Despite its effectiveness, combination antiretroviral therapy often fails to suppress HIV replication durably, as a result of poor adherence (often related to adverse effects) and the emergence of drug-resistant genetic variants [1,2]. Drug resistance increases markedly with successive antiretroviral regimens and limits treatment options. Several strategies to overcome drug resistance have been explored, including drug-resistance testing, scheduled treatment interruptions and the use of new drug combinations that incorporate pharmacokinetic boosting .
In the Puzzle 1-ANRS 104 Study, a randomized, open-label, multicentre clinical trial, patients were randomly assigned centrally to receive a ritonavir dose of 200 or 400 mg/day. In each of the two arms, patients were further randomly assigned to either amprenavir (1200 mg/day) or lopinavir (800 mg/day) for 2 weeks before the second protease inhibitor (PI) was added and the nucleoside reverse transcriptase inhibitors (NRTI) were optimized on the basis of viral genotyping and previous antiretroviral exposure . At week 26, the 400 mg/day ritonavir group experienced a significantly greater reduction of HIV RNA (−2.5 log10 copies/ml) than the 200 mg/day group (−1.4 log10 copies/ml). Although not significant, the difference in the proportion of HIV-RNA levels below 50 copies/ml was 61 and 32%, respectively. After week 26, all patients were followed for a further period of 26 weeks. We report the efficacy and safety of the combinations tested at the end of this extended period of follow-up. We used Mann–Whitney two-sample statistics to compare changes in plasma HIV-1-RNA levels from baseline to week 52, and Fisher's exact test for the comparison of the proportion between groups. The Kaplan–Meier method was used to estimate probability of B or C classifying events and the log rank test to compare groups. P values are based on two-sided tests.
Nineteen patients were initially allocated to a regimen containing a total daily ritonavir dose of 200 mg and 18 to a total daily ritonavir dose of 400 mg. The two arms did not differ in terms of baseline characteristics . Overall, the mean CD4 cell count was 207 × 106/l, and the mean plasma HIV-1-RNA level was 4.7 log10 copies/ml. The median length of previous exposure to antiretroviral therapy was 94 months. In addition to NRTI, 35 patients had been exposed to at least two PI and one non-nucleoside reverse transcriptase inhibitor (NNRTI), and two patients had been exposed to two PI only, with median exposure times of 53 months for PI, 28 months for NNRTI, and 94 months for NRTI. The median number of NRTI, NNRTI and PI administered was five, one and three, respectively. The mean number of baseline PI and RT mutations was 7 and 6.9, respectively.
At week 52, the reduction in HIV RNA remained significantly higher in those initially randomly assigned to receive 400 mg/day: −2.0 log10 copies/ml [95% confidence interval (CI) −2.7, −1.3] than 200 mg/day: −1.1 log10 copies/ml (95% CI −1.7, −0.3, P = 0.05; Fig. 1). The HIV-RNA level was below 50 copies/ml in 39 and 11% of patients, respectively (P = 0.12). The mean increase in the CD4 cell count was +156 cells/μl (95% CI +67, +240) and +100 cells/μl (95% CI +18, +182), respectively (P = 0.50). The median CD4 cell count at one year was 353 cells/μl [interquartile range (IQR) 235–491] and 256 cells/μl (IQR 68–479), respectively (P = 0.18).
Over the one-year period of follow-up, six out of 19 patients in the 200 mg/day ritonavir group experienced 10 events classified as Centers for Disease Control and Prevention stage B or C (oral candidiasis, three; herpes, two; ocular toxoplasmosis, one; progressive multifocal leukoencephalopathy, one; Kaposi's sarcoma, one; Burkitt's lymphoma, one; non-Hodgkin's lymphoma leading to death, one) compared with two out of 18 in the 400 mg/day ritonavir group, who experienced four events (oral candidiasis, two; herpes, one; zoster, one). Although not significant (P = 0.13), the difference in the rate of clinical events is in line with the virological results.
Tolerability was similar in the two groups. During the one-year follow-up, six out of 18 patients (33%) in the 400 mg/day ritonavir group discontinued treatment compared with eight out of 19 patients (42%) in the 200 mg/day ritonavir group. The incidence of grade 4 side effects was also similar: 10 out of 19 patients in the 200 mg/day ritonavir group experienced 11 events (hypertriglyceridemia, four; neutropenia, three; vasculitis with spondyloarthropathy, one; pancytopenia, one; gamma-glutamyltransferase elevation, one; creatine phosphokinase elevation, one) compared with seven out of 18 in the 400 mg/day ritonavir group, who experienced eight events (hypertriglyceridemia, five; creatine phosphokinase elevation, one; vomiting and myalgia, one; peripheral neuropathy, one).
The combination of amprenavir and lopinavir with 400 mg/day ritonavir had substantial and sustained antiviral efficacy as well as good tolerability in these heavily pretreated patients, despite extensive genotypic and phenotypic resistance at baseline. The durable efficacy of the combination is reflected by the relatively high proportion of patients (39% in the intent-to-treat analysis) in whom the plasma HIV-1-RNA load was below 50 copies/ml after 52 weeks of treatment and the lower rate of B or C grade clinical events. Our results compare very favourably with those of other clinical trials of salvage regimens for patients in whom multiple antiretroviral agents had failed [5–7]. ACTG 359 was a randomized comparison of six oral antiretroviral regimens based on combinations of saquinavir soft gel with ritonavir or nelfinavir, together with delavirdine or adefovir dipivoxil, or both, in indinavir-experienced patients with virological failure . The most effective regimen yielded HIV-RNA values below 500 copies/ml at week 16 in 40% of patients. In the ACTG 398 trial, Hammer et al.  examined whether adding a second PI improved the antiviral efficacy of a four-drug combination in patients in whom virological failure had occurred on a PI-containing regimen. The proportion of patients with HIV-RNA levels below 200 copies/ml at 24 weeks was only 35%. In an uncontrolled trial, Montaner et al.  tested a ‘multiple drug rescue therapy’ containing five to nine antiretroviral agents in 106 heavily pretreated patients. In the intention-to-continue analysis, approximately 28% of patients had HIV-RNA values below 50 copies/ml at one year.
Pharmacokinetics, drug metabolism, and drug–drug interactions are important factors when selecting combinations for patients with virological failure. Unfortunately, pharmacokinetic data are rarely available for interactions between drugs used in antiretroviral combinations. We found that the median minimal concentration of amprenavir was significantly lower after the addition of lopinavir, confirming the existence of a pharmacokinetic interaction between the two drugs [8,9]. The strong antiviral effect observed in this study, despite the decrease in amprenavir concentrations, shows that the clinical significance of a given drug–drug interaction cannot be predicted only from the magnitude of the change in drug concentrations. The latter should always be determined in clinical studies, along with their impact on virological–immunological outcomes.
In conclusion, a combination of amprenavir (1200 mg/day), lopinavir (800 mg/day) and ritonavir (400 mg/day) is durably potent in patients in whom multiple antiretroviral agents have failed, yielding sustained virological responses.
The authors would like to thank Estelle Noé for data monitoring and Cécile Droz-Perroteau for analysis of the first period of the trial.
Sponsorship: This study was supported by ANRS (National Agency for AIDS Research), Paris, France.
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The following institutions and investigators participated in the Puzzle 1 ANRS 104 Study: W. Rozenbaum, L. Naït-Ighil, T.H. Nguyen, L. Slama, P.M. Girard, Hôpital Rothschild, Paris; J.M. Molina, D. Sereni, N. Colin de Verdière, C. Lascoux-Combes, C. Pintado, D. Ponscarme, F. Prevoteau de Clary, M. Tourneur, Hôpital Saint-Louis, Paris; M. Bentata, L. Guillevin, O. Launay, R. Mansouri, F. Rouges, Hôpital Avicenne, Bobigny; M. Kazatchkine, A. Aouba, M. Azizi, J.N. Fiessinger, P. Le Houssine, Hôpital Européen Georges Pompidou, Paris; D. Sicard, C. Bernasconi, D. Salmon, B. Silbermann, Hôpital Cochin, Paris; J.P. Cassuto, C. Ceppi, Dr Poiree, Hôpital de l'Archet, Nice; G. Raguin, M. Merad, Hôpital de la Croix Saint Simon, Paris; J.F. Delfraissy, C. Goujard, Y. Quertainmont, Hôpital de Bicêtre, Le Kremlin-Bicêtre; C. Perronne, P. de Truchis, Hôpital Raymond Poincaré, Garches; B. Dupont, J.L. Bresson, I. Calatroni, Hôpital Necker, Paris; F. Raffi, J.L. Esnault, S. Leautez, Hôtel Dieu, Nantes.