(T1030-05-32) Gender Differences in the Pharmacokinetics, Tissue Distribution and Metabolism of a Potent Pulmonary Arterial Hypertension Therapeutic Drug ZEX in Mice

Purpose: We previously identified ethyl (Z)-4-oxo-4-(9H-pyrido[3,4-b]indol-1-yl)but-2-enoate (ZEX, Fig. 1), a novel Chinese herbal component derived from Codonopsis Radix, as a promising candidate for treating abnormal vascular remodeling. We have demonstrated that ZEX could significantly alleviate hypoxia-and monocrotaline-induced pulmonary arterial hypertension in rodent models with minimal toxicity. In addition, we revealed that ZEX exhibited moderate in vitro intestinal absorption, and extensive hydrolysis mediated by human liver microsomes, carboxylesterase (CES) 1 and 2 to generate 4-oxo-4-(9H-pyrido[3,4-b]indol-1-yl)but-2-enoic acid (ZEXM, Fig. 1). Our preliminary pharmacokinetic profiling in male mice also revealed rapid absorption with a time taken to reach maximum plasma concentration (C_max) of 1h and elimination with a half-life (t_1/2) of 2.9 h after oral administration^[1]. To further validate its druggability and address potential gender-based therapeutic variability, the current study aimed to characterize gender differences in pharmacokinetics and tissue distributions of ZEX in male and female mice and explore the related mechanisms.

Methods: The C57BL/6 mice (8-week-old; 12 male and 12 female per time point, across 8 time points, in total 192 mice) received a single oral gavage dose of ZEX (100 mg/kg). At 0, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 hours after administration, the mice were sacrificed to gather blood and tissues including the heart, liver, spleen, lung, kidney, intestine, stomach, muscle and brain for concentration analysis of ZEX and its metabolite ZEXM by the method we previously developed^[1]. After obtaining related plasma concentration versus time profiles, pharmacokinetic parameters including C_max, the time taken to reach C_max (T_max), the area under the plasma concentration-time curve from zero to 8 hours (AUC_0-8h) and t_1/2 were calculated with WinNonlin through a non-compartmental model. In addition, tissue concentrations at different time intervals were compared. Unpaired t-tests were used to compare all pharmacokinetic parameters except T_max, which was compared with a nonparametric Wilcoxon two sample test. To further explore potential gender-specific activities of CES1 and CES2, their activities in blank plasma and tissue homogenate samples were determined using our previously developed bioluminescence method and a reported fluorescence method, respectively [2, 3]. In brief, the samples were incubated with NLMe/NCEN (specific probe substrates for CES1/CES2) followed by monitoring formation rates of their hydrolyzed metabolites NL/NAH. To evaluate differences between the females and males in CESs activities and metabolisms of ZEX, which were expressed as concentration ratios of ZEXM to ZEX, a two-way analysis of variance was performed using Prism with a p of less than 0.05 considered as statistically significant.

Results: The plasma concentration versus time profiles of ZEX and ZEXM were depicted in Fig. 1. The males demonstrated a trend of longer T_max of ZEX (1.2 ± 0.9 h) than females (0.9 ± 0.2h), however, with no significant difference. The T_max of ZEXM was the same in both genders (4.9 ± 1.8h). In addition, no significant difference was observed between the females and males in pharmacokinetic parameters including C_max of ZEX (female: 529.2 ± 259.3 ng/mL, male: 610.3 ± 319.5 ng/mL) and ZEXM (females: 903.7 ± 369.3 ng/mL, male: 877.1 ± 359.0 ng/mL), AUC_0-8h of ZEX (female: 1234.2 ± 395.4 ng·h/mL, male: 1332.6 ± 500.6 ng·h/mL) and ZEXM (female: 3826.1 ± 962.8 ng·h/mL, male: 3688.6 ± 946.9 ng·h/mL), t_1/2 of ZEX (female: 3.2 ± 1.3 h, male: 2.5 ± 1.2 h) and ZEXM (female: 4.5 ± 1.4 h, male: 6.5 ± 4.5 h). Tissue concentrations of ZEX or ZEXM after administrations were shown in Fig. 2, suggesting their high accumulations in intestine followed by kidney, stomach and plasma in both genders. Compared with females, significant differences on concentrations of ZEX (higher in kidney at 2, 4 h and in brain at 0.5 h; lower in intestine at 0.75 h and in lung at 0.25, 1, 2, 6 h) and ZEXM (higher in intestine at 2 h and in brain at 0.75 h; lower in lung at 0.25, 1, 2 h and in muscle at 0.75, 1 h), and on concentration ratios of ZEXM to ZEX (higher in intestine at 0.75, 8 h and in liver at 1, 2, 4, 6 and 8 h; lower in stomach at 0.5 h, in kidney at 1, 2, 4, 6 h and in brain at 0.5h) were observed in males. Further mechanistic investigations on gender-specific activities of CES revealed significantly higher CES1 and CES2 activities in males than in females. Specifically, CES 1 in plasma, lung, liver, intestine and CES2 in intestine of males were all higher than those in females.

Conclusion: In this study, the gender-specific pharmacokinetics of a potent pulmonary arterial hypertension therapeutic candidate ZEX in mice were investigated for the first time. Significantly higher CES1 and CES2 in the liver and intestine of male mice could lead to statistically significantly higher metabolisms of ZEX in these tissues.

References: [1] J. Zhang1, Y. F. Zhang, Q. Huang, J. J. Zou, Q. B. Song, J. H. Li, L. He, L. J. Luo, D. M. Gou, Y. Huang, Z. Zuo. Druggability Evaluation of ZEX, a Novel Derivative of Component from Codonopsis Radix. American Association of Pharmaceutical Scientists 2024 PharmSci 360, Salt Lake City, UT, United States.
[2] J. Zhang, D. D. Wang, L. W. Zou, M. Xiao, Y. F. Zhang, Z. W. Li, L. Yang, G. B. Ge, Z. Zuo. Rapid Bioluminescence Assay for Monitoring Rat CES1 Activity and Its Alteration by Traditional Chinese Medicines. Journal of Pharmaceutical Analysis 2020, 10 (3), 253–262.
[3] J. Qiang, L. Feng, D. D. Wang, Z. Dai, P. Wang, L. W. Zou, Z. H. Liu, J. Y. Wang, Y. Yu, G. B. Ge, J. N. Cui, L. Yang. A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues. ACS Applied Materials & Interfaces 2015, 7(51), 28474–28481.

Acknowledgements: This work is financially supported by Guangdong-Hong Kong Technology Cooperation Funding Scheme (GHP/209/20SZ, SGDX 20201103095404019).


Fig. 1 Mean plasma concentration versus time profile of ZEX (Upper) and ZEXM (Bottom) in female and male mice after oral administrations of ZEX at 100 mg/kg. Data were expressed as mean ± SD, n = 12.


Fig. 2 Tissue concentrations of ZEX (Upper)/ZEXM (Middle) and ratio of ZEXM to ZEX (Bottom) at 0.25, 0.5, 0.75, 1, 2, 4 and 8 h in plasma and tissues of female and male mice after oral administrations of ZEX at 100 mg/kg. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with those of female mice. Data were expressed as mean ± SD, n = 12.


Fig. 3 Comparison of CES1 (Upper) and CES2 (Bottom) activities in plasma and tissues of female and male mice. *p < 0.05, ***p < 0.001 and ****p < 0.0001 compared with those of female mice. Data were expressed as mean ± SD, n = 3.