BackgroundAnamorelin HCl (ANAM) is a novel, orally active, ghrelin receptor agonist in clinical development for the treatment of cancer cachexia. We report in vitro and in vivo studies evaluating the preclinical pharmacologic profile of ANAM.MethodsFluorescent imaging plate reader and binding assays in HEK293 and baby hamster kidney cells determined the agonist and antagonist activity of ANAM, and its affinity for the ghrelin receptor. Rat pituitary cells were incubated with ANAM to evaluate its effect on growth hormone (GH) release. In vivo, rats were treated with ANAM 3, 10, or 30 mg/kg, or control orally, once daily for 6 days to evaluate the effect on food intake (FI) and body weight (BW), and once to assess GH response. In pigs, single (3.5 mg/kg) or continuous (1 mg/kg/day) ANAM doses were administered to assess GH and insulin-like growth factor (IGF-1) response.ResultsANAM showed significant agonist and binding activity on the ghrelin receptor, and stimulated GH release in vitro. In rats, ANAM significantly and dose-dependently increased FI and BW at all dose levels compared with control, and significantly increased GH levels at 10 or 30 mg/kg doses. Increases in GH and IGF-1 levels were observed following ANAM administration in pigs.ConclusionANAM is a potent and highly specific ghrelin receptor agonist with significant appetite-enhancing activity, leading to increases in FI and BW, and a stimulatory effect on GH secretion. These results support the continued investigation of ANAM as a potential treatment of cancer anorexia-cachexia syndrome.

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.

Objective: Osimertinib is the 3rd generation EGFR inhibitor, which has been approved for the treatment of NSCLC patients with EGFRT790M. More recently, a tertiary EGFRC797S mutation was reported as the dominant resistance (40~20%) mechanism to Osimertinib. The emergence of C797S mutation prevent covalent bond formation with Osimertinib, and caused the drug resistance. So, it’s an urgent demand for new EGFR inhibitors that can effectively inhibit EGFR triple mutant, d746-750/T790M/C797S & L858R/T790M/C797S. Here, we disclose our clinical candidate, TQB3804, as a novel 4th generation inhibitor that potently inhibits triple mutants. Methods: The enzyme activities of TQB3804 for EGFRd746-750/T790M/C797S, EGFRL858R/T790M/C797S, EGFRd746-750/T790M, EGFRL858R/T790M, and EGFRWT were measured with corresponding kinase assays. The anti-proliferative activity was evaluated in Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750), and A431 (EGFRWT) cell lines, and the phosphorylation of EGFR was also evaluated in Ba/F3 (EGFRd746-750/T790M/C797S) cell line. Antitumor activity of TQB3804 was evaluated in three triple mutant cell-derived tumor xenograft (CDX) models Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), and PC9 (EGFRd746-750/T790M/C797S) and one Osimertinib resistant patient-derived xenograft (PDX) model of NSCLC (LUPF104, EGFRd746-750/T790M/C797S). Results: TQB3804 displayed potent enzymatic activities for EGFRd746-750/T790M/C797S, EGFRL858R/T790M/C797S, EGFRd746-750/T790M, and EGFRL858R/T790M with IC50 of 0.46, 0.13, 0.26, and 0.19 nM respectively, and has similar enzymatic activity for EGFRWT (IC50 = 1.07) to Osimertinib. It also showed expected anti-proliferative activity in 4 cell lines, Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750), and A431 (EGFRWT), with IC50 of 26.8, 163, 45, and 147 nM, respectively. The phosphorylation for EGFR in Ba/F3 (EGFRd746-750/T790M/C797S) cell line was potently inhibited with IC50 =18.5 nM. TQB3804 significantly inhibited tumor growth in the triple mutant Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), and PC9 (EGFRd746-750/T790M/C797S) CDX models, as well as in the LUPF104 PDX model. Western blot analysis of the tumor samples in the Ba/F3 (EGFRd746-750/T790M/C797S) CDX model showed that TQB3804 inhibited p-EGFR, p-AKT and p-ERK indicating that the tumor growth inhibition was through inhibition of the resistant triple mutant EGFR. Conclusions: We have identified a potent orally active 4th generation EGFR inhibitor, TQB3804. It can inhibit the activity of Osimertinib resistant triple mutant EGFR, and showed strong antitumor activity in corresponding in vitro and in vivo preclinical assays. These results are considered highly promising and warrant moving the compound forward to clinical investigation. Citation Format: Xile Liu, Xiquan Zhang, Ling Yang, Xin Tian, Tiantian Dong, Charles Z Ding, Lihong Hu, Lingyu Wu, Lele Zhao, Jun Mao, Qusheng Ji, Shaoyu Yan, Zhenzhen Zhu, Yuanfeng Xia, Chichung Chan, Shuhui Chen. Preclinical evaluation of TQB3804, a potent EGFR C797S inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1320.

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