The study drugs were withdrawn in two patients: one because of virological failure and one by personal choice.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 Ctrough and Cmin 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, Cmax, Ctrough, and Cmin, 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 Ctrough and C1 ATV concentrations were significantly higher in arm A (Fig. 2). Specifically, median ATV Ctrough (1.14 versus 0.61 μg/ml; P = 0.020) and median ATV Cmin (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 Cmax between arms A and D. In arm A we observed a moderate correlation between the RTV Cmin, and the ATV AUC0–24 (r, 0.57; P = 0.020) and Cmin (r, 0.46; P = 0.042), and in arm D, between the RTV Cmin and ATV Cmin (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, Cmax and Ctrough 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, Cmax and Ctrough 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, Cmax, Cmin, 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, Cmax, 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 Cmax 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 Ctrough and Cmin 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 Cmax as compared to 400 mg of unboosted ATV, but it did produce a five- to sevenfold increase in the Ctrough . It is likely that the Cmin 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 Cmin than those in arm D. There was a significant correlation between RTV Cmin and ATV Cmin. 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 Cmax 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 Ctrough 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 Cmin 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.
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