Preclinical studies of YK-4-272, an inhibitor of class II histone deacetylases by disruption of nucleocytoplasmic shuttling

Abstract

Purpose: The HDAC shuttling inhibitor, YK-4-272 functions by restricting nuclear shuttling of Class II HDACs. Pre-clinical investigations of YK-4-272 bioavailability, pharmacokinetics, in vivo toxicity and tumor growth inhibition were performed to determine its potential as an HDAC shuttling disruptor for use in clinical applications.

Methods: The solubility, lipophilicity, in vitro metabolic stability, in vitro intestinal permeability, and in vivo pharmacokinetics of YK-4-272 were determined by HPLC methods. The anti-tumor activity of YK-4-272 was determined by monitoring athymic Balb/c nude mice bearing PC-3 xenografts.

Results: Oral bioavailability of YK-4-272 is supported by its solubility (0.537 mg/mL) and apparent partition coefficient of 2.0. The compound was chemically and metabolically stable and not a substrate for CYP450. In Caco-2 cell transport studies, YK-4-272 was highly permeable. The time-concentration profile of YK-4-272 in plasma resulted in a C ( max ) of 2.47 μg/mL at 0.25 h with a AUC of 3.304 μg × h/mL. Treatment of PC-3 tumor xenografts with YK-4-272 showed significant growth delay.

Conclusions: YK-4-272 is stable and bio-available following oral administration. Growth inhibition of cancer cells and tumors was observed. These studies support advancing YK-4-272 for further evaluation as a novel HDAC shuttling inhibitor for use in cancer treatment.

Fig 1

(a) Chemical structure of YK-4-272. (b) Metabolic stability of YK-4-272 in human liver microsomes. 10 µM of YK-4-272 was incubated with 10-donor mixed gender pooled human liver microsomes (1 mg/mL) for 2 h. (c) Chemical stability of YK-4-272. YK-4-272 was incubated in pH 1.2 hydrochloric acid buffer or pH 6.8 isotonic phosphate buffer at 37°C for 24 h. At the appropriate time intervals, depletion of YK-4-272 was monitored by analyzing the concentration of YK-4-272 through HPLC. Each bar represents the mean ± S.D. (n=3).

Fig 2

Cytochrome P450 metabolism of YK-4-272. Specific activity (%) of YK-4-272 and reference compounds (furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole) on CYP1A2, 2 C9, 2 C19, 2D6, and 3A4 high throughput inhibition screening is shown in a dose range from 0.001 to 1000 µM (logarithmic scale). (a) % specific activity of YK-4-272 and furafylline on CYP1A2. (b) % specific activity of sulfaphenazole, tranylcypromine, quinidine, and ketoconazole on CYP2C9, 2 C19, 2D6, and 3A4, respectively. (c) % specific activity of YK-4-272 on CYP2C9, 2C19, 2D6, and 3A4. The mean of duplicate analysis is shown.

Fig 3

Plasma concentration of YK-4-272 after oral administration. 100 mg/kg YK-4-272 suspension was prepared with 0.5% Tween 80 aqueous solution and administered to rats (225 ± 10 g) by a gastric intubation. After an appropriate time interval, blood was collected and the concentration of YK-4-272 in the plasma was determined by HPLC. Each bar represents the mean ± S.D. (n=5).

Fig 4

Antitumor activity of YK-4-272 on the growth of human prostate PC-3 tumor xenografts. Male athymic Balb/c nude mice were injected with PC-3 cells and the resulting tumors allowed growing for a week before i.p. injection once a day every other day with 10 mg/kg of YK-4-272 or vehicle in 1:1 PEG/ PBS solution. (a) The tumor volumes were monitored up to 30 day. The tumor size of each mouse was measured by caliper and calculated by the formula: Length×width×height/2. Each bar represents the mean ± SEM (n=4). (b) Expression of HDAC4 in PC-3 tumor either 1) control (untreated) or 2) YK-4-272 treated. HDAC4 protein levels in tumor were analyzed by Western blotting. Actin expression is shown as a loading control. Each data point represents the mean value of optical density for HDAC4 from three experiments with similar results. (c, d) Tumors were stained with HDAC4 antibody and counterstained with Hematoxylin. (c) control (d) treated with YK-4-272.