Pacritinib

Kinase experiment:

All assays are carried out in 384-well white microtiter plates. Compounds (e.g., Pacritinib) are 4-fold serially diluted in 8 steps, starting from 10 µM. The reaction mixture consist of 25 µL assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 5 mM β-glycerol phosphate). For FLT3 assays, the reaction contain 2.0 µg/mL FLT3 enzyme, 5 µM of poly(Glu,Tyr) substrate and 4 µM of ATP. For JAK1 assays, the reaction contain 2.5 µg/mL of JAK1 enzyme, 10 µM of poly(Glu,Ala,Tyr) substrate and 1.0 µM of ATP. For JAK2 assays, the reaction contain 0.35 µg/mL of JAK2 enzyme, 10 µM of poly (Glu,Ala,Tyr) substrate and 0.15 µM of ATP. For JAK3 assays, the reaction contain 3.5 µg/mL of JAK3 enzyme, 10 µM of poly (Glu,Ala,Tyr) substrate and 6.0 µM of ATP. For TYK2 assays, the reaction contain 2.5 µg/mL of TYK2 enzyme, 10 µM of poly (Glu,Ala,Tyr) substrate and 0.15 µM of ATP. The reaction is incubated at room temperature for 2 h prior to addition of 13 µL PKLight detection reagent. After 10 min incubation luminescent signals are read on a multi-label plate reader[1].

Cell experiment:

SET-2 and Karpas 1106P cells, and Ba/F3-JAK2V617F-GFP-Luc cells are used. For proliferation assays in 96-well plates, cells are seeded at 30-50% confluency and are treated the following day with compounds (e.g., Pacritinib) (in triplicate) at concentrations up to 10 μM for 48 h. Cell viability is monitored using the CellTiter-Glo assay. Dose-response curves are plotted to determine IC50 values for the compounds using the XL-fit software[1].

Animal experiment:

Mice[1] Female athymic BALB/c nude mice (BALB/cOlaHsd-Foxn1nu) of age 12 weeks are used; and female SCID Beige mice (CB17.Cg-PrkdcscidLystbg/Crl) of age 9-10 weeks are used. For the SET-2 leukemia model, 5×106 tumor cells are injected subcutaneously in the right flank of severe combined immunodeficient beige mice. The cells are resuspended in 50 μL serum-free growth medium, mixed 1:1 with Matrigel and injected in a total volume of 100 μL. Tumor volumes are determined by caliper measurements and drug treatment is initiated after 31 days when tumors have reached a mean volume of 280 mm3 (tumor volume (mm3)=(w2×l)/2). This study is performed using 12 mice per group and animals are killed 3 h post-dose on day 18. Tumor growth inhibition is calculated. For the efficacy studies, mice are treated by oral gavage (10 mL/kg body weight) with doses from 50 to 150 mg/kg SB1518. Rats and Dogs[3] Male Wistar rats (aged 6-8 weeks, weighing 270 to 325 g) and male Beagle dogs (6 to 7 months of age, weighing 9-11 kg) are used in this study. The oral doses for dogs and rats are 3, and 10 mg/kg, respectively. The doses are administered, by gavage, as suspensions (0.5 % methylcellulose and 0.1%tween 80) to rats, and as gelatin capsules to dogs. Following oral dosing, serial blood samples are collected (jugular vein in dogs, and superior vena cava in rats) at different time points (0 to 24 h) in tubes containing K3EDTA as anticoagulant, and centrifuged, the plasma is separated and stored at -70°C until analysis. Plasma samples are processed and analyzed by LC/MS/MS. Pharmacokinetic parameters are estimated by noncompartmental methods using WinNonlin.

References:

[1]. Hart S, et al. SB1518, a novel macrocyclic pyrimidine-based JAK2 inhibitor for the treatment of myeloid and lymphoid malignancies. Leukemia. 2011 Nov;25(11):1751-9.
[2]. Hart S, et al. Pacritinib (SB1518), a JAK2/FLT3 inhibitor for the treatment of acute myeloid leukemia. Blood Cancer J. 2011 Nov;1(11):e44.
[3]. William AD, et al. Discovery of the macrocycle 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (SB1518), a potent Janus kinase 2/fms-like tyrosine kinase-3 (JA