AZD9291 (Osimertinib)

Biological Activity

AZD9291 is an oral, irreversible, and mutant-selective EGFR inhibitor with IC50 of 12.92, 11.44 and 493.8 nM for Exon 19 deletion EGFR, L858R/T790M EGFR, and WT EGFR, respectively. In vivo, AZD9291 shows significantly more potent inhibition of proliferation in mutant EGFR cell lines when compared with wild-type. AZD9291(5mg/kg p.o.) causes profound regression of tumors across EGFRm+ (PC9) and EGFRm+/T790M (H1975) tumor models with profound inhibition of EGFR phosphorylation and key downstream signaling pathways such as AKT and ERK .

Product Citations

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  Mol Cancer Ther. 2022 Jul 5;21(7):1060-1066.

  HJM-561, a Potent, Selective, and Orally Bioavailable EGFR PROTAC that Overcomes Osimertinib-Resistant EGFR Triple Mutations
  AZD9291 (Osimertinib) purchased from AbMole

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  Chem Res Toxicol. 2021 Nov 15;34(11):2309-2318.

  Quantification of Osimertinib and Metabolite–Protein Modification Reveals Its High Potency and Long Duration of Effects on Target Organs
  AZD9291 (Osimertinib) purchased from AbMole

• 

  Bioengineered. 2021 Dec;12(1):8679-8689.

  AZD3759 inhibits glioma through the blockade of the epidermal growth factor receptor and Janus kinase pathways
  AZD9291 (Osimertinib) purchased from AbMole

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  Bioengineered. 2022 Jan;13(1):331-344.

  AZD3759 enhances radiation effects in non-small-cell lung cancer by a synergistic blockade of epidermal growth factor receptor and Janus kinase-1
  AZD9291 (Osimertinib) purchased from AbMole

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  Clin Cancer Res. 2018 Nov 15;24(22):5658-5672.

  Increased Synthesis of MCL-1 Protein Underlies Initial Survival of EGFR-Mutant Lung Cancer to EGFR Inhibitors and Provides a Novel Drug Target.
  AZD9291 (Osimertinib) purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Clinical Cancer Research (2018 Nov). Figure 1. osimertinib (Abmole Bioscience)
	Method	Western Blot
	Cell Lines	EGFR mutant NSCLC cell lines
	Concentrations	50 nM
	Incubation Time	72 h
	Results	In both sets of DTCs, other than BCL-2 in the PC9 osimertinib-DTCs, other anti-apoptotic BCL-2 family members were not upregulated, and, with the exception of NOXA in the HCC827 gefitinib-DTCs, expression of the key pro-apoptotic BCL-2 family members BIM and NOXA was not markedly diminished.

	Source	Nat Med (2015). Figure 1. AZD9291
	Method	digital PCR (ddPCR) assay
	Cell Lines	Ba/F3 cells
	Concentrations	1.0 μM
	Incubation Time	72 h
	Results	Although a more comprehensive analysis of plasma and biopsy tissue collected after resistance develops will be needed to provide greater clarity on C797S mutation incidence, the emergence of this mutation in a marked proportion of AZD9291-resistant patients suggests that development of targeted therapies with the ability to overcome the C797S mutation is warranted.

Protocol (for reference only)

Cell Experiment
Cell lines	PC-9, H1975, LOVO, H1650 and H3255 lines
Preparation method	In vitro EGFR phosphorylation assays Cells were treated for 2 h with a dose-response of each drug. Wild-type cells were stimulated for 10 minutes with 25 ng/ml of EGF before lysis. Level of EGFR phosphorylation was quantified in cell extracts using a modified R&D Systems DuoSet Human phospho-EGFR ELISA (36).
Concentrations	0-1000nM
Incubation time	2h

Animal Experiment
Animal models	PC-9 and H1975 cells subcutaneous xenograft models
Formulation	not mentioned
Dosages	5 mg/kg/day QD
Administration	oral

Chemical Information

Molecular Weight	499.61
Formula	C28H33N7O2
CAS Number	1421373-65-0
Solubility (25°C)	DMSO 90 mg/mL (Need warming and ultrasonic)
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Xiao-E Yan, et al. Structural Basis of AZD9291 Selectivity for EGFR T790M

[2] Alessandro Leonetti, et al. Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer

[3] Kenneth S Thress, et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M

[4] Pasi A Jnne, et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer

[5] Darren A E Cross, et al. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer