylase, lipase], and serum lipids (triglycerides, cholesterol). Toxicity was graded according to the World Health Organization (WHO) classification system.
CD4 cell counts and plasma HIV-RNA (polymerase chain reaction, Roche Amplicor, Roche Diagnostic Systems, Branchburg, New Jersey, USA) were determined every 4 weeks. The limit of detection for HIV-RNA was 200 copies/ml.
For pharmacological examinations, plasma samples were taken 4h post dosing and stored at -20ΥC until shipment to the laboratory. Plasma concentrations of ritonavir and saquinavir were determined as published previously [5,6]. Out of serial plasma samples over time (weeks 4 to 12), the respective minimal, median and maximal concentrations of saquinavir and ritonavir were determined for each patient.
The rate of patients with virological success at week 12 was the primary endpoint. Partial virological success was defined as a decrease of HIV-RNA by at least 1 log10 copies/ml at week 12, and complete virological success as a value below the limit of detection at week 12.
Secondary endpoints were the number of CD4 cells at week 12, AIDS-defining clinical events, toxicity and plasma concentrations of ritonavir and saquinavir.
For group comparisons the chi-square test (categorical variables) and the Mann-Whitney-Wilcoxon test (continous variables) were used. All analyses were performed with SPSS version 6.1.3 software (SPSS Inc., Chicago, Illinois, USA). Two sided P-values of less than0.05 were considered as statistically significant.
Thirty persons with symptomatic HIV infection were included in this study (for baseline characteristics see Table 1). Patients were pretreated for a median time of 3 years (range: 1.8-6.9 years) with antiretroviral drugs including reverse transcriptase inhibitors and protease inhibitors. Only two patients were naive for ritonavir and saquinavir. In 28 patients new drugs could be used. Twelve patients treated with NNRTIs had been naive for this class: nevirapine was newly introduced in 11 patients (and continued in four previously treated individuals), and delavirdine in one patient. (see Table 2). Twelve patients took three, sixteen patients four and two patients five antiretrovirals during the study.
Twenty-five patients completed 12 weeks of therapy. Discontinuation was caused by gastrointestinal intolerance (three), suspected pregnancy (one), and patient‚s decision (one).
Virological and immunological response
After 12 weeks, six patients had a virus load <200copies/ml (complete virological response). In addition, three patients exhibited a decrease of HIV-RNA by at least 1log10 (partial response). Thus, rates for complete and partial response were 20 and 10%, respectively (intent-to-treat analysis). In the 25 patients completing 12 weeks, the CD4 cell count was increased by week 12 from 209 × cells/l (mean; SD: ±113 ×cells/l ) to 237 × cells/l (mean; SD: ±132 × cells/l ). The mean increase of CD4 cells was 66 × cells/l (SD: ±54 × cells/l) in patients with virological response as compared to 6 × cells/l (SD: ±45 × cells/l ) in patients without virological response (P=0.01).
After an observation time of 6.3 months (median; range: 4.4-7.5 months), all six patients with complete virological response remained responders, and CD4 cells remained unchanged up to week 12. No clinical events were observed.
Plasma concentrations of ritonavir and saquinavir
Plasma concentrations of saquinavir and of ritonavir are shown in Table 3. Out of the 30 patients included into the study, drug concentrations were available in 24 patients; they were not determined in five persons with preliminary study termination; and in one person who was not compliant with the 4 h post dosing interval. Maximal saquinavir concentrations were higher in eight patients with complete or partial virological success at week 12 (median 3254ng/ml, range 724-6785ng/ml) than in 16 patients without success (median 1130ng/ml; range 0-6393ng/ml), but this difference was not statistically significant (P=0.086; Mann- Whitney-Wilcoxon test). At week 8 there was a significant correlation between virological response and saquinavir concentrations (P=0.03). There was no correlation between median or minimal saquinavir concentrations and virological response, and saquinavir concentrations were independent from ritonavir concentrations. Ritonavir concentrations showed no influence on virological outcome.
No other risk factors for virological failure at week 12 could be identified. Baseline HIV-RNA, baseline CD4 cells, number of drugs prescribed previously, pretreatment with saquinavir or ritonavir, number of new drugs and treatment with nevirapine as a new drug were all not significant.
Side effects were common in this study. Seventeen patients exhibited gastrointestinal discomfort, and eight patients complained of perioral paresthesia. Elevations of serum lipids, transaminases and/or gamma-GT were observed in the majority of participants. Most severe toxicities (WHO grade 3 or 4) occurred with triglycerides (39%), followed by gamma-GT (22%) and transaminases and cholesterin (7%) (see Table 4). During follow-up a lipodystrophy syndrome developed in one patient.
In this study salvage therapy for HIV-infected patients failing HAART was based on a combination of ritonavir and saquinavir. We chose a saquinavir dose of 600mg twice daily instead of the usually administered 400mg twice daily in order to achieve saquinavir concentrations as high as possible. Indeed, it was speculated that by increasing drug exposure it may be possible to overcome moderate phenotypic resistance to saquinavir and thereby to increase response rates of antiretroviral salvage therapy.
Participants were extensively pretreated with antiretrovirals: The mean drug history per patient included 2.1 protease inhibitors and combinations of protease inhibitors, respectively, and 3.9 nucleoside analogues. Therefore, it was only possible for one-half of the patients to introduce more than one new antiretroviral to the salvage regimen. This illuminates the difficulties to apply consensus recommendations for changing all or at least two drugs in the case of virological failure in the setting of extensively pretreated patients [3,4].
Only 20 per cent of the patients achieved complete virological susppression (which was sustained during follow-up for 6 months). Partial virological response was present in another 10 per cent. These figures differ significantly from studies in naive patients treated with a combination of ritonavir and saquinavir. In the study of Cameron et al. 89 out of 141 patients (63%) had a complete virological suppression after 2 years . In an unselected cohort the risk of virological failure was seven-fold for pretreated patients as compared with naive patients. Half of the patients in the whole cohort had HIV-RNA levels below the limit of detection . A similar result was obtained by Kaufman et al. who reported a rate of 49% of patients with undetectable viral load after 48 weeks. Pretreatment with protease inhibitors was a risk factor for virological failure .
When the combination of ritonavir and saquinavir was exclusively prescribed for pretreated patients, response rates considerably lower than 50% have been described. In a cohort of 18 protease-inhibitor-naive patients with very advanced HIV infection Lorenzi et al. found partial virological suppression after 13 weeks in 33% and complete virological suppression in 11% . Treatment response was even poorer in a study of Deeks et al., who found decreases (>0.5log10) of HIV-RNA after 24 weeks only in four out of 18 patients .
Very high response rates (85%) were reported in a small study of 13 patients receiving a NNRTI in addition to the combination of ritonavir and saquinavir . We could not confirm these results in our study. No difference in virological outcome was observed between NNRTI naive patients treated with a NNRTI (n=12) and patients without NNRTI treatment (n=16; four patients treated with nevirapine had experienced this drug). However, due to the small numbers and to the heterogenous patient population one cannot conclude from this study that the concept of using NNRTIs for salvage therapy may be invalid. More favourable results have been published recently on the use of efavirenz in salvage therapy by other authors [13,14].
Median post-dosing plasma concentrations of saquinavir were above 1000ng/ml during the entire study. Comparable results have been reported by other groups examining ritonavir/saquinavir combination therapy [15,16]. In contrast, maximal saquinavir concentrations between 146ng/ml and 242ng/ml are achieved when saquinavir hard gelatin capsule were given alone [16,17].
No clear correlation between plasma concentrations of saquinavir and virological response could be demonstrated in our study. Since we observed a trend towards a better outcome in patients with higher concentrations of saquinavir, the lack of a significant correlation could be due to the small numbers, and more extensive studies may be necessary to clarify this issue. At least it seems justified to further investigate the concept of high drug exposure in patients who failed initial antiretroviral therapy. Preliminary results from studies with combinations of five or more drugs suggest that such ‚Mega-HAART‚ may be a more successful approach to achieve virological suppression in extensively pretreated patients than currently applied regimens .
There is no other study which evaluated the influence of drug concentrations on outcome in protease-inhibitor-experienced patients. Lorenzi et al. found a correlation of saquinavir concentrations and virological response . However, their patients were naive for protease inhibitors. The probability of resistance mutations for saquinavir is much lower under such conditions
The toxicity of the protease inhibitor combination ritonavir and saquinavir was high in our cohort and included gastrointestinal discomfort, perioral paresthesia, elevations of serum lipids, transaminases, and gamma-GT. These side effects fit well into the toxicity profile which has been described either for ritonavir alone or for the combination of ritonavir and saquinavir [7,19-21]. Other known adverse effects of ritonavir such as nephrotoxicity [22,23] or induction of diabetes  have not been observed in our patients. One patient developed peripheral lipodystrophy during follow-up, which has been observed under various protease inhibitors .
In conclusion, salvage therapy with ritonavir and saquinavir in combination with reverse transcriptase inhibitors in extensively pretreated patients was virologically successful only in a minority of patients. Therefore, new concepts of salvage therapy are urgently required.
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Keywords:© 1999 Lippincott Williams & Wilkins, Inc.
HIV; AIDS; protease inhibitors; ritonavir; saquinavir; salvage therapy; virological failure; plasma concentrations; drug monitoring