EGFR^L858R (IC50 = 12 nM); EGFR^L858R/T790M (IC50 = 1 nM)

Osimertinib (AZD-9291) (5 mg/kg/day) is administered to the tumor-bearing mice for a duration of one to two weeks. After receiving Osimertinib (AZD-9291) therapy, 5 out of 5 C/L858R mice showed an almost 80% reduction in tumor volume by magnetic resonance imaging (MRI) within days of treatment, whereas 5 out of 5 mice given a vehicle treatment showed tumor growth[1]. Osimertinib (AZD-9291) is chosen for additional research because it exhibits better rat PK, decreased hERG affinity, and improved IGF1R margins in comparison to the previously reported compounds. Comparing osimertinib (AZD-9291) to the previously reported lead compounds, it also provides a further level of wider chemical and profile diversity. In three efficacy models, Osimertinib (AZD-9291) doses comparatively well; comparable efficacy is seen at doses of 10 mg/kg per day. When Osimertinib (AZD-9291) is dosed at 5 mg/kg per day, even greater efficacy is seen[2].

Osimertinib, formerly known as mereletinib and AZD-9291, is a third generation EGFR inhibitor that is available orally and is irreversible. It selectively targets specific mutants of EGFR, with IC50 values of 493.8 nM for wild-type EGFR in LoVo cells, L858R/T790M EGFR, and Exon 19 deletion EGFR, respectively. It reduces the side effects associated with currently available medications by inhibiting both activating and resistant EGFR mutations while protecting the normal form of EGFR found in normal skin and gut cells.

PC-9 cells are cultured at 37°C with 5% CO₂ after being seeded into T75 flasks (5×10⁵ cells/flask) with RPMI growth media. The medium is changed the next day to one supplemented with an EGFR inhibitor at a concentration equivalent to the predetermined EC50 in PC-9 cells. Every two to three days, the medium is changed, and resistant clones are allowed to reach 80% confluency before the cells are trypsinized and reseeded in the same amount of media that contains twice as much EGFR inhibitor. Until a final concentration of 1.5 μM ZD1839, 1.5 μM BIBW 2992, 1.5 μM WZ4002, or 160 nM Osimertinib (AZD-9291) is reached, dose escalations are carried out[1].

Mice: The mice used are male and female EGFR^L858R and EGFR^L858R+T790M mice. Oral gavage is used to administer osimertinib (AZD-9291) at a dose of 7.5 mg/kg and 5 mg/kg, respectively. The drug is suspended in 1% Polysorbate 80. Every week, the Vanderbilt University Institute of Imaging Science images mice. Prior to lung dissection and flash freezing, mice are given a drug treatment for eight hours in preparation for immunoblot analysis. Liquid nitrogen is used to grind the lungs before lysis.
Rats: The 10-week-old male RccHan:WIST rats are given a single oral dose of 200 mg/kg of osimertinib (AZD-9291). Accuchek Active meters are used to measure blood glucose levels.

[1]. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014 Sep;4(9):1046-61.

[2]. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistancemutations that spares the wild type form of the receptor. J Med Chem. 2014 Oct 23;57(20):8249-67.

The first generation EGFR TKIs gefitinib and erlotinib provide significant clinical benefit in patients with advanced lung adenocarcinoma harbouring activating EGFR mutants (EGFRm+), but patients will ultimately develop disease progression due to acquired resistance. Acquisition of the EGFR T790M mutation is the most common mechanism of drug resistance, detected in more than 50% of gefitinib/erlotinib resistant patients. Current therapeutic strategies are limited for advanced lung adenocarcinoma patients with EGFR T790M (EGFRm+/T790M), so this remains a key area of unmet need. AZD9291 (structure to be disclosed at meeting) is an oral, irreversible, third generation, selective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations. The mechanistic and functional activity of AZD9291 was characterised in vitro and in vivo across a number of cell lines harbouring various EGFR-mutations or wild type EGFR. Presented data shows AZD9291 potently inhibits EGFR phosphorylation in EGFRm+ (e.g. PC9; <25nM) and EGFRm+/T790M (e.g. H1975; <25nM) cell lines in vitro, whilst demonstrating much less activity against wild-type EGFR lines (e.g. LoVo; >500nM). Consistently, AZD9291 showed significantly more potent inhibition of proliferation in mutant EGFR cell lines compared to wild-type in vitro. In addition, AZD9291 administered once daily orally at 5mg/kg caused profound regression of tumours across EGFRm+ (PC9; 178% growth inhibition) and EGFRm+/T790M (H1975; 119% growth inhibition) tumour models in vivo, after 14 days dosing. Furthermore 5mg/kg AZD9291 was sufficient to cause significant shrinkage of EGFRm+ and EGFRm+/T790M transgenic mouse lung tumours. Tumour growth inhibition was associated with profound inhibition of EGFR phosphorylation and key downstream signaling pathways such as AKT and ERK. Chronic long-term treatment of PC9 and H1975 xenograft tumours with AZD9291 led to a complete and sustained macroscopic response, with no visible tumours after 40 days dosing, and being maintained beyond 100 days. Furthermore, pre-clinical data also indicates that AZD9291 could target tumours that have acquired resistance to the more recently identified HER2-amplification mechanism, thus potentially extending its benefit in TKI resistant patients. Taken together, preclinical data demonstrates that AZD9291 is a potent and effective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations whilst sparing wild-type EGFR. These data support the further clinical investigation of AZD9291 in advanced EGFR mutant lung adenocarcinoma.

C30H41N7O8S2

691.818644285202

691.245

2070014-82-1

Osimertinib;1421373-65-0;Osimertinib mesylate;1421373-66-1

92044416

Light yellow to yellow solid

4

13

10

47

845

0

RPUCCTLBBCSFEX-UHFFFAOYSA-N

InChI=1S/C28H33N7O2.2CH4O3S/c1-7-27(36)30-22-16-23(26(37-6)17-25(22)34(4)15-14-33(2)3)32-28-29-13-12-21(31-28)20-18-35(5)24-11-9-8-10-19(20)24;2*1-5(2,3)4/h7-13,16-18H,1,14-15H2,2-6H3,(H,30,36)

N-[2-[2-(dimethylamino)ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide;methanesulfonic acid

AZD-9291 dimesylate; AZD9291; AZD 9291; Mereletinib dimesylate; Trade name: Tagrisso

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)