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Antimicrobial Agents and Chemotherapy, May 2009, p. 1739-1746, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01479-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Single-Dose Escalation and Multiple-Dose Safety, Tolerability, and Pharmacokinetics of IDX899, a Candidate Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor, in Healthy Subjects{triangledown}

Xiao-Jian Zhou,* Keith Pietropaolo, David Damphousse, Bruce Belanger, Jie Chen, John Sullivan-Bólyai, and Douglas Mayers

Idenix Pharmaceuticals Inc., One Kendall Square, Building 1400, Cambridge, Massachusetts 02139

Received 5 November 2008/ Returned for modification 16 December 2008/ Accepted 4 February 2009


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ABSTRACT
 
IDX899 is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with potent in vitro activity against wild-type and NNRTI-resistant strains of human immunodeficiency virus type 1 (HIV-1) and with a high genetic barrier to resistance. Single rising doses of 50 and 100 (given by use of a 50-mg capsule) and 200, 400, 800, and 1,200 mg (given by use of a 200-mg capsule) of IDX899 or matching placebo were administered sequentially to cohorts of healthy male subjects, followed by the administration of multiple doses of 800 mg once daily (QD) or 400 mg twice daily (BID) for 7 days. A single dose of 400 mg was also administered to a cohort of females. IDX899 was administered orally under fasted (50- to 400-mg doses) and then fed (≥200-mg doses) conditions. Exposure to IDX899 was dose proportional and comparable in males and females. With a different drug-to-excipient ratio, the 50-mg capsule led to a higher exposure but a shorter mean terminal half-life (t1/2) of 6.2 to 6.8 h. The 200-mg capsule resulted in a more sustained exposure with a longer mean t1/2 of 7.9 to 14.6 h. Food enhanced absorption by approximately twofold, while it delayed the time to the maximum concentration. The mean concentration at 24 h following the administration of a single 200-mg dose under fed conditions exceeded the in vitro protein binding-adjusted 90% inhibitory concentration by fourfold. The levels of plasma exposure were similar between the single dosing and the repeat dosing with 800 mg QD and was approximately twofold higher with 400 mg BID. Mean steady-state trough levels were 0.9 µg/ml (range, 0.2 to 2.5 µg/ml) and 2.1 µg/ml (range, 0.5 to 4.5 µg/ml) for the 800-mg QD and 400-mg BID regimens, respectively. The level of excretion of unchanged drug in urine was negligible. IDX899 was well tolerated; and no serious adverse events, dose-dependent adverse events, or laboratory abnormalities were detected. These favorable safety and pharmacokinetic results support further clinical studies with patients with HIV-1 infection by the use of a QD regimen.


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INTRODUCTION
 
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a key component of combination antiretroviral drug regimens used to treat human immunodeficiency virus (HIV) type 1 (HIV-1) infection. NNRTI-based regimens are commonly prescribed as initial therapy for treatment-naïve patients. The major disadvantage of the early NNRTIs, including delavirdine, efavirenz, and nevirapine, is their low genetic barrier to the development of resistance (5). As drug resistance increases among patients newly infected with HIV, there is an urgent need for agents that are active against drug-resistant HIV-1 with better tolerability and safety.

IDX899 (Fig. 1) is a new NNRTI with a high genetic barrier to the emergence of resistance in vitro (1, 3, 6). It is a potent and selective inhibitor of HIV-1 replication in vitro and has potency against wild-type HIV-1 replication at nanomolar concentrations: the estimated mean 50% inhibitory concentration (IC50) was 1.2 nM. This compound retains marked activity against a broad range of HIV-1 strains, including NNRTI-resistant mutants with single mutations (K103N or Y181C) and double mutations, as well as clinical isolates with documented efavirenz resistance-conferring mutations (3, 6; D. Standring, unpublished data).


Figure 1
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  		FIG. 1. Chemical structure of IDX899. Me, methoxy.

IDX899 was not inhibitory to human cellular DNA polymerase alpha, beta, or gamma at clinically relevant concentrations; had a favorable cytotoxicity profile; and was not genotoxic in vitro (6; J. Selden, unpublished data). IDX899 exhibited a favorable preclinical safety profile following the oral administration of single doses of up to 1,000 mg/kg of body weight and repeat doses of up to 450 mg/kg/day in monkeys and rats for 14 days (6; Selden, unpublished).

Preclinical pharmacokinetic studies showed that IDX899 is highly protein bound (>99.8%); undergoes both cytochrome P450 (CYP450)-mediated biotransformation and phase II metabolism through conjugation; and inhibits several human CYP450s, including 3A4, 2C8, and 2C9 (6).

Clinical results from a microdose study with healthy male subjects demonstrated that orally administered IDX899 is rapidly and well absorbed and has an oral bioavailability of 61% and sustained plasma exposure. IDX899 was extensively metabolized and was excreted at low levels in urine (X. J. Zhou, unpublished data).

The favorable preclinical activity and safety profile of IDX899, along with the encouraging data obtained from the human microdose study, prompted the phase I study with healthy subjects at the pharmacologically relevant doses described here. The objectives were to assess the safety, tolerability, and pharmacokinetics of single rising doses of IDX899 as well as multiple doses in male subjects. This study also evaluated the effect of food and gender on the pharmacokinetics of IDX899.


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MATERIALS AND METHODS
 
This study was conducted in accordance with Good Clinical Practice procedures, the principles of the Declaration of Helsinki, and U.S. Food and Drug Administration regulations. Approval for the study was obtained from an independent institutional review board (IntegReview Ltd., Austin, TX).

The clinical study was conducted at Pharmaceutical Product Development in Austin, TX. The first subject was screened on 14 May 2007, and the last subject completed the study on 21 September 2007.

Study population. All subjects voluntarily gave written informed consent after the nature of the study was fully explained. Eligible subjects were healthy adult nonsmoking male subjects (for cohorts with all doses) and women of nonchildbearing potential (for the 400-mg single-dose cohort only) from the general population. They were 18 to 65 years of age; had body mass indexes between 18 and 32 kg/m2; and had no evidence of clinically significant abnormalities on medical history, physical examination, 12-lead electrocardiogram (ECG), or clinical laboratory testing during screening. The male subjects were required to use an acceptable double-barrier method of birth control during the study and for at least 30 days after they received the last dose of the study drug. Subjects were excluded if they had received prescription medications for chronic conditions within the previous 3 months, prescription drugs for acute conditions within the previous 14 days, and systemic over-the-counter medications (including aspirin, vitamins, and herbal supplements) or alcohol-containing beverages within 2 days of reporting to the clinic. Subjects were also excluded if they tested positive for HIV, hepatitis C virus, or hepatitis B virus; tested positive for drugs of abuse or alcohol; or had participated in a clinical study within 30 days prior to study drug administration.

Study design. The trial was a single-center, randomized, double-blind, placebo-controlled, single-dose-escalation and multiple-dose (7 days) study with healthy males and women of nonchildbearing potential.

IDX899 and matching placebo were supplied as a dispersion in the vehicle (Gelucire 44/14) filled into hypromellose hard capsules. Both a 50-mg capsule and a 200-mg capsule were provided. The two capsules had different ratios of active pharmaceutical ingredient (API) to excipient. The ratio in the 200-mg capsule was reduced compared to that in the 50-mg capsule formulation.

The single-dose-escalation phase started at 50 mg, which escalated to 100, 200, 400, 800, and 1,200 mg. The cohorts with the 50- and 100-mg doses were completed under fasted conditions, while the cohorts with the 200- and 400-mg doses were completed under fasted and then fed conditions and the cohorts with the 800- and 1,200-mg doses were completed under fed conditions. Among the eight male subjects within each cohort, six were randomly assigned to receive the active drug and two were randomly assigned to receive the placebo. Safety and plasma pharmacokinetic evaluations were performed before escalation to the next dose level. The cohorts receiving the 50- and 100-mg doses used the 50-mg capsules, while the cohorts receiving doses of 200 mg and above employed the 200-mg capsules.

The effect of food was evaluated with the 200- and 400-mg doses. Subjects were administered the dose within 5 min after the consumption of a test meal. The high-fat (~55 g) and high-calorie (~950 kcal) test meal was composed of two eggs fried in butter, two strips of bacon, two slices of toast with butter, 4 oz of hash brown potatoes, and 8 oz of whole milk, which provided approximately 150, 250, and 500 to 600 cal of protein, carbohydrate, and fat, respectively. For the 200-mg dose, the same cohort received the fed treatment according to the same assignment (active or placebo) 7 days following administration of the dose in the fasted state. For the 400-mg dose, a separate cohort of eight male subjects was enrolled for treatment in the fed state.

At the conclusion of the single-dose-escalation study, a cohort of eight women of nonchildbearing potential was enrolled; six women were randomly assigned to receive the active drug and two were randomly assigned to receive the placebo. The active drug consisted of a single dose of 400 mg IDX899 administered under fed conditions.

The multiple-dose phase of the study consisted of daily dosing with two doses (400 mg twice daily [BID] and 800 mg once daily [QD]) under fed conditions for 7 days. The doses were selected on the basis of the safety and the plasma pharmacokinetics of the single-dose-escalation study. For each regimen, 10 male subjects were enrolled; 8 were randomly assigned to receive the active drug and 2 were randomly assigned to receive the placebo.

For treatment in the fasted state, the study drug was given on an empty stomach after a fasting period of approximately 10 h prior to dosing and for an additional 4 h postdosing. All single-dose treatments in the fed state were administered with the high-fat and high-calorie meal described above. The 800-mg QD and 400-mg BID regimens were administered with a standard meal. Grapefruit juice was not allowed from 24 h before the subject reported to the study center until the subject was released. Xanthine- or caffeine-containing substances were not allowed from 10 h prior to and until at least 4 h after dosing. For each dose, at least 240 ml (8 fluid oz) of water was given with the study dose. Water was permitted ad libitum from 1 h postdosing. The subjects were asked to remain upright (sitting or standing) for the first 4 h following dosing and not to engage in any strenuous activity during their stay at the study center.

Safety evaluation. The safety assessments consisted of the collection of all adverse events (AEs) and serious adverse events (SAEs), along with their severities and their relationship to the study drug. Safety assessments also included regular monitoring by hematologic analysis, blood chemistry analysis, and urinalysis, as well as determination of vital signs and performance of 12-lead ECGs and physical examinations. The medications that the subjects were concomitantly taking were also noted.

Pharmacokinetic sampling. For the single-dose studies, intensive sampling of plasma for pharmacokinetic analysis was performed over a period of 48 h after dosing. Samples were obtained at the following time points: time zero (predosing) and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 16, 24, 28, 32, 36, and 48 h. For the subjects in the 200-mg dose group, the sampling was repeated after administration of the dose under fed conditions for evaluation of the effect of food. Intensive sampling was also performed for the multiple-dose regimens. Blood was collected at the following time points, as appropriate, after administration of the first dose over 12 h and 24 h for the 400-mg BID and 800-mg QD regimens, respectively, and over 48 h after administration of the last dose for both regimens: time zero (predosing) and 1, 2, 4, 6, 8, 12, 16, 24, 28, 32, 36, and 48 h. A single blood sample was collected before the administration of each dose to monitor the trough drug levels. Blood samples were collected in Vacutainer tubes containing EDTA as the anticoagulant and within a ±5-min window of the time points specified above. Plasma was obtained by centrifugation at 2,000 x g for 15 min at 4°C and was stored frozen at –20°C or below until analysis.

Sampling of urine for pharmacokinetic analysis was performed over 24 h for subjects receiving single doses of 200 mg (fasted state), 400 mg (fasted and fed states), and 800 mg (fed state). Urine samples were collected according to the following time intervals: –2 to 0 h (predosing), 0 to 4, 4 to 8, 8 to 12, and 12 to 24 h. Urine samples were stored frozen at –20°C or below until analysis.

IDX899 has been shown to remain stable in plasma and urine during storage and assay. The short-term stability of the analyte in plasma has been documented when spiked samples were subject to three freeze-thaw cycles (–20°C to room temperature), storage at ambient temperature for 24 h, and after preparation for 57 h for plasma samples and 101 h for urine samples. The long-term storage stability was 86 days in plasma and 20 days in urine at temperatures of –20°C or below. Study samples were analyzed without exceeding the limits on the long-term or short-term storage stability, freeze-thaw stability, or postpreparation stability of IDX899.

Sample analysis. Plasma and urine samples were analyzed for IDX899 by validated high-performance liquid chromatography and tandem mass spectrometry methodologies. Briefly, an aliquot of 50 µl of the internal standard, IDX989 (an analogue of IDX899), was added at 2,000 ng/ml to 50 µl of the calibration standards (5 to 2,500 ng/ml), quality controls (5 to 1,950 ng/ml), and unknown plasma or urine samples. For plasma samples, protein was precipitated by adding acetonitrile (300 µl) and was removed by centrifugation. The recovery of both analytes by extraction from plasma was nearly complete. The plasma supernatant (100 µl) or urine (50 µl) was diluted with 400 µl of water-acetonitrile (60:40, vol/vol). A 25-µl volume of the final prepared sample was analyzed by high-performance liquid chromatography with tandem mass spectrometry detection. For plasma samples, chromatography was performed on a Polar reversed-phase column (50 mm by 2 mm; particle size, 4 µm; Phenomenex, Torrance, CA) preceded by a Javelin Hypersil C18 guard column (20 mm by 2.1 mm; Thermo Fisher Scientific, Waltham, MA). For urine samples, a Synergi Polar reversed-phase analytical column (50 mm by 2.0 mm; particle size, 4 µm) coupled with a SecurityGuard AQ C18 guard column (4 mm by 2.0 mm) was used (Phenomenex). Elution was carried out isocratically at a constant flow rate of 0.5 ml/min with a mobile phase of water-methanol-ammonium acetate (1.0 M, pH 4.5) (27.2:71.8:1, vol/vol/vol). Under these conditions, the retention times were approximately 2.1 and 2.2 min for IDX899 and IDX989, respectively. The analytes were monitored with PE Sciex API 4000 triple-quadrupole mass analyzer at a mass transition of 411.7 to 369.1 m/z for IDX899 and 429.8 to 387.4 m/z for IDX989. There was no interference at the specific mass transitions, indicating the absence of a matrix effect. The mass analyzer was operated under negative ion mode with electrospray ionization. These assays have a lower limit of quantitation of 5 ng/ml with a calibration curve ranging from 5 to 2,500 ng/ml. The intra- and interday precisions (coefficient of variation) and accuracies (percent deviation) ranged from 2.47 to 6.28% and –9.34 to 3.19%, respectively, on the basis of the results obtained with quality control samples spiked with concentrations ranging from 5 to 1,950 ng/ml.

Pharmacokinetic and statistical analyses. The plasma concentration-time data of IDX899 were analyzed by noncompartmental methods. The maximum drug concentration in plasma (Cmax), the time to Cmax (Tmax), the concentration 24 h after the administration of a single dose (C24), and the steady-state trough (minimum) concentration (Cmin) were directly obtained from the plasma concentration-time profiles. The area under the plasma concentration-time curve (AUC) from time zero to time t or {tau} (AUC0-t and AUC{tau} [for multiple doses], respectively), where t is the time at which the last sample with a measurable concentration was obtained and {tau} is the dose interval (12 h for the BID regimen or 24 h for the QD regimen), was calculated according to the linear trapezoidal rule. The AUC from time zero to infinity (AUC0-{infty}) was estimated as AUC0-t + Ct/ke, where Ct is the concentration in the last plasma sample in which a measurable concentration was obtained, and ke is the slope of the linear portion of the natural log-transformed postpeak plasma drug concentration-time curve estimated by linear regression. The terminal half-life (t1/2) over the sampling period was calculated as 0.693/ke. Apparent total plasma clearance (CL/F) was calculated as dose/AUC0-{infty} or dose/AUC{tau}, and the apparent total volume of distribution (V/F) was calculated as CL/ke.

For the evaluation of the effect of food, the pharmacokinetic parameters underlying plasma exposure (Cmax, AUC0-t, and AUC0-{infty}) were log transformed. The results for the fasted and fed groups were compared by using an analysis of variance model, and the treatment (dosing condition) was used as a fixed effect and the subject was used as a random effect. Analysis was performed by using the Generalized-Additive-Model procedure in SAS software (version 8.2; SAS Institute Inc., Cary, NC). The results for Cmax and AUC were reported as 90% confidence intervals (CIs) for the ratios of the geometric means of the pharmacokinetic measures between treatments in the fed (test) and the fasted (reference) states. The analysis of variance-based confidence limits for the differences in the mean values of the log-transformed parameters were exponentiated and are reported as ratios (test treatment to reference treatment). It was concluded that food has no significant effect on pharmacokinetics if the 90% CI for the ratio of geometric means was contained within the critical range of 80 to 125% for bioequivalence for Cmax and AUC.

The principal parameters underlying plasma drug exposure, including Cmax and AUC0-{infty}, were assessed for dose proportionality after administration of a single dose under fed conditions during the single-dose-escalation phase in the 200- to 1,200-mg dose range by using the following power model: Yij = a x Djb x eij, where Dj is the dose D at level j; Yij is the pharmacokinetic parameter for subject i at dose level j; a and b are the mean intercept and the slope, respectively; and eij is the residual error for subject i at dose level j. For practical reasons, this model is log linearized as log(Yij) = a + b x log(Dj) + eij and is fit by linear regression by using the Generalized-Additive-Model procedure in SAS software (version 8.2; SAS Institute Inc.). A dose-proportional relationship is concluded if the 95% CI of the mean of b includes unity and was contained within a critical range of 0.70 to 1.30.

The cumulative excretion in urine was calculated as the sum of the amount excreted during each interval and is expressed as a percentage of the administered dose.


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RESULTS
 
Subject characteristics and disposition. All males in the treatment groups had comparable demographics at the baseline. The female subjects enrolled in the study were older (mean age, 53.3 years) than the male subjects (mean age range for the males in the single-dose cohorts, 26.0 to 36.0 years). This was expected, because only women of nonchildbearing potential were enrolled. Table 1 summarizes the characteristics of the subjects at the baseline.


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  		TABLE 1. Subject characteristics

Two subjects prematurely withdrew from the study, as described below; one of those two subjects was replaced. Data for all randomized subjects were included in the safety analyses.

Safety and tolerability. There were no deaths, SAEs, or dose-dependent AEs or abnormal vital signs, ECG results, or laboratory test results. A maximum tolerated dose of IDX899 was not identified in this study. For repeat doses, there were no clinically relevant changes over time. The values of all principal hematologic parameters (hematocrit, platelet, and white blood cell counts) and serum chemistries (alanine transaminase, aspartate transaminase, bilirubin, blood urea nitrogen, and creatinine) were within the normal ranges.

The AEs that occurred in more than one subject are summarized by the preferred term in Table 2. The most common AE in all IDX899 groups was headache (5/65 subjects). Abnormal or vivid dreams occurred in 2 of 65 subjects (3.1%) exposed to IDX899 across all cohorts. Vivid dreams lasting 2 days occurred in one female subject 3 days after she received a single 400-mg dose. Another subject in the cohort receiving the 800-mg QD regimen also reported vivid dreams that began on day 2 and that lasted 6 days. Two of 65 subjects (3.1%) exposed to IDX899 reported a skin and subcutaneous tissue disorder. Both were male and were in the cohort receiving the 800-mg QD regimen. One subject discontinued the study after the first IDX899 dose due to mild pruritus and urticaria to the inner aspect of his right arm and popliteal fossa of the right knee. The AE was treated with a single dose of 50 mg diphenhydramine (Benadryl) and resolved. The other subject reported contact dermatitis.


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  		TABLE 2. AEs reported in more than one subjecta

With the exception of a female subject in the 400-mg cohort who experienced moderate vomiting and was withdrawn at the sponsor's request, all other AEs were mild in severity and resolved by the end of the study.

Plasma pharmacokinetics. The single-dose escalation study was initially performed with IDX899 dosed under fasted conditions, followed by dosing under fed conditions, once the effect of food was confirmed. Mean (+standard deviation [SD]) plasma concentration-time curves for IDX899 administered under fasted conditions are depicted in Fig. 2 (left panel), and a summary of the values of the pharmacokinetic parameters is presented in Table 3. Following oral administration of a single dose under fasted conditions to healthy male subjects, IDX899 was rapidly absorbed, and the median Tmax was 1.5 to 1.8 h, regardless of the formulation or the dose. In contrast, the two formulations differed substantially in the extent of absorption, with the 50-mg capsule achieving higher levels of exposure in the 50- and 100-mg dose cohorts. Nevertheless, within each formulation, the level of exposure to IDX899 under fasted conditions was proportional to the dose administered, with AUC0-{infty} and Cmax doubling as the dose was doubled from 50 to 100 mg with the 50-mg formulation and from 200 to 400 mg with the 200-mg formulation (Table 3). The 200-mg formulation resulted in a longer mean t1/2 of 11.9 to 14.6 h in the 200- and 400-mg dose cohorts, whereas the mean t1/2 was 6.2 to 6.8 h with the 50-mg capsule.


Figure 2
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  		FIG. 2. Single-dose plasma concentration-time profiles of IDX899. (Left panel) Profiles for doses of 50 to 400 mg administered to healthy male subjects under fasted conditions; (right panel) profiles for doses of 200 to 1,200 mg administered to healthy male subjects under fed conditions and a dose of 400 mg administered to women of nonchildbearing potential under fed conditions. The mean (+SD) is shown.


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  		TABLE 3. Pharmacokinetic parameters of IDX899 for the single-dose cohortsa

The effect of food on the absorption of IDX899 was initially evaluated with the 200-mg dose cohort. As shown in Table 3, a high-fat, high-calorie test meal enhanced absorption, with the mean AUC0-{infty} increasing from 7.1 to 16.8 µg·h/ml (ratio of AUC0-{infty} in fed state to AUC0-{infty} in fasted state, 2.60; 90% CI, 1.62 to 4.16) and the mean Cmax increasing from 0.7 to 1.3 µg/ml (ratio of Cmax in fed state to Cmax in fasted state, 1.91; 90% CI, 1.57 to 2.32). The effect of food was further evaluated by enrolling an additional 400-mg cohort to which IDX899 was administered immediately after the subjects consumed a high-fat, high-calorie test meal. As presented in Table 3, similar to the data for the 200-mg dose cohort, the test meal essentially doubled the level of exposure to 400 mg of IDX899, with the mean AUC0-{infty} increasing from 14.7 to 36.0 µg·h/ml (ratio of AUC0-{infty} in fed state to AUC0-{infty} in fasted state, 2.46) and Cmax increasing from 1.1 to 2.4 µg/ml (ratio of Cmax in fed state to Cmax in fasted state, 2.18), respectively. In addition, food also resulted in a higher C24 (with 24 h being the anticipated dosing interval), with the mean C24 for the 200-mg dose increasing from 0.07 to 0.19 µg/ml (ratio of C24 in fed state to C24 in fasted state, 2.71) and with the mean C24 for the 400-mg dose increasing from 0.16 to 0.36 µg/ml (ratio of C24 in fed state to C24 in fasted state, 2.25). While food enhanced the overall absorption, food largely delayed Tmax to a median of 7.0 h for both doses. The elimination of IDX899 remained unaffected by food intake, with comparable t1/2s being obtained under the fasted and the fed conditions for the 200- and 400-mg doses (Table 3).

With the effect of food confirmed, the remaining planned single doses of 800 and 1,200 mg were continued under the same fed conditions. The mean (+SD) plasma concentration-time curves for IDX899 administered under fed conditions are depicted in Fig. 2 (right panel). As can be seen in Table 3, the level of exposure to IDX899 under fed conditions in male subjects increased, with increases in mean values of from 16.8 to 106.4 µg·h/ml for AUC0-{infty}, 1.3 to 5.6 µg/ml for Cmax, and 0.19 to 2.02 µg/ml for C24 as the dose was increased from 200 to 1,200 mg. The C24 for a single 200-mg dose exceeded the in vitro protein-binding-adjusted IC90 of 0.046 µg/ml by fourfold (3; Standring, unpublished). The pharmacokinetic dose proportionality of IDX899 in the 200- to 1,200-mg dose range was evaluated by regression analyses of log-transformed parameters of exposure and doses. The model estimate of b was close to unity (b, 0.98; 95% CI, 0.75 to 1.22) for AUC0-{infty}, the primary measure of overall exposure. For Cmax and C24, the point estimates of b were 0.77 (95% CI, 0.62 to 0.92) and 1.30 (95% CI, 0.81 to 1.79), respectively. Absorption under fed conditions was delayed, with the median Tmaxs ranging from 6.0 to 10.1 h, which are regarded as being consistent given the large dose range used in the study. Similarly, IDX899 exhibited dose-independent values of t1/2 (mean, 7.9 to 11.5 h), CL/F (mean, 11.8 to 17.8 liters/h), and V/F (mean, 133.7 to 301.0 liters) in male subjects under fed conditions.

Following completion of the single-dose-escalation phase of the study with demonstrated safety and tolerability in male subjects, a cohort of women of nonchildbearing potential was enrolled and administered a single dose of 400 mg (n = 6 for the population for pharmacokinetic analysis) or matching placebo (n = 2) of IDX899 under fed conditions. As depicted in Fig. 2 (right panel), the mean (+SD) plasma concentration-time curve for this group of female subjects closely paralleled the mean kinetic profile for the male group treated with the same 400-mg single dose under fed conditions. As presented in Table 3, the mean values of the principal pharmacokinetic parameters were comparable in the male and the female subjects.

After completion of the single-dose-escalation phase of the study, healthy male subjects were enrolled to receive multiple doses of IDX899 either as 400 mg BID or 800 mg QD for 7 days, and the subjects within each cohort were randomized so that eight received the active drug and two subjects received placebo. The subjects were dosed under fed conditions after they consumed a standardized meal throughout the study period. Figure 3 depicts the mean (+SD) plasma concentration-time curves after administration of the first dose and at steady state with the daily Cmins. The values of the pharmacokinetic parameters obtained after administration of the first dose and at steady state are summarized in Table 4. With the 400-mg BID regimen for 7 days, the steady-state plasma exposure was about twice as high as that resulting from the first dose on the basis of Cmax (means, 4.0 and 2.0 µg/ml, respectively) and Cmin (means, 2.1 and 0.8 µg/ml, respectively). With the 800-mg QD regimen, the values of Cmax at steady state (mean, 4.6 µg/ml) and after administration of the first dose (mean, 4.9 µg/ml) were comparable, and both were in the range of the data for the 400-mg BID regimen at steady state. Both multiple-dose regimens resulted in stable Cmins over the entire dosing period (Fig. 3, insets). The 800-mg QD regimen also achieved a high Cmin after administration of the first dose (mean, 0.9 µg/ml) which was maintained through the administration of the last dose. The Cmin obtained with the 800-mg QD regimen exceeded by 20-fold the in vitro protein-binding-adjusted IC90 of 0.046 µg/ml. The steady-state AUC{tau} obtained with the 800-mg QD regimen (mean, 54.6 µg·h/ml) was similar to the AUC0-{infty} obtained with an 800-mg single dose (mean, 68.0 µg·h/ml), and the values for both parameters were in close agreement with the data for the 800-mg single dose in Table 3. The same was also true for the 400-mg BID regimen when the values of the steady-state AUC{tau} were compared to the data for the 400-mg single dose in Table 3. In addition, the total daily exposure to IDX899 from the 400-mg BID regimen at steady state, i.e., 2 x 400 mg resulting in 2 x AUC{tau} (expected mean, 2 x 35.2 = 70.4 µg·h/ml), was equivalent to that from the 800-mg QD regimen. Finally, the steady-state pharmacokinetic parameters were comparable between the two regimens for the other key parameters t1/2 (mean, 12.3 to 13.6 h), CL/F (mean, 16.1 to 17.5 liters/h), and V/F (mean, 235.6 to 366.1) and were in close accordance with the data obtained during the single-dose-escalation studies (Table 3). Together, these data indicate that IDX899 exhibits linear pharmacokinetics.


Figure 3
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  		FIG. 3. Multiple-dose plasma concentration-time profiles of IDX899 as 800 mg QD and 400 mg BID administered for 7 days under fed conditions to healthy subjects. (Insets) Mean C24/Cmin plotted against time. The mean (+SD) is shown. IC90, 90% inhibitory concentration.


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  		TABLE 4. Pharmacokinetic parameters for the multiple-dose cohortsa

Urinary excretion. The amount of IDX899 excreted unchanged in urine was monitored over a 24-h interval after administration of the 200-mg (fasted), 400-mg (fasted and fed), and 800-mg (fed) doses only. For the 200-mg and 400-mg doses under fasted conditions, no IDX899 could be detected in the urine. Excretion in urine became measurable for the 400-mg and 800-mg doses under the fed condition, but the amount excreted was extremely low. The mean (±SD) cumulative amounts excreted were 1.69 ± 2.93 and 4.18 ± 3.72 µg for the 400- and 800-mg doses, respectively; these amounts represent less than 0.001% of the doses administered. The renal clearance of unchanged IDX899 was negligible (<0.0001 liter/h).


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DISCUSSION
 
HIV infection is a chronic disease that requires lifelong management with antiretroviral drugs. Despite the availability of approximately 30 approved drugs or drug combinations, long-term therapy for HIV infection continuously requires the development of new drugs with improved safety and tolerability, a higher barrier to the emergence of resistance, and convenient dosing. IDX899, a candidate NNRTI, exhibited favorable preclinical toxicological and antiviral properties (3, 6).

Resistance to NNRTIs emerges quickly when complete viral suppression is not achieved. In addition, HIV-1 strains typically require only a single mutation to confer high-level resistance to the early NNRTIs, such as nevirapine and efavirenz, often resulting in cross-resistance to the entire NNRTI class (2). Therefore, a rational NNRTI clinical development program would first determine the safe and well-tolerated single dose of a drug and would then determine the multiple doses expected to achieve relevant antiretroviral exposures in healthy subjects prior to the evaluation of the antiretroviral activity of the drug in HIV-1-infected patients. In that context, the current study consisted of a single-dose-escalation phase with doses ranging from 50 to 1,200 mg and with an embedded evaluation of the effects of food and gender. This was followed by the administration of multiple doses given as both 800-mg QD and 400-mg BID regimens for 7 days, a duration suitable for a short-term proof-of-concept study with HIV-1-infected patients.

The findings from the preclinical toxicology data fully supported the single and multiple doses studied, with the safety margin in rats and monkeys exceeding 80-fold for the 50-mg starting dose (Selden, unpublished). The clinical results of the current study revealed that overall, IDX899 administered as single oral doses of 50 mg to 1,200 mg and multiple doses of 400 mg BID and 800 mg QD for 7 days was safe and well tolerated by the healthy male subjects as well as by a cohort of women of nonchildbearing potential. AEs were generally transient and mild in intensity and did not have a clear relationship to IDX899. No risks were identified by evaluation of vital sign measurements, ECG measurements, or physical examination findings. No patterns of laboratory abnormalities, either by system organ class or dose, were observed by hematologic or chemical analysis or urinalysis.

Neurologic side effects and rash are commonly reported by patients taking NNRTIs (5, 7). Up to 50% of patients receiving efavirenz reported a central nervous system side effect, including about 6% who reported abnormal dreams. Rash is commonly reported after 2 to 6 weeks of exposure to NNRTIs and was not expected in this study with up to 7 days of drug exposure. In the current study, abnormal dreams and rash were reported in only 3% of the subjects. While larger and longer trials will be conducted to thoroughly define the safety of the drug, data from the current study suggest that IDX899 may have an improved safety profile which could allow it to be used in both treatment-naïve and treatment-experienced HIV-infected subjects.

The single-dose-escalation phase of this study covered a large dose range spanning from 50 to 1,200 mg, resulting in a maximum 24-fold increase in dose. Two oral formulations were used to minimize the pill burden, with a 50-mg capsule used for doses up to 100 mg and a 200-mg capsule used for doses ≥200 mg. The two capsules are of equal size and appearance, but they differ in their amounts of API and excipient. The 50-mg capsule led to a higher but transient exposure and had a high fluctuation index (estimated as the mean Cmax divided by mean C24, on the basis of the data for the 50- and 100-mg doses in Table 3) of approximately 30. The 200-mg capsule achieved more sustained plasma concentrations and had a reduced fluctuation index of approximately 7 to 10 on the basis of the data for the 200- and 400-mg doses obtained under fasted conditions (Table 3). Use of the 200-mg capsule also resulted in a longer elimination t1/2. The pharmacokinetic profile associated with the 200-mg capsule is clearly more desirable with regard to maintaining continuous suppressive pressure on viral replication. The different drug-to-excipient ratios may account for the observed differences in the pharmacokinetics of IDX899 between the two capsules; i.e., the higher excipient content may have facilitated the more extensive dissolution of IDX899 from the 50-mg capsule.

The absorption of IDX899 from the 200-mg capsule could be improved by food intake. A high-fat, high-calorie meal enhanced the level of exposure by approximately twofold. In fact, a standard meal that had a reduced fat and calorie content and that was served during the multiple-dose step also enhanced the level of absorption comparable to that achieved with the high-fat, high-calorie meal. In contrast, while this study did not evaluate the effect of food on the 50-mg dose, a follow-up study showed that food failed to increase the absorption of IDX899 from the 50-mg capsule (9). These data suggest that maximum dissolution may have already been achieved with the 50-mg capsule under fasted conditions, while food enhanced the dissolution of the 200-mg capsule. The absorption process was prolonged under fed conditions, as evidenced by a delay in Tmax. The elimination process was, as expected, not affected by food intake: the long t1/2 associated with the 200-mg capsule was maintained when IDX899 was administered with food.

IDX899 exhibited dose-proportional pharmacokinetics. This was initially demonstrated within each formulation under fasted conditions, as the level of exposure doubled when the doses were doubled. The pharmacokinetic dose proportionality was confirmed for total exposure (AUC0-{infty}) by using the single-dose data for the 200- to 1,200-mg doses administered under fed conditions. Cmax and C24 appeared to be somewhat less and more than dose proportional, respectively. While no formal statistical tests were performed, other pharmacokinetic observations that are consistent with dose proportionality include (i) the dose-independent t1/2; (ii) the dose-independent CL/F and V/F; and (iii) the dosing interval-independent exposure; i.e., the daily exposure associated with the 400-mg BID regimen was equivalent to that associated with the 800-mg QD regimen, albeit with some interindividual variability (Table 4). IDX899 also exhibited time-independent pharmacokinetics, as indicated by (i) the fact that the single-dose AUC0-{infty} was predictive of the steady-state AUC{tau} and (ii) the consistent values of t1/2 and CL/F over time. These favorable pharmacokinetic properties led to a predictable systemic exposure to IDX899.

Multiple-dose pharmacokinetics are more relevant to the clinical use of the drug. For NNRTIs, among the pharmacokinetic parameters, Cmins have been shown to be associated with antiretroviral activity in HIV-1-infected patients (4). In the current study, both the 400-mg BID and the 800-mg QD regimens of IDX899 were evaluated for 7 days. On the basis of the principal of superposition for linear pharmacokinetics, higher Cmins were expected with the 400-mg BID regimen. The 800-mg QD nevertheless also achieved steady-state Cmins that exceeded by 20-fold the in vitro protein binding-adjusted IC90 (0.046 µg/ml) or by 40-fold the IC50 (0.023 µg/ml) of IDX899 (3; Standring, unpublished). While a single dose of 200 mg produced a mean C24 that was fourfold the IC90, it can be predicted, on the basis of dose proportionality from the data for the 800-mg QD regimen, that even with a lower 200-mg QD regimen, the steady-state Cmin would be approximately 5-fold the IC90 or 10-fold the IC50. The large exposure margin in conjunction with the convenience of QD dosing supports dosing of IDX899 QD as a proposed regimen in future clinical studies.

Preclinical data showed that IDX899 underwent extensive metabolism via CYP450 systems and conjugation, suggesting that hepatic clearance may be the major elimination pathway for IDX899. The latter appears to be consistent with the findings of the current study. The urinary excretion of unchanged IDX899 was extremely small: less than 0.001% for the single doses tested in this study. The complete metabolic profile of IDX899 in humans is still under investigation. However, the time-independent pharmacokinetics of IDX899 observed in this short-term study suggest that IDX899 did not inhibit or induce its own metabolism. The potential for IDX899 to interact with other antiretroviral drugs metabolized via the CYP450 system will be evaluated in future drug-drug interaction studies.

Finally, women constitute approximately half of all adults currently living with HIV globally (8). Therefore, it is important to enroll female subjects early in the drug development process to assess any gender-related differences in pharmacokinetics. Because supportive reproductive and developmental toxicology data were not available, only women of nonchildbearing potential were enrolled in the current study. Despite a mean age that was twice that of the male comparator group, the pharmacokinetics of IDX899 following administration of a single dose of 400 mg were comparable in the female and the male subjects. Considering the small sample size used in this study, however, data from more subjects will be required to confirm the absence of gender differences, and preferably, these data will be obtained by using a population pharmacokinetic approach.

In summary, single oral doses of 50 mg to 1,200 mg of IDX899 and multiple doses of 400 mg BID and 800 mg QD for 7 days were safe and well tolerated by the healthy subjects in this study. No relationship between adverse events and dose was observed. Clinical laboratory assessments showed no signs of liver or kidney toxicity. On the basis of the favorable pharmacokinetic and safety data for IDX899 administered over 7 days to healthy subjects, a dose regimen of 800 mg QD was selected as the first dose for a 7-day proof-of-concept study with HIV-1-infected treatment-naïve patients. The favorable safety profile and predictable pharmacokinetics warrant further clinical studies for IDX899.


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ACKNOWLEDGMENTS
 
We thank the healthy volunteers and the staff of Pharmaceutical Product Development.


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FOOTNOTES
 
* Corresponding author. Mailing address: Idenix Pharmaceuticals Inc., One Kendall Square, Building 1400, Cambridge, MA 02139. Phone: (617) 995-9805. Fax: (617) 995-9817. E-mail: zhou.xj{at}idenix.com Back

{triangledown} Published ahead of print on 17 February 2009. Back


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Antimicrobial Agents and Chemotherapy, May 2009, p. 1739-1746, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01479-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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