INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Amprenavir (141W94) is a novel anti-human immunodeficiency virus (anti-HIV) agent recently approved for treatment of HIV infection. The mechanism of antiviral activity of amprenavir is inhibition of viral aspartic protease, with a K_i of 0.6 nM (7). Amprenavir is a potent inhibitor of HIV type 1 (HIV-1) replication in vitro, with 50% inhibitory concentrations of 0.084 and 0.080 mM for virus in human MT-4 cells and peripheral blood lymphocytes, respectively (15). Clinical studies have evaluated single doses of amprenavir over a range of 150 to 1,200 mg in HIV-1-infected adults and have shown that in the range evaluated, plasma amprenavir concentrations increased linearly with dose but the increases were slightly greater than dose proportional (13). Following administration of a single, oral dose of 1,200 mg of amprenavir, the mean plasma amprenavir concentration 12 h after drug administration was fourfold greater than the in vitro 50% inhibitory concentration for HIV in peripheral blood lymphocytes (13). The dose approved for treatment of HIV-1 infection in adults is 1,200 mg twice daily.

Liver dysfunction resulting from coinfection with hepatitis B or hepatitis C virus may be a complication of HIV infection (F. Moretti, R. Novati, G. Morsica, and A. Poli, Abstr. 12th Int. Conf. AIDS, p. 1124-1125, 1998). Like the rest of the currently licensed HIV-1 protease inhibitors used as antiviral drugs for HIV-1-infected subjects, amprenavir is extensively metabolized in the liver by cytochrome P450 enzymes, specifically, through the CYP3A4 pathway (7; J. Woolley, S. Studenberg, C. Boehlert, G. Bowers, A. Sinhabaru, and P. Adams, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-60, p. 12, 1997). Drug metabolism by cytochrome P450 enzymes has important clinical consequences because of the possibility of reduced drug metabolism and subsequent increased plasma drug concentrations in subjects with liver dysfunction (3). In addition, the potential for protease inhibitors to induce or inhibit specific P450 isozymes has clinical significance due to the possibility of drug-drug interactions in a patient population likely to require multiple concomitant medications. In vitro studies have shown that amprenavir inhibits CYP3A4 (K_i = 0.06 mM) at clinically achievable concentrations and CYP2C19 (K_i = 47 mM) at much higher levels of exposure but does not inhibit CYP1A2, CYP2C9, CYP2D6, or 2 CYP2E1 (Woolley et al., 37th ICAAC).

Like other HIV-1 protease inhibitors, amprenavir is bound to albumin and to ₁-acid glycoprotein (AAG), both of which are synthesized in the liver. In vitro studies with the protease inhibitors indinavir, saquinavir, and ritonavir, as well as with investigational protease inhibitors A77003, A-80987, KN1-272, and CGP 61755, have shown that addition of AAG decreases in vitro drug activity (4, 5, 8, 10). In vivo, the situation is more complex as AAG is an acute-phase protein whose levels are changed in certain disease states (9; K. Stellrecht, G. L. Drusano, D. S. Stein, and J. A. Bilello, 3rd Conf. Retroviruses and Opportunistic Infections, abstract, p. 84, 1996). In addition, amprenavir and other protease inhibitors are hepatically cleared.

In order to investigate the effect of hepatic impairment on amprenavir pharmacokinetics, we undertook a phase I clinical trial (Glaxo Wellcome study PROB-1008) designed to compare the pharmacokinetics of amprenavir in healthy volunteers to that in subjects with moderate or severe cirrhosis. Because amprenavir is extensively metabolized in the liver and is approximately 90% bound to AAG, amprenavir pharmacokinetics might be significantly altered in subjects with liver disease, in whom both P450 enzyme activities and AAG concentrations might be reduced. For safety, the dose of amprenavir administered in this study was 600 mg, or one-half the 1,200-mg dose under clinical evaluation in phase III trials. The primary goal of this trial was to evaluate the differences in pharmacokinetics of amprenavir among the three groups of subjects and thereby to determine whether a dose reduction is necessary for subjects with cirrhosis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Study design. This study was an open-label, single-period, single-dose, parallel-group, phase I study conducted at seven study centers in France during the period from March through November 1997. The study was designed to include 30 subjects, both male and female, divided equally among three groups. The three groups were composed of 10 subjects with severe cirrhosis, 10 subjects with moderate cirrhosis, and 10 healthy volunteers who were chosen as controls to match the subjects with moderate cirrhosis for gender, smoking status, weight, and age. Subjects with cirrhosis (moderate or severe) were enrolled at five study centers: Hôpital Avicenne, Bobigny; Hôpital Broussais, Paris; Hôpital Dupuytren, Limoges; Hôpital de Rangueil, Toulouse; and Hôtel-Dieu, Nantes. Healthy volunteers were enrolled at Therapharm Recherche in Boulogne and at ASTER in Paris. In accordance with French law, the study was registered with the French Ministry of Health and was approved by the authorized Ethics Review Board prior to initiation. Subjects provided written informed consent prior to enrollment in the study.

Subjects. Ten subjects were enrolled in each of the three groups: healthy volunteers, subjects with moderate cirrhosis, and subjects with severe cirrhosis. All subjects were required to meet the following criteria for enrollment: agreed to consume no more than 4 units of alcohol per day until the end of the study (1 unit = 1/2 pint of beer, 1 glass of wine, or 1 measure of spirits); were capable of giving informed consent; and was affiliated with the French Social Security System for health care coverage. Healthy male and female volunteers were eligible for the study if they met the following criteria: were 18 to 65 years of age; had body weight (±10 kg), age (±5 years), gender, and smoking habits matched to those of subjects with moderate cirrhosis; and had good general health and were free from significant disease as determined by physical examination, medical history, and screening assessments. Healthy volunteers were ineligible if they were considered unfit by the investigator or if any of the following criteria were met: history of drug allergy or other allergy that contraindicated participation; blood donation within the previous month; current use of medication; current daily consumption of more than 4 units of alcohol; participation in an investigational drug study within the previous 3 months; positive antibody screen for HIV, hepatitis B virus, or hepatitis C virus; pregnant; breast-feeding female; or female of childbearing potential not using effective contraception.

Drug supply and administration. Drug was supplied by Laboratoire Glaxo Wellcome Evreux. Individual 600-mg doses were provided in plastic bottles containing four soft gelatin capsules of 150 mg of amprenavir free base. On the morning of the dosing day, subjects ingested the four capsules with 200 ml of water.

Clinical procedures. At the screening assessment the following procedures were performed: medical history and full physical examination; 12-lead electrocardiogram (ECG); blood collection for clinical chemistry (including AAG) and hematology (complete blood count and differential); urinalysis; urine screen for illicit drugs; screens for hepatitis B virus, hepatitis C virus, and HIV; thyroid function tests; and urine pregnancy test, if appropriate. Procedures and assessments performed at 30 min predosing, at 24 h postdosing, and at 96 h postdosing (cirrhotic subjects only) included clinical chemistry, hematology, ECG, and vital signs. Female subjects of childbearing potential were given a urine pregnancy test on the evening prior to dosing. Adverse events were monitored throughout the dosing period by means of subject interviews and physical examination at the end of the study period.

Sample collection. Blood samples taken predosing and during the first 24 h postdosing were drawn through an intravenous cannula; other samples were taken by venipuncture. For amprenavir assays, 3-ml blood samples were drawn into EDTA-containing tubes; the plasma was separated by centrifugation and was stored at 20°C until analysis. For hematology and clinical chemistry, 2-ml blood samples were drawn into EDTA-containing tubes and lithium heparin-containing tubes, respectively. For thyroid function tests, 3-ml blood samples were drawn into EDTA-containing tubes. For determination of amprenavir levels, plasma samples were collected from all subjects at 0.5 h predosing (baseline) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 15, and 24 h postdosing. An additional five plasma samples were collected from subjects with cirrhosis, at 34, 48, 58, 72, and 96 h postdosing.

Plasma assay conditions. Amprenavir concentrations were determined by liquid chromatography-mass spectrometry in the Department of Bioanalysis, Glaxo Wellcome, Research Triangle Park, N.C. Aliquots of 200 µl of acetonitrile containing 0.5 ng of internal standard per µl were dispensed into a clean 96-well plate. Aliquots (100 µl) of plasma samples and appropriate standards or controls were added to the wells, and the plate was mixed by vortexing. Following centrifugation at 2,500 rpm for 2 min, the supernatants were transferred to a separate 96-well plate containing 100 µl of 0.1% formic acid. Samples were mixed by vortexing and were injected (10 to 40 µl) at 4-min intervals onto a Waters Symmetry C₁₈ analytical column (Waters Inc., Milford, Mass.). For the first 2 min the samples were eluted to the mass spectrometer in mobile phase B (55% acetonitrile and 45% water; vol/vol) at a flow rate of 0.35 ml/min. After 2 min the pumps were switched to mobile phase A (55% acetonitrile and 45% water with 0.1% formic acid; vol/vol) at the same flow rate. An API-300 triple quadruple mass spectrometer (PE Sciex, Toronto, Ontario, Canada) operated in the positive-ion multiple-reaction-monitoring mode was used to detect amprenavir and the internal standard by atmospheric pressure chemical ionization tandem mass spectrometry.

Data analyses. Pharmacokinetic calculations for determination of plasma amprenavir concentrations were performed for each subject by using WinNonlin (version 1.5; Scientific Consulting, Inc., Cary, N.C.). Model-independent methods were used to estimate the maximum concentration of amprenavir (C_max), the time associated with C_max (T_max), the area under the concentration-time curve (AUC) from time zero to time t (AUC_0-t), the half-life (t_1/2), the apparent total clearance (CL/F), and the apparent volume of distribution during the elimination phase (V_Z/F). The apparent terminal elimination rate constant (_Z) was estimated by log-linear regression of the terminal portions of the concentration-versus-time curves. For consistency with previously reported data, the AUC_0-t was calculated by using the linear trapezoidal rule. AUC from time zero to infinity (AUC_0-) was determined by extrapolation from AUC_0-t with the addition of C_last/_Z, where C_last is the last measured concentration in plasma.

0-

H  L

0-

0-

0-

(1)

(2)

0-

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Subject enrollment and baseline characteristics. Thirty subjects were enrolled in the study and completed the study. One of the subjects in the group with moderate cirrhosis was enrolled twice. After initial enrollment he was discontinued when it was discovered that he was taking disulfiram, a concomitant medication whose use was prohibited in this study. Treatment with disulfiram was interrupted for 2 weeks, and the subject was enrolled again and completed the study. Data from the subject's first enrollment are not included in the pharmacokinetic analysis.

Safety assessments. Five subjects reported a total of five adverse events, of which three were considered possibly drug related: one episode of rhinitis that occurred 10 h postdosing resolved after 4 days and was experienced by a 43-year-old female in the group with moderate cirrhosis; one episode of moderate epigastric pain that occurred 10 h postdosing resolved after 16 h and was experienced by a 32-year-old female in the group with severe cirrhosis; and one episode of thrombocytopenia (platelet count, 146,000/mm³) was experienced at 96 h postdosing, was resolved at the end of the study but was clinically insignificant, and was experienced by a 59-year-old male with severe cirrhosis. The remaining two adverse events reported in the study were considered not drug related and consisted of one episode of diarrhea, experienced by a subject in the group with severe cirrhosis, and one episode of palpitations, experienced by a subject in the group with moderate cirrhosis. No adverse events were reported by the healthy subjects.

Pharmacokinetic analysis. Median plasma amprenavir profiles for each group are shown in Fig. 1. For all subjects, plasma amprenavir concentrations reached a peak within 0.25 to 3 h postdosing and declined in a biphasic pattern with a t_1/2 value of approximately 5 to 8 h. Although blood samples for pharmacokinetic analysis continued to be obtained through 96 h for cirrhotic subjects, median concentrations of amprenavir were below the lower limit of quantification (10 ng/ml) at 34 and 48 h postdosing for subjects with moderate and severe cirrhosis, respectively. Median concentrations of amprenavir were detectable at 24 h postdosing (end of study) in the control group.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Median plasma amprenavir concentration profiles.

0-

0-

0-

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Relationship between amprenavir AUC0- and Child-Pugh score. No subjects with a Child-Pugh score of >12 were enrolled; therefore, extrapolation of results to subjects with higher Child-Pugh scores should be made with caution.

0-

0-

0-

0-

0-

0-

0-

0-

0-

View this table: [in this window] [in a new window]   TABLE 2.   Estimated amprenavir AUC0- for each Child-Pugh score and the proposed dosage required to achieve equivalence with the recommended 1,200-mg dosea

0-

0-

0-

0-

0-

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Plot of amprenavir AUC0- versus mean baseline total bilirubin concentration.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This study was designed to assess the impact of liver disease on amprenavir pharmacokinetics. Thirty subjects completed the study, including 10 subjects with moderate cirrhosis and 10 subjects with severe cirrhosis. No new or unexpected adverse events were attributed to amprenavir, and the majority of laboratory abnormalities that occurred in subjects with moderate or severe cirrhosis were consistent with those associated with serious liver disease. The pharmacokinetic profile was altered for subjects with cirrhosis, with higher AUC_0- values and two- to fourfold lower CL/F values for cirrhotic subjects relative to those for healthy subjects. Although serum AAG concentrations were reduced in cirrhotic subjects, there was no apparent increase in total clearance of amprenavir. Because amprenavir is extensively metabolized by CYP3A4, the findings are consistent with a significant reduction in CYP3A4 activity, porto-caval shunting, or both.

A linear relationship was observed between AUC_0- and the Child-Pugh score. Because there is also a linear relationship between the amprenavir dose administered and AUC_0- (13), we were able to construct a table that estimates the dose of amprenavir for subjects with a given Child-Pugh score required to obtain AUC levels comparable to those obtained with a 1,200-mg dose administered to a subject without liver disease. Mean baseline total bilirubin concentrations were significantly correlated with AUC values, suggesting that it may be possible to use bilirubin concentration to predict the initial dose of amprenavir appropriate for a patient with liver disease. This relationship suggests that there may be common transport mechanisms (e.g., p-glycoprotein) or metabolic pathways that involve both amprenavir and bilirubin.

As would be expected, the total clearance of amprenavir was reduced in subjects with hepatic impairment, consistent with a decrease in unbound (intrinsic) clearance from a loss of hepatic CYP3A4. Consistent with another clinical study of subjects with liver disease (Stellrecht et al., 3rd Conf. Retroviruses and Opportunistic Infections), there was also a decrease in the serum AAG concentrations. By itself, the decrease in AAG would result in a decrease in the total drug concentration in plasma and therefore an apparent increase in the total clearance. However, the opposite trend was observed. The percentage of unbound drug would vary inversely with the AAG concentration, although the absolute free drug concentrations would not be affected in the absence of a change in intrinsic clearance (12). These data therefore indicate that the decrease in intrinsic clearance outweighs the decrease in AAG concentrations with regard to its effect on the apparent clearance of total drug.

In summary, results from this study indicate that the dosing should be reduced in subjects with liver disease to obtain plasma amprenavir levels comparable to those achieved in healthy subjects given a 1,200-mg oral dose twice daily. For subjects with moderate cirrhosis and Child-Pugh scores of 5 to 8, the equivalent dose of amprenavir is estimated to be 450 mg twice daily. For subjects with severe cirrhosis and Child-Pugh scores of 9 to 15, the equivalent dose of amprenavir is estimated to be 300 mg twice daily.