(T1530-02-12) Druggability Evaluation of ZEX, a Novel Derivative of Component from Codonopsis Radix

Purpose: As a new derivative of the Chinese herbal component from Codonopsis Radix developed by us, ethyl (Z)-4-oxo-4-(9H-pyrido[3,4-b]indol-1-yl)but-2-enoate (ZEX, Fig. 1) could significantly alleviate vascular remodeling in rodents. To further explore the druggability of ZEX, the current study was proposed aiming to evaluate the absorption, metabolism, plasma pharmacokinetics and tissue distribution of ZEX in mice.

Methods: A LC-MS/MS method was first developed for analysing ZEX concentrations in buffer/plasma/tissue homogenate. Briefly, about 100 μL of the sample was added with 200 μL of acetonitrile containing 150 ng/mL diclofenac (internal standard). followed by centrifugation to obtain the supernatant for LC-MS/MS analyses with Agilent 1290 UPLC coupled to an Agilent 6430 Triple Quadrupole mass spectrometer via a Xbridge C18 column (250 mm×4.6 mm, 5 µm). The mobile phase composed of 0.1% formic acid in water/acetonitrile (45/55, v/v) at a flow rate of 0.6 mL/min was employed, and multiple reaction monitoring detections with transitions of m/z 295.1→167.0 for ZEX and m/z 296→214 for diclofenac were operated in a positive ion mode. The intestinal permeability of ZEX was studied using the Caco-2 cell monolayer model by adding 10 μg/mL ZEX solutions to either the apical or basolateral side. After incubation at 37 °C for 180 min, samples from the receiver chamber were collected for LC-MS/MS analyses of ZEX concentrations. To estimate its metabolisms, ZEX at 2, 10 or 15 μg/mL was incubated with rat liver microsome (RLM)/human liver microsome (HLM), human carboxylesterase 1 (hCES1)/human carboxylesterase 2 (hCES2), HLM in the presence of NADPH and magnesium chloride  at 37 °C in 100 mM phosphate buffered saline (pH 7.4) for 60 min. To terminate the reaction, about threefold volumes of acetonitrile was added followed by centrifugation of the mixture at 13000 rpm for 10 min at 4°C. About 20 μL of the supernatants were subject to LC-MS/MS for analysis of the remaining ZEX concentrations. The human plasma protein binding of ZEX was investigated by rapid equilibrium dialysis. Briefly, ZEX at 5, 10 and 20 μM were incubated with human plasma in dialysis buffer at 37°C for 4 h, followed by adding threefold volumes of acetonitrile containing with diclofenac 300 ng/mL and centrifugation at 13000 rpm for 10 min at 4 °C. About 20 μL of the supernatants were injected into LC-MS/MS for analyses of ZEX concentrations. The plasma protein binding of ZEX was estimated by the percentages of bound concentrations of ZEX to its total concentrations. To further evaluate its plasma pharmacokinetics and tissue distributions, eighteen (3 mice/time point for 6 time points) 8-week-old BL/C57 male mice received a single oral dose of 100 mg/kg ZEX. Three mice were sacrificed at 0, 0.5, 1, 2, 4 and 8 hours post dosing to collect blood and tissues including heart, liver, spleen, lung, kidney, intestine, stomach, testicular fat, muscle and brain for analyses of ZEX concentrations by above-mentioned LC-MS/MS assay. Finally, ZEX pharmacokinetic parameters including maximum plasma concentration (C_max), time to reach C_max (T_max), elimination half-life (t_1/2) and the area under the plasma concentration versus time curve from zero to 8 hours (AUC_0-8h) were calculated by WinNonlin software using a non-compartmental model.

Results: ZEX in Caco-2 model demonstrated a permeability coefficient value between 10^-6 and 10^-5 cm/s, suggesting its medium extent of intestinal absorption. As shown in Fig. 2i/ii, incubation of ZEX at 15 μg/mL with the RLM/HLM/hCES1/hCES2 resulted in significant decrease of concentrations, suggesting a potential significant in vivo CES mediated hydrolysis of ZEX to ZEXM (Fig. 1). Since NADPH had no in impact on ZEX concentration when it was incubated with HLM (Fig. 2iii), minimum oxidation/reduction metabolism of ZEX was suggested despite its significant hydrolytical metabolism in HLM. ZEX was found to have a high human plasma protein bindings ranging from  94.0 ± 2.0% to 97.9 ± 2.1% at the concentrations of 5, 10 and 20 µg/mL. After oral administration of ZEX at 100 mg/kg, the plasma concentration versus time profile of ZEX shown in Fig. 3i indicated that its C_max of 638.9 ± 60.1 ng/mL could be achieved at 1 hour post dosing with the AUC_0-8h of 1453.0 ± 55.6 ng·h/mL and a t ½ of 2.9 ± 1.7 h. Tissue distributions of ZEX at 8h after oral administration in Fig. 3ii suggested its high accumulation in the kidney followed by intestine, stomach and plasma.

Conclusion: ZEX has demonstrated a medium extent of intestinal absorption with high plasma protein binding and extensive hydrolysis in liver microsome. After oral administration, ZEX could reach its maximum plasma concentration in 1 h and eliminate from body with a t_1/2 of 2.9 h. Our current study for the first time provide scientific evidances for the druggability of ZEX as a new therapeutic drug for the treatment of abnormal vascular remodeling.

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

Fig. 1 Chemical structures of ZEX and ZEXM

Fig. 2 Concentrations of ZEX after its incubation at 37 °C for 60 min i) at 2/10 μg/mL without/with human liver microsome (HLM, 1 mg/mL) in the absence/presence of NADPH (5 mM) and ii) at 15 μg/mL with human carboxylesterase 1 (hCES1, 1 mg/mL)/human carboxylesterase 2 (hCES2, 1 mg/mL). Data were expressed as mean ± SEM, n = 3. ** p < 0.01, *** p < 0.001; ns: no significant difference.

Fig. 3 Mean plasma concentration versus time profile of ZEX (i) and tissue distributions of ZEX at 0.5, 1, 2, 4 and 8 h (ii) in mice after oral administrations of ZEX at 100 mg/kg. Data were expressed as mean ± SEM, n = 3.