Subject eligibility and recruitment
Healthy adults between the age of 18 and 65 years with an Eastern Cooperative Oncology Group (ECOG) performance status of 1 or less than 1 (Karnofsky performance status ≥ 80%) were eligible for this study. See . Subjects were required to have the following: WBC ≥3,000/ μL; platelets ≥100,000/μL, hemoglobin >10 g/dL, bilirubin ≤1.4 mg/dL; aspartate aminotransferase (AST) ≤1.5 × normal; TG ≤1.5 × ULN, and cholesterol ≤1.5 × ULN as well as normal renal and electrolyte levels. Participants were excluded if they were taking St. John’s wort, ketoconazole, vitamin A, tetracycline, lipid-lowering agents, oral corticosteroids or any other RA. Subjects were also excluded if they were taking other investigational agents, had a history of allergic reactions attributed to compounds similar in composition to 9cUAB30, had an uncontrolled intercurrent illness, or were pregnant. The study was approved by the Institutional Review Board, and all subjects provided written informed consent.
Baseline characteristics of enrolled patientsa
This pilot clinical trial was designed to characterize the single dose pharmacokinetics of 9cUAB30 in humans at a single dose of 5, 10, or 20 mg of 9cUAB30 given orally in the morning on an empty stomach. The first subject was to receive the 5-mg dose, and after 30 days without a grade 2 or higher toxicity, 3 more subjects were to also receive the 5-mg dose. Without grade 2 or higher toxicities observed among the initial 4 subjects, the 4 subject cohorts of 10 mg and 20 mg were then to be dosed sequentially. Following dosing, subjects were to stay for 24 hours as inpatients, during which time urine samples were collected at 0, 6, 12, 18, and 24 hours after 9cUAB30 administration. Plasma samples were collected at 0, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 16, 20, and 24 hours postdose. All samples were stored at −70 °C for less than 3 months. Subjects returned on day 8 for pharmacokinetic sample collection and toxicity evaluation and were contacted on day 30 to assess health status and any adverse events related to the study agent. Toxicity was graded using the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE; ref. 13
). All adverse events were followed until resolution to grade 1 or less.
9cUAB30 was quantitated in heparinized plasma and human urine after storage of up to 3 months using LC-MS/MS detection with a method similar to Kane et al. (14
). Liquid-liquid extraction and sample preparation was performed as follows: added 10 μL of 5 μg/mL of 9-cis-RA (internal standard) to 0.5 mL sample, vortexed, added 1 mL of 0.025 mol/L of KOH in ethanol, vortexed, added 5.0 mL of hexane, agitated on an orbital shaker for 10 minutes, centrifuged at 1,200 rpm for 10 minutes, removed and discarded the top organic layer, added 1 mL of acetonitrile and 60 μL of 4 mol/L of HCl, vortexed, added 5 mL of hexane, agitated on an orbital shaker for 10 minutes, centrifuged at 1,200 rpm for 10 minutes, transferred the organic layer to a glass tube, and dried down under N2
, reconstituted residue with 50 μL of acetonitrile, vortexed, added 50 μL of 0.5% formic acid, vortexed, placed into autosampler vial fitted with an insert for small volumes.
LC-MS/MS data were obtained on an Applied Biosystems/ MDS Sciex API 4000 in positive ion mode for 9cUAB30 and 9-cis-RA (internal standard). The multiple reaction monitoring (mrm) transitions were m/z 295.1 → m/z 165.1 for UAB30, m/z 301.15 → m/z 201.2 for 9-cis-RA. The following settings for the MS/MS were used. Positive ion instrument parameters were: curtain gas (CUR) 12 units, gas1 (GS1) 40 units, gas2 (GS2) 42 units, collision gas (CAD) 7 units, interface heater (IHE) on, nebulizer current 5.00, declustering potential (DP) 40 V, exit potential (EP) 15 V. For 9cUAB30, collision exit potential (CXP) 30 V, collision energy (CE) 71 V, dwell 210 milliseconds. For 9-cis-RA, CXP 10 V, CE 25, dwell 1,000 milliseconds.
A 15 × 4.6-mm SB C18 (Agilent) 1.8-micron HPLC column was used as the analytical column, and 5 μL samples were injected. The mobile phase solvents were A) 0.1% formic acid in water, and B) 0.1% formic acid in acetonitrile. These solvents were mixed and delivered as a gradient at 800 μL/min as follows: 80% A/20% B for 0.5 minutes; ramped to 25% A/75% B over 1.5 minutes and held for 2.0 minutes; ramped to 80% A/20% B over 0.5 minute and held for 2.0 minutes.
Samples were quantitated by linear regression from a 6-point standard curve ranging from 1.56 to 100 ng/mL with a trend line r2 of 0.998 over the range. This quantitative method’s lower limit of quantitation (LLOQ) was 1.56 ng/ mL, and the lower limit of detection (LOD) was 0.78 ng/ mL. Recovery of 9cUAB30 from plasma was greater than 80% compared to water standards. Intraday variability was 0.7% for low-standard triplicates (1.56 ng/mL) and 7.2% for high-standard triplicates (50 ng/mL). The interday variability over 35 days was 14% for a low standard (3.125 ng/mL), and 11% for a high standard (50 ng/ mL). Low, medium, and high concentration quality control (QC) samples were prepared by spiking plasma or urine with a known amount of 9cUAB30, and analytical runs were rejected if QC samples varied by less than 15% from the expected concentration.
To assess stability, blank human urine and plasma were spiked with a 9cUAB30 stock standard solution (0.2 mL of 1 μg/mL UAB30 + 4.8 mL blank plasma) to obtain con-centrations of 10 ng/mL (plasma and urine), 40 ng/mL (plasma and urine), and 100 ng/mL (urine) and stored at −70 °C. Both the plasma and urine standards were stored for 12 weeks and analyzed in triplicate to assess stability of 9cUAB30 over time.
Pharmacokinetic variables were determined by noncom-partmental methods with WinNonlin Pro version 5.2 (Pharsight Corporation). Area under the plasma concentration-time curve (AUC) was estimated using the trapezoidal rule from time 0 to peak concentration, and the trapezoidal rule from the peak concentration to the last measurable plasma concentration (AUClast). AUC (0-∞) was then calculated from the time of dosing and extrapolated to infinity. Renal clearance was calculated with the following equation Clr = X/24h/AUC0-24h, where X24h was the total amount excreted in nanograms over 24 hours.
The primary objective was to characterize the single-dose pharmacokinetics of 9cUAB30 in normal volunteers. Each of the pharmacokinetic parameters were examined by dose level and in aggregate with basic statistics including means and standard deviations. In general, Jonckheere-Terpstra trend test was performed to determine the significance of the association between increasing dose level and each of the pharmacokinetic measures. The relationships between the parameters AUC and Cmax with dose were especially of interest and were examined using regression models. These measures are by definition 0 for an undosed subject. To fulfill this constraint (that a subject receiving no dose would be predicted to have no measurable Cmax or AUC), models with no intercept were used.
The secondary objectives were to determine the toxicities of 9cUAB30 after administration of a single dose and to correlate the pharmacokinetics of 9cUAB30 with toxicity. All grade II and any grade I toxicities that occur in more than 1 patient have been recorded in . As only 1 patient had any grade II adverse events, correlation of pharmacokinetics with maximum grade of adverse event was not feasible. A relationship between dose and change in TG levels was tested using the Jonckheere-Terpstra test.
All grade II adverse events and any grade I adverse events occurring in more than 1 patient (n = 14)