Biotransformation And Excretion Study Of MSY-001in Rats

Cancer, one of the most dreaded diseases, continues to spread with increasing incidence. Of all available anti-cancer treatment, chemotherapy remains the most important therapeutic method. In recent years, screening of the anti-cancer ingredients from herb plants have become an important approach for drug discovery and development, since natural products represent an unparalleled source of molecular diversity. MSY-001is an active ingredient of the traditional Chinese medicine, Rhizoma Curcumae.The anti-tumor effect and the molecular mechanism of MSY-001have attracted great attention. In vitro studies, MSY-001showed stronger growth inhibitions on Hela, Hep-2, HL-60, PC3, SGC-7901and HT-1080cells and its activity was far more stronger than5-Fu and roughly equal with cisplatin. It also exhibited inhibitory effects on the growth of uterine cervical (U14) and sarcoma180(S180) tumors in mice. Thus, there is a need to explore the metabolic and excretive properties of MSY-001in vivo and eventually to support its pre-clinical ADME studies. The main objectives of the present study were to characterize the metabolites of MSY-001in rats and establish a series of LC-MS analytical methods for excretion study. 1. Identification of urinary metabolites of MSY-001in ratsObjective:To characterize the metabolism of MSY-001after oral administration in rats, including isolation and identification of metabolites by chemical and spectral methods.Methods:By contrasting the urine samples after dose of the oil formulation of MSY-001to rats, six major distinctive metabolite peaks were observed in the sample group but not in the control group. Using macroporous adsorption resin chromatography, Sephadex LH-20and preparative high-performance liquid chromatography,8metabolites were isolated. All of the metabolites were characterized on the basis of UV, HR-ESI-MS,1H NMR,13C NMR and two-dimensional NMR information. And the metabolic pathways of MSY-001in rats was proposed.Results:From the rat urine after oral administration of MSY-001, eight phase I metabolites were identificated, with four new metabolites,2a-hydroxyl-aeruginolactone (M2),14-hydroxyl-aeruginolactone (M3), aeruginolactone B (M6), and1β,8β-dihydroxyeudesm-4,7(11),-dien-8a,12-olide (M4-la), and four known metabolites,1β,8β-dihydroxyeudesm-4(14),7(11)-dien-8a,12-olide (M4-lb), aeruginolactone (M5),1β,8β-dihydroxyeudesm-3,7(11),-dien-8a,12-olide (M4-2) and1β,10a,4a,5β-diepoxy-8ahydroxy-glechoman-8a,12-olide (Ml). Hydroxylation, epoxidation and oxidative skeletal rearrangement seemd to be the main metabolic pathways of MSY-001and M5might be precursors of the others.2. Development of LC-MS method for determination of MSY-001and its metabolites in urine, bile and fecesObjective:To develop rapid and sensitive LC-MS analytical methods for application in the excretion study of MSY-001in rats. Methods:On the basis of the metabolites isolated, a series of LC-MS analytical methods were established and validated for determination of MSY-001and its metabolites in rat urine, bile and feces. Liquid-liquid extraction and solid-liquid extraction were employed for sample preparation.Results:Four LC-MS analytical methods were established and validated for determination of MSY-001and its metabolites in rat urine, bile and feces including:(1) In urine samples, the method was validated over the range of16.68-1668ng/mL,31.25-3125ng/mL,23.62-2362ng/mL,28.93-2893ng/mL and16.35-1635ng/mL for Ml to M5respectively with a correlation coefficient>0.99. The lower limit of quantification was16.68ng/mL for Ml (RSD<5%),31.25ng/mL for M2(RSD<6%),23.62ng/mL for M3(RSD<5%),28.93ng/mL for M4(RSD<7%) and16.35ng/mL for M5(RSD<10%). Inter-day precision were all less than10%. For each metabolite the method recovery was between93%and108%and the extraction recovery was between50%and95%. Samples were considered to be stable under conditions including-80℃storage in stock solution for3,20days and two cycles of freeze-thaw;(2) In feces samples, the methods were validated over the range of0.4675-116.9ng/mg and2-500ng/mg for M5and MSY-001respectively with a correlation coefficient>0.99. The lower limit of quantification was0.4675ng/mL for M5(RSD<5%) and2ng/mg for MSY-001(RSD<5%). Inter-day precision was less than5%and10%for M5and MSY-001respectively. For M5, the method recovery was above90%and the extraction recovery was above95%. For MSY-001, the method recovery was above95%and the extraction recovery was above60%;(3) In bile samples, the method was validated over the range of53.38-3336ng/mL,100-6250ng/mL,75.59-4724ng/mL,370.3-23143ng/mL and209.2-13076ng/mL for M1to M5respectively with a correlation coefficient>0.99. The lower limit of quantification was53.38ng/mL for Ml (RSD<5%),100ng/mL for M2(RSD<5%),75.59ng/mL for M3(RSD<5%),370.3ng/mL for M4(RSD<5%) and209.2ng/mL for M5(RSD<5%). Inter-day precision were all less than10%. For each metabolite the method recovery was between88%and107%and the extraction recovery was between75%and95%.3. Excretion study of MSY-001after oral administrationObjective:To determine the excretion rate after a single administration of MSY-001and eventually to support the mass balance study of MSY-001formulation.Methods:The LC-MS analytical methods were applied to measure the concentration of MSY-001and metabolites urine, bile and feces from rats after a single administration of MSY-001.Results:The metabolites, including M1to M5, isolated from urine were also detected in bile partly as glucuronic acid and sulfate conjugates, and extremely low amounts of parent drug were detected in urine and bile. The total excretion rate of MSY-001and metabolites from urine and feces was41.87±9.3%(n=6), while the total excretion from bile was6.430±2.5%(n=6). The major route of excretion for MSY-001and its metabolites was through feces in the form of MSY-001and M5.