| AIM: (1) To investigate the metabolic characteristics of the analogues of m-Nif in the liver microsomes, rat hepatocytes, and Beagle dog. (2) To explore new ideas and methods for seeking new drugs.METHODS: (1) One-step method was adopted to synthesize m-Nif, while three-steps method was introduced to synthesize MN9201 and MN9202. First, propyl 3-aminocrotonate and amyl 3-aminocrotonate were synthesized with propyl acetoacetate or amyl acetoacetate. Then, under sulfuric acid catalyzed, 2-acetyl-3-arly-methyl acrylate was synthesized with methyl acetoacetate and meta-nitrobenzaldehyde. Finally, MN9201 and MN9202 were synthesized by using the methods described by Hanstzsch. These synthesized chemicals were validated by melting point, the content and ultraviolet scan graph. (2) Conditions of chromatography and the extraction of the samples were selected by orthogonal experimental design. Orthogonal experimental design with L16 (45) was introduced, the factors on the selection of m-Nif and MN9201 were the volum of alkalization, the extraction times, the ratio of methanol, the concentration of buffer and pH of thebuffer. Orthogonal experimental design with L9( 34)was used, there were four factors on the selection of MN9202 and MN9203, including the volum of alkalization, the extractable times, the ratio of acetonitrile and pH of the buffer.(3) m-Nif,MN9201, MN9202 and MN9203 were intravenously administered to the Beagle dogs at the dose of 0.288, 0.312, 0.335 and 0.358 mg-kg"1 respectively. After iv administered, .the blood samples were collected at 0,5,10,20,40,60,120,180,240,300,360,420,480 and 600 min. m-Nif was orally administered at the dose of 1.152, 3.456 and 10.370 mg-kg-1 respectively. After ig administered, the blood samples were drawn at 0,5,15,30,45,60,90,120, 180,240,300 and 420min.All collected blood samples were centrifuged to obtain plasma and the concentration of the analogue of m-Nif in plasma was detected by RP-HPLC. The pharmacokinetic parameters and PK model were analysed by using the 3P97 software. (4) Beagle dog liver microsomes were prepared by using ultracentrifuge method. The incubation mixture consisted of 0.15mol-L-'KCl-O.lmol-L"1 PBS (pH 7.4), NADPH generating system, the analogue of m-Nif 0.4 y mol-L"'and microsomal protein 1 g-L'1 in a final volume of 0.5 mL. After being incubated for different time (10-50 min) at 37 C, the reaction was terminated by adding 0.5 mL alkalization. The RP-HPLC was used to determine the concentration of drug in the incubation mixture. The analogues of m-Nif (0.4 n mol-L"1 ) were incubated in different amount of microsomal protein (0.5 - 2.5 g-L"1 ) at 37 C for 30 min , the concentration of the analogues of m-Nif in the incubation mixture was determined by the RP-HPLC; The different concentration of the analogues of m-Nif were incubated with 1 g-L"1 microsomalprotein at 37 C for 30 min, and the RP-HPLC was used to determine the concentration of the analogues of m-Nif in the incubation mixture. The Michaelis-Menten parameters (Michaelis constant [Km] and maximal velocity [Vmax]) in Beagle dog liver microsomes were determined. The values of Km and Vmax were initially estimated by analysing Lineweave-Brurk plot. (5) The analogues of m-Nif ( 0.4 u mol-L-1 ) were incubated with lg-L-1 microsomal protein and different kind of selective CYP inhibitor at 37 C for 30 min in order to investigate its effect on the metabolism of the drug. After incubation, the RP-HPLC was used to assay the concentration of analogues of m-Nif in the incubation mixture. (6) Same concentration (0.8 u mol-L-1) of the analogue of m-Nif was incubated with 1 106 cell m L-1 isolated rat hepatocytes at 37 C. The concentration of drug in the supernatant was determined by the RP-HPLC at proper time intervals. The Cl in vitro and Cl in vivo predicted for isolated rat hepatocytes were calculated. The analogues of m-Nif ( 0.8n mol-L"1 ) were incubated with different density of rat hepatocytes (from 1 105 to 1 107 cells-mL-1 ) at 37 C for 15 min. The concentration of the drug in the supernatant was... |
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