The study drugs were withdrawn in two patients: one because of virological failure and one by personal choice. Another patient had an undetectable viral load at 3 months of treatment, but presented HIV RNA rebound at 6 months. This patient continued with the same treatment, waiting for new therapeutic options. After 6 months of follow-up, 13 of 16 patients (81%) had HIV RNA values less than 50 copies/ml (intent-to-treat). At week 24, the mean reduction in plasma HIV-1 RNA in patients who initiated treatment with a detectable viral load was 2.9 log10 copies/ml and the mean increase in CD4 cell count was 118 cells/μl. In the two patients who initiated treatment with a viral load of < 50 copies/ml, viral load remained undetectable after 24 weeks of treatment and CD4 lymphocyte count remained steady. No deaths occurred during the study period.
Plasma RTV concentrations were similar in the three arms (A, B, and C) where the same dose was administered (100 mg b.i.d.). Patients in arm D received 100 mg q.d. and the median C trough and C min for RTV were significantly lower than in the other three groups. The AUC0–24 of RTV in group D was similar to the AUC0–12 of RTV in the other three groups. (Table 3). In arm A no significant correlations were found for any of the pharmacokinetic parameters between LPV and RTV. In arms B and C there was a strong positive linear correlation between the two drugs for the AUC0–12, C max, C trough, and C min, as reported previously .
Comparison of the concentration–time profiles for ATV when ATV was administered in combination with LPV/RTV (400/100 mg b.i.d.) (arm A) or with RTV (100 mg q.d.) (arm D), revealed that C trough and C1 ATV concentrations were significantly higher in arm A (Fig. 2). Specifically, median ATV C trough (1.14 versus 0.61 μg/ml; P = 0.020) and median ATV C min (1.07 versus 0.58 μg/ml; P = 0.017) showed higher values in arm A as compared with arm D. There were no significant differences in ATV AUC0–24 or in ATV C max between arms A and D. In arm A we observed a moderate correlation between the RTV C min, and the ATV AUC0–24 (r, 0.57; P = 0.020) and C min (r, 0.46; P = 0.042), and in arm D, between the RTV C min and ATV C min (r, 0.62; P = 0.015). No significant correlations were found for any of the pharmacokinetic parameters between LPV and ATV.
Seven of 16 patients in arm A were taking tenofovir as part of their treatment. The median AUC, C max and C trough values for ATV in these patients as compared to those who were not taking tenofovir were 47.8 (IQR, 25.6–58.5) and 48.6 (IQR, 36.4–55.5) μg/ml/h (P = 0.68), 3.87 (IQR, 2.36–4.17) and 3.98 (IQR, 2.56–4.27) μg/ml (P = 0.54), and 0.98 (IQR, 0.77–1.28) and 1.14 (IQR, 0.52–2.04) μg/ml (P = 0.68), respectively. Nine of 15 patients in arm D were taking tenofovir. The median AUC, C max and C trough values for ATV in these patients as compared to those who were not taking tenofovir in arm D were 38.3 (IQR, 20.5–45.7) versus 49.3 (IQR, 31.2–78.5) μg/ml/h (P = 0.020), 3.82 (IQR, 2.36–4.17) versus 5.46 (IQR, 2.56–4.27) μg/ml (P = 0.011), and 0.45 (IQR, 0.28–0.72) versus 0.68 (IQR, 0.43–1.03) μg/ml (P = 0.14), respectively.
The combination of ATV (300 mg q.d.) and LPV/RTV (400/100 mg b.i.d.) was generally well tolerated. None of the patients discontinued treatment due to adverse effects. Six patients presented grade 1 diarrhoea, which was self-limited or improved without withdrawal of treatment. The most common adverse event was mild hyperbilirubinaemia. All patients experienced an increase in total bilirubin greater than the upper normal limit (UNL), with levels of 1.1–1.5 × UNL (grade 1) in one case, 1.6–2.5 × UNL (grade 2) in seven cases and 2.6–5 × UNL (grade 3) in eight cases. At different points along the follow-up period, four patients (25%) developed scleral icterus or jaundice. In contrast, none of the patients showed clinical symptoms suggesting acute hepatitis, and none had grade 3–4 hepatic transaminase elevations (> 5.1 × UNL). Median levels of bilirubin (IQR) at baseline and at months 1, 3, and 6 were 0.47 (0.38–0.67), 1.92 (1.48–2.39), 2.74 (1.80–3.53), and 2.50 (1.83–3.21) mg/dl, respectively. The lipid profile changes were mild, with a slight elevation of total cholesterol and no triglyceride changes.
In the present study, administration of LPV/RTV (400/100 mg b.i.d.) with ATV (300 mg q.d.) achieved high LPV and ATV plasma levels. Co-administration of ATV and LPV/RTV substantially increased LPV exposure, producing statistically significant increases in the LPV AUC0–12, C max, C min, and Ctrough. RTV is a potent inhibitor and ATV a modest inhibitor of CYP3A4 metabolism [18,20]. However, ATV seems to be able to further enhance LPV exposure in the presence of RTV. Boffito et al.  reported that the addition of ATV (300 mg q.d.) to SQV/RTV (1600/100 q.d.) further increased SQV and RTV AUC0–24, C max, and Ctrough, and they speculated that inhibition of P-glycoprotein mediated drug transport may be responsible for the increase in SQV and RTV exposure. Among currently available PI, ATV is the only one that exerts a clinically significant inhibiting effect while producing plasma increases in RTV-boosted PI. In this setting, SQV [15,16] or indinavir  do not seem to modify plasma concentrations of LPV, whereas with APV, [10,11,13,14] FPV,  or nelfinavir,  decreases in plasma LPV concentrations to varying extents have been documented.
The mechanism by which ATV further boosts LPV is unknown. LPV is metabolized by CYP3A4 and is a substrate for P-glycoprotein and other drug efflux pumps. An increase in the RTV dose produces a further increase in plasma LPV concentration [27,28]. In the present study, no increases were found in RTV concentrations in the presence of ATV, but it is possible that ATV might have produced some additional CYP3A4 inhibition, resulting in a slight increase in plasma LPV. It was demonstrated recently that ATV is an inhibitor of P-glycoprotein and multidrug resistance-associated protein, and that the inhibitory effect is greater when ATV is combined with RTV than when these drugs are used alone [29,30]. This may be the main mechanism responsible for the increase in LPV exposure when ATV is co-administered with LPV/RTV.
When double-RTV-boosted PI combinations are utilized, the subsequent effects of LPV on exposure to other PI vary. The combination of LPV/RTV plus APV or FPV has resulted in important reductions in plasma concentrations of APV [10–14]. The combination of LPV/RTV and SQV has shown favourable pharmacokinetic profiles, without any apparent modification of SQV exposure [15,16]. In our study, the AUC0–24 and C max values of ATV were similar in patients with LPV (48.2 μg/h/ml and 3.95 μg/ml, respectively) and in patients without LPV (45.2 μg/h/ml and 4.44 μg/ml, respectively). Plasma C trough and C min ATV values were almost twofold higher in patients receiving ATV plus LPV/RTV than in patients receiving ATV plus RTV (1.14 versus 0.61 μg/ml, P = 0.008; and 1.07 versus 0.58 μg/ml, P = 0.007, respectively). However, patients with LPV/RTV received 200 mg of RTV (100 mg b.i.d.), whereas those with RTV alone received only 100 mg. It has been observed that the association of 300 mg of ATV with 100 mg of RTV did not significantly increase the C max as compared to 400 mg of unboosted ATV, but it did produce a five- to sevenfold increase in the C trough . It is likely that the C min increase in arm A as compared to arm D in our study was due to the fact that patients in arm A received 100 mg more RTV and had a higher RTV C min than those in arm D. There was a significant correlation between RTV C min and ATV C min. In this setting, the combination of LPV with ATV did not appear to negatively influence exposure to ATV. Winston et al.  also observed significantly higher plasma trough ATV levels in nine patients on LPV/RTV/ATV regimens than in 72 patients on RTV-boosted ATV regimens without LPV (median values: 1.457 versus 0.604 μg/ml, P = 0.032).
When ATV was co-administered with LPV/RTV in arm A, plasma ATV concentrations in patients taking tenofovir were similar to those in patients who were not taking tenofovir. However, when ATV was co-administered with RTV at standard boosting dose (100 mg q.d.), without LPV, plasma ATV AUC0–24 and C max were significantly lower in patients taking tenofovir than in patients not taking this drug. The data in the literature about the interaction between tenofovir and ATV are controversial. In healthy individuals, a significant 25% reduction in the ATV C trough was observed when tenofovir was added to a regimen with ATV/RTV (300/100 mg) . After adding tenofovir to a regimen containing ATV/RTV (300/100 mg) in HIV-infected patients, Taburet et al.  found a trend toward lower ATV concentrations, but the decrease in the ATV AUC was the only difference that reached statistical significance. In other studies in HIV-infected patients, tenofovir had no effect on RTV-boosted ATV trough concentrations [17,32,35–37]. In any case the magnitude of the interaction between tenofovir and boosted ATV seems to be small and it is not necessary to increase ATV dose when it is administered with RTV boosting.
The combination of ATV and LPV/RTV was well tolerated, despite the high plasma concentrations of both ATV and LPV. None of the 16 patients had to discontinue study medication because of adverse events . Digestive tolerance was good in our patients. Nevertheless, a possible selection bias in the participating patients could have led to better tolerance of LPV/RTV: a large number of patients included had already received or were receiving LPV/RTV with good tolerance, whereas patients who had previously shown poor tolerance to these drugs were not considered for this treatment. Digestive tolerance to ATV is generally good [38–41]. The most common adverse event seen after ATV/LPV/RTV administration was an elevation in total bilirubin level, predominantly unconjugated. ATV plasma concentration plays a role in causing hyperbilirubinaemia. The frequency of grade 3 or 4 elevation of total bilirubin was 20–40% in non-boosted ATV regimens [38–40,42] and 30–50% in RTV-boosted ATV regimens [39,41]. The effect of jaundice is judged by patients on an individual basis, and less than 2% of patients in clinical trials discontinued therapy because they found it cosmetically unacceptable.
The combination of ATV plus LPV/RTV had substantial antiviral efficacy in these heavily pre-treated patients, with a reduction of 2.9 log10 in HIV RNA load and an increase of 118 CD4 cells/μl after 24 weeks of treatment. The study was not designed to assess therapeutic efficacy and does not have sufficient statistical power for this purpose; nevertheless, it is worthy of mention that a very high proportion of patients (13/16 in the intent-to-treat analysis) showed a plasma HIV-1 RNA load < 50 copies/ml after 24 weeks of treatment. In intensively pre-treated patients it is difficult to achieve lasting treatment efficacy. In the majority of studies, the percentage of patients with undetectable viral load after 24–48 weeks of rescue treatment following numerous treatment failures ranges from 20% to 55% [22,43–48]. The substantial efficacy in the present study can be attributed to the elevated concentrations of LPV and ATV achieved, the good tolerability of the treatment and the effect of the other associated antiretroviral drugs.
In summary, the combination of ATV and LPV/RTV provided high plasma concentrations of both PI. ATV seems to be able to further enhance LPV exposure in the presence of RTV. The C min of ATV was higher with the ATV/LPV/RTV combination than with standard boosting, probably because a higher dose of RTV was used when ATV was combined with LPV/RTV than in the standard boosting regimen. These high plasma concentrations of LPV and ATV seemed to be appropriate for combining these agents in a dual PI-based antiretroviral regimen for patients in whom multiple antiretrovirals had failed, yielding good tolerability and substantial antiviral efficacy. Further studies are required to confirm these encouraging preliminary results.
We thank, Sofia Garcia, Dolors Palau and the other members of the nursing staff for technical advice. The authors thank Celine L. Cavallo for English language editing.
1. Iribarren JA, Labarga P, Rubio R, Berenguer J, Miro JM, Antela A, et al
. Spanish GESIDA/Nacional AIDS Plan Recommendations for antiretroviral therapy in HIV-infected Adults (October 2004). Enferm Infecc Microbiol Clin 2004; 22:564–642.
2. Struble K, Murray J, Cheng B, Gegeny T, Miller V, Gulick R. Antiretroviral therapies for treatment-experienced patients: current status and research challenges. AIDS 2005; 19:747–756.
3. Gallant JE. Protease-inhibitor boosting in the treatment-experienced patient. AIDS Rev 2004; 6:226–233.
4. Boffito M, Maitland D, Samarasinghe Y, Pozniak A. The pharmacokinetics of HIV protease inhibitor combinations. Curr Opin Infect Dis 2005; 18:1–7.
5. King JR, Wynn H, Brundage R, Acosta EP. Pharmacokinetic enhancement of protease inhibitor therapy. Clin Pharmacokinet 2004; 43:291–310.
6. Andrew D. Luber. Double-boosted protease inhibitor regimens: a pharmacologic and pharmacokinetic perspective
. Clinical Care Options Web Site, available at: http://clinicaloptions.com/04doubleboost
7. Molla A, Mo H, Vasavanonda S, Han L, Lin CT, Hsu A, Kempf DJ. In vitro antiviral interaction of lopinavir with other protease inhibitors. Antimicrob Agents Chemother 2002; 46:2249–2253.
8. Pirmohamed M, Back DJ. The pharmacogenomics of HIV therapy. Pharmacogenomics J 2001; 1:243–253.
9. Jackson A, Taylor S, Boffito M. Pharmacokinetics and pharmacodynamics of drug interactions involving HIV-1 protease inhibitors. AIDS Rev 2004; 6:208–217.
10. De Luca A, Baldini F, Cingolani A, Di Giambenedetto S, Hoetelmans RM, Cauda R. Deep salvage with amprenavir and lopinavir/ritonavir: correlation of pharmacokinetics and drug resistance with pharmacodynamics. J Acquir Immune Defic Syndr 2004; 35:359–366.
11. Taburet AM, Raguin G, Le Tiec C, Droz C, Barrail A, Vincent I, et al
. Interactions between amprenavir and the lopinavir-ritonavir combination in heavily pretreated patients infected with human immunodeficiency virus. Clin Pharmacol Ther 2004; 75:310–323.
12. Kashuba AD, Tierney C, Downey GF, Acosta EP, Vergis EN, Klingman K, et al
. Combining fosamprenavir with lopinavir/ritonavir substantially reduces amprenavir and lopinavir exposure: ACTG protocol A5143 results. AIDS 2005; 19:145–152.
13. Khanlou H, Graham E, Brill M, Farthing C. Drug interaction between amprenavir and lopinavir/ritonavir in salvage therapy. AIDS 2002; 16:797–798.
14. Mauss S, Scholten S, Wolf E, Berger F, Schmutz G, Jaeger H, et al
. A prospective, controlled study assessing the effect of lopinavir on amprenavir concentrations boosted by ritonavir. HIV Med 2004; 5:15–17.
15. Ribera E, Lopez RM, Diaz M, Pou L, Ruiz L, Falco V, et al
. Steady-state pharmacokinetics of a double-boosting regimen of saquinavir soft gel plus lopinavir plus minidose ritonavir in human immunodeficiency virus-infected adults. Antimicrob Agents Chemother 2004; 48:4256–4262.
16. Stephan C, Hentig N, Kourbeti I, Dauer B, Mosch M, Lutz T, et al
. Saquinavir drug exposure is not impaired by the boosted double protease inhibitor combination of lopinavir/ritonavir. AIDS 2004; 18:503–508.
17. Boffito M, Kurowski M, Kruse G, Hill A, Benzie AA, Nelson MR, et al
. Atazanavir enhances saquinavir hard-gel concentrations in a ritonavir-boosted once-daily regimen. AIDS 2004; 18:1291–1297.
18. Cvetkovic RS, Goa KL. Lopinavir/ritonavir: a review of its use in the management of HIV infection. Drugs 2003; 63:769–802.
19. Le Tiec C, Barrail A, Goujard C, Taburet AM. Clinical pharmacokinetics and summary of efficacy and tolerability of atazanavir. Clin Pharmacokinet 2005; 44:1035–1050.
20. Goldsmith DR, Perry CM. Atazanavir. Drugs 2003; 63:1679–1693.
21. de Mendoza C, Martin-Carbonero L, Barreiro P, Diaz B, Valencia E, Jimenez-Nacher I, et al
. Salvage treatment with lopinavir/ritonavir (Kaletra) in HIV-infected patients failing all current antiretroviral drug families. HIV Clin Trials 2002; 3:304–309.
22. Ruiz L, Ribera E, Bonjoch A, Romeu J, Martinez-Picado J, Paredes R, et al
. Role of structured treatment interruption before a 5-drug salvage antiretroviral regimen: the Retrogene Study. J Infect Dis 2003; 188:977–985.
23. Brundage MD, Pater JL, Zee B. Assessing the reliability of two toxicity scales: implications for interpreting toxicity data. J Natl Cancer Inst 1993; 85:1138–1148.
24. Droste JA, Aarnoutse RE, Koopmans PP, Hekster YA, Burger DM. Evaluation of antiretroviral drug measurements by an interlaboratory quality control program. J Acquir Immune Defic Syndr 2003; 32:287–291.
25. Isaac A, Taylor S, Cane P, Smit E, Gibbons SE, White DJ, et al
. Lopinavir/ritonavir combined with twice-daily 400 mg indinavir: pharmacokinetics and pharmacodynamics in blood, CSF and semen. J Antimicrob Chemother 2004; 54:498–502.
26. Klein C, Bertz R, Ashbrenner E. Assessment of the multiple-dose pharmacokinetic interaction of lopinavir/ritonavir with nelfinavir
. Tenth Conference on Retroviruses and Opportunistic Infections
. Boston, February 2003 [abstract 536].
27. Flexner C, Chiu YL, Foit C, Perez P, Tillmann E, Podzamzcer D, et al
. Steady-state pharmacokinetics and short-term virologic response of two lopinavir/ritonavir (LPV/r) high-dose regimens in multiple antiretroviral-experienced subjects (Study 049). In: Second IAS Conference on HIV Pathogenesis and Treatment
. Paris, July, 2003 [Abstract 843].
28. Murphy RL, Brun S, Hicks C, Eron JJ, Gulick R, King M, et al
. ABT-378/ritonavir plus stavudine and lamivudine for the treatment of antiretroviral-naive adults with HIV-1 infection: 48-week results. AIDS 2001; 15:F1–F9.
29. Perloff ES, Duan SX, Skolnik PR, Greenblatt DJ, von Moltke LL. Atazanavir: effects on P-glycoprotein transport and CYP3A metabolism in vitro. Drug Metab Dispos 2005; 33:764–770.
30. Lucia MB, Golotta C, Rutella S, Rastrelli E, Savarino A, Cauda R. Atazanavir inhibits P-glycoprotein and multidrug resistance-associated protein efflux activity. J Acquir Immune Defic Syndr 2005; 39:635–637.
31. Busti AJ, Hall RG, Margolis DM. Atazanavir for the treatment of human immunodeficiency virus infection. Pharmacotherapy 2004; 24:1732–1747.
32. Winston A, Bloch M, Carr A, Amin J, Mallon PW, Ray J, et al
. Atazanavir trough plasma concentration monitoring in a cohort of HIV-1-positive individuals receiving highly active antiretroviral therapy. J Antimicrob Chemother 2005; 56:380–387.
33. Agarwala S, Eley T, Villegas C, Wang Y, Hughes E, Xie J, et al
. Pharmacokinetic interaction between tenofovir and atazanavir coadministered with ritonavir in healthy subjects
. Sixth International Workshop on Clinical Pharmacology of HIV Therapy
. Quebec, Canada, April, 2005. [abstract 16].
34. Taburet AM, Piketty C, Chazallon C, Vincent I, Gerard L, Calvez V, et al
. Interactions between atazanavir-ritonavir and tenofovir in heavily pretreated human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 2004; 48:2091–2096.
35. Guiard-Schmid JB, Poirier JM, Bonnard P, Meynard JL, Slama L, Lukiana T, et al
. Proton pump inhibitors do not reduce atazanavir concentrations in HIV-infected patients treated with ritonavir-boosted atazanavir. AIDS 2005; 19:1937–1938.
36. Von Hentig N, Haberl A, Lutz T, Klauke S, Kurowski M, Harder S, et al
. Concomitant intake of tenofovir disoproxil fumarate does not impair the plasma exposure of ritonavir boosted atazanavir in HIV-1 infected adults. American Society for Clinical Pharmacology and Therapeutics
. Orlando, March 2005 [abstract PI-40.]
37. Kruse G, Stocker H, Breske A, Aratesh K, Plettenberg A, Stazewski S, et al
. Trough levels of six different atazanavir regimens in HIV-infected patients
. Fifth International Workshop on Clinical Pharmacology of HIV Therapy
. Rome, April 2004. 2005 [Poster 6.6].
38. Haas DW, Zala C, Schrader S, Piliero P, Jaeger H, Nunes D, et al
. Therapy with atazanavir plus saquinavir in patients failing highly active antiretroviral therapy: a randomized comparative pilot trial. AIDS 2003; 17:1339–1349.
39. Johnson M, Grinsztejn B, Rodriguez C, Coco J, Dejesus E, Lazzarin A, et al
. Atazanavir plus ritonavir or saquinavir, and lopinavir/ritonavir in patients experiencing multiple virological failures. AIDS 2005; 19:685–694.
40. Sanne I, Piliero P, Squires K, Thiry A, Schnittman S. Results of a phase 2 clinical trial at 48 weeks (AI424-007): a dose-ranging, safety, and efficacy comparative trial of atazanavir at three doses in combination with didanosine and stavudine in antiretroviral-naive subjects. J Acquir Immune Defic Syndr 2003; 32:18–29.
41. Squires K, Lazzarin A, Gatell JM, Powderly WG, Pokrovskiy V, Delfraissy JF, et al
. Comparison of once-daily atazanavir with efavirenz, each in combination with fixed-dose zidovudine and lamivudine, as initial therapy for patients infected with HIV. J Acquir Immune Defic Syndr 2004; 36:1011–1019.
42. Cohen C, Nieto-Cisneros L, Zala C, Fessel WJ, Gonzalez-Garcia J, Gladysz A, et al
. Comparison of atazanavir with lopinavir/ritonavir in patients with prior protease inhibitor failure: a randomized multinational trial. Curr Med Res Opin 2005; 21:1683–1692.
43. Delaugerre C, Peytavin G, Dominguez S, Marcelin AG, Duvivier C, Gourlain K, et al
. Virological and pharmacological factors associated with virological response to salvage therapy after an 8-week of treatment interruption in a context of very advanced HIV disease (GigHAART ANRS 097). J Med Virol 2005; 77:345–350.
44. Montaner JS, Harrigan PR, Jahnke N, Raboud J, Castillo E, Hogg RS, et al
. Multiple drug rescue therapy for HIV-infected individuals with prior virologic failure to multiple regimens. AIDS 2001; 15:61–69.
45. Mazzotta F, Lo CS, Torti C, Tinelli C, Pierotti P, Castelli F, et al
. Real versus virtual phenotype to guide treatment in heavily pretreated patients: 48-week follow-up of the Genotipo-Fenotipo di Resistenza (GenPheRex) trial. J Acquir Immune Defic Syndr 2003; 32:268–280.
46. Saracino A, Monno L, Locaputo S, Torti C, Scudeller L, Ladisa N, et al
. Selection of Antiretroviral Therapy Guided by Genotypic or Phenotypic Resistance Testing: An Open-Label, Randomized, Multicenter Study (PhenGen). J Acquir Immune Defic Syndr 2004; 37:1587–1598.
47. Casau NC, Glesby MJ, Paul S, Gulick RM. Brief report: efficacy and treatment-limiting toxicity with the concurrent use of lopinavir/ritonavir and a third protease inhibitor in treatment-experienced HIV-infected patients. J Acquir Immune Defic Syndr 2003; 32:494–498.
48. Lazzarin A, Clotet B, Cooper D, Reynes J, Arasteh K, Nelson M, et al
. Efficacy of enfuvirtide in patients infected with drug-resistant HIV-1 in Europe and Australia. N Engl J Med 2003; 348:2186–2195.