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.]

Absorption: After oral administration, Ceralasertib is rapidly absorbed, with a median time to peak plasma concentration (tmax) of approximately 1 hour. Bioavailability: Preclinical studies in mice have shown that the oral bioavailability of Ceralasertib is dose-dependent. With increasing dose, the increase in drug exposure is greater than the dose-proportional increase, resulting in a roughly two-fold increase in bioavailability between the lowest and highest doses studied. This non-linear behavior is attributed to saturable first-pass metabolism. Distribution: In mice, Ceralasertib is rapidly and extensively distributed in most tissues, except the brain and spinal cord. Metabolism: Ceralasertib undergoes significant first-pass metabolism. Metabolic analyses show that the metabolic rate decreases with increasing dose, further supporting the hypothesis of first-pass metabolic saturation, which involves intestinal and mesenteric metabolism. An ongoing clinical study aims to identify and quantify its major metabolites in plasma and excrement. Elimination: The terminal plasma half-life (t1/2) in patients is approximately 8 to 11 hours. A clinical ADME study is investigating the primary route of excretion, which will measure the recovery of the radiolabeled dose in urine and feces. Linearity: Similar to bioavailability, Ceralasertib exhibits nonlinear pharmacokinetic characteristics; the increase in exposure observed in population pharmacokinetic models is not dose-proportional across the dose range of 20 to 320 mg. This behavior is described using a relative bioavailability parameter that decreases linearly with increasing dose.

Hematologic Toxicity (Clinical): The primary dose-limiting toxicity (DLT) of ceralasertib is thrombocytopenia (low platelet count). In a Phase I study of ceralasertib in combination with carboplatin, the most common ≥ Grade 3 adverse events were anemia (39%), thrombocytopenia (36%), and neutropenia (25%). Population pharmacokinetic-safety models showed that platelet and neutrophil recovery requires a two-week withdrawal period. A shorter withdrawal period may lead to incomplete recovery and increase the probability of serious (≥ Grade 3) adverse events in subsequent treatment cycles. With monotherapy, a dose of 160 mg or 240 mg twice daily for 14 days is expected to result in a lower probability of serious hematologic toxicity (<20%). The PATRIOT Phase I study confirmed that intermittent dosing regimens (e.g., 14 days in a 28-day cycle) are better tolerated than continuous dosing regimens due to their lower hematologic toxicity.
Cardiotoxicity (Preclinical): A comparative in vivo toxicology study in mice revealed that Ceralasertib possesses a unique toxicity profile. While no other ATR inhibitors (ATRi) exacerbated the toxicity of total body irradiation (TBI), cardiotoxicity was observed following a single injection of Ceralasertib, whereas it was not observed with other tested ATRis (elimusertib or berzosertib). This effect may be related to the high free plasma drug concentration of Ceralasertib.
Other Toxicity (Preclinical): In the same study, all three tested ATRis, including Ceralasertib, induced neutrophilia in mice within 48 hours of administration.

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

Background: Defects in DNA damage response (DDR), particularly TP53 and biallelic ataxia-telangiectasia mutations (ATM), are associated with genomic instability, clonal evolution, and chemotherapy resistance in chronic lymphocytic leukemia (CLL). Currently, there is a lack of therapies capable of long-term control of DDR-deficient CLL patients. We investigated ATR pathway inhibition as a synthetic lethal strategy targeting CLL cells with these defects using a novel ATR inhibitor, AZD6738. Methods: The role of AZD6738 was evaluated by detecting key DDR proteins using Western blotting and immunofluorescence. Cytotoxicity was assessed using the CellTiter-Gloluminescence assay (Promega, Madison, Wisconsin, USA) and the propidium iodide exclusion assay. Primary CLL cells inactivated by biallelic TP53 or ATM were xenografted into NOD/Shi-scid/IL-2Rγ mice. After treatment with AZD6738 or its vector, the tumor burden infiltrating the spleen was analyzed by flow cytometry, and subclonal composition was analyzed by fluorescence in situ hybridization to detect 17p (TP53) or 11q (ATM) deletions. Results: Under genotoxic stress, AZD6738 potently and specifically inhibited the ATR signaling pathway and compensatorily activated the ATM/p53 pathway in proliferating CLL cells. In p53 or ATM-deficient cells, AZD6738 treatment led to replication fork arrest and the accumulation of unrepaired DNA damage, manifested as the formation of γH2AX and 53BP1 clusters. These damages entered mitosis, ultimately leading to cell death due to mitotic catastrophe. AZD6738 exhibited selective cytotoxicity against ATM or p53-deficient CLL cells and showed a high synergistic effect when used in combination with cytotoxic chemotherapeutic agents. This finding has been confirmed in a primary xenograft model of DDR-deficient CLL, where AZD6738 treatment reduced tumor burden and selectively reduced CLL subclones with ATM or TP53 alterations. Conclusion: We provide mechanistic insights and confirm the efficacy of a novel treatment approach in vitro and in vivo that specifically targets CLL cells lacking p53 or ATM. This approach holds promise for helping to avoid clonal evolution, which is a major cause of treatment resistance and disease relapse. [3] Understanding the therapeutic effects of drug dosage and administration regimens is crucial for guiding the design and implementation of clinical trials. The increasing complexity of monotherapy, especially combination therapy, poses a significant challenge to the clinical phase of oncology drug development. We used a systems pharmacology approach to construct a cell cycle mechanism model based on existing tumor growth PK-PD models, which was able to simulate monotherapy and combination therapy with the ATR inhibitor AZD6738 and ionizing radiation. Using AZD6738, we developed a multi-parameter cell detection method to measure DNA damage and cell cycle transition and provide quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model can predict tumor growth observed in in vivo mouse xenograft studies. This model is being used in the design of a Phase I clinical trial of AZD6738 to improve patient treatment by quantitatively predicting dosage and administration regimens. [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.)