Figure 1.
Biochemical and Cellular HDAC inhibition by 4b.
(A) % inhibition of human recombinant Class I enzymes HDAC1 (red), HDAC2 (green), HDAC3 (black) and HDAC8 (blue) by 4b. (B) No inhibition of ClassIIa/b enzymes by 4b; HDAC 4(green), HDAC5 (red), HDAC7 (purple), HDAC9 (orange), HDAC6 (brown). (C) Time-dependence of human recombinant HDAC3 inhibition by varying preincubation time of 4b with enzyme (as shown). (D) Cellular inhibition of endogenous Class I HDACs/HDAC6 using Boc_Lys_Ac (black traces) or Class IIa/HDAC8 HDACs using Boc_Lys_TFA substrate (red traces) by 4b (closed circles) or reference compounds SAHA and Compound 26. (E) Time- dependence of cellular Class I HDAC inhibition by varying preincubation of 4b with cells (as shown). (F) Plot of IC50 values versus compound-cell preincubation time for SAHA (green) and 4b (black).
Figure 2.
In vitro metabolite product identification of 4b and C1.
LC-MS identification of 4b (red) and C1 (green) metabolite products after incubation in mouse plasma and mouse hepatic microsomes. Metabolites of 4b included amide hydrolysis products M1 and M2, and the NADPH-dependent hydroxylated metabolites M4, M5 and M6. Metabolites of C1 included the amide hydrolysis product M3, the NADPH-dependent mono-hydroxylated products M7 and M8 and di-hydroxylated products M9 and M10. Boxed metabolites were present in both plasma and hepatic microsomal incubations, while the hydroxylated products were only present in hepatic microsomal incubations.
Figure 3.
Instability of 4b and C1 in mouse plasma and hepatic microsomes.
(A) Time course of metabolism of 4b (5 µM), and generation of metabolites M1 and M2 in mouse plasma. (B) Time course of metabolism of C1 (3 µM), and generation of metabolites M3 in mouse plasma. (C and D) Time course of metabolism of 4b (1 µM), and generation of metabolites M1, M2 and M4 in mouse hepatic microsomes, in presence (C) and absence (D) of NADPH. The dashed black line indicates the sum of 4b and metabolites measured at each time point.
Figure 4.
Pharmacokinetic evaluation of 4b in male C57BL/6NCRL mice.
(A) Plasma concentrations of 4b were monitored between 5 min and 24 h after a single subcutaneous (4.22 mg/kg eq) or single oral dose (50 mg/kg) of 4b. Amount of drug in plasma was below the limit of quantitation (LLQ = 3.07 nM eq.) after 4 h (sc) and 8 h po. (B) Equivalent brain concentrations of 4b were monitored at 0.5, 1, 8 and 24 h in the same study. Amount of 4b in brain was much lower than plasma and below the limit of quantitation (LLQ = 2.3 nM eq.) after 8 h by both routes.
Table 1.
Pharmacokinetic parameters following a single SC or PO dose to mice.
Table 2.
Brain-to-plasma concentration ratio (B:P) following a single SC or PO administration to mice.
Figure 5.
4b treatment does not affect histone acetylation in mouse brain.
(A) Representative immunoblot showing histone acetylation in mouse brain in response to 4b treatment. Mice treated with SAHA were used as a positive control (B). Acetylation at specific lysine residues on histone 3 (H3K4, H3K9, H3K14) and Histone 4 (H4K5) as well as global acetylation of H3 (Ac-H3) and H4 (Ac-H4) were studied using specific antibodies. Acetylation level was normalized to H3 and H4 expression level. (C) and (D) Quantification of (A) and (B) respectively. **P<0.01, *P<0.05 versus vehicle (veh). n = four per treatment. Error bars indicate SEM.