In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combimolecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice

Abstract Introduction. Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumours with an average survival rate of 12-18 months. The existing treatment (chemotherapy with temozolomide (TMZ)+radiotherapy) increases the median survival by 2-3 months. One protein, the epidermal growth factor receptor (EGFR) is expressed in 57.4% of GBM and half of them expresses its aggressive mutant, EGFRvIII, whose expression is associated with resistance to apoptosis, activation of DNA repair and ultimately resistance to chemotherapy. In the context of a strategy termed “combi-targeting”, we developed molecules designed to block EGFRvIII while being capable of inducing DNA damage. In vivo analysis suggests that the first prototype of such a class ZR2002 loses its DNA damaging function through metabolic oxidation. Recently, we surmised that a molecule designed to be metabolically stable may require a lower dose to be active in vivo. To test this hypothesis, we measured the cytotoxicity of the new combi-molecule, called "AF143", in a panel of glioblastoma cell lines and then we determined its efficacy and drug metabolism in an in vivo model of glioblastoma derived from human glioblastoma stem cells (GSC) with known resistance to TMZ. Material and Methods. Three glioblastoma cells line (U87-wt, U87-EGFR and U87-EGFRvIII) and the human GSC (1123IC7R) were used to determine IC50 of AF143 and ZR2002 with SRB or alamarBlue assay. Xenograft of tumors were obtained by intracranial injection of 1123IC7R-Luc GSC into the head of NSG female mice. One group of mice was treated with ZR2002 (N=8), the other with AF143 (N=7), the last with vehicle (N=8). Mice received 10 total doses: 2 cycles of 1 dose (100mg/kg) daily for 5 consecutive days followed by 2 days off, by oral gavage. The tumour burden was measured by bioluminescent imaging and drug metabolism by LC-MS. Body weights, mortality and toxicity were monitored. Survival curves and statistical analysis calculated. Results. In vitro growth inhibitory analysis showed that AF143 was 2-fold more potent than ZR2002 in U87-EGFR and 1123IC7R GSC and 3-fold more potent in U87-EGFRvIII. In vivo analysis of AF143 given alone showed significant antitumor activity (p<0.05) after 5 doses treatment, while ZR2002 induced an apparent tumour delay (p>0.05). Remarkably, AF143 and ZR2002 treatment significantly prolonged the overall survival of mice compared with the control group (p= 0.0004 for AF143 and p=0.04 for ZR2002). Analysis of drug metabolism revealed that in contrast to ZR2002, AF143 remained intact after 2h treatment, with moderate levels of N-acetyl and N6-dealkylated metabolites. Conclusion. The results in toto suggest that the intact structure of AF143 is stable to metabolism, can cross the blood brain barrier and induce antitumour activity in a EGFRvIII expressing glioblastoma stem cell model. Citation Format: Caterina Facchin, Ana Belen Fraga Timiraos, Brian Meehan, Nadia Babaa, Lata Adnani, Anne-Laure Larroque, Janusz Rak, Bertrand J Jean-Claude. In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combi-molecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1647.