mTOR (IC50 = 0.13 nM); mTOR (IC50 = 0.8 nM)

AZD8055 shows low activity (∼1,000-fold) against all PI3K isoforms (α, β, γ, δ) and other members of the PI3K-like kinase family (ATM and DNA-PK). AZD8055 prevents the phosphorylation of mTORC1 (p70S6K and 4E-BP1), mTORC2 (Akt), and downstream proteins. A significant amount of cap-dependent translation can be inhibited by AZD8055 because it completely inhibits the rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1. With IC50 values of 53, 50, and 20 nM, respectively, AZD8055 potently inhibits proliferation in U87MG, A549, and H838 cells. In addition, autophagy and elevated LC3-II levels are induced by AZD8055 in H838 and A549 cells.[1] AML blast cell proliferation and cell cycle progression are reduced, leukemic progenitors' clonogenic growth is inhibited, and AZD8055 induces caspase-dependent apoptosis in leukemic cells but not in healthy, immature CD34+ cells.[2] With an IC50 of 24.7 nM, AZD8055 exhibits inhibitory activity against the pediatric preclinical testing program (PPTP) cell lines and causes appreciable variations in EFS distribution. [3]

AZD8055 inhibits the pS6 and pAkt in U87MG and A549 xenografts at 2.5/10 mg/kg, which leads to tumor growth inhibition. At doses of 10–20 mg/kg, AZD8055 significantly inhibits tumor growth in a variety of xenografts, including U87MG, BT474c, A549, Calu-3, LoVo, SW620, PC3, and MES-SA. [1] A 40% reduction in tumor volume is brought about by AZD8055, and Akt, S6K, and SGK protein kinase phosphorylation are also eliminated. By inhibiting mTORC1 and mTORC2 signaling, the administration of AZD8055 (5 mg/kg, Bid) and SAHA (100 mg/kg/d) completely inhibits tumor growth in PTEN+/−LKB1+/hypo xenografts in mice. [4]

In order to identify mTORC1 and mTORC2 activity, a high-throughput screening cell-based assay is created using MDA-MB-468 cells. Increasing amounts of AZD8055 are applied to cells for two hours. Cells are fixed, cleaned, and then probed with S473 pAkt or S235/236 phosphorylated S6 (pS6) antibodies at the conclusion of the incubation period. Utilizing an Acumen laser scanning cytometer, phosphorylation levels are measured. cells to detect mTORC1 and mTORC2 activity. Cells are exposed for 2 hours to increasing concentrations of AZD8055. At the end of the incubation period, cells are fixed, washed, and probed with antibodies against S473 pAkt or against S235/236 phosphorylated S6 (pS6). Levels of phosphorylation are assessed using an Acumen laser scanning cytometer.

Cell nuclei are stained for (0.03 mg/mL Hoechst 33342) and acidic vesicles (1 g/mL acridine orange) in cells that have been exposed to AZD8055 for 72 to 96 hours. On an ArrayScan II platform, images are taken at 450 and 536 nm, and the proportion of acidic vesicles and the number of cells are counted. Before being incubated with AZD8055, cells are exposed to e64d/pepstatin (10 g/mL) for 30 to 90 min in order to assess LC3. After being lysed on ice, cells are examined using immunoblotting.

U87MG, BT474c, A549, Calu-3, LoVo, SW620, PC3 and MES-SA U87-MG and A549 are established in pathogen-free, female nude mice (nu/nu:Alpk).
2.5-20 mg/kg
Oral gavage once or twice daily

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In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials.[1]

Tumor cells (106 for U87-MG, 5 × 106 for A549) were injected s.c. in a volume of 0.1 mL, and mice were randomized into control and treatment groups when tumor size reached 0.2 cm3. AZD8055 was formulated in 30% (w/v) captisol (pH 3.0). The control group received the vehicle only. Tumor volumes (measured by caliper), animal body weight, and tumor condition were recorded twice weekly for the duration of the study. The tumor volume was calculated (taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter) using the following formula: (length × width) × √(length × width) × (π/6).[1]

For pharmacodynamic studies, animals were randomized when tumor size reached 0.5 cm3. The treatment groups received a single dose of AZD8055 and the control group received vehicle only. Tumor samples and blood were collected at various times after drug administration. The expression of pAKT and pS6 was determined in xenograft tissue by immunoblotting as described above. Ki67 nuclear staining was carried out using formalin-fixed, paraffin-embedded A549 xenografts.[1]

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In vivo AZD8055 induced significant differences in EFS distribution compared to controls in 23 of 36 (64%) evaluable solid tumor xenografts, and 1 of 6 evaluable ALL xenografts. Intermediate activity for the time to event activity measure (EFS T/C >2) was observed in 5 of 32 (16%) solid tumor xenografts evaluable. The best response was stable disease. PD2 (progressive disease with growth delay) was observed in 20 of 36 (55.6%) evaluable solid tumor xenografts. AZD8055 significantly inhibited 4E-BP1, S6, and Akt phosphorylation following day 1 and day 4 dosing, but suppression of mTORC1 or mTORC2 signaling did not predict tumor sensitivity.[3]

In Vivo Tumor Growth Inhibition Studies[3]
CB17SC scid−/− female mice (Taconic Farms, Germantown NY), were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas, neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously 24. Female mice were used irrespective of the patient gender from which the original tumor was derived. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Ten mice were used in each control or treatment group. Tumor volumes (cm3) [solid tumor xenografts] or percentages of human CD45-positive [hCD45] cells [ALL xenografts] were determined as previously described 25 and responses were determined using three activity measures as previously described 25. An in-depth description of the analysis methods is included in the supplemental response definitions.[3]

Drugs and Formulation[3]
AZD8055 was provided to the PPTP by Astrazeneca, through the Cancer Therapy Evaluation Program (NCI). AZD8055 was dissolved in 0.5% hydroxypropylmethylcellulose containing 0.1% Tween 80 in water, sonicated and stirred overnight. AZD8055 was administered P.O. daily for 28 days at 20 mg/kg per day.

[1]. Cancer Res . 2010 Jan 1;70(1):288-98.

[2]. Leukemia . 2012 Jun;26(6):1195-202.

[3]. Pediatr Blood Cancer . 2012 Feb;58(2):191-9.

AZD-8055 is a pyridopyrimidine that is pyrido[2,3-d]pyrimidine which is substituted at positions 2 and 4 by (3S)-3-methylmorpholin-4-yl groups and at position 5 by a 3-(hydroxymethyl)-4-methoxyphenyl group. It is an mTOR complex 1/2 (mTORC1/2) dual inhibitor [mTOR = mammalian target of rapamycin]. It has a role as a mTOR inhibitor, an apoptosis inducer and an antineoplastic agent. It is a member of benzyl alcohols, a tertiary amino compound, a pyridopyrimidine and a member of morpholines.
AZD8055 has been used in trials studying the treatment of Cancer, Lymphomas, Solid Tumors, MALIGNANT GLIOMA, and brainstem glioma, among others.
mTOR Kinase Inhibitor AZD8055 is an inhibitor of the mammalian target of rapamycin (mTOR) with potential antineoplastic activity. mTOR kinase inhibitor AZD8055 inhibits the serine/threonine kinase activity of mTOR, resulting in decreased expression of mRNAs necessary for cell cycle progression, which may induce cell cycle arrest and tumor cell apoptosis. mTOR phosphorylates transcription factors, such as S6K1 and 4E-BP1, which stimulate protein synthesis and regulate cell growth, proliferation, motility, and survival.

C25H31N5O4

465.5447

465.237

C, 64.50; H, 6.71; N, 15.04; O, 13.75

1009298-09-2

25262965

Yellow solid powder

1.2±0.1 g/cm3

694.3±65.0 °C at 760 mmHg

373.7±34.3 °C

0.0±2.3 mmHg at 25°C

1.609

0.27

1

9

5

34

659

2

O1C([H])([H])C([H])([H])N(C2C3C([H])=C([H])C(C4C([H])=C([H])C(=C(C([H])([H])O[H])C=4[H])OC([H])([H])[H])=NC=3N=C(N=2)N2C([H])([H])C([H])([H])OC([H])([H])[C@]2([H])C([H])([H])[H])[C@@]([H])(C([H])([H])[H])C1([H])[H]

KVLFRAWTRWDEDF-IRXDYDNUSA-N

InChI=1S/C25H31N5O4/c1-16-14-33-10-8-29(16)24-20-5-6-21(18-4-7-22(32-3)19(12-18)13-31)26-23(20)27-25(28-24)30-9-11-34-15-17(30)2/h4-7,12,16-17,31H,8-11,13-15H2,1-3H3/t16-,17-/m0/s1

[5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol

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: ~50 mg/mL (~107.4 mM)
Water: <1 mg/mL
Ethanol: ~3 mg/mL (~6.4 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (10.74 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.37 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 3: ≥ 2.5 mg/mL (5.37 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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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.37 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (5.37 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 4% DMSO+30% PEG 300+ddH2O: 5mg/mL

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Solubility in Formulation 7: 50 mg/mL (107.40 mM) in 30 % SBE-β-CD (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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