Ceralasertib（AZD6738）

Biological Activity

AZD6738 is an orally active, selective ATR kinase inhibitor with an IC50 of 1 nM. AZD6738 inhibits ATR kinase activity and impels cell viability. AZD6738 induces rapid cell death in tumor cells with an ATM gene defect.

In vitro studies showed that AZD6738 inhibited ATR kinase activity and impaired cell activity in four Kras mutant cell lines H23, H460, A549 and H358. In ATM deficient H23 cells, AZD6738 strongly enhanced the effect of cisplatin on rapid cell death. In cells with p53 or ATM deletion, AZD6738 treatment causes replication fork stagnation and accumulation of unrepaired DNA damage, leading to mitosis disorder and thus cell death.

In vivo studies, AZD6738 (50 mg/kg, P.O.) induced tumor growth inhibition (TGI) in nude mice burdened with H460 and H23 tumors, and combined with cisplatin induced rapid degeneration of ATM-deficient H23 tumors. The combination of AZD6738 (50 mg/kg)+IR (2 Gy) in nude mice loaded with LoVo xenografts avoided toxicity while maintaining efficacy.

Customer Product Validations & Biological Datas

	Source	Oncotarget (2015 Dec). Figure 5. AZD6738
	Method	Western blot
	Cell Lines	H460 and A549 cells
	Concentrations	0.3 or 1.0 μM
	Incubation Time	48 hour
	Results	In H460 shATM cells, 1.0 μM AZD6738 shifted cisplatin IC50 from 2.77 μM to 0.22 μM (12.59-fold), compared to 2.36 μM to 0.43 μM (5.44-fold) and 1.83 uM to 0.84 (2.18-fold) in wildtype and scrambled control cells, respectively. Similar results were seen in A549 shATM cells. Treatment with 1.0 μM AZD6738 reduced cisplatin IC50 from 19.84 μM to 1.02 μM (19.45-fold) versus reductions from 7.79 μM to 2.61 μM (2.98-fold) and 9.68 μM to 3.48 μM (2.78- fold) in wildtype and scrambled control cells, respectively. Treatment with 0.3 μM AZD6738 also resulted in greater shifts in cisplatin IC50 and lower net IC50 values in shATM cell lines compared to wildtype and scrambled control lines.

	Source	Sci Rep (2015 Aug). Figure 6. AZD6738
	Method	multi-parametric cell based assays
	Cell Lines	LoVo cells
	Concentrations	80 mg/kg
	Incubation Time	3 weeks
	Results	From tumor volume simulations an efficacious starting dose of 80 mg/kg AZD6738 was identified, providing clinicians with an indication of the expected rate of tumor regression and recovery.

Protocol

Cell Experiment
Cell lines	H23, H460, A549, and H358 cells
Preparation method	Cells are treated in white walled, clear bottom 96-well plates with the indicated doses of AZD6738, cisplatin, gemcitabine, or combination for 48 h. ATP levels are assessed as surrogate measure of viability is assessed using the CellTiter-Glo Luminescent Cell Viability Assay and Safire2 plate reader. Raw data are corrected for background luminescence prior to further analysis. For AZD6738 treatment, log dose response curves are generated in GraphPad Prism 6 by nonlinear regression (log(inhibitor) vs. response with variable slope) of log-transformed (x = log(x)) data normalized to the mean of untreated controls. GI50 values, defined as the dose X at which Y = 50%, were extrapolated from dose response curves.
Concentrations	~30 μM
Incubation time	48 h

Animal Experiment
Animal models	Female athymic nude mice bearing H23 or H460 xenografts
Formulation	10% DMSO, 40% propylene glycol, and 50% sterile dH2O
Dosages	25 or 50 mg/kg
Administration	p.o.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

[1] Kim HJ, et al. Int J Cancer. Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

[2] Vendetti FP, et al. Oncotarget. The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo.

[3] Kwok M, et al. Lancet. Synthetic lethality in chronic lymphocytic leukaemia with DNA damage response defects by targeting the ATR pathway.

[4] Checkley S, et al. Sci Rep. Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738.