INTRODUCTION

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Abacavir (formerly 1592U89), ()-(1S,4R)-4-[2-amino-6-(cy-clopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a synthetic carbocyclic nucleoside analog that inhibits human immunodeficiency virus type 1 (HIV-1) replication with low levels of cytotoxicity to MT4 cells (a transformed human leukemic cell line), peripheral blood lymphocytes, and macrophages (3). Abacavir is phosphorylated in a unique stepwise manner to produce the active moiety, carbocyclic guanosine triphosphate (3, 5). The mechanism of anti-HIV activity for abacavir has been shown to be substrate inhibition of HIV reverse transcriptase (RT) by carbocyclic guanosine triphosphate, resulting in chain termination and interruption of the viral replication cycle. When tested with normal human peripheral blood lymphocytes against fresh clinical isolates of HIV-1 obtained from antiretroviral drug-naive patients, the mean 50% inhibitory concentration (IC₅₀) was 0.26 µM (0.07 µg/ml); the corresponding IC₅₀s of zidovudine (ZDV), didanosine (ddI), and zalcitabine (ddC) were 0.23, 0.49, and 0.03 µM, respectively (3). Abacavir has been shown to have similar activities against HIV strains that were resistant to ZDV, lamivudine (3TC), ddI, ddC, and a number of nonnucleoside RT inhibitors. Studies have shown that abacavir synergistically inhibits HIV-1 IIIB in MT4 cells when it is combined with ZDV, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor amprenavir (141W94) (3, 11). Combinations of abacavir with 3TC, ddI, ddC, or stavudine were additive to synergistic (3).

Abacavir is a low-molecular-weight compound (molecular weight, 281.4) that is lipophilic (the 1-octanol-0.1 M sodium phosphate [pH 7.4] partition coefficient [log P] is 1.22) and that is a weak base (pK_a = 5.01). It has good solubility in water (>80 mM at 25°C) and is not protonated at a neutral pH. Abacavir is significantly more lipophilic than ZDV (log P, 0.09), the most lipophilic of the currently approved nucleoside RT inhibitors. Studies have shown that abacavir, administered as the succinate salt, has high oral bioavailability (>76%) in mice and monkeys and can penetrate the blood-brain barrier as well as ZDV can (6). Abacavir is primarily eliminated by metabolism, with only approximately 11 to 13% of the dose being recovered as unchanged drug in the urine of mice and monkeys (7). The two principal metabolites of abacavir identified in monkey urine were 5'-carboxylate (20% of the dose administered) and 5'-glucuronide (32% of the dose administered).

In preclinical studies, abacavir has been shown to have minimal toxicity in in vitro cytotoxic, cytogenetic, and mutagenic assays and in animals (2, 6). In monkeys dosed orally with abacavir (50, 140, and 420 mg/kg of body weight/day) for 30 and 90 days, reversible increases in serum triglyceride levels were noted at the intermediate and highest doses. In addition, the toxicities observed with other nucleoside RT inhibitorsneurophysiological deterioration and renal, cardiac, and hematopoietic toxicitieswere not observed with the highest dose tested.

The first phase I study (Glaxo Wellcome protocol 131-001), described in this report, was conducted to determine the safety and pharmacokinetics of single escalating oral doses of abacavir in HIV-infected subjects.

(This work was presented in part at the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 17 to 20 September 1995 [10].)

	MATERIALS AND METHODS

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Study population. Eligible subjects included HIV-positive, asymptomatic male and female subjects of any race between 13 and 55 years of age. Written informed consent was obtained from all participants, and the study was approved by the institutional review boards of the Georgetown University Medical Center and the University of Kansas. Each center enrolled nine subjects. The subjects had tested positive for antibody to HIV-1 or had clinical evidence of HIV infection as defined by the Centers for Disease Control and Prevention (CDC) HIV classification guidelines. Subjects were excluded from enrollment if they had a history of pancreatitis or hepatitis within the last 5 years, an absolute neutrophil count of <1,500 cells/mm³, a hemoglobin level of <10 g/dl (for women) or <11 g/dl (for men), a platelet count of <100,000 cells/mm³, serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels more than three times the upper limit of normal, and an estimated creatinine clearance of <50 ml/min. Subjects were also excluded from the study if they had debilitating HIV infection or a malabsorption disorder, were active substance or alcohol abusers, or were pregnant or nursing. All prescription and over-the-counter medications were withheld for 48 h (or 24 h for antiretroviral agents) prior to dosing and during the day of abacavir dosing.

Study design. This was a double-blind, placebo-controlled, parallel, rising-dose study. The subjects were randomly assigned 2:1 to receive abacavir or matching placebo for all caplet doses. Each subject received five single escalating oral doses of abacavir separated by at least a 6-day washout period and were given the option to receive a sixth dose. The abacavir doses administered sequentially were 100, 300, 600, 900, and 1,200 mg as caplets and 300 mg as a solution. Abacavir was supplied as 100-mg white, biconvex caplets or as a 50-ml solution by Glaxo Wellcome Inc., Research Triangle Park, N.C. Abacavir placebo was supplied as matching caplets, but no placebo solution was supplied. Each caplet contained 100 mg of abacavir (free base) as the succinate salt, and the oral solution was prepared as abacavir succinate dissolved in water to a concentration of 6 mg/ml (as the free base content). Each subject took the abacavir doses (1 to 12 caplets) with 200 ml of water and fasted for another 3 h postdosing.

Blood samples. Blood samples (5 ml each) were collected by venipuncture and placed into heparin-containing Vacutainer tubes immediately prior to dosing and at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, and 24.0 h after the administration of each dose. Blood samples were kept at 4°C upon collection and were centrifuged within 1 h of collection to separate the plasma, which was stored at 40°C until it was analyzed. The stability of abacavir in plasma samples has been validated at 40°C for 11 months, which covered the period from the time of sample collection to the time of assay.

Assay for plasma samples. Plasma abacavir concentrations were determined by a validated reversed-phase high-performance liquid chromatography (HPLC) assay with UV detection. Briefly, analytical stock standard and control solutions were prepared separately in HPLC-grade water. The appropriate volumes of the stock solutions were spiked into normal, blank, pooled human plasma to provide working standards or controls. The quantifiable range was 25 to 5,000 ng/ml, and the control concentrations were 40, 250, and 2,500 ng/ml. To 0.2 ml of standard, control, or unknown samples, 0.1 ml of 10% trichloroacetic acid was added, and the components were mixed by vortexing and were centrifuged at 8,800 × g for 10 min. The supernatants were transferred into injection vials (containing limited-volume inserts) and were placed in an autosampler. The supernatants (0.1 ml) were injected at 15-min intervals, and the chromatographic separation was achieved on a Rainin C₁₈ Microsorb MV column. The mobile phase consisted of 40% methanol and 0.3% (vol/vol) triethylamine (TEA) at a constant flow rate of 1.0 ml/min. Abacavir was detected by measuring the UV absorbance at 284 nm. The approximate retention time for abacavir was 9 min under these conditions. The interday precisions (percent coefficients of variation) calculated from the quality control samples were 7.7% at 0.04 µg/ml, 3.6% at 0.25 µg/ml, and 3.0% at 2.50 µg/ml; and the interday variabilities (biases) were 2.0, 2.4, and 5.8%, respectively.

Safety evaluation. The safety and tolerability of single escalating doses of abacavir were evaluated on the basis of adverse experience reports, measurements of vital signs and clinical laboratory test values and the results of physical examinations and electrocardiograms. In each dosing period, the severity (mild, moderate, or severe), duration, and potential relationship to the study drug (unrelated or possibly, probably, or almost certainly related, according to the investigator) of any adverse events were recorded. Vital sign determinations (sitting blood pressure and sitting pulse), routine hematologic studies (complete blood count with differential, mean corpuscular volume, and platelet count), serum chemistry studies (electrolyte, AST, ALT, total bilirubin, creatinine, albumin, glucose, alkaline phosphatase, and serum amylase levels), and urinalysis (dipstick for protein and blood) were performed at screening, prior to the administration of study drug in each dosing period, and at a follow-up visit.

Pharmacokinetic analysis. The plasma concentration-time data for abacavir were analyzed by standard noncompartmental pharmacokinetic methods. The peak concentration in plasma (C_max) and the time to C_max (T_max) were obtained from direct inspection of the plasma concentration-time profile. Estimates of the apparent terminal elimination half-life (t_1/2) were calculated as ln(2)/_z, where _z is the terminal elimination rate constant and is a first-order rate constant determined from the negative of the slope of the linear regression line of the apparent terminal linear portion of the log concentration-versus-time curve. The data points for inclusion in the linear regression line were selected by starting with the last three measurable concentrations, and points were added on the basis of changes in the regression slope, regression R², negative regression residual, and C_max. These points were visually inspected, with no changes made to the selected points. The area under the plasma concentration-time curve from time zero to time t (AUC_0-t), where t is the last time point with a measurable concentration of the compound of interest, was calculated by using the linear trapezoidal method. The AUC from time zero to infinity (AUC_0-) was then determined as AUC_0-t + C_last/_z, where C_last is the last measurable concentration of the compound of interest. The apparent clearance from plasma (CL/F) was calculated as the dose divided by AUC_0- and was then normalized to body weight.

Statistical analysis. C_max and AUC_0- values were dose normalized to 100 mg, and all pharmacokinetic parameters (except T_max) were log transformed (base e) prior to analysis. A power model was used to assess the extent of dose proportionality for C_max and AUC_0- across treatments, as follows: Y_ij = _i (D_j)^_i. The log-transformed model is log (Y_ij) = log (_i) + _i log (D_j) + e_ij, where D_j is dose level j and Y_ij is the value of the pharmacokinetic parameter for subject i at dose level j; _i and _i are the intercept and slope for subject i, respectively; and e_ij is the residual error. The power model was fitted by restricted maximum likelihood methods with unrestricted variance structure by using SAS PROC MIXED (version 6.09; SAS Institute, Inc., Cary, N.C.). A population average estimate of  and its 90% confidence interval (CI) were calculated from the individual  values of both parameters for all doses and for doses from 600 to 1,200 mg. The degree of departure of the slope  from unity was the primary assessment of nonproportionality. Parameters were considered dose proportional if the resultant 90% CI of the population average estimate of  included 1.0.

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	RESULTS

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Subject demographics and characteristics. Eighteen subjects (15 men and 3 women) were enrolled in this study. Four subjects in the abacavir-treated group were prematurely discontinued from the study for reasons unrelated to treatment with the study drug. The reasons for premature discontinuation included loss of intravenous access (one subject after the first dose), withdrawal of consent (two subjects after the second dose), and failure to return for treatment after a brief hiatus to recover from giardiasis (one subject after the fourth dose). Two subjects elected not to receive the 300-mg dose of abacavir in solution. All subjects in the placebo group completed the study. The treatment groups were comparable at the baseline with respect to demographic variables, HIV risk factors, and CDC classification (Table 1).

Safety evaluation. Abacavir was well tolerated at single oral doses of up to 1,200 mg. There were no significant differences between groups in the type or frequency of adverse events. Ten of 12 subjects in the abacavir group and 4 of 6 subjects in the placebo group reported at least one adverse event during the study. None were serious, and there were no withdrawals due to adverse events. Most were mild to moderate in intensity, and those assessed as possibly related to abacavir included asthenia (33%), abdominal pain (33%), headache (25%), diarrhea (17%), and dyspepsia (17%). Three subjects each reported one adverse event that was classified as severe in intensity: headache (possibly related to abacavir), diarrhea (unrelated to abacavir), and diarrhea (possibly related to placebo).

Pharmacokinetic evaluation. Mean plasma abacavir concentration-versus-time profiles for the 100- to 1,200-mg doses are depicted in Fig. 1. Following oral administration, abacavir was rapidly absorbed, achieving measurable concentrations in plasma by the first sampling time (15 min). In general, plasma abacavir concentrations fell below detectable concentrations (0.025 µg/ml) at or after 12 h postdosing. In addition, all abacavir doses resulted in mean concentrations in plasma that exceeded the mean IC₅₀ for clinical HIV isolates in vitro (0.07 µg/ml) (Fig. 1).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   (A) Mean ± standard deviation plasma abacavir concentration-versus-time curves following oral administration of single escalating doses under fasting conditions. (B) Logarithmic plot of mean plasma abacavir concentration-time profiles for each dose. The lower limit of quantitation was 0.025 µg/ml.

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View larger version (12K): [in this window] [in a new window]   FIG. 2.   (A) Mean ± standard deviation estimates of AUC0- under fasting conditions versus dose. A linear but greater than dose-proportional relationship was noted between AUC0- and dose. For example, a 4-fold increase in dose from 300 mg resulted in a 5.88-fold increase in AUC0-. (B) Mean ± standard deviation estimates of Cmax under fasting conditions versus dose. A linear and almost dose-proportional relationship was noted between Cmax and dose. For example, a 4-fold increase in dose from 300 mg resulted in a 3.70-fold increase in Cmax.

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	DISCUSSION

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This is the first study to evaluate the safety and pharmacokinetics of abacavir in humans. The results of this study indicate that abacavir is well tolerated and rapidly absorbed following the administration of single oral doses ranging from 100 to 1,200 mg to HIV-infected adults. Single oral doses of abacavir of up to 1,200 mg were well tolerated by HIV-infected subjects. No significant changes in hematologic parameters and no significant laboratory abnormalities were observed throughout the study. This favorable safety profile of abacavir is well supported by preclinical toxicology studies with animals (2, 3, 6, 7).

The findings of this single-dose pharmacokinetic study indicate that abacavir reaches a maximum concentration rapidly (within 2 h), has adequate bioavailability (as indicated by concentrations in plasma), and has a relatively short t_1/2 (<2 h). These values indicate that abacavir has absorption characteristics comparable to those of currently available RT inhibitors and a t_1/2 that is similar to those of the other RT inhibitors except for 3TC (1, 4, 8, 9, 12).

The results of the statistical analysis across all five doses demonstrated the lack of strict dose proportionality for AUC_0- and C_max, although the three highest doses were shown to be dose proportional. While the cause is unclear at this stage, the lack of proportionality in AUC_0- and C_max over the lower dose range may be due to a possible first-pass effect which is saturated at higher doses, or it may be the result of an observational artifact associated with the assay of lower concentrations. Despite the overall lack of exact dose proportionality, abacavir dose is highly predictive of AUC_0- and C_max due to the linear relationship. The deviation from dose proportionality over the entire dose range was not considered clinically significant for abacavir because only the 300-mg dose will be used clinically.

The intersubject variabilities in the pharmacokinetic parameter estimates were highest at the lower doses and tended to decrease with increasing dose. The variability at the lowest dose may be explained in part by higher assay variability at low drug concentrations. The variability in the pharmacokinetic data may be attributed to intersubject differences in the metabolism of abacavir or in the first-pass effect in the gastrointestinal tract or in the liver.

The bioavailability of the 100-mg caplet formulation relative to that of the solution was high (almost 100%), indicating that the disintegration and dissolution of abacavir from the caplet formulation were rapid and complete.

Administration of all single oral doses resulted in mean plasma abacavir concentrations that exceeded the IC₅₀ for clinical HIV isolates (0.26 µM or 0.07 µg/ml) for almost 3 h (Fig. 1), and the mean plasma abacavir concentration for the 300-mg dose exceeded twice the IC₅₀ (0.14 µg/ml) for over 6 h, i.e., over half the dosing interval for twice-daily administration. However, the in vivo antiviral effect is also dependent on factors such as distribution into target cells, kinetics of intracellular phosphorylation, and disposition of the active metabolite, carbocyclic guanosine triphosphate. It is noteworthy that the reported intracellular half-life of the active moiety of abacavir, carbocyclic guanosine triphosphate, was 3.3 h in CD4⁺ CEM cells (3), which is much longer than the plasma t_1/2 observed in the current study (0.9 to 1.7 h). This difference in half-lives should sustain the intracellular concentrations of the active moiety. The longer intracellular half-life should also contribute to greater intracellular accumulation of the active moiety.

In summary, this study confirms the desirable pharmacokinetic properties and the favorable safety profiles of single oral doses of abacavir in HIV-infected individuals. The results of this study have supported the design of subsequent single- and multiple-dose studies with adults and children (with an oral solution) to evaluate the safety, antiviral activity, and pharmacokinetics of abacavir as monotherapy and in combination therapy with other antiretroviral agents for the treatment of HIV infection.