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

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].

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The combination of AZD6738 and cisplatin has efficacy in NSCLC xenograft models and causes rapid regression of ATM-deficient NSCLC tumors Next we assessed the efficacy of AZD6738 alone and in combination with cisplatin in vivo. Effects on food consumption and body weight are dose limiting for AZD6738 in mice, rats and dogs, and are typically accompanied by atrophic/degenerative histopathology in the gastrointestinal tract after repeated dosing (AstraZeneca, personal communication). AZD6738 caused hypocellularity in multiple lymphoid tissues and bone marrow toxicity correlated with a decrease in all cell lineages in the peripheral blood. There was a minimal increase in alveolar macrophages. Recovery of these effects was seen after cessation of dosing.[1]
We treated nude mice bearing H460 tumors with 50 mg/kg AZD6738 (PO) and mice bearing ATM- deficient H23 tumors with 25 mg/kg AZD6738 (PO) and for 14 consecutive days. Mice received 3 mg/kg cisplatin (IP) on days 1 and 8 of the two week treatment cycle. Body weight loss was the dose limiting toxicity with daily administration of 50 mg/kg AZD6738, alone and in combination with cisplatin. However, body weights remained within protocol guidelines for the duration of treatment, and no animal on study lost greater than 14.3% BW at any point during treatment (Figure 6A). Conversely, 25 mg/kg AZD6738 was well tolerated, with mean body weight (BW) losses of less than 2.7% and 4.8% in the single agent and combination arms, respectively (Figure 6B). Mice treated with the combination exhibited BW loss similar to those that received cisplatin alone [1]. The combination of 50 mg/kg AZD6738 and cisplatin resulted in a 75.5% mean tumor growth inhibition (TGI) of H460 xenografts at day 14 (P ≤ 0.0001 compared to vehicle) (Figure 6C). Growth of tumors treated with the combination was also significantly different from that of tumors treated with cisplatin or AZD6738 alone (P ≤ 0.01 or P ≤ 0.05, respectively). Growth delay for the combination treatment was 12 days (day 26 vs. day 14), although only one of seven tumors had reached the 2000 mm3 endpoint on day 26. While modest growth inhibition was observed in the single agent AZD6738 and cisplatin treatment arms, the differences in growth were not statistically significant (P ≥ 0.05). [1]
Strikingly, the combination of 25 mg/kg AZD6738 and cisplatin resulted in rapid and near complete tumor regression (84.8%) of ATM-deficient H23 tumors by day 29 (Figure 6D). The mean change in tumor growth was significantly different than that of the mock, cisplatin, and AZD6738 treatment arms (P ≤ 0.001, P ≤ 0.01, and P ≤ 0.05, respectively). Treatment with cisplatin or AZD6738 alone did not result in significant inhibition of tumor growth (P > 0.05). After day 29, mice in the combination treatment arm were observed weekly for tumor regrowth. Of the six mice that received combination treatment, three exhibited complete tumor resolution by days 43, 64, and 92, respectively. There was no visual or palpable evidence of tumor out to a final observation on day 113. In the remaining three mice, tumors began to slowly regrow within 3–5 weeks of the end of treatment.[1]
We confirmed by immunohistochemistry that 25 mg/kg AZD6738 inhibits ATR activity in H23 xenografts. Mice were treated with 25 mg/kg AZD6738 daily for 8 consecutive days, 3 mg/kg cisplatin on days 1 and 8, combination, or vehicle, and tumors were harvested six hours following the final dose on day 8. Tumors from mice treated with AZD6738 exhibited reduced phosphorylation of T1989 (Supplementary Figure S6), a marker of active ATR.

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].

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Cell viability assays [1]
Cells were treated in white walled, clear bottom 96-well plates with the indicated doses of AZD6738, cisplatin, gemcitabine, or combination for 48 h. ATP levels were assessed as surrogate measure of viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay and Safire2 plate reader. Raw data were corrected for background luminescence prior to further analysis. For AZD6738 treatment, log dose response curves were 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. For combination treatments, data were normalized to the mean of untreated controls. Loewe excess matrices were generated using Chalice Analyzer Online and mean normalized inhibition values. For AZD6738 + cisplatin curve shift experiments, data were normalized to the mean of 0 μM cisplatin controls within each AZD6738 treatment condition. Log dose response curves were generated in GraphPad Prism 6 by nonlinear regression (log(inhibitor) vs. normalized response with variable slope) of log-transformed (x = log(x)), normalized data. IC50 values were calculated by Prism 6.

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Immunoblotting [1]
Cells were treated with the indicated doses of AZD6738, cisplatin, combination, or mock for 24 h. Protein lysates were generated by scraping adherent cells in lysis buffer (150 mM NaCl, 50 mM Tris-HCL, 5 mM NaF, 1% Tween 20, 0.5% IGEPAL CA-630, protease inhibitor cocktail, pH 7.5) and incubating on ice for 30 min. For AZD6738 + cisplatin experiments, detached cells were pelleted from the media and combined with the adherent cell lysate. SDS-PAGE using 4–12% Bis-Tris gels and Western blotting were performed using standard techniques. Antibody details are provided in the supplementary methods. Following detection of phospho-proteins, membranes were stripped for 25 min at room temperature in Restore stripping buffer and re-probed for corresponding total protein. Images of blots were acquired at 24-bit depth using a Canon LiDE110 scanner and were processed (converted to 8-bit, cropped) using ImageJ.

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Crystal violet colony formation and senescence assays [1]
Cells were treated (in triplicate) in 12-well plates with 0.3 μM, 1 μM AZD6738, or mock for 48 h. Following treatment, AZD6738 was removed, and cells were cultured an additional 2–4 days in fresh media. Colony formation was visualized by staining with 0.5% crystal violet in 95% EtOH. Images were captured with an Olympus SZX10 stereo microscope and DP26 camera. Unprocessed images were resized for inclusion in figures. Experiments were repeated at least three times to ensure consistent results. Senescence-associated β-galactosidase activity was assessed using the Biovision Senescence Detection Kit. Images were acquired using a Leica DMI3000B inverted microscope (20X objective) and DFC420C camera. Unprocessed images were resized for inclusion in figures.

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Replating assays for long term cell viability [1]
Cells were treated with 0.3 μM, 1 μM AZD6738, or mock for 48 h. Following treatment, cells were seeded in 96 well plates (4 replicates) at equal density per condition and grown for an additional 6 days. Viability was assessed on day 8 using the CellTiter-Glo Luminescent Cell Viability Assay and Safire2 plate reader. Background corrected data were normalized to the mean of untreated controls.

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]

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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].

[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.

Ceralasertib is under investigation in clinical trial NCT03682289 (Phase II Trial of AZD6738 Alone and in Combination With Olaparib).
Ceralasertib is an orally available morpholino-pyrimidine-based inhibitor of ataxia telangiectasia and rad3 related (ATR) kinase, with potential antineoplastic activity. Upon oral administration, Ceralasertib selectively inhibits ATR activity by blocking the downstream phosphorylation of the serine/threonine protein kinase CHK1. This prevents ATR-mediated signaling, and results in the inhibition of DNA damage checkpoint activation, disruption of DNA damage repair, and the induction of tumor cell apoptosis. In addition, AZD6738 sensitizes tumor cells to chemo- and radiotherapy. ATR, a serine/threonine protein kinase upregulated in a variety of cancer cell types, plays a key role in DNA repair, cell cycle progression and survival; it is activated by DNA damage caused during DNA replication-associated stress.
See also: Ceralasertib (annotation moved to).

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Drug Indication
Treatment of lung carcinoma (small cell and non-small cell carcinoma)

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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. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.[1]

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Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment.[2]

C20H24N6O2S

412.51

412.168

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

1352226-88-0

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

54761306

White solid powder

1.5±0.1 g/cm3

1.750

0.54

2

7

4

29

724

2

O=[S@@](C1(CC1)C2=NC(C3=C4C(NC=C4)=NC=C3)=NC(N5CCOC[C@H]5C)=C2)(C)=N

OHUHVTCQTUDPIJ-JYCIKRDWSA-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

AZD6738; AZD-6738; AZD 6738; AZD6738; Ceralasertib; 1352226-88-0; CHEMBL4285417; AZD 6738; BDBM50468001; 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;

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)

Solubility in Formulation 1: 6.67 mg/mL (16.17 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 66.7 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: ≥ 2.08 mg/mL (5.04 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 20.8 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% propylene glycol+ddH2O: 10mg/mL

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Solubility in Formulation 5: 10 mg/mL (24.24 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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