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 (Lincoln NE).
Clinical conduct for the study took place at MDS Pharma Services in Lincoln, NE. The first subject was dosed on 27 July 2008, and the last subject completed the study on 10 October 2008.
All subjects voluntarily gave written informed consent after the nature of the study was fully explained. Eligible subjects were healthy adult nonsmoking male or female subjects from the general population. They were 19 to 65 years of age, with body mass indices between 18 and 32 kg/m2 and no evidence of clinically significant abnormalities in medical history, physical examination, 12-lead electrocardiogram (ECG), or clinical laboratory testing during screening. Subjects of childbearing potential were required to use an acceptable double-barrier method of birth control during the study and for up to 90 days after the last dose of the study drug. Female subjects were excluded if they were pregnant or breastfeeding. Subjects were excluded if they had donated blood or had a significant blood loss within 56 days or donated plasma within 7 days of screening or had used a known inhibitor and/or inducer of CYP450 3A4 (CYP 3A4) within 30 days of reporting to the clinic. Subjects were excluded if they received any chronic prescription medications within 3 months, acute prescription drugs within 14 days, or systemic over-the-counter medications (including aspirin, vitamins, and herbal supplements) within 7 days of reporting to the clinic. Subjects were excluded if currently abusing alcohol or illicit drugs or having a history of alcohol or illicit drug abuse within the preceding 2 years. 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 participated in a clinical study within 30 days prior to screening.
The trial was a single-center, randomized, double-blind, placebo-controlled, single-dose-escalation study in healthy male or female subjects.
IDX184 and matching placebo were supplied as white opaque capsules. Both a 5-mg and a 25-mg capsule were provided. The single-dose escalation started at 5 mg, which escalated to 10, 25, 50, 75, and 100 mg. The 5- and 10-mg cohorts used the 5-mg capsules, while other doses employed the 25-mg capsules. Eight subjects were randomly assigned at a 6:2 active/placebo ratio within each cohort. Safety and plasma pharmacokinetic evaluations were performed before escalation to the next dose level.
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. At least 240 ml (8 fluid oz.) of water was given with the study dose. A standard lunch was served no less than 4 h after dosing. Meals and beverages served within 24 h prior to and after dosing were free of xanthine and caffeine. Grapefruit juice was not allowed within 24 h prior to and after dosing. Water was permitted ad libitum except for 1 h prior to and after dosing. Subjects were asked to remain upright (sitting or standing) for the first 3 h following dosing and not to engage in any strenuous activity during their stay at the study center.
Safety and tolerability evaluation.
Safety assessments consisted of collecting all adverse events (AEs) and serious adverse events (SAEs), along with determining their severity and relationship to the study drug. Safety assessments also included regular monitoring of hematology, blood chemistry, and urinalysis, as well as vital signs, 12-lead ECG, and physical examination. Concomitant medications were also collected.
Intensive plasma pharmacokinetic sampling was performed over a period of 120 h after dosing at the following time points: 0 (predose) and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 h. Blood samples were collected in Vacutainer tubes containing EDTA as an anticoagulant at the time points specified above within a time window of up to ±10 min. Plasma was obtained by centrifugation at 1,500 × g for 10 min at 4°C and stored frozen at a nominal temperature of −80°C until analysis.
Urine samples were collected over 120 h according to the following time intervals: −2 to 0 (predose), 0 to 4, 4 to 8, 8 to 12, 12 to 24, 24 to 48, 48 to 72, 72 to 96, and 96 to 120 h. Urine samples were stored frozen at a nominal temperature of −80°C until analysis.
Plasma and urine IDX184 and 2′-MeG have been shown to remain stable during storage and assay. The short-term stability of both analytes has been documented when spiked samples were subjected to 6 freeze and thaw cycles (−80°C to 8°C in plasma and −20°C to ambient temperature in urine), storage for at least 24 h at 8°C in plasma and at ambient temperature in urine, and postpreparative for 153 h for plasma samples and at least 108 h for urine samples. Long-term storage stability was 441 days in plasma at −80°C and 86 days in urine under −20°C. Study samples were analyzed without exceeding freeze-thaw/postpreparative stability or long-term/short-term stability.
Plasma and urine samples were analyzed for IDX184 and 2′-MeG using validated high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) methodologies. Concentrations of the two analytes in a sample were simultaneously measured in a single run. For plasma samples, an aliquot of 25 μl of an internal standard working solution containing 16 ng/ml 13CD3-IDX184 and 8 ng/ml D3-2′-MeG was added to 200 μl of calibration standards (0.1 to 20 ng/ml for IDX184 and 0.2 to 40 ng/ml for 2′-MeG), quality controls (QCs) (0.3 to 15 ng/ml for IDX184 and 0.6 to 30 ng/ml for 2′-MeG), and unknown plasma samples. For urine samples, an aliquot of 25 μl of an internal standard working solution containing 40 ng/ml 13CD3-IDX184 and D3-2′-MeG was added to 100 μl of calibration standards (1 to 200 ng/ml), QCs (3 to 150 ng/ml), and unknown urine samples. Plasma and urine samples were then diluted with 1 ml and 0.5 ml, respectively, with a 100 mM ammonium acetate solution. After mixing, diluted samples (approximately 600 μl) were subjected to solid-phase extraction on a 96-well plate (Bond Elut PBA, 100 mg; Varian, Palo Alto, CA). The plate was preconditioned and thereafter successively washed after sample loading with acetonitrile and water. Analytes were eluted with 0.8 ml 5% formic acid in methanol. Eluent was evaporated to dryness under a gentle nitrogen flow at 40°C. The residual was reconstituted with 200 μl acetonitrile, and a 25-μl volume of the final prepared sample was analyzed via HPLC with MS/MS detection. Under these conditions, average extraction recovery from plasma and urine was 76% and 59%, respectively, for IDX184 and 79% and 69%, respectively, for 2′-MeG. For both matrices, chromatography was performed on a Zorbax 300-SCX column (50 mm by 3 mm; particle size, 5 μm; Agilent Technologies, Santa Clara, CA) preceded by a SecurityGuard ODS C18 guard column (4 mm by 3 mm; particle size, 5 μm; Phenomenex, Torrance, CA). Elution was carried out isocratically at a constant flow rate of 1.5 ml/min with a mobile phase of 95:5 (vol/vol) acetonitrile/ammonium formate (100 mM, pH 3.8). Under these conditions, the retention times were approximately 1.4 and 1.0 min for IDX184 and 2′-MeG, respectively. The analytes were monitored using a PE Sciex API 5000 triple quadrupole mass analyzer at a mass transition of 627.2 → 316.1 m/z and 631.2 → 320.1 m/z for IDX184 and 13CD3-IDX184, respectively, and 298.2 → 152.1 m/z and 301.2 → 152.1 m/z for 2′-MeG and D3-2′-MeG, respectively. The mass analyzer was operated in positive ion mode using electrospray ionization. This assay has a lower limit of quantitation (LOQ) of 0.1 ng/ml for IDX184 and 0.2 ng/ml for 2′-MeG in plasma and 1 ng/ml for both analytes in urine. The intra- and interday precisions (coefficient of variation) and accuracies (percent deviation) for plasma and urine assays were from 2.5 to 16.1% and −13.3 to 10.3%, respectively, for IDX184 and from 0.8 to 6.6% and −7.2 to 5.0%, respectively, for 2′-MeG.
Pharmacokinetic and statistical analyses.
The plasma concentration-time data for IDX184 and 2′-MeG were analyzed using noncompartmental methods. The maximum drug concentration in plasma (Cmax), time to Cmax (Tmax), and concentration 24 h after dosing (C24) were obtained directly from the plasma concentration-time profiles. The area under the plasma concentration-time curve (AUC) from time zero to time t (AUC0-t), where t is the time at which the last sample with a measurable concentration was obtained, was calculated according to the linear trapezoidal rule. The AUC from time zero to infinity (AUC0-∞) 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 liner regression. The terminal half-life (t1/2) over the sampling period was calculated as 0.693/ke. For IDX184, apparent total plasma clearance (CL/F) was calculated as dose/AUC0-∞, and the apparent total volume of distribution (Vd/F) was calculated as CL/ke. Both parameters were further normalized to body weight.
Principal parameters underlying plasma drug exposure, including Cmax and AUC0-∞, were assessed for dose proportionality in the 5- 100-mg dose range by using the following log-linearized power model: log (Yij) = a + b × log(Dj) + eij, where Dj is the dose at level j, Yij is the pharmacokinetic parameter for subject i at dose level j, a and b are the mean intercept and slope, respectively; and eij is the residual error for subject i at dose level j. Fitting was carried out by linear regression using the GLM procedure in SAS (version 9.2; SAS Institute Inc., Cary, NC). A dose-proportional relationship is concluded if the 95% confidence interval (CI) of the mean slope (b) includes unity and was contained within a critical range of 0.70 to 1.30.
Cumulative urine excretion (Au0-t) was calculated as the sum of the amounts excreted during each interval and expressed as a percentage of the administered dose. Renal clearance (CLR) was calculated as Au0-t/AUC0-t, where the time interval 0-t was the same for the numerator and the denominator.