Zotiraciclib

SB1317/TG02 inhibits the proliferating of all the cell lines tested including solid tumor cell lines such as colon (HCT-116, COLO205) and prostate (DU145) with IC50 of 33 nM, 72 nM and 140 nM, respectively. SB1317/TG02 potently inhibits the CDK2 biomarker pRb (phospho-Rb, retinoblastoma tumor suppressor protein) in HCT-116, and effects can be detected at the 40 nM with the protein phosphorylation being completely inhibited at 200 nM. SB1317/TG02 is potent against pRb in MV4-11 cells with IC50 of 0.13 μM and also inhibits pFLT3 and pSTAT5 in the same cell line. SB1317/TG02 results in the permeability (Papp) of 26h in the apical to basolateral (Papp,A→B) direction and in the basolateral to apical (Papp,B→A) direction of 28.0 × 10^-6 cm/s and 27.4 ×10^-6 cm/s, respectively, in the Caco-2 bidirectional permeability assays. SB1317/TG02 is found to be stable with a half-life of 45 min in human liver microsomes (HLM), is moderately stable in DLM (t_1/2 = 33 min), and is quite rapidly cleared in MLM (t_1/22 = 12 min) and in RLM (t_1/2 = 11 min). ^[1] TG02 most potently inhibits CDK isoforms, inhibits CDK1, CDK2, CDK3, CDK5 and CDK9 with IC50 of 9 nM, 5 nM, 8 nM, 4 nM and 3 nM, respectively. TG02 also inhibits Lck, TYK2, Fyn, JAK2 and FLT3 with IC50 of 11 nM, 14 nM, 15 nM, 19 nM and 19 nM, respectively. TG02 has more potent anti-proliferative effects than SNS-032 in tumor cell lines. TG02 shows a stronger inhibition of the liquid tumor panel with IC50 of 0.13 μM compared with the solid tumor panel with IC50 of 0.30 μM. TG02 (100 nM) induces cell cycle arrest and apoptosis in MV4-11 cells. TG02 inhibits pRb, pFLT3 and pSTAT5 with IC50 of 125 nM, 4.7 μM and 560 nM, respectively, in MV4-11 cells. TG02 inhibits pJAK2 (Y1007/8) and pSTAT3 with IC50 of 63 nM and 53 nM, respectively, in Karpas 1106P. TG02 (300 nM) exposure leads to CDK9 inhibition, followed by G1 phase arrest and apoptotic induction, in HL-60 cells. ^[2]

SB1317/TG02 is highly bound to plasma proteins in human, mouse, and dog plasma with PPB ranging between 99.4% to 99.9%. SB1317/TG02 shows oral bioavailability (F) of 4% and 37% in rats and dogs, respectively. SB1317/TG02 (75 mg/kg, orally) shows rapid absorption (t_max = 0.5 h) and a mean Cmax and AUC of 1029 ng/mL and 2523 ng•h/mL, respectively, with a mean terminal half-life of 6.1 hours, and oral bioavailability of 24% in mice. SB1317/TG02 (75 mg/kg po q.d. 3×/week) significantly inhibits the growth of tumors with a mean TGI of 82% in a murine sc xenograft model of human colon cancer (HCT-116), while the lower dose of 50 mg/kg po 3×/week is marginally effective. SB1317/TG02 (75 mg/kg po, 15 mg/kg ip) significantly inhibits the growth of tumors with mean TGIs of 42% and 63% for the oral and ip delivery methods, respectively, in a murine sc xenograft model of human B-cell lymphoma (Ramos). ^[1] TG02 (60 mg/kg) is selectively retained at supra-therapeutic levels and effectively inhibits CDK2, CDK9 and FLT3 in vivo, causing apoptosis in the tumor tissues, in tumor tissues in MV4-11 tumor-bearing nude mice. TG02 (10 mg/kg, 20 mg/kg and 40 mg/kg) leads to a tumor growth inhibition of 53%, 61% and 113%, respectively, in MV4-11 tumor-bearing nude mice. ^[2]

• Enzyme Assays

  All assays are carried out in 384-well white microtiter plates using the PKLight assay system. TG02 are tested at eight concentrations prepared from 3- or 4-fold serial dilution starting at 10 μM. For CDK2/cyclin A assay, the reaction mixture consist of the following components in 25 μL of assay buffer (50 mM Hepes, pH 7.5, 10 mM MgCl₂, 5 mM MnCl₂, 5 mM BGP, 1 mM DTT, 0.1 mM sodium orthovanadate), 1.4 μg/mL of CDK2/cyclin A complex, 0.5 μM RbING substrate, and 0.5 μM ATP. The mixture is incubated at room temperature for 2 hours. Then 13 μL of PKLight ATP detection reagent is added and the mixture is incubated for 10 min. Luminescence signals are detected on a multilabel plate reader. The other kinase assays are similar, with the following differences in reagents: For FLT3 assays, the mixture contains 2.0 μg/mL FLT3 enzyme, 5 μM poly(Glu,Tyr) substrate, and 4 μM ATP. For JAK2 assays, the reaction contains 0.35 μg/mL JAK2 enzyme, 10 μM poly(Glu,Ala,Tyr) substrate, and 0.15 μM ATP. The analytical software Prism 5.0 is used to generate IC50 values from the data.

• Cell lines

  HCT-116, COLO205 and DU145 cell lines

• Concentrations

  ~10 μM

• Incubation Time

  48 hours

• Method

  For proliferation assays in 96-well plates, 2×10⁵ cells are seeded in 100 μL of medium and treated the following day with TG02 (in triplicate) at concentrations up to 10 μM for 48 hours. Cell viability is monitored using the CellTiter-96 Aqueous One solution cell proliferation assay. Dose-response curves are plotted to determine IC50 values for TG02 using the XL-fit software.

• Animal Models

  Murine sc xenograft model of human colon cancer (HCT-116)

• Dosages

  75 mg/kg

• Administration

  orally