A non-michaelian behavior of the in vitro metabolism of the pentacyclic triterpene alfa and beta amyrins by employing rat liver microsomes

• Published in: Journal of pharmaceutical and biomedical analysis 2013-10, Vol.84, p.14-19
• Main Authors: Moreira, Fernanda de Lima, de Souza, Gustavo Henrique Bianco, Rodrigues, Ivanildes Vasconcelos, Lopes, Norberto Peporine, de Oliveira, Anderson Rodrigo Moraes
• Format: Article
• Language: English
• Subjects: Amyrins; Animals; Atypical enzymatic kinetics; Drug Discovery - methods; drugs; gas chromatography; Gas Chromatography-Mass Spectrometry - methods; Hill profile; In vitro metabolism; Isomerism; isomers; Kinetics; liver microsomes; Male; mass spectrometry; metabolism; Microsomes, Liver - metabolism; Natural product; Oleanolic Acid - analogs & derivatives; Oleanolic Acid - chemistry; Oleanolic Acid - metabolism; Pentacyclic Triterpenes - chemistry; Pentacyclic Triterpenes - metabolism; Rats; Rats, Wistar; temperature; triterpenoids
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2013.05.038

Pharmacological studies employing alpha and beta amyrin have demonstrated potential application in several biological activities suggesting their application as promising drugs. In the early drug development, metabolism studies may give important parameters regarding the efficacy and safety of the drug candidate. Therefore, the aim of this work was to determine the enzymatic kinetic parameters of these pentacyclic triterpenes. Chromatographic analyzes were performed using a Shimadzu GC–MS system. The resolution of amyrins was achieved with a DB5-MS column of 0.25μM film thickness, 30.0cm length and 0.25mm diameter. At this condition, the retention times of beta- and alpha-amyrin were 21.3 and 20.2min, respectively. The proposed method showed to be linear over the concentration range of 0.16–42.18μM for beta amyrin and 0.11–28.12μM for alpha amyrin. The lowest concentration quantified by the validated method was 0.16μM for beta and 0.11μM for alpha amyrin. The stability study showed that amyrins were stable at room temperature for 12h and at 37°C for 1h. The absolute recovery of the amyrin isomers from the rat microsome was 54.3–59.2%. The enzymatic kinetics presented sigmoidal plots. It was observed a Vmax=0.698±0.022μmol/mg protein/min, S50=4.4μM and Hill coefficient of 2.7±0.17 for alpha amyrin and a Vmax=0.775±0.034μmol/mg protein/min, S50=7.0μM and Hill coefficient of 2.5±0.21 for beta amyrin. The obtained results give the first clues regarding amyrin metabolism and suggests a more detailed study conducted employing isolated CYP isoforms.