In non-small cell lung cancer (NSCLC), multiple classes of activating mutations have been identified in EGFR and HER2 that vary widely in their sensitivity to available tyrosine kinase inhibitors (TKIs). Erlotinib, gefitinib, and afatinib are approved for use in patients with the most common forms of EGFR activating mutations (ie, exon 19 deletions or L858R substitutions). However, no TKIs are approved for patients with EGFR activated by any other mutation, including exon 20 insertions or other uncommon substitutions, or for patients with any class of HER2 activating mutation (including exon 20 insertions). As inhibition of wild-type (WT) EGFR is associated with dose-limiting toxicities, a TKI that inhibits oncogenic EGFR and HER2 variants more potently than WT EGFR is more likely to be able to be dosed to efficacious levels. AP32788 is a potent inhibitor of all oncogenic forms of EGFR and HER2, including exon 20 insertions, with selectivity over WT EGFR. Activity of AP32788 and other TKIs was assessed by measuring viability of Ba/F3 cell lines engineered to express 20 mutant variants of EGFR (n = 14) or HER2 (n = 6): 4 EGFR variants containing a common activating mutation with or without a T790M resistance mutation, 8 EGFR/HER2 variants containing an exon 20 activating insertion (eg, EGFR ASV, HER2 YVMA), and 8 EGFR/HER2 variants containing other uncommon activating mutations (eg, EGFR G719A, HER2 G776V). Inhibition of WT EGFR was assessed by measuring effects on EGFR phosphorylation in cells (A431) that over-express WT EGFR. Consistent with their clinical activity, erlotinib and gefitinib generally only inhibited the 2 EGFR variants with common activating mutations more potently than WT EGFR (IC50s 71 and 56 nM, respectively), and afatinib generally only inhibited EGFR with common activating mutations or uncommon substitutions more potently than WT EGFR (IC50 4 nM). In contrast, AP32788 inhibited all 14 mutant variants of EGFR (IC50s 2.4-22 nM), and all 6 mutant variants of HER2 (IC50s 2.4-26 nM), more potently than it inhibited WT EGFR (IC50 35 nM), including all 8 variants with exon 20 activating insertions. In mice implanted with a patient-derived tumor containing an EGFR exon 20 activating insertion, or with engineered Ba/F3 cells containing a HER2 exon 20 activating insertion, once daily oral dosing of AP32788 induced regression of tumors at doses that were well tolerated (30-100 mg/kg). In vivo efficacy was associated with inhibition of EGFR signaling in the tumor. AP32788 potently inhibited all activated forms of EGFR and HER2 tested, including exon 20 insertions, more potently than WT EGFR, suggesting it may have the selectivity necessary to achieve efficacious levels of exposure in patients. A phase 1/2 clinical trial of AP32788 in NSCLC patients is planned. Citation Format: Francois Gonzalvez, Xiaotian Zhu, Wei-Sheng Huang, Theresa E. Baker, Yaoyu Ning, Scott D. Wardwell, Sara Nadworny, Sen Zhang, Biplab Das, Yongjin Gong, Matthew T. Greenfield, Hyun G. Jang, Anna Kohlmann, Feng Li, Paul M. Taslimi, Meera Tugnait, Yongjin Xu, Emily Y. Ye, Willmen W. Youngsaye, Stephan G. Zech, Yun Zhang, Tianjun Zhou, Narayana I. Narasimhan, David C. Dalgarno, William C. Shakespeare, Victor M. Rivera. AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2644.
High DGAT1 expression levels in the small intestine highlight the critical role this enzyme plays in nutrient absorption. Identification of inhibitors which predominantly inhibit DGAT1 in the gut is an attractive drug discovery strategy with anticipated benefits of reduced systemic toxicity. In this report we describe our discovery and optimization of DGAT1 inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented. KEYWORDS: DGAT1, triglyceride synthesis, efflux O rally ingested triglycerides (TG) undergo hydrolysis and then are reassembled within enterocytes into TG-rich chylomicrons destined for systemic circulation. The final committed step in triglyceride biosynthesis is known to be mediated by at least two distinct intracellular acyl-coA diacylglycerol acyltransferases (DGATs), namely DGAT1 1 and DGAT2. 2 Since the development of whole-body knockout models of these enzymes, there has been intense evaluation of pharmacological approaches to modulate their activity. 3−8 For DGAT1, this interest is inspired by the favorable metabolic phenotype of DGAT1 −/− mice, 9 which are resistant to dietinduced body weight gain, 10 are more insulin-sensitive relative to wild-type littermates, 11 and exhibit a reduced rate of chylomicrons formation when challenged with lipid nutrients. 12 Interestingly, all aspects of this phenotype are lost when DGAT1 is reintroduced via a tissue-specific promoter into the intestines of female DGAT1 −/− mice, implying that intestinal DGAT1 plays a crucial role in the effects observed in the whole-body knockout model. 13 Indeed, DGAT1 mRNA expression levels have been shown to be high in regions of the small intestine in mice and humans. 14,15 Selective inhibition of intestinal DGAT1 therefore becomes an intriguing drug discovery approach to recapitulate aspects of the DGAT1 −/− mouse, especially if this gut-specific inhibition reduces the potential risk of on-and off-target activity for candidate molecules. Particularly relevant for DGAT1 pharmacological inhibition is the observation of functional and morphological abnormalities in the fur and sebaceous glands of DGAT1 −/− mice. 16 In this report we describe a novel approach to specifically inhibit intestinal DGAT1, and demonstrate the potential viability of this strategy with regard to efficacy and safety in multiple preclinical models.High-throughput screening efforts using recombinant human DGAT1 enzyme identified the benzimidazole 1 (DGAT1 IC 50 = 1.3 μM; DGAT2 IC 50 > 20 μM; Figure 1) as a potential starting point for optimization. Initial structural modifications demonstrated that both the ethyl carbamate and the 2,6-dichlorophenyl substituents on the benzimidazole core could be replaced without substantial loss of activity (i.e., 2, DGAT1 IC 50 = 1.4 μM), and in fact introducing an additional substituent at the 4-position of the 2,6-dimethylphenyl ring led to an im...
Modification of a gut restricted class of benzimidazole DGAT1 inhibitor 1 led to 9 with good oral bioavailability. The key structural changes to 1 include bioisosteric replacement of the amide with oxadiazole and α,α-dimethylation of the carboxylic acid, improving DGAT1 potency and gut permeability. Since DGAT1 is expressed in the small intestine, both 1 and 9 can suppress postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only 9 was found to be effective in suppressing body weight gain relative to control in a diet-induced obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight control. 9 has advanced to clinical investigation and successfully suppressed postprandial triglycerides during an acute meal challenge in humans.
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