PMC

Mice with xenograft (A) A4573, * control vs. racemic p=0.008, * control vs. (S)-YK-4-279 p=0.005, and (B) SK-ES tumors were treated for 3 days BID with IP injections of vehicle control, racemic, (S)-, or (R)-YK-4-279 when tumors reached 200 mm³. ** Control vs. racemic p=0.003, * control vs. (S)-YK-4-279 p=0.006 (○ Control, ■ racemic YK-4-279, ▲ (S)-YK-4-279 and ◊ (R)-YK-4-279). (C) Examples of H&E staining from animals treated in (A). Scale bar = 50 ¼m. (D) TUNEL staining from animals treated in (A). Scale bar = 50 ¼m. (E) Quantifcation of TUNEL positive cells out of 500 counted on each slide from each tumor, ** control vs. racemic p=0.004, ** control vs. (S)-YK-4-279 p=0.002, * (R)-YK-4-279 vs. racemic p=0.03, * (R)-YK-4-279 vs. (S)-YK-4-279 p=0.01.

Effects of nilvadipine enantiomers on Aβ production, sAPPβ secretion, and BACE-1 expression. A, dose-dependent inhibition of Aβ₄₀ production by 7W CHO cells overexpressing Aβ. ANOVA reveals a significant main effect of (−)-nilvadipine (p < 0.001), (+)-nilvadipine (p < 0.001), and racemic nilvadipine (p < 0.001) on Aβ₄₀ production. Post hoc comparisons show significant differences in Aβ₄₀ levels between control and (−)-nilvadipine (p < 0.001), control and (+)-nilvadipine (p < 0.001), and control and racemic nilvadipine (p < 0.001) for doses of nilvadipine greater than 1 μm. No significant difference in Aβ₄₀ values was observed among (−)-nilvadipine, (+)-nilvadipine, and racemic nilvadipine treatments for all doses tested (p > 0.05) showing that (−)-nilvadipine, (+)-nilvadipine, and racemic nilvadipine dose-dependently inhibit Aβ₄₀ to the same extent. B, representative Western blots showing that (−)-nilvadipine reduces the secretion of sAPPβ in 7W CHO cells overexpressing APP showed an inhibition of the β-cleavage of APP, whereas sAPPα secretion is not significantly impacted. C, effects of (−)-nilvadipine and racemic nilvadipine on TNFα-induced BACE-1 transcription in human neuron-like cells SH-SY5Y. SH-SY5Y cells were treated with (−)-nilvadipine, racemic nilvadipine, and TNFα alone and in combination for the period of time indicated in the bar graph. BACE-1 transcription was quantified by quantitative real time RT-PCR using the housekeeping GAPDH mRNA as a reference. ANOVA reveals a significant main effect of treatment duration (p < 0.001), (−)-nilvadipine (p < 0.001) and racemic nilvadipine (p < 0.001) on BACE-1 mRNA levels as well as an interactive term between treatment duration and (−)-nilvadipine (p < 0.006) and between treatment duration and racemic nilvadipine (p < 0.003). Post hoc comparisons show significant differences between the control conditions and TNFα treatment for all the time points studied (p < 0.001) showing that TNFα is stimulating BACE-1 transcription. BACE-1 mRNA levels are also significantly reduced in SH-SY5Y cells co-treated with TNFα and (−)-nilvadipine (p < 0.01) and in cells co-treated with TNFα and racemic nilvadipine (p < 0.001) compared with the TNFα treatment conditions for all the time points tested showing that (−)-nilvadipine and racemic nilvadipine antagonize BACE-1 transcription induced by TNFα. * denotes statistically significant differences compared with the control conditions, and $ indicates statistically significant differences compared with the TNFα treatment conditions. D, effects of racemic nilvadipine and (−)-nilvadipine on basal BACE-1 protein expression. HEK293 cells were treated for 24 h with 20 μm racemic nilvadipine or (−)-nilvadipine. BACE-1 protein expression was analyzed by Western blots in cellular lysates, and actin was used as a reference protein. Quantification of BACE-1/actin chemiluminescent signals reveal a significant decrease in BACE-1 protein expression following (−)-nilvadipine (p < 0.01) and racemic nilvadipine (p < 0.01) treatments.

(A) Plasma concentrations of (S)-YK-4-279 were simulated for a dosing regimen of BID IP injections of YK-4-279 (375 mg/kg) for 3 days followed by 2 days of no therapy. This cycle was repeated for another 5-day period, in order to maintain plasma concentrations of (S)-YK-4-279 greater than 3 ¼M. (B) A4573, (* p=0.04) or (C) SK-ES xenograft tumors were treated BID with IP injections of 400 mg/kg racemic YK-4-279 when tumors reached 200 mm³ (O Control and ■ racemic YK-4-279) (* p=0.02). (D) Mice with xenograft SK-ES tumors were treated BID with IP injections of 400 mg/kg racemic-, (S)-, or (R)-YK-4-279 over a 10-day period after tumors reached 200 mm³ (○ Control, ■ racemic YK-4-279, ▲ (S)-YK-4-279 and ◊ (R)-YK-4-279). ** Control vs. racemic, p=0.003, * control vs. (S)-yk-4-279 p=0.02. (E) Racemic YK-4-279 (red) treated SK-ES xenograft mice show survival benefit compared to those of control (black), (S)-YK-4-279 (green), and (R)-YK-4-279 (blue) treated mice. * Control vs. racemic p=0.013. (F) (S)-YK-4-279 xenograft tumor (SK-ES) tissue levels of racemic YK-4-279 treated mice showed higher (S)-YK-4-279 concentration than those of control, (S)-YK-4-279, (R)-YK-4-279 (◀ Control, ■ racemic YK-4-279, ▲(S)-YK-4-279 and ◊ (R)-YK-4-279).

Comparisons of efficacy (A) and safety (B) for all single-enantiomer and racemic drug pairs.