Mide (4)–The final compound is made by deprotection of Boc group from tert-butyl (2-(pyrazine-2carboxamido)phenyl)carbamate using dichloromethane and trifluoroacetic acid (1:1) mixture at room temperature for 30 min, which was then made free base by suspending the crude mixture into aqNaHCO3 solution and extraction into dichloromethane. The organic layer was evaporated to obtain the pure final compound with quantitative yield (0.016 g). Inhibitory activity of BG45 against individual HDAC isoforms was determined as previously described 12. Murine xenograft models CB17 SCID mice (484 days old) were purchased from Charles River Laboratories (Wilmington, MA). All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Dana-Farber Cancer Institute. After irradiation (200cGy), mice were subcutaneously injected with 506 MM.1S cells in the right flank. BG45 and bortezomib were dissolved in 10 Dimethylacetamide (DMSA; Sigma-Aldrich) in 10 KolliphorHS15 (Sigma-Aldrich) in phosphate buffered saline (PBS) and 0.9 saline solution, respectively. When tumors were measurable, mice were treated with intraperitoneal injection (IP) of vehicle control, BG45 (15 mg/kg), or BG45 (50mg/kg) 5 days a week for 3 weeks (n=6/group). Additionally, mice were also treated with 50 mg/kg BG45 in combination with 0.5 mg/kg (subcutaneous injection) bortezomib twice a week. Tumor size was measured every three days, and tumor volume was calculated with the formula: V=0.Tezepelumab 5(a 2), where “a” is the long diameter of the tumor and “b” is the short diameter of the tumor.Iptacopan Mice were sacrificed when the tumor reached 2cm in length or 2cm3 volume, or if mice appeared moribund to prevent unnecessary morbidity. Survival was evaluated from the first day of the treatment until death. Statistical analysis The combined effect of drugs was analyzed by isobologram analysis using the Compusyn software program (ComboSyn, Inc.); a combination index (CI) 1 is indicative of a synergistic effect. In the murine xenograft studies, statistical significance was determined by Student t test. The minimal level of significance was p 0.05.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLeukemia. Author manuscript; available in PMC 2014 September 16.Minami et al.PageResultsMS275 is more cytotoxic than Merck60 in MM cells Non-selective HDACi have demonstrated variable anti-MM activity in preclinical studies. We first examined the growth inhibitory effect of Merck60 (HDAC1, 2 inhibitor previously reported as compound #60 by Method et al. PMID 18182289) versus MS275 (HDAC1, 2, 3 inhibitor) in MM cell lines using MTT assay.PMID:23671446 MS275 triggered significant MM cell growth inhibition, whereas Merck60 induced only a modest growth inhibition effect (Figure 1A). Immunoblotting confirmed that all MM cell lines express HDAC1, 2, and 3 proteins (Figure 1B). We next examined the effects of these agents on acetylation of histones in RPMI8226 MM cells. Importantly, MS275 in a dose-dependent manner more potently induced acetylation of histones (H2A, H2B, H3 and H4) and increased p21WAF1 expression than Merck60 (Figure 1C). These results suggest that HDAC3 plays an important role in MM cell growth and/or survival. HDAC3 knockdown inhibits MM cell growth To determine that the MM cell growth inhibitory effect of MS275 is predominantly due to HDAC3 inhibition, we next performed knockdown of HDAC isoforms (HDAC 1, 2, and 3) using a lentiviral shRNA infection.
