Pre-clinical ADME Study of Kinsenoside, an Antihyperlipidemic Drug Candidate

Abstract

Kinsenoside, the herb derived medicine mainly isolated from Anoectochilus (Orchidaceae), a medicinal plant traditionally used in Asian countries. Kinsenoside possessed diverse pharmacological activities, including antihyperlipidemic, antihyperglycemic, anti-inflammatory, antioxidant, immunostimulating, hepatoprotective, anti-osteoporosis, and vasculoprotective, effects. Kinsenoside is considered as a promising drug candidate, due to its diverse pharmacological activities, particularly, antihyperlipidemia drug candidate, and currently being extensively investigated for its therapeutic efficacy. In the current study, the chemical and enzymatic (metabolic) stability of kinsenoside were evaluated, as an essential screening for a potential drug candidate. Results indicated that kinsenoside stability was good in solvent as well as in human and rat liver microsomes. Then kinsenoside pharmacokinetic (PK) study has also been investigated. A sensitive, rapid, and reliable bioanalytical method using hydrophilic interaction liquid chromatography (HILIC), tandem mass spectrometry (MS/MS) was developed for kinsenoside. The column retention and ex-vivo stability of kinsenoside were optimized. In sample preparation, the plasma sample was pre-treated with 1% acetic acid, then plasma proteins were precipitated with acetonitrile and methanol (70:30, A:M). An HILIC silica column (2.1 mm × 100 mm, 3 µm) was used for chromatographic separation. The mobile phases (A) consisted of 0.1% acetic acid in water, and (B) consisted of 0.1% acetic acid in acetonitrile. Step-gradient elution at a flow rate of 0.2 mL/min was adapted. For mass spectrometric detection, the multiple reaction monitoring mode (MRM) transitions were m/z 265.2 → m/z 102.9 for kinsenoside, and m/z 163.3 → m/z 132.1 for the internal standard (IS) nicotine in the positive ionization mode. A calibration curve in the range of 2–500 ng/mL was constructed. The intraday and interday accuracy and precision were within 5%. The HILIC-MS/MS method was specific, selective, accurate, and reproducible, which was successfully applied in a pharmacokinetic study of kinsenoside in rats. To predict the potential for kinsenoside-drug interactions, the effects of kinsenoside on CYP enzyme-mediated drug metabolism was investigated. Kinsenoside was tested at different concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 µM in human liver microsomes. The cocktail probe assay based on liquid chromatography-tandem mass spectrometry was conducted to measure the CYP inhibitory effect of kinsenoside. Subsequently, the metabolism profiles of amlodipine and lovastatin in human liver microsomes were analyzed following co-incubation with kinsenoside. The concentration levels of the parent drug and the major metabolites were compared with the kinsenoside co-treated samples. The effect of kinsenoside was negligible on the enzyme activity of all the CYP isozymes tested even though CYP2A6 was slightly but non-significantly inhibited at higher concentrations. The drug-drug interaction assay also showed that the concomitant use of kinsenoside has a non-significant effect on the concentration of lovastatin or amlodipine, and their major metabolites. So, it was concluded that kinsenoside has good metabolic stability in rat and human liver microsomes. The pharmacokinetic study reveals that kinsenoside is well distributed to the tissues (Vd: 19.9 L/kg), and smoothly clear from the body (Cl: 20.8 L/min/kg; T(1/2) 0.68 h). Furthermore, its concomitant use is considered safe with conventional medicine, as there is almost no risk of drug interaction between kinsenoside and CYP drug substrates via CYP inhibition.