| A novel natural tyrosinase inhibitor for skin hyperpigmentation UP302, 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl) propane, was isolated from Dianella ensifolia (Liliacea), a native plant of Southeast Asia. The activity of tyrosinase is closely related to dermatological disorders, the browning of food etc.. Inhibition of activity of tyrosinase is significant for the treatment of dermatosis, food fresh-keeping and pesticides in agriculture. UP302 is being developed into a new drug for the treatment of dermatological disorders, therefore it is necessary to study pharmacokinetics and toxicokinetics of UP302.In this investigation, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated to determine UP302 in plasma, skin, urine, feces and microsomes system. The method was successfully applied to a preclinical pharmacokinetics of UP302 in rats.Firstly, biological samples were precipitated by methanol using daidzein as an internal standard. On Hypersil Gold C18 column, separation was achieved by a gradient mobile phase of methanol and 5 mmol·L-1 ammonium formate solution at a flow rate of 0.2 mL·min-1 at 30°C. The total analysis time was 6 min. Selection-reaction monitoring mode transitions at m/z 301.1→135.2 for UP302, m/z 252.9→132.0 for daidzein in negative-mode electrospray ionization was used to detect UP302 in biological matrix. The specificity, sensitivity, detection range, accuracy and precision of this method can meet the requirements of the pharmacokinetic studies.Secondly, pharmacokinetics was carried out in rats intravenously injected with UP302 at a single dose of 5 mg·kg-1. At designated time points after dosing, 0.3 mL of blood samples were taken and centrifuged in heparin-coated microcentrifuge tubes to obtain plasma and measured by UPLC-MS/MS. Pharmacokinetic parameters of UP302 were calculated by WinNonLin 5.2.1 at noncompartmental model. The result indicated that UP302 was widely distributed and eliminated rapidly.Thirdly, absorption and excretion of UP302 cream applied to rats in two groups after 10 mg·cm-2 transdermal administration was investigated. One group was used to collect blood samples at 1,2,4,6,8,12,16,20 and 24 h and the skin at 24 h, and the other was used to collect urine and feces. UPLC-MS/MS was employed to determine UP302 in plasma, skin, feces and urine. The distribution percentage values of UP302 in plasma, skin and feces were 8.07%, 0.087% and 0.091%, respectively. No UP302 was found in urine. There was 78.96% of UP302 cream remained on skin.Fouthly, based on the short half-life of UP302 in plasma, it was assumed that UP302 was maybe metabolized by liver P450 enzyme, therefore it is necessary to study the stability of UP302 in microsomes system. A UPLC-MS/MS method was fast, simple, specific, sensitive and successively applied to investigate enzyme kinetics of UP302 metabolism in rat liver microsome. After optimization of incubation time, substrate concentration and enzyme concentraion, enzyme kinetics of UP302 metabolism in rat liver microsomes was studied. Enzyme kinetic parameter are obtained:maximum reaction speed Vmax of 196.08μmol·L-1·min-1·g-1, Km of 14.98μmol·L-1, CLint of 13.09 mL-1·min-1·g-1.Finally, stability of UP302 in rat, dog, monkey and human plasma was investigated. UP302 was found to decline quickly at first four hours and then keep stable.
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