The inhibitory quotient (IQ) of human immunodeficiency virus (HIV) protease inhibitors (PI) that is the ratio of drug concentration to viral susceptibility is considered to be predictive of the virological response. We used several approaches to calculate the IQs of amprenavir and lopinavir in a subset of heavily pretreated patients participating in the ANRS-104 trial, then compared their potential for predicting changes in the plasma HIV-RNA level. Thirty-seven patients were randomly assigned to receive either amprenavir (600mg BID) or lopinavir (400mg BID) plus ritonavir (100 or 200mg BID) for two weeks, before combining the two PIs. The IC₉₀ was measured using a recombinant assay with or without additional human serum (IC_90+serum). Total and unbound PIs concentrations in plasma were measured. Univariate linear regression was used to estimate the relation between the change in viral load and the IC₉₀ or IQ values. The amprenavir phenotypic IQ values were very similar when measured with the standard and protein binding-adjusted IC₉₀. No relationship was found between the viral load decline and the lopinavir IQ. During combination therapy the amprenavir and lopinavir genotypic IQ values were predictive of the viral response at week 6 (p=0.03). The number of protease mutations (< or ≥ 5) was related to the virological response throughout the study. These findings suggest that the combined genotypic IQ and the number of protease mutations are the best predictors of virological response. High amprenavir and lopinavir concentrations in these patients might explain why plasma concentrations and the phenotypic IQ have poor predictive value.

The virological efficacy of protease inhibitors (PI) in patients infected by the Human Immunodeficiency Virus (HIV) is dependent on both their pharmacodynamic and pharmacokinetic properties. In vitro high and sustained plasma (or cell) concentrations are needed to maximally suppress viral replication. The dose administered in HIV-infected patients should provide such concentrations.

The inhibitory quotient (IQ), defined as the ratio of the trough drug concentration to viral susceptibility expressed as an inhibitory concentration (ideally the IC₉₀), has been used to estimate the antiviral potency of PIs in vivo (24). This parameter has also been proposed to optimize the dosing regimen of treatment-experienced patients (12, 20). Although there is a strong theoretical rationale for using the inhibitory quotient, the practical value of this parameter is controversial. Firstly, there are few prospective studies of the relationship between IQ and virological response (22). Secondly, there is no consensus method for calculating this parameter (1, 24, 27). Standard calculations estimate IQ from both the plasma drug concentration and virus susceptibility. However, several pharmacokinetic and virologic issues remain unsolved. In most pharmacokinetic studies the total drug concentration in plasma is measured, whereas the active component is the free (protein-unbound) fraction. Furthermore, the addition of human albumin to the cell culture medium increases the IC₉₀ in vitro. In summary, either the total concentration or the protein-adjusted concentration, and either the standard or the serum-adjusted IC₉₀, can be used to calculate the IQ. More recently, a “genotypic inhibitory quotient” was proposed, as the ratio of the plasma concentration to the number of mutations on the viral protease gene. Indeed, genotypic resistance assays can be performed rapidly and are less costly than phenotypic resistance assays (11). Both genotypic and phenotypic IQs are predictive of changes in the HIV RNA level in treatment-experienced patients (5, 9, 11, 17, 22, 23, 31), but only one study of the predictive value of the IQ is available in patients treated with two ritonavir-boosted PIs (8).

We aimed at estimating the relationship between IQs of amprenavir and lopinavir and virological response after 2, 6 and 26 weeks of treatment in a group of heavily pretreated patients who participated in the ANRS-104 trial. Several methods for calculating IQs were compared with the viral phenotype and genotype for their ability to predict changes in plasma viral load.

Overall, 37 patients were enrolled in the ANRS 104 study. Their baseline characteristics are shown in table 1. Twenty three patients started their study antiretroviral drug regimen with lopinavir/ritonavir (group 1) and 14 patients started with amprenavir (group 2).

Amprenavir and lopinavir Cmin values are reported in detail elsewhere (26, 29) and were not related to the virologic response. The median unbound amprenavir Cmin_u at week 2 was 177 ng/mL and was not predictive of the virologic response at week 2.

In a subgroup of 12 patients who had phenotypic studies (group 2), the amprenavir IC₉₀ was 57.8 ng/mL (8.7–150.9), and increased to 453 ng/mL (33–1105) when measured in the presence of 50% human serum (IC_90+serum). The IC_90+serum was a good predictor of the early virologic response (w2 p=0.006), whereas the IC₉₀ was not. Amprenavir IQs adjusted for protein binding (IQu and IQ_serum) were rather similar (2.5 and 3.6, respectively). The relationship with viral load decline at week 2 was slightly better with IQ_u than IQ_serum (r²=0.45, p=0.02 versus r²=0.31, p=0.06), although those results might be explained by an outlier patient (r²=0.24, p=0.12 versus r²=0.11, p=0.31 without the outlier patient).

In the 21 patients treated with lopinavir during the first period, the lopinavir IC₉₀ was 34.2 ng/mL (0.7–330.4) and was a good predictor of the virologic response at weeks 2, 6 and 26 (r²=0.37, p=0.003; r²=0.23, p=0.03; r²=0.33, p=0.006 respectively). The number of protease mutations (< or > 5) was related to the virologic response throughout the study (at week 2, r²=0.18, p=0.008; at week 6 r²=0.11, p=0.046; and at week 26 r²=0.12, p=0.034 - figure 1).

Table 2 shows the virologic responses throughout the study as a function of the amprenavir and lopinavir phenotypic IQ and GIQ values measured at week 2 or week 6. The amprenavir genotypic IQ (corrected amprenavir Cmin at week 2/number of protease mutations at week -2) was related to the virologic response at week 2 (r²=0.66, p=0.0004) and at week 6 (r²=0.38, p=0.02). Patients who had a Cmin corrected GIQ above 75 had a median decline in HIV-RNA of 1.23 log10 copies/mL (−2.27 ; −0.97) versus 0.19 log10 copies/mL (−1.07 ; 0.15) in patients with a GIQ below 75 (p=0.005). In contrast, none of the lopinavir IQs was predictive of antiretroviral activity.

None of the phenotypic or genotypic IQs of amprenavir or lopinavir (determined with the Cmin corrected for protein binding and measured at week 6) was predictive of the viral load decline. However, a combination of the amprenavir and lopinavir genotypic IQs, whether measured either with the standard equation or adjusted for protein binding, and with or without ritonavir, was predictive of the viral response at week 6 but not at the end of the study as shown on figure 2. Patients who responded at week 6 (plasma HIV-RNA < vs ≥ 10000 copies/mL) had a higher median combined genotypic IQ measured at week 6 (65 vs 29 p=0.01).

A significant proportion of antiretroviral-experienced patients never have optimal viral suppression or experience a viral rebound shortly after starting a new treatment. Our population was heavily pretreated with a high proportion of baseline NRTIs genotypic resistance, consequently, the efficacy of the combination results essentially from both PIs. Treatment thus needs to be optimized according to viral susceptibility and the plasma PI concentration. This study provides IQ data for two currently used ritonavir-boosted PIs, amprenavir and lopinavir, when administered alone (first two weeks of the study) or in combination, in heavily pretreated HIV-infected patients. This is one of the few studies to show that the combined IQ can be a useful predictor in patients who receive ritonavir-boosted dual-PI therapy (8).

We compared two methods for calculating the phenotypic IQ, which incorporates protein binding. Amprenavir and lopinavir are highly bound to plasma proteins, and especially alpha1 acid glycoprotein (90% and 98–99% respectively). As only free drug inhibits viral replication, protein binding affects the potency of these two PIs. ICs measured in vitro must be adjusted for protein binding before extrapolation to the clinical setting. Moreover, ICs suffer from variability due to a lack of standardisation of phenotyping methods (14). There are three methods for adjusting the in vitro IC for protein binding: multiplication of the IC by the free fraction measured in vivo; measurement of the IC in the presence of 50% human serum albumin; and multiplication of the IC by a constant to adjust for assay variations (20), but none of these methods is clinically validated. Our results show that, whatever the equation used to calculate IQ (Cmin_u/standard IC₉₀ or Cmin/IC_90+serum), the relationship with viral load decline is very similar. The best correlation was obtained with the IQ_u (Cmin_u/IC90). It has previously been demonstrated that measuring and calculating the Cmin_u gives similar results (29). However, it remains to be determined whether these findings can be extrapolated to PIs other than amprenavir.

We found no relationship between lopinavir or amprenavir exposure and the decline in viral load, in agreement with previous studies (6, 7, 13, 16, 30). Amprenavir and lopinavir Cmin values obtained with the ritonavir boost were far higher than the IC₅₀ reported for wild-type virus (28) and the IC₉₀ measured at screening. This might explain why plasma concentrations were poorly predictive of the decline in plasma HIV RNA. Compliance with medication was not measured in our study; the amprenavir and lopinavir Cmin values suggested that it was maximal at week 2 and week 6 but declined thereafter. Thus, none of the phenotypic IQ values based on concentrations measured at week 6 was predictive of antiviral efficacy.

The genotypic IQ is simpler to measure than the phenotypic IQ. We found that the genotypic IQ of amprenavir was associated with the virologic response at week 2 and week 6 but not at week 26. Furthermore the amprenavir GIQ cut off of 75 could be a useful tool in clinical practice as previously demonstrated by Marcelin et al. (18). Our findings confirm that the genotypic IQ, which incorporates both baseline viral resistance and the level of drug exposure in plasma, is superior to drug exposure alone in predicting the virologic response to a salvage regimen (18). However, lopinavir IQs did not correlate with virologic efficacy, and our data do not support those reported elsewhere (5, 9, 16). This is probably because the lopinavir Cmin values in our patients were sufficiently high not to restrict efficacy (13), whereas lopinavir concentrations were lower in other studies (5, 9). In these latter studies, the Cmin was determined as part of routine therapeutic drug monitoring or observational studies, settings in which compliance is important for overall exposure (5, 9, 16). One limitation of therapeutic drug monitoring is the wide intra- patient variability of trough concentrations, as recently demonstrated by Nettles et al.(21) and Goujard et al. (10). Poor compliance and an effect of food may be involved in this variability. We acknowledge that one limitation of our study is that compliance was not measured. However, comparison of trough concentrations measured at weeks 6, 14 and 26 clearly shows that compliance is decreasing from week 6, where concentrations of lopinavir and amprenavir were in the expected range in all patients, in contrast to weeks 14 and 26 where 7 and 6 patients respectively, had concentrations below the limit of quantification of the assay (26).

Interestingly, the combined GIQ was predictive of the virologic response at week 6, as in the GigHAART trial (8). This suggests that a combination of two PIs has a strong antiviral effect that might overcome resistance in these strongly pretreated patients. As expected, ritonavir did not participate in the drug effect, as the concentrations used to boost PIs are too low (even though some patients received 200 mg BID). The combined IQ of amprenavir+lopinavir+ritonavir was close to the combined IQ of amprenavir+lopinavir and had the same predictive potential. However, the predictive value of this parameter disappeared at the end of the study, for several possible reasons: in particular, the Cmin tends to fall, as a negative initial interaction between the two PIs and a decreased compliance tends to reduce their concentrations and exposure levels; second, the viral resistance profile is continually evolving (19). Further studies are needed to determine which viral mutations have the biggest impact on the genotypic IQ, and how these mutations can be taken into account in the calculations (30).

The combined genotypic IQ did not have better predictive value than the number of mutations. In these highly pretreated patients with high PI Cmin values, the number of PI resistance mutations is a major determinant of virologic outcome (5, 13, 16) and, in our study, was the only factor predictive of the virologic response at week 26. Finally, as previously demonstrated (26), patients with more than five protease resistance mutations or a lopinavir mutation score (13,14) higher than 5 at baseline had a significantly poorer virological response than other patients (p=0.04 and p=0.006, respectively).

Thus, this study suggests that when treatment compliance is optimal and high PI concentrations are achieved, the viral genotype is the best predictor of virologic outcome.