ATR ( IC50 = 1 nM ); PI3Kδ ( IC50 = 6.8 μM ); DYRK ( IC50 = 10.8 μM )

Ceralasertib (AZD6738) is a potent inhibitor of ATR kinase activity, with an IC50 of 0.001 μM against the isolated enzyme and 0.074 μM against the phosphorylation of CHK1 in cells that is dependent on ATR kinase. In non-small cell lung cancer (NSCLC) cell lines, celeralasertib (AZD6738) causes senescence and cell death. Four Kras mutant cell lines are less viable when ceralasertib (AZD6738) is used; H23, H460, A549, and H358 have the lowest GI50 and the largest maximal inhibition (1.05 μM, 88.0% and 2.38 μM, 86.2%, respectively). In NSCLC cell lines with intact ATM kinase signaling, ceralasertib (AZD6738) amplifies the cytotoxicity of CDDP and NSC 613327, and in ATM-deficient NSCLC cells, it potently synergizes with CDDP[1]. With IC50 values less than 1 μM, ceralasertib (AZD6738) inhibits human breast cancer cell lines using the MTT assay. Ceralasertib (AZD6738) causes apoptosis and cell cycle arrest. It suppresses cell proliferative signaling molecules and DNA damage response molecules[2].

Ceralasertib (AZD6738) and ATR kinase inhibition given daily for 14 days in a row improves CDDP's therapeutic efficacy in xenograft models and is well tolerated by mice. It's amazing how well CDDP and Ceralasertib (AZD6738) work together to treat ATM-deficient lung cancer xenografts[1].

AZD6738 is a potent inhibitor of ATR kinase activity, with an IC50 of 0.001 μM against the isolated enzyme and 0.074 μM against the phosphorylation of CHK1 in cells that is dependent on ATR kinase. ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic.

Ceralasertib (AZD6738) is diluted in DMSO to the appropriate working concentrations after being dissolved at a 30 mM concentration. For Ceralasertib (AZD6738) dose response experiments, the final DMSO concentration in media for all conditions and controls is 0.1%; for Ceralasertib (AZD6738) + chemotherapy viability experiments, it is 0.05%; and for all experiments involving 0.3 μM and 1.0 μM doses of Ceralasertib (AZD6738), it is 0.025%[1].

Mice: Ceralasertib (AZD6738) is diluted 1:5 in propylene glycol after being dissolved in DMSO at a concentration of 25 mg/mL or 50 mg/mL. Ceralasertib (AZD6738) is given orally as a gavage for 14 days at a dose of 25 mg/kg (H23) or 50 mg/kg (H460). 10 mL/kg is the dosage volume.[1].

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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.[1]

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]. The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of CDDP to resolve ATM-deficient non-small cell lung cancer in vivo.Oncotarget. 2015 Dec 29;6(42):44289-305.

[2]. Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells. Int J Cancer. 2017 Jan 1;140(1):109-119.

[3]. Synthetic lethality in chronic lymphocytic leukaemia with DNA damage response defects by targeting the ATR pathway. Lancet.2015 Feb 26;385 Suppl 1:S58.
[4]. Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738. Sci Rep.2015 Aug 27;5:13545.

ATR and ATM are DNA damage signaling kinases that phosphorylate thousands of substrates. ATR kinase activity is enhanced at damaged replication forks and excised DNA double-strand breaks (DSBs). ATM kinase activity is enhanced at DSBs. ATM has been extensively studied because patients with ataxia-telangiectasia (ATM) (who do not express ATM proteins) are the most radiosensitive known patient group. Since ATM is not an essential protein, ATM kinase inhibitors are generally considered to be well-tolerated clinically. ATR has also been extensively studied, but progress on ATR research has been more complex due to the discovery that ATR is an essential protein and the general perception that ATR kinase inhibitors are clinically toxic. We describe an orally active and bioavailable ATR kinase inhibitor, AZD6738. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 enhanced the cytotoxicity of cisplatin and gemcitabine in non-small cell lung cancer (NSCLC) cell lines with intact ATM kinase signaling pathways and produced a significant synergistic effect with cisplatin in ATM-deficient NSCLC cells. Contrary to expectations, mice tolerated AZD6738 and the ATR kinase inhibitor well after 14 consecutive days of daily administration and enhanced the therapeutic effect of cisplatin in xenograft models. Notably, the combination of cisplatin and AZD6738 cured ATM-deficient lung cancer xenografts. [1]

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Ataxia-telangiectasia and Rad3-associated protein (ATR) are sensors of DNA damage that can induce homologous recombination (HR)-dependent repair. ATR is a major regulator of DNA damage repair (DDR) and controls DNA replication, DNA repair and apoptosis through signal transduction. Therefore, the ATR pathway may be a very attractive target for new drug development. This study aimed to investigate the antitumor effect and potential mechanism of the ATR inhibitor AZD6738 in human breast cancer cells. The growth-inhibiting effect of AZD6738 on human breast cancer cell lines was detected using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide). In addition, cell cycle analysis, Western blotting, immunofluorescence, and comet assays were performed to elucidate the mechanism of action of AZD6738. The results showed that AZD6738 had anti-proliferative and anti-DNA damage response (DDR) inhibitory effects in human breast cancer cell lines. Among 13 cell lines, the IC50 values of 9 cell lines measured by the MTT assay were all less than 1 μmol/L. We selected two cell lines, SK-BR-3 and BT-474, for further evaluation, focusing on human epidermal growth factor receptor 2 (HER2) positive breast cancer cells. Drug-sensitive SK-BR-3 breast cancer cells (rather than less sensitive BT-474 breast cancer cells) showed increased apoptosis and S-phase arrest, as well as decreased expression levels of phosphorylated checkpoint kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression led to homologous recombination (HR) inactivation, which in turn induced DNA damage accumulation. AZD6738 had a synergistic effect with cisplatin. Understanding the antitumor activity and mechanism of AZD6738 in HER2-positive breast cancer cells lays the foundation for future clinical trials targeting DNA damage response (DDR) in the treatment of HER2-positive breast cancer. [2]

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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 that can control the disease in CLL patients with DDR deficiency in the long term. We investigated ATR pathway inhibition as a synthetic lethal strategy for targeted therapy of CLL cells with these defects using a novel ATR inhibitor, AZD6738. Methods: The effect of AZD6738 was assessed by Western blotting and immunofluorescence analysis of key DDR proteins. Cytotoxicity was assessed using CellTiter-Gloluminescence assay (Promega, Madison, Wisconsin, USA) and propidium iodide exclusion assay. Primary CLL cells with biallelic TP53 or ATM inactivation were xenografted into NOD/Shi-scid/IL-2Rγ mice. Tumor burden in infiltrating spleens was analyzed by flow cytometry after treatment with AZD6738 or the vector, and subclonal composition was analyzed by fluorescence in situ hybridization to detect 17p (TP53) or 11q (ATM) deletion. Results: Under genotoxic stress, AZD6738 effectively 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. This damage persisted into 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 was 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 elucidated the mechanism of a novel therapeutic approach and demonstrated its efficacy in vitro and in vivo, specifically targeting p53-deficient or ATM-deficient CLL cells. This approach may help avoid clonal evolution, which is one of the main causes of treatment resistance and disease relapse. [3] Understanding the therapeutic effects of drug dosage and dosing 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 have expanded existing tumor growth PK-PD models using a systems pharmacology approach and incorporated a cell cycle mechanism model 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 transitions, providing quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model was able to 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 dosing regimens. [4]

458.1736245

C, 55.01; H, 5.72; N, 18.33; O, 13.96; S, 6.99

1352280-98-8

1352226-88-0;1352280-98-8 (formate);1352226-97-1 (racemic);

154701782

Typically exists as solid at room temperature

3

9

4

32

734

2

JOKLXYXIZOXQHY-FQAMYIAXSA-N

InChI=1S/C20H24N6O2S.CH2O2/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)3-7-22-182-1-3/h3-4,7-8,11,13,21H,5-6,9-10,12H2,1-2H3,(H,22,23)1H,(H,2,3)/t13-,29-/m1./s1

4-[4-[1-[[S(R)]-S-Methylsulfonimidoyl]cyclopropyl]-6-[(3R)-3-methyl-4-morpholinyl]-2-pyrimidinyl]-1H-pyrrolo[2,3-b]pyridine, formic acid

AZD-6738 formate; AZD6738; 1352280-98-8; Ceralasertib formate; AKOS040748106; AZD 6738

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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