ATM ( IC50 = 0.78 nM )

AZD1390 exhibits superior oral bioavailability in preclinical species (64 percent in rats and 74 percent in dogs). PET studies on non-human primates have shown that it can effectively cross the blood-brain barrier. When AZD1390 is combined with radiation therapy for just two or four days, as opposed to radiotherapy alone, profound tumor regressions and longer animal survival (>50 days) have been seen in orthotopic xenograft models of brain cancer[1]. AZD1390 dosed in conjunction with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induces tumor regressions and increased animal survival compared to IR treatment alone in in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models. For clinical applications requiring exposures inside the central nervous system, AZD1390 offers advantageous physical, chemical, PK, and PD properties[2].

AZD1390 belongs to the same exquisitely potent series of ATM inhibitor as the clinical development compound AZD0156 (Fig. 1). However, AZD1390 was discovered following a series of in vitro assays designed to screen for (i) ATM autophosphorylation activity; (ii) selectivity against closely related PIKK family kinases ATR, DNA-PK, and mTOR activity and (iii) broader kinase panels; and (iv) lack of substrate activity in novel dual-transfected human MDR1 and BCRP efflux transporters assays. AZD1390 was screened against ATM [modulation of purified ATM-dependent phosphorylation of glutathione S-transferase (GST)–p53 Ser15] with activity defined as ≥50% [median inhibitory concentration (IC50)] of 0.00009 μM (0.00004 μM corrected for tight binding). IC50 activity against closely related and purified PIKK family enzymes was never more potent than 1 μM. In broader purified kinase screening panels, AZD1390 was tested at two concentrations, 1 and 0.1 μM, against the Thermo Fisher Scientific kinase panel. At the very high concentration of 1 μM, AZD1390 showed ≥50% inhibition against 3 targets (CSF1R, NUAK1, and SGK), with no activity against the remaining 118 targets tested. At 0.1 μM, no activity was found (<50% inhibition) against 354 kinases. We also tested activity and selectivity of AZD1390 against a panel of kinases run by Eurofins Panlabs. AZD1390 showed activity (>50% inhibition at 1 μM) against 1 kinase, FMS, and showed no activity (<50% inhibition at 1 μM) against 124 other kinases from the panel (Table 1). [2]

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Brain and plasma binding of AZD1390[2]
Rat brain binding (fubrain) was determined using the rat brain slice binding method, as detailed by Fridén et al.. Plasma binding (rat, mouse, dog, monkey, and human) was determined by equilibrium dialysis using a rapid equilibrium device. The compound in plasma at 1 or 0.1 μM was dialyzed with buffer at pH 7.4 and 37°C for 16 hours. After incubation, aliquots of both plasma and buffer were added to equal volumes of blank buffer and plasma, respectively, before precipitation with acetonitrile prior to centrifugation and analysis of the supernatants by UPLC-MS/MS. Fuplasma was determined by dividing the concentration in the buffer chamber by the concentration in the plasma chamber.

In an RPMI format, 3000 cells per well are seeded using 10% fetal bovine serum in a 384-well format.Plates are Echo-dosed with a semi-log dose dilution of each compound after a 24-hour period, starting at a top concentration of 1250 nM. After compound dosing, plates are exposed to 0, 2.5, or 4 Gy of radiation for one hour. After the plates are fixed at 1, 6, 24, and 48 hours after the radiation, they are incubated for 30 minutes at room temperature and then three times with phosphate-buffered saline solution (PBSA). This is done by directly adding a 1:1 volume of 8% PFA to the medium, resulting in a final concentration of 4% PFA.

In vivo H2228 model efficacy[2]
Bioluminescence signaling of implanted 3 × 105 NCI-H2228-Luc cells was measured using an IVIS Xenogen imaging machine to monitor tumor growth. When the signal reached the range of 107 to 108, the mice were randomized into different treatment groups and treated orally with either vehicle or AZD1390 QD or BID + IR at 2.5 Gy daily for four consecutive days. AZD1390 or vehicle was dosed at 1 hour before IR on each dosing day. The bioluminescence signals and body weight of the mice were measured once weekly, and the raw data were recorded according to their study number and measurement date in the in vivo database. TGI from the start of treatment was assessed by comparison of the mean change in bioluminescence intensity for the control and treated groups and presented as % of TGI. The calculation of inhibition and regression was based on the geometric mean of relative tumor volume (RTV) in each group. “CG” means the geometric mean of RTV of the control group, whereas “TG” means the geometric mean of RTV of the treated group. On specific day, for each treated group, the inhibition value was calculated using the following formula: Inhibition% = (CG − TG) * 100/(CG − 1). CG should use the corresponding control group of the treated group during calculation. If inhibition was >100%, then regression was calculated using the following formula: Regression = 1 – TG. Statistical significance was evaluated using a one-tailed t test. Survival benefit was measured by Kaplan-Meier plots at the end of the study.[2]

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In vivo efficacy studies in syngeneic glioma model[2]
GL261_Luc cells (1.6 × 105) were implanted into mice through ICB injection, as described above. An IVIS Xenogen imaging machine used to monitor tumor growth measured the bioluminescence signals. When the signals reached the range of 107 to 108, the mice were randomized into treatment groups and treated orally with either vehicle, AZD1390, IR at 2.5 Gy per day for four consecutive days, IR + AZD1390, or AZD1390 + TMZ. AZD1390, TMZ, or vehicle was dosed 1 hour before IR on each dosing day. The bioluminescent signals and body weight of the mice were measured once a week. TGI from the start of treatment was assessed by comparison of the mean change in bioluminescence intensity for the control and treatment groups, and data are presented as % of TGI. The calculation of inhibition and regression was based on the geometric mean of RTV in each group. CG means the geometric mean of RTV of the control group, whereas TG means the geometric mean of RTV of the treated group. On specific day, for each treated group, inhibition value was calculated using the following formula: Inhibition% = (CG − TG) * 100/(CG − 1). CG should use the corresponding control group of the treated group during calculation. If Inhibition was >100%, then regression was calculated using the following formula: Regression = 1 − TG. Statistical significance was evaluated using a one-tailed t test. A Kaplan-Meier curve was generated to calculate the survival benefit of mice treated with compounds.[2]

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In vivo PDX efficacy studies[2]
Human tumor tissue fragments were taken from TMZ-resistant or TMZ-sensitive GBM patients, derived from START (http://startthecure.com/preclinical_services_research.php), and implanted subcutaneously in female NMRI nude mice (Janvier Labs) between 7 and 11 weeks of age to establish the GBM PDX models. Animals were enrolled into the study when their tumor volume was approximately 200 mm3 and randomized into four groups: vehicle, 0.5% (w/v) HPMC, and 0.1% (w/v) Tween 80 given QD for 5 days by oral gavage; 2-Gy XRT given QD for 5 days; AZD1390 (20 mg/kg) given QD for 5 days by oral gavage; and AZD1390 + XRT given QD for 5 days. XRT was performed with X-RAD 320 (Precision X-Ray) to the whole head, and AZD1390 was administered 1 hour before XRT in the combination group. Animals were observed daily, and tumor volume and body weight were measured twice per week. Tumor volumes were calculated using the following formula: 0.52 (width × length2). All animal experiments were performed under a protocol approved by the Danish Animal Experiments Inspectorate. [2]

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Bioluminescent imaging (BLI) is performed after intracranial implantation of mouse GL261 glioma (p53 mutant) cells into immunocompetent, syngeneic C57/bl6 mice, before the mice are randomly assigned. Before receiving several fractions of 2-3 Gy of radiation over the course of two to four days, AZD1390 is given orally via gavage. Radiation therapy is applied to the tumor site using a 5 x 5 mm lateral field using the Small Animal Radiation Research Platform (SARRP).

BBB penetration[2]
The endothelial cells of the BBB contain efflux transporters MDR1 (Pgp) and BCRP, which serve to actively exclude the compound from the brain (32). In vitro efflux assays were set up using Madin-Darby canine kidney (MDCK) cells dual-transfected with human MDR1 and BCRP efflux transporters to identify compounds without substrate activity. In vitro MDCK_MDR1_BCRP studies at both 1 and 0.1 μM suggest that AD1390 is not a substrate for the human Pgp and/or BCRP efflux transporters (efflux ratio, <2); however, it does have a higher efflux rate in rodent species, as lower Kp,uu values were observed in rat and mouse (0.17 and 0.04, respectively). This reflects that, in rodents, AZD1390 is seen to be an efflux substrate with increased brain exposure (Kp,uu, 0.85 and 0.77) on administration of the chemical efflux transporter knockout elacridar (Fig. 1C) and an efflux ratio of 3.2 in the rat transporter-transfected in vitro LLC-PK1-rMdr1a assay at 1 μM. In contrast, AZD0156 at 0.1 μM has an efflux ratio of 23, indicating that it is a human efflux transporter substrate (Fig. 1, B and C). This BBB permeability difference is also reflected in vivo with AZD1390 rat and mouse brain Kp,uu values six- and sevenfold higher, respectively, than AZD0156.

Cynomolgus macaque positron emission tomography (PET) images for the two compounds (Fig. 1D) show that only AZD1390 gives significant brain penetration with a Cmax (%ID) of 0.68 ± 0.078 (n = 5) [compare AZD0156 Cmax %ID 0.15 ± 0.036 (n = 3, P < 0.01)]. Two-tissue compartment (2-TC) modeling of AZD1390 PET data yielded a VT (equivalent to Kp) of 5.8 ± 1.2 (n = 5) and a calculated Kp,uu of 0.33 ± 0.068 (n = 5). It was not possible to accurately determine Kp for AZD0156 in cynomolgus macaques. Here, the 2-TC model showed poor identifiability with very high SEs in VT. We observed lower Kp,uu values in rat and mouse for AZD1390 (0.17 and 0.04, respectively). This reflects that, in rodents, AZD1390 appears to be an efflux substrate with increased brain exposure (Kp,uu, 0.85 and 0.77) on administration of the chemical efflux transporter knockout elacridar (Fig. 3B) and an efflux ratio of 3.2 in the rat transporter-transfected in vitro LLC-PK1-rMdr1a assay at 1 μM. Despite the lower rodent Kp,uu values in mouse at 2 to 20 mg/kg, free brain exposure is achieved, with pATM inhibition and efficacy observed.[2]

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PD and PK of AZD1390 in vivo[2]
Researchers performed an extensive assessment of the relationship between PK and PD of AZD1390 in plasma, brain, and tumor samples from our orthotopic brain tumor model, NCI-H2228, implanted in the brain. The data show that the combination of pharmacologically active doses of AZD1390 from the in vitro and cell potency assays inhibited the IR-induced PD biomarkers pATM (Ser1981) and phospho-Rad50 (pRad50) (Ser635) in vivo in a dose- and time-dependent manner (Fig. 3, A and B). The antibody used to detect the latter is being used in clinical trials, and the data in Fig. 4B show the staining levels correlating with PK observations in Fig. 2A. The combination of AZD1390 with IR also significantly increased the apoptotic marker CC3 (cleaved caspase-3) compared to IR alone in NCI-H2228 lung cancer brain metastasis (LC-BM) model, suggesting that the combination is inducing tumor cell death (Fig. 3C). The data reveal a correlation between PK and PD modulation, with AZD1390 free brain levels peaking within 1 hour of dosing and dissipating over a 24-hour period, correlating with ATM inhibition activity (see fig. S4, A and D, for further details on PK and PD analyzed).

The IC50 against the cardiac ion channel hERG was also confirmed as minimal for both AZD0156 and AZD1390: >33.3 and 6.55 μM, respectively. (A similar IC50 for AZD1390 of 7.99 μM against hERG was generated using an alternative assay with improvements in compound handling and data processing).[2]

[1]. AACR Mol Cancer Ther. 2018, 17(1 Suppl):Abstract nr A124.

[2]. Science Advances. 2018, 4(6): eaat1719.

ATM Kinase Inhibitor AZD1390 is an orally bioavailable inhibitor of ataxia telangiectasia mutated (ATM) kinase, with potential antineoplastic activity. Upon oral administration, AZD1390 targets and binds to ATM, thereby inhibiting the kinase activity of ATM and ATM-mediated signaling. This prevents DNA damage checkpoint activation, disrupts DNA damage repair, induces tumor cell apoptosis, and leads to cell death in ATM-overexpressing tumor cells. AZD1390 hypersensitizes tumors to chemo/radiotherapy. In addition, AZD1390 is able to cross the blood-brain barrier (BBB). ATM, a serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases, is upregulated in a variety of cancer cell types. It is activated in response to DNA double-strand breaks (DSB) and plays a key role in DNA repair.

C27H32FN5O2

477.5737

477.25

C, 67.90; H, 6.75; F, 3.98; N, 14.66; O, 6.70

2089288-03-7

126689157

White to off-white solid powder

4.2

0

6

7

35

720

0

VQSZIPCGAGVRRP-UHFFFAOYSA-N

InChI=1S/C27H32FN5O2/c1-18(2)33-26-21-14-20(22(28)15-23(21)29-17-24(26)31(3)27(33)34)19-8-9-25(30-16-19)35-13-7-12-32-10-5-4-6-11-32/h8-9,14-18H,4-7,10-13H2,1-3H3

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)

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.)