GNE-781

Dog PK: [1]
Twelve non-naïve male beagle dogs were used. All animals were 6 months to 2 years old at the time of study and weighed between 6 and 10 kg. Animals (n = 3 per dosing route) were dosed with 10 or 19 at 1 mg/kg iv (in ethanol (20% v/v), propyl ethylene glycol 400 (15% v/v) and water (65% v/v)) or 5 mg/kg po (suspended in 0.5% w/v methylcellulose, 0.2% w/v Tween 80). Food and water were available ad libitum to animals in the iv groups. Animals in po groups were fasted overnight and food withheld until 4 h postdose. Approximately 800 μL of blood was collected from a peripheral vessel at predose, 0.033, 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h after the intravenous administration and predose, 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h after the oral administration. All blood samples were collected into tubes containing 10 μL of 0.5 M K2EDTA and processed for plasma. Samples were centrifuged (3000g for 10 min at 4 °C) within 1 h of collection, and plasma samples were kept at −80 °C until analysis.

Monkey PK: [1]
Twelve non-naïve male cynomolgus monkeys were used. All animals were at least 2 years old at the time of study and weighed between 2 and 5 kg. Animals (n = 3 per dosing route) were dosed with 10 or 19 1 mg/kg iv (in ethanol (20% v/v), propyl ethylene glycol 400 (15% v/v) and water (65% v/v)) or 5 mg/kg po (suspended in 0.5% w/v methylcellulose, 0.2% w/v Tween 80). Food and water were available ad libitum to animals in the iv groups. Animals in po groups were fasted overnight and food withheld until 4 h postdose. Approximately 800 μL of blood was collected from a peripheral vessel at predose, 0.033, 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h after the intravenous administration and predose, 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h after the oral administration. All blood samples were collected into tubes containing 10 μL of 0.5 M K2EDTA and processed for plasma. Samples were centrifuged (3000g for 10 min at 4 °C) within 1 h of collection, and plasma samples were kept at −80 °C until analysis.

Bio-analytical method of PK samples: [1]
Concentrations of 10 and 19 were determined by a nonvalidated LC-MS/MS assay. The diluted blood samples were prepared for analysis by placing a 25 μL aliquot into a 96-well plate followed by the addition of 200 μL of acetonitrile containing an internal standard mixture (0.1 μg/mL indomethacin). The samples were vortexed and centrifuged at 4000 rpm for 10 min at 4 °C; 50 μL of the supernatant was diluted with 150 μL of water, and 10 μL of the solution was injected onto an analytical column. An Acquity UPLC system coupled with an API 4000 mass spectrometer was used for sample analysis. The mobile phases were 0.025% FA and 1 mM NH4OAc in water/ACN (v:v, 95:5) (A) and 0.025% FA and 1 mM NH4OAc in ACN/water (v:v, 95:5) (B). The gradient was as follows: starting at 10% B and increased to 65% B for 1.2 min, then to 95% for 0.6 min, maintained at 95% B for 0.2 min, then decreased to 10% B within 0.01 min. The total flow rate was 0.6 mL/min, and samples were injected onto an ACE 3 AQ (2.1 mm × 100 mm, 3 μm) analytical column with a total run time of 2 min. Data were acquired using multiple reactions monitoring (MRM) in positive ion electrospray mode with an operating source temperature of 550 °C. The MRM transition was m/z 511.400 → 471.400 for 10, 526.400 → 486.400 for 19, and 357.900 → 139.000 for indomethacin. The lower and upper limits of quantitation of the assay for 10 were 0.002 and 13.1 μM, respectively. The lower and upper limits of quantitation of the assay for 19 were 0.002 and 12.7 μM, respectively.

In Vivo Evaluation of 19 in MOLM-16 AML PK/PD and Antitumor Efficacy Model[1]
Female C.B-17 SCID.bg mice that were 8–9 weeks old and weighed 20–24 g were used. They were inoculated with five million MOLM-16 leukemia acute myelogenic cells (suspended in a 1:1 mixture of Hank’s Balanced Salt Solution containing Matrigel at a 1:1 ratio) in the right flank subcutaneously. Tumors were monitored until they reached a mean tumor volume of 130–300 mm3. The mean tumor volume across all eight groups was 260 ± 34.5 mm3 (mean ± SD) at the initiation of dosing. Mice were given 0 (vehicle–0.5% methylcellulose, 0.2% Tween-80), 3, 10, and 30 mg/kg of compound 19 by gavage, twice daily (BID) for 21 days in a volume of 100 μL. Tumor volumes were measured in two dimensions (length and width) using Ultra Cal-IV calipers (model 54-10-111; Fred V. Fowler Co., Newton, MA) and analyzed using Excel, version 11.2. The tumor volume was calculated with the following formula: tumor size (mm3) = (longer measurement × shorter measurement2) × 0.5. Animal body weights were measured using an Adventura Pro AV812 scale. Percent weight change was calculated using the following formula: group percent weight change = (new weight – initial weight)/initial weight) × 100. Plasma, tumor, and brain samples were collected at 2 h postdose. To analyze the repeated measurement of tumor volumes from the same animals over time, a mixed-modeling approach was used. This approach addresses both repeated measurements and modest dropouts due to any nontreatment related removal of animals before the end of study. Cubic regression splines were used to fit a nonlinear profile to the time courses of log 2 tumor volume at each dose level. The nonlinear profiles were then related to dose within the mixed model. Tumor growth inhibition as a percentage of vehicle was calculated as the percentage of the area under the fitted tumor volume–time curve (AUC) per day for each dose group in relation to the vehicle, using the following formula: %TGI = 100 × [1 – (AUCdose per day/AUCvehicle per day)].

Concentrations of 19 were determined by a nonvalidated LC-MS/MS assay. The plasma samples were prepared for analysis by placing a 25 μL aliquot into a 96-well plate. The tumor samples were collected and weighed. Four volume of water by tissue weight was added. Using a bead beating homogenizer, the tissue samples were homogenized and 25 μL of each was aliquoted into a 96-well plate. A volume of 200 μL of acetonitrile containing an internal standard (labetalol) was added to the sample. The samples were vortexed and centrifuged at 4000 rpm for 10 min, and 50 μL of the supernatant was diluted with 150 μL of water. A 10 μL injection volume was used for analysis on a SIL-30ACMP autosampler system was linked to LC-30AD pumps, coupled with an API 5500 QTrap mass spectrometer, was used for sample analysis. The mobile phases were 0.1% FA (formic acid) in water (A) and 0.1% FA in MeCN (B). The gradient was as following: starting at 10% B and increased to 90% B in 0.6 min, maintained at 90% B for 0.2 min, then decreased to 10% B within 0.1 min. The total flow rate was 1.2 mL/min and column for separation was Kinetex XB-C18 column (50 mm × 2.1 mm, 2.6 μm) with a total run time of 1 min. Data were acquired using multiple reactions monitoring (MRM) in positive ion electrospray mode with an operating source temperature of 550 °C. The MRM transition was m/z 526.1 → 486.2 for 19 and 329.076 → 294.1 for labetalol. The lower and upper limits of quantitation of the assay for 19 were 0.002 and 39 μM, respectively.

配方 3 中的溶解度: ≥ 1.67 mg/mL (3.18 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 16.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。