JAC Advance Access originally published online on August 7, 2007
Journal of Antimicrobial Chemotherapy 2007 60(4):831-836; doi:10.1093/jac/dkm282
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Amprenavir and ritonavir plasma concentrations in HIV-infected patients treated with fosamprenavir/ritonavir with various degrees of liver impairment
1 Infectious Disease Department, San Raffaele Scientific Institute and Università Vita-Salute San Raffaele, Via Stamina d'Ancona 20, 20122 Milan, Italy 2 Laboraf, San Raffaele Scientific Institute and Università Vita-Salute San Raffaele, Milan, Italy
* Corresponding author. Tel: +39-02-26437934; Fax: +39-02-26437903; E-mail: elena.seminari{at}hsr.it
Received 23 April 2007; returned 8 June 2007; revised 26 June 2007; accepted 3 July 2007
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Abstract |
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Objectives: The purpose of this study was to evaluate the steady-state pharmacokinetics of amprenavir and ritonavir in HIV-infected patients with different degrees of hepatic impairment.
Methods: HIV-positive patients receiving fosamprenavir/ritonavir (700/100 mg twice daily) were included. Patients were classified into three groups: (i) chronic hepatitis; (ii) liver cirrhosis; (iii) normal liver function. Serial blood samples for steady-state amprenavir and ritonavir pharmacokinetics (>14 days on treatment) were collected in the fasting state before the morning dose (Ctrough) and then 1, 2, 3, 4, 6, 8, 10 and 12 h after drug intake. Amprenavir and ritonavir plasma concentrations were determined by HPLC.
Results: Twenty-one HIV-infected patients were included. Seven had chronic hepatitis, eight had liver cirrhosis and six patients were in the control group. Amprenavir AUC0–12, AUC0–
, Cmax and Css were increased by 50% to 60% in the cirrhotic group when compared with controls, whereas CL/F was decreased by 40%. Patients with chronic hepatitis showed a significant increase in AUC0–12, Cmax and Css values when compared with controls. Ritonavir pharmacokinetics was different only in cirrhotic patients when compared with controls. Liver function parameters at weeks 4, 12 and 24 were not different from baseline in any of the groups. Overall, a significant correlation between amprenavir AUC0–12 and total bilirubin values on the day of pharmacokinetic analysis was found (r = 0.64, P = 0.003).
Conclusions: On the basis of these data and also of data available in the literature, it seems reasonable to adapt the dose of fosamprenavir and/or ritonavir exclusively in the presence of adverse events, possibly related to protease inhibitors (i.e. liver toxicity), in subjects with high drug plasma levels. Therapeutic drug monitoring is advised in the management of these patients.
Keywords: cirrhosis , HCV , HBV , pharmacokinetics
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Introduction |
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Like most protease inhibitors, amprenavir undergoes extensive hepatic metabolism primarily via cytochrome P450 (CYP) 3A4 isoenzymes and is largely bound to albumin and
1-acid glycoprotein, which are produced by the liver. Liver diseases, including hepatitis C virus (HCV) infection, significantly decrease the amount and function of CYP enzymes. Only a few studies have so far examined the effect of liver disease on antiretroviral drug metabolism,1–3 and amprenavir pharmacokinetics has only been investigated in a single-dose pharmacokinetics study involving HIV-negative patients with severe liver disease, who showed a significant increase in the AUC0– of amprenavir in comparison with controls.1 A recent study showed reduced plasma concentrations of amprenavir and ritonavir in patients with hepatic impairment treated with different doses of fosamprenavir and ritonavir.4 As the acute liver toxicity observed in 6% to 30% of the patients treated with antiretrovirals is primarily associated with HCV co-infection,5 it has been hypothesized that it might be due to increased drug exposure in patients with liver disease.
The aim of this study was to evaluate the steady-state pharmacokinetics of amprenavir and ritonavir in HIV-infected patients with different degrees of hepatic impairment.
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Methods |
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Study design
HIV-positive patients received fosamprenavir within the ‘Fosamprenavir Expanded Access Program (EAP)’ (GlaxoSmithKline APV102027). The study was approved by Ethics Committee of San Raffaele Hospital, Milan, and patients gave their informed written consent.
Patients included in this study received a single boosted protease inhibitor-based regimen containing fosamprenavir/ritonavir (700/100 mg twice daily).
The patients were divided into three groups: (i) HIV-infected patients with chronic hepatitis [documented by detectable plasma HCV-RNA/hepatitis B virus (HBV)-DNA and liver histology]; (ii) HIV-infected patients with liver cirrhosis (determined by selected imaging modalities: CT scan, ultrasound and/or liver histology); (iii) HIV-infected patients with normal liver function test results and without history of HCV or HBV co-infection (control group).
Patients with cirrhosis were eligible provided that they did not have stage 3 or 4 encephalopathy, severe ascites or oedema or bleeding from oesophageal varices in the 2 months preceding enrolment and had no history of clinically significant malabsorption or gastrointestinal surgery or renal or cardiac dysfunction. Concomitant treatment with non-nucleoside reverse transcriptase inhibitors was not allowed, nor was treatment with non-antiretroviral drugs potentially capable of interfering with the CYP enzymatic system or the consumption of alcohol.
Serial blood samples for steady-state amprenavir and ritonavir pharmacokinetics (>14 days on treatment) were collected in the fasting state before the morning dose (Ctrough) and then 1, 2, 3, 4, 6, 8, 10 and 12 h after drug intake. Fosamprenavir and ritonavir were given together after a meal (standard continental breakfast). On the day of pharmacokinetic assessment, aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholinesterases, total bilirubin, albumin, 1-acid glycoprotein, creatinine, creatinine clearance, CD4+ and plasma HIV-RNA were tested and patients' medical histories and concomitant therapies were assessed.
The liver function parameters were evaluated also on the day the patients started fosamprenavir/ritonavir (baseline) and after 4, 12 and 24 weeks on therapy.
High-performance liquid chromatography
Amprenavir and ritonavir plasma concentrations were both determined by HPLC. The assay was adapted as required from a previously reported method by Hugen et al.6
Plasma for drug measurement was separated by centrifugation and frozen at –20°C until analysed.
The calibration curves were calculated by the response ratio of the compound's peak height to the internal standard peak height plotted versus the analytes concentration. The assay was linear over the validated concentration range of 100–20 000 ng/mL for amprenavir and of 50–12 000 ng/mL for ritonavir. Variation of calibration standard fittings was usually below 7%. The regression coefficients of determination (r2) values of the calibration curves were >0.998.
The inter-assay and intra-assay precision and accuracy were determined using control samples at low, medium and high concentrations. The precision was calculated as the relative standard deviation within a single run (intra-assay) and between different assays (inter-assay), expressed as coefficient of variation (CV%). The accuracy was defined as the percentage of the nominal and the measured concentrations [(measured concentration–nominal concentration)/nominal concentration x 100]. Control samples were prepared by addition of a known amount of the drugs to blank plasma. Three different concentrations (1000, 5000 and 10 000 ng/mL) were used in replicates of six on 6 separate days. The method showed inter-assay and intra-assay precision and accuracy always <15%, for both amprenavir and ritonavir, in accordance with published recommendations.7 The limit of detection (the lowest detected drug concentration, giving a signal-to-noise ratio of >3:1) was 
50 ng/mL for amprenavir and 35 ng/mL for ritonavir. The limit of quantification, defined as the lowest concentration in plasma sample such that the deviation between measured and nominal concentration is <20%, was set at 100 ng/mL for amprenavir and 50 ng/mL for ritonavir.
Amprenavir and ritonavir were extracted from 1 mL of plasma spiked with 50 µL of internal standard (10 mg of A86093.0 ABBOTT in 10 mL of methanol and diluted with a solution of methanol/H2O, 1:1, to give a 100 000 ng/mL concentration). Plasma samples were buffered with 1 mL of 0.1 N NH4OH and extracted with 5 mL of methyl tert-butyl ether. Samples were shaken for 5 min and then centrifuged at 3000 rpm for 5 min. The organic supernatant was transferred and evaporated under high vacuum centrifuge at 37°C. The residues were dissolved in 600 µL of eluent (acetonitrile/50 mM KH2PO4 pH 5.63, 40:60, v/v), vortexed and put in an ultrasonic bath for 15 min. These solutions were vortexed a second time and then washed with 2.5 mL of hexane. Samples were shaken for 1 min and then centrifuged for 5 min at 3000 rpm. An aliquot of the lower layer (200 µL) was transferred to an autosampler vial with insert for injection into the HPLC system and analysed.
Chromatographic analysis was performed at ambient temperature with gradient elution at a wavelength of 215 nm. During gradient elution, the acetonitrile content of the mobile phase was increased linearly from 36% to 64% during the first 23 min at a flow rate of 1.5 mL/min. In 2 min, the acetonitrile content was returned to 34% and semi-equilibrated for 3 min before the next injection. Aliquots of 100 µL were injected into the chromatograph. Under these chromatographic conditions, the retention times of amprenavir, internal standard and ritonavir were 8, 16 and 13 min, respectively.
The liquid chromatographic unit consisted of a VARIAN ProStar HPLC system formed by two Binary Gradient Pumps, UV–Vis Detector Model 310 and Auto Sampler Model 410.
Chromatographic separation was performed by a CHROPACK INTERTSIL ODS-2 C18 analytical column (4.6 x 150 mm, 5 µm; CPS Analitica s.r.l, Milan, Italy).
Pharmacokinetic calculations on plasma amprenavir and ritonavir concentrations were performed using WinNonlin 4.1. The pharmacokinetic parameters were estimated from the plasma concentration versus time data using non-compartmental methods: observed peak concentration (Cmax), time to Cmax (Tmax), AUC0–12 calculated using the linear trapezoidal rule, apparent oral plasma clearance (CL/F) calculated as dose/AUC0–12 and adjusted for body weight and Css calculated as AUC0–12 divided by the dosing interval. The data are expressed as median values and ranges.
The Ctrough, Cmax, AUC0–12, AUC0–, CL/F, Css values of each group were log transformed, and the resulting estimates of mean values and mean differences were exponentiated to express the results as geometric means and geometric mean ratios on the original scale of measurement together with their 95% confidence intervals (CIs). The differences in the pharmacokinetic parameters between each experimental group and the controls were considered statistically significant if the 95% CI did not include 1.
The Tmax distribution values of the three groups were compared using the non-parametric Mann–Whitney rank sum test; Wilcoxon's signed rank test was applied within each group to assess significant variations in liver function variables from baseline to weeks 4, 12 and 24.
Covariance analysis on amprenavir CL/F was performed using 1-acid glycoprotein as covariate; the adjusted means were estimated for each of the three groups. Pair wise comparisons of the adjusted means were calculated.
To assess linear correlation between the pharmacokinetics and the liver function parameters, the Spearman rank correlation coefficient was estimated.
Bonferroni correction was applied when appropriate.
All the statistical tests were two-sided at the 5% level and performed using SAS Software, release 8.2.
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Results |
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The study involved 21 HIV-infected patients (Table 1): 7 with chronic hepatitis (6 with HCV and 1 with HBV/HCV co-infection) as determined by liver biopsy, 8 with liver cirrhosis (7 with HCV and 1 with HBV/HCV co-infection) and 6 controls. Cirrhosis was verified by liver biopsy in five patients (all in Child–Pugh class A) and determined by imaging modalities in the remaining three (one class B and two class C). The three groups were comparable in terms of weight, age and renal function, as well as CD4+ cell counts and HIV-RNA (Table 1). The median length of treatment with fosamprenavir was 77 days (14–183).
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The details of the amprenavir and ritonavir pharmacokinetic parameters are shown in Tables 2 and 3. Amprenavir AUC0–12, AUC0–
, Cmax and Css were 50% to 60% higher in the cirrhotic group than in the controls, whereas CL/F was 40% lower, thus indicating a reduction in apparent oral clearance. The reduction in the crude mean apparent oral clearance of amprenavir in the cirrhotic patients (193 ± 21.5 versus 355 ± 109 mL/h/kg) was maintained after adjusting for 1-acid glycoprotein (73 ± 98 versus 454 ± 77 mL/h/kg) (P = 0.02).
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Patients with chronic hepatitis showed a significant (roughly 20%) increase in AUC0–12, Cmax and Css in comparison with controls, but there was no difference in Ctrough or CL/F between the two groups.
In comparison with the controls, the cirrhotic patients had higher ritonavir AUC0–12, AUC0–, Cmax and Css values and a lower CL/F, whereas the concentration–time profiles overlapped in the chronic hepatitis and control groups.
The only correlation among the pharmacokinetic variables was between amprenavir AUC0–12 and total bilirubin values performed on the day of pharmacokinetic analysis (r = 0.64, P = 0.003). The absence of correlation was observed between amprenavir and ritonavir plasma concentrations.
Liver function parameters at weeks 4, 12 and 24 were no different from baseline in any of the groups. One grade 3 AST value was observed at week 12 in a patient with chronic hepatitis who had a grade 2 baseline value.
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Discussion |
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In comparison with controls, our cirrhotic patients had substantially increased plasma amprenavir concentrations and, although the difference was less marked, the patients with chronic hepatitis also had significantly higher AUC0–12, Cmax and Css values. The plasma amprenavir concentrations in the control group were comparable with those that have been published previously.8 The differences in plasma amprenavir concentrations in patients with liver disease were associated with their lower apparent oral clearance, which was not significant in the group with chronic hepatitis (GMR 0.83), but significant in the group with cirrhosis (GMR 0.62).
1-Acid glycoprotein concentrations, which inversely correlate with the apparent oral clearance of amprenavir,9 were decreased in the cirrhotic patients without any increase in amprenavir clearance; the observed reduction in amprenavir clearance could therefore reasonably be ascribed to liver decompensation.
Plasma ritonavir concentrations were only increased in the cirrhotic group; in the group with chronic hepatitis, they were comparable to those of the controls. Ritonavir undergoes extensive hepatic metabolism primarily through CYP3A4 isoforms.10 The higher plasma ritonavir concentrations observed in our patients with liver cirrhosis may have been due to the combined effect of hepatic deficiency and, possibly, additional CYP3A4 inhibition by amprenavir.11 The pharmacokinetics of various ritonavir doses has been studied in subjects with mild or moderate hepatic disease, but the results are conflicting insofar as ritonavir concentrations have been found to be both increased12 and decreased.13
However, the increased ritonavir values found in our patients should not have affected the pharmacokinetics of amprenavir, as it has been shown that increasing the ritonavir dose (from 100 to 200 mg twice daily) leads to additional CYP3A4 induction rather than inhibition and to a decrease in plasma amprenavir concentrations.11
The impact of an advanced liver disease such as cirrhosis on the pharmacokinetics of amprenavir has been previously described,1 but not that of chronic viral hepatitis. It has been reported that patients with chronic hepatitis C show significantly reduced CPY3A4 and CYP2D6 activity in comparison with healthy volunteers,14 and the same has been found in HIV-infected patients with HCV or HBV co-infection;15 this might explain the altered pharmacokinetic parameters observed in our patients. In the same way, the pharmacokinetics of lopinavir and ritonavir has been found to be different in patients with HCV infection and mild–moderate hepatic impairment.15
The clinical significance of the altered pharmacokinetics of amprenavir in patients with chronic hepatitis or cirrhosis remains unclear. It is known that hepatitis B or C co-infection is a risk factor for developing hepatotoxicity during HAART,16,17 but no association has been found between the plasma Ctrough concentrations of some protease inhibitors (lopinavir and ritonavir) and the development of hepatotoxicity.18 However, it is possible that an analysis of the variables that better reflect drug exposure, such as the AUC, could lead to different results.
We did not observe any increase in AST/ALT concentrations during the 24 week follow-up of the study patients, but cannot exclude the possibility that a longer follow-up may lead to different results. Liver toxicity can be observed up to 60 months after starting a protease inhibitor,16 and so our follow-up may have been too short. Other potential limitations of this study are the small number of subjects evaluated and some small (but not significant) differences in the demographic characteristics of the three groups.
In conclusion, patients with liver cirrhosis have high protease inhibitor plasma concentrations; they did not develop liver toxicity over a 24 week follow-up. On the basis of these data and also of data available in the literature, it seems reasonable to adapt the dose of fosamprenavir and/or ritonavir exclusively in the presence of adverse events, possibly related to protease inhibitors (i.e. liver toxicity), in subjects with high drug plasma levels. Therapeutic drug monitoring is advised in the management of these patients.
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None to declare.
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Acknowledgements |
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This work was presented in part at the Seventh International Congress on Drug Therapy in HIV Infection, Glasgow, 2004 (Poster 87).
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References |
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