In Vitro Trial On Biotransformation Of Mptp To MPP～+ Catalyzed By Mao-b And Studies Of Mao-B Inhibition Of Isatin

Isatin(ISA) has long been known as a pharmacologically active agent which exerts a number of in vitro and in vivo effects, especially as an MAO-B inhibitor. The molecular basis of this inhibition is unknown. l-methyl-4- phenyl-l,2,3,6-tetrahydropydine (MPTP) is a toxin well-known to cause neuro-degeneration in the dopaminergic system of the nigro-striatum of primates and rodents, with symptoms very similar to human parkinsonism. The metabolism of MPTP to MPP+ proved to be carried out by MAO-B. So MPTP is a prime tool for the study of PD and MAO-B inhibitor.Objective: To establish a method for the study of the biotransforma-tion of MPTP in vitro. To investigate the inhibition effect of ISA on MAO-B. To explain the molecular mechanism of ISA in the treatment of PD.Methods: MPTP of various concentration(0-40 g/ml) was incubated with mice brain homogenates at 37 C for 60min. All incubations were terminated by the addition of an equal volumn of cold acetonitrile and the mixtures were centrifuged , the supernatant fractions were analyzed by HPLC to determine the concentration of MPP+. A Shimaz LC-6A HPLC system was used. The precolumn (4.6mm 2cm) and an analytical column (4.6mm 20cm) were packed with 5 Hypersil ODS2 C18. The mobile phase consisted of acetonitril/0.1M acetic acid (85:15,v/v) adjusted to pH5.6 withtriethlamine; the flow rate was 1.3ml/min. To observe the inhibiting effect of ISA on MAO-B, ISA of various concentrtion was preincubated with mice brain homogenates at 37 C for 10min using deprenyl(DEP) as control.Results: The results showed that MPTP was converted to MPP+ by MAO-B in vitro. Over the range 0.1-10ug/ml MPP+ homogenate concentration, the peak area show good linear relationship with concentration (r= 0.9998). The recovery and pricision were both very good. The selectivity of the assay was good with no interfering peaks.The data showed that MPP+ concentration decreased with the increase of ISA concentration. The efficacy of DEP is superior to that of ISA. But in this particular experiment(concentration of ISA is 10 times of that of DEP), there were no remarkable defferences between the regression coefficients of the two kinds of drug. These observations led to the hypothsis that a similar MAO-B inhibition might be involved in the treatment of PD with ISA and DEP.Conclusion: This paper described a new procedure for the study of interference of ISA in the metabolism of MPTP in vitro for the first time in the world. The method was simple and applicable. The quantitation of MPP+ in crude samples by HPLC with a UV detector could be used widely. The specific MAO-B inhibition of ISA was estabished and a potential therapeutic use in PD's treatment is proposed for ISA.