ATR; PI3Kδ

AZD6738 provided potent and specific inhibition of ATR signalling with compensatory activation of ATM/p53 pathway in cycling CLL cells in the presence of genotoxic stress. In p53 or ATM defective cells, AZD6738 treatment resulted in replication fork stalls and accumulation of unrepaired DNA damage, as evidenced by γH2AX and 53BP1 foci formation, which was carried through into mitosis, resulting in cell death by mitotic catastrophe. AZD6738 displayed selective cytotoxicity towards ATM or p53 deficient CLL cells, and was highly synergistic in combination with cytotoxic chemotherapy. This finding was confirmed in primary xenograft models of DDR-defective CLL, where treatment with AZD6738 resulted in decreased tumour load and selective reduction of CLL subclones with ATM or TP53 alterations[1].

Understanding the therapeutic effect of drug dose and scheduling is critical to inform the design and implementation of clinical trials. The increasing complexity of both mono, and particularly combination therapies presents a substantial challenge in the clinical stages of drug development for oncology. Using a systems pharmacology approach, we have extended an existing PK-PD model of tumor growth with a mechanistic model of the cell cycle, enabling simulation of mono and combination treatment with the ATR inhibitor AZD6738 and ionizing radiation. Using AZD6738, we have developed multi-parametric cell based assays measuring DNA damage and cell cycle transition, providing quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model is predictive of tumor growth observed in in vivo mouse xenograft studies. The model is being used for phase I clinical trial designs for AZD6738, with the aim of improving patient care through quantitative dose and scheduling prediction[2].

DNA damage response (DDR) defects, particularly TP53 and biallelic ataxia telangiectasia mutated (ATM) aberrations, are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukaemia (CLL). Therapies capable of providing long-term disease control in CLL patients with DDR defects are lacking. Using AZD6738, a novel ATR inhibitor, we investigated ATR pathway inhibition as a synthetically lethal strategy for targeting CLL cells with these defects[1].

The effect of AZD6738 was assessed by western blotting and immunofluorescence of key DDR proteins. Cytotoxicity was assessed by CellTiter-Gloluminescence assay (Promega, Madison, WI, USA) and by propidium iodide exclusion. Primary CLL cells with biallelic TP53 or ATM inactivation were xenotransplanted into NOD/Shi-scid/IL-2Rγ mice. After treatment with AZD6738 or vehicle, tumour load was measured by flow cytometric analysis of infiltrated spleens, and subclonal composition by fluorescence in-situ hybridisation for 17p(TP53) or 11q(ATM) deletion[1].

Female athymic nude (Foxn1nu) mice, 6–7 weeks old, were purchased from Harlan Laboratories. H23 (3 × 106 cells) or H460 (7 × 105 cells) were injected subcutaneously into the right hind flank in a volume of 100 μL (equal parts 1x PBS and Matrigel). Cells were tested for mycoplasma prior to inoculation in mice. Mice began receiving treatment once tumors reached approximately 220 mm3 (± 15%) for H23 or 180 mm3 (± 15%) for H460. Tumor volume was calculated as (L × W2)/2. AZD6738 was administered by oral gavage (qd × 14) at 25 mg/kg (H23) or 50 mg/kg (H460). Cisplatin was administered intraperitoneally (q7d × 2) at 3 mg/kg. The dosing volume was 10 mL/kg. Growth curves depict mean (± SEM) tumor volume over time. Mean tumor growth inhibition was calculated as TGI = (1–(Tf–T0)/(Cf–C0))*100, where Tf and T0 represent final and initial mean tumor volumes in the treatment arm, respectively, and Cf and C0 represent final and initial mean tumor volumes in the vehicle control arm, respectively. Mean tumor regression was calculated as % Regression = ((T0–Tf)/T0)*100. For H460 xenografts, the experimental endpoint was defined as the day on which any single tumor within the treatment arm reached 2000 mm3. Tumor growth delay is defined as the difference in the number of days to reach the endpoint for a given treatment arm compared to vehicle control.[Oncotarget. 2015 Dec 29;6(42):44289-305.]

[1]. Lancet.2015 Feb 26;385 Suppl 1:S58
[2]. Sci Rep.2015 Aug 27;5:13545.

Background: DNA damage response (DDR) defects, particularly TP53 and biallelic ataxia telangiectasia mutated (ATM) aberrations, are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukaemia (CLL). Therapies capable of providing long-term disease control in CLL patients with DDR defects are lacking. Using AZD6738, a novel ATR inhibitor, we investigated ATR pathway inhibition as a synthetically lethal strategy for targeting CLL cells with these defects. Methods: The effect of AZD6738 was assessed by western blotting and immunofluorescence of key DDR proteins. Cytotoxicity was assessed by CellTiter-Gloluminescence assay (Promega, Madison, WI, USA) and by propidium iodide exclusion. Primary CLL cells with biallelic TP53 or ATM inactivation were xenotransplanted into NOD/Shi-scid/IL-2Rγ mice. After treatment with AZD6738 or vehicle, tumour load was measured by flow cytometric analysis of infiltrated spleens, and subclonal composition by fluorescence in-situ hybridisation for 17p(TP53) or 11q(ATM) deletion. Findings: AZD6738 provided potent and specific inhibition of ATR signalling with compensatory activation of ATM/p53 pathway in cycling CLL cells in the presence of genotoxic stress. In p53 or ATM defective cells, AZD6738 treatment resulted in replication fork stalls and accumulation of unrepaired DNA damage, as evidenced by γH2AX and 53BP1 foci formation, which was carried through into mitosis, resulting in cell death by mitotic catastrophe. AZD6738 displayed selective cytotoxicity towards ATM or p53 deficient CLL cells, and was highly synergistic in combination with cytotoxic chemotherapy. This finding was confirmed in primary xenograft models of DDR-defective CLL, where treatment with AZD6738 resulted in decreased tumour load and selective reduction of CLL subclones with ATM or TP53 alterations. Interpretation: We have provided mechanistic insight and demonstrated in-vitro and in-vivo efficacy of a novel therapeutic approach that specifically targets p53-null or ATM-null CLL cells. Such an approach can potentially help to avert clonal evolution, a major cause of therapeutic resistance and disease relapse.[3]

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Understanding the therapeutic effect of drug dose and scheduling is critical to inform the design and implementation of clinical trials. The increasing complexity of both mono, and particularly combination therapies presents a substantial challenge in the clinical stages of drug development for oncology. Using a systems pharmacology approach, we have extended an existing PK-PD model of tumor growth with a mechanistic model of the cell cycle, enabling simulation of mono and combination treatment with the ATR inhibitor AZD6738 and ionizing radiation. Using AZD6738, we have developed multi-parametric cell based assays measuring DNA damage and cell cycle transition, providing quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model is predictive of tumor growth observed in in vivo mouse xenograft studies. The model is being used for phase I clinical trial designs for AZD6738, with the aim of improving patient care through quantitative dose and scheduling prediction.[4]

C20H24N6O2S

412.5086

412.17

C, 58.23; H, 5.86; N, 20.37; O, 7.76; S, 7.77

1352226-87-9

Ceralasertib;1352226-88-0; 1352280-98-8 (formate); 1352226-87-9 (S-isomer); 1352226-97-1 (racemic)

54761305

Typically exists as light yellow to yellow solids at room temperature

2.6

2

7

4

29

724

2

C[C@@H]1COCCN1C2=NC(=NC(=C2)C3(CC3)[S@@](=N)(=O)C)C4=C5C=CNC5=NC=C4

OHUHVTCQTUDPIJ-MUWSIPGASA-N

InChI=1S/C20H24N6O2S/c1-13-12-28-10-9-26(13)17-11-16(20(5-6-20)29(2,21)27)24-19(25-17)15-4-8-23-18-14(15)3-7-22-18/h3-4,7-8,11,13,21H,5-6,9-10,12H2,1-2H3,(H,22,23)/t13-,29+/m1/s1

imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-lambda6-sulfane

(S)-Ceralasertib; 1352226-87-9; (S)-AZD6738; imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-lambda6-sulfane; BDBM60432; AZD6738; AZD 6738; SCHEMBL9979159;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~242.42 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.06 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)