Indinavir protein-free concentrations when used in indinavir/ritonavir combination therapy

Objective: To describe the in vivo protein-binding characteristics of indinavir (IDV) in the presence of ritonavir (RTV) relative to total IDV plasma concentrations.

Design: The ACTG protocol 5055 was a multicenter study comparing the safety and pharmacokinetics of IDV/RTV at doses of 800/200 and 400/400 mg twice daily in HIV-infected adults.

Methods: Forty-four patients underwent a 12-h intensive pharmacokinetic assessment after 2 weeks of therapy. Three plasma samples from 35 patients at C_max, 6 and 12 h post dose were used to determine the unbound IDV concentrations. Unbound IDV was separated in plasma samples using ultra-filtration and measured using high-performance liquid chromatography with UV detection.

Results: Mean IDV protein-bound fraction across all time points in the 800/200 and 400/400 arm were 53.4 and 51.8%, respectively. In the 800/200 arm, percentage binding at C_max was 50% compared with 56% at 12 h (P = 0.008). In the 400/400 arm, percentage binding at C_max was 49% compared with 54% at 12 h (P = 0.008).

Conclusions: The extent of plasma protein binding of IDV in this study was less than in previously published data with IDV alone. Although IDV concentrations differed across the arms, the percentage of IDV protein binding at all time points was not different between the 800/200 and 400/400 arms. However, the percentage of IDV protein binding at C_max was significantly lower compared with 12 h in each arm, possibly suggesting that IDV protein binding is concentration-dependent. These data suggest that RTV affects IDV protein-binding characteristics and IDV also exhibits concentration dependent binding when administered with RTV.

Introduction

Traditional assays used to quantify protease inhibitors (PIs) in plasma measure the total drug concentration, but it is the unbound fraction that penetrates cells and elicits a pharmacologic effect. All PIs are extensively bound to proteins at varying degrees; nelfinavir (NFV), lopinavir (LPV) and ritonavir (RTV) are the highest (> 98%) followed by amprenavir (APV), atazanavir, and indinavir (IDV). In vitro data indicate that IDV is approximately 60% plasma protein bound, primarily to alpha-1 acid glycoprotein (AAG) ^[1]. IDV is commonly administered with RTV, which has a higher binding affinity for AAG ^[2]. When co-administered, RTV may displace IDV from AAG although the IDV steady-state unbound concentration may not be altered. As a result, interpretation of the total IDV concentration may be inaccurate. The goals of this study were to examine the magnitude of difference between total and unbound IDV concentrations at select time points throughout a steady-state dosing interval in HIV-infected adults, and to estimate the change in percentage IDV bound in the presence of RTV as compared to IDV alone from historical data.

Methods

The ACTG protocol 5055 was a multicenter, open-label, 24-week comparative study of two IDV/RTV regimens plus two nucleoside reverse transcriptase inhibitors (NRTI) in HIV-infected adults ^[3]. Patients were randomized to either IDV/RTV 800/200 mg twice daily (Arm A) or 400/400 mg twice daily (Arm B). Forty-four subjects participated in a 12-h pharmacokinetic assessment at steady state. Blood samples were collected at baseline and 0.5, 1, 2, 3, 4, 5, 6, 8, 10 and 12 h post dose. IDV and RTV pharmacokinetic parameters were determined using non-compartmental techniques ^[4]. Three plasma samples from each patient at the maximum IDV concentration (C_max) and approximately 6 and 12 h post dose were used to determine the unbound IDV concentrations.

Unbound IDV was separated in plasma samples using ultra-filtration. One milliliter of plasma was added to an Amicon Centrifree YM-30 filter system (Millipore Corp., Bedford, Massachusetts, USA). Samples were equilibrated for 15 min at 37°C and centrifuged at 300 g for 30 min. IDV concentrations were quantifed with a validated high-performance liquid chromatography assay ^[5]. The lower limit of quantification was 20 ng/ml. Inter-day and intra-day coefficients of variation were less than 7 and 5%, respectively. Microsoft Excel-2000 (Microsoft, Redmond, Washington, USA) software was used for all calculations. Statistical comparisons were made using non-parametric t-tests. Differences in variability between treatment arms were tested using the comparison of variance in independent samples.

Results

Plasma samples from 35 of 44 subjects were used to determine the unbound IDV concentrations; 83% of samples were from males and 17% from females. The median (range) age and weight were 39 (30-61) years and 75.4 (58.5-101) kg. Sufficient amounts of plasma were unattainable in nine patients. Total and unbound IDV concentrations were determined for 17 patients in the 800/200 arm and 18 patients in the 400/400 arm. Table 1 describes the total and unbound IDV concentrations and the percentage binding; Fig. 1 depicts the percentage binding at 12 h and at C_max.

Table 1

Fig. 1

Binding characteristics between the 800/200 and 400/400 arm

Total IDV concentrations at each time point were significantly different between treatment arms. Unbound IDV concentrations at C_max and 6 h post dose for the 800/200 arm were significantly higher in comparison with the 400/400 arm but did not differ 12 h post dose. Percentage protein binding was similar at each time point between the treatment arms. Variability in total and unbound IDV concentrations was greater for the 800/200 arm compared with the 400/400 arm (P < 0.05). Variability in percentage protein binding was significantly higher in the 800/200 arm compared with the 400/400 arm at 12 h post dose only (P < 0.05).

Binding characteristics within the 800/200 and 400/400 arms

The mean ± SD IDV percentage protein binding across all concentrations in the 800/200 arm was 53.4 ± 5.7%. Percentage protein binding 12 h post dose was significantly higher compared with C_max (56 versus 50%, P = 0.008). The variability in percentage protein binding was similar across all time points and the variability between total and unbound concentrations was similar at each time point. For example, the coefficient of variation (CV%) at C_max was 34% for total and 35% for unbound concentrations.

The mean ± SD IDV percentage protein bound across all concentrations in the 400/400 arm were 51.8 ± 4.4%. Percentage protein binding 12 h post dose was significantly higher compared with C_max (54 versus 49%, P = 0.008). The variability in percentage protein binding was similar across all time points and the variability between total and unbound concentrations was also similar at each time point; the CV% at C_max was 24% for total and 30% for unbound concentrations.

Discussion

Data describing in vivo protein-binding characteristics of IDV are limited. These data are the first to describe IDV protein-binding characteristics in a large number of patients receiving IDV and RTV at different doses. Overall, protein binding for IDV was 53% for the 800/200 arm and 52% for the 400/400 arm. The percentage of protein binding at any time point between the two arms was not significantly different. Within both arms, however, the percentage of IDV protein binding at 12 h post dose was significantly higher compared with C_max. The percentage binding 12 h post dose was 56 versus 50% at C_max for 800/200 (P = 0.008), and 54 versus 49% at C_max for 400/400 (P = 0.008). Variability in percentage protein binding was present as demonstrated by the range of 42-69% in the 800/200 arm and 40-60% in the 400/400 arm (Fig. 1). This variability is likely not accounted for by assay or ultrafiltration methodology since the lowest concentration measured was four times above the assay limit of detection. Total IDV concentrations were different between the two arms at all time points. Unbound IDV concentrations in the 800/200 arm were significantly higher than the 400/400 arm for C_max and 6 h post dose, but not 12 h post dose.

Protein-binding characteristics of IDV when administered with RTV in our HIV-infected subjects differ slightly from previously reported in vivo and in vitro data. IDV protein binding in our patients was considerably lower than the in vitro estimation of 60% ^[1] and the in vivo estimation of 61% ^[6]. These estimations, however, were from IDV administered alone. These differences suggest that RTV may affect IDV protein-binding characteristics relative to IDV alone. Protease inhibitors bind primarily to AAG, which binds drug molecules with low capacity but high affinity and makes dissociation or displacement the most common cause of altered affinity. For example, the addition of RTV to NFV in vitro showed that RTV displaces NFV from AAG although the NFV unbound fraction in plasma is not altered ^[7]. RTV has been reported to have a higher affinity for AAG compared with IDV ^[2], and may displace IDV from AAG. In contrast, one study reported no difference in SQV or IDV protein-binding characteristics in vivo with the addition of RTV ^[8]. The median percentage protein binding in six HIV-infected patients receiving SQV twice daily was 98.5% compared with 98.9% in 12 patients receiving SQV and RTV at undefined doses (P = 0.069). The median percentage binding in eight patients receiving IDV three times daily was 64.8% compared with 58.7% in nine patients receiving IDV and RTV at undefined doses (P = 0.069). Although not statistically different, there was a trend toward decreased percentage binding with the addition of RTV, and the small sample size of this study probably decreased the power to detect a statistical difference between groups.

The protein-binding characteristics of IDV in our study suggests the potential for concentration-dependent protein binding in accordance to previously published data from eight HIV-infected men receiving IDV 800 mg three times daily ^[6]. The mean IDV percentage protein binding 8 h post dose was 66 ± 5.9% compared with 57 ± 5.2% at 1 h post dose (P = 0.0006). Likewise, in both arms of our study, IDV percentage protein binding at concentrations 12 h post dose was significantly higher compared with C_max. This is in contrast to a previous report in which the percentage of IDV protein binding remained constant over the full dosing interval in 17 HIV-infected patients receiving an undefined dose of IDV ± RTV ^[9]. Concentration-dependent protein binding has also been noted with LPV, which is more than 99% protein bound ^[10]. The unbound percentage of LPV was higher 2 h post dose (1.05 ± 0.32%) compared with 12 h post dose (0.84 ± 0.31%, P = 0.0009) in 23 HIV-infected patients receiving LPV/RTV 400/100 mg twice daily. Whether IDV displacement or concentration-dependent binding is occurring, these changes will likely be clinically insignificant, as altered protein binding will not appreciably change the unbound concentration at steady state. However, these data do have potential implications for monitoring drug levels. Since therapeutic and toxic concentrations of PIs are dependent on unbound drug concentrations, interpretation of total plasma concentrations of PIs may be misleading because at a given total concentration there could be higher unbound concentrations. Furthermore, multiple protease inhibitor use has become more common in highly treatment experienced patients, and complex drug-drug interactions have resulted. By measuring total drug concentrations in these regimens, we may be misinterpreting the clinical significance of the interaction as the ratio of free to unbound drug might be increased. An example of this has recently been published ^[11]. Nine patients receiving APV 600 mg twice daily plus RTV 100-200 mg twice daily for 2 weeks then added LPV/RTV 400/100 mg twice daily for 4 more weeks. The median APV-free drug fraction increased from 8.9 to 11.4% after the addition of LPV/RTV; the total and unbound APV concentrations both decreased due to the induction effect of LPV. These data suggest that displacement interactions probably play some role when multiple highly-bound PIs are used concomitantly, and imply that measuring only total drug concentrations when evaluating PI interactions may be ambiguous in terms of the clinical efficacy of the regimen.

Greater variability in total and unbound concentrations was seen in the 800/200 arm compared with the 400/400 arm. Studies of dual protease inhibitor regimens have shown that higher RTV doses decrease variability in plasma concentrations of the concomitantly administered protease inhibitor ^[12,13]. The decreased variability seen in these concentrations between the treatment arms is presumably due to the more potent CYP450 3A4 inhibition of IDV with the higher 400 mg dose of RTV. The range of variability in protein binding (42-69% in the 800/200 arm and 40-60% in the 400/400 arm; Fig. 1) was similar to the range of 54-70% reported previously with IDV alone ^[6] and was significantly different between the treatment arms at 12 h post dose only.

In conclusion, IDV protein binding in this study appeared lower when administered with RTV in comparison with previous data using IDV alone. Results from this study suggest that RTV may displace IDV from AAG, thus decreasing protein binding and increasing the unbound drug levels. The difference in IDV protein binding when co-administered with RTV is probably clinically insignificant. However, studies comparing the protein binding characteristics of other dual PI combinations with drugs that exhibit more extensive protein binding are warranted.

Acknowledgements

Sponsorship: This study was supported by grants UO1 AI38858 from the National Institute of Allergy and Infectious Diseases.