Application of LC/MS/MS in the quantitation of SU101 and SU0020 uptake by 3T3/PDGFr cells.
SU101 or leflunomide, has been studied extensively because of its anti-cancer and immunomodulating properties. The parent isoxazole compound is converted in vitro and metabolized in vivo to an open ring isomeric form, SU0020. Several pharmacological activities have been reported for the parent and metabolite compounds including inhibition of platelet-derived growth factor (PDGF)-mediated signaling for the parent compound and inhibition of de novo pyrimidine biosynthesis for the metabolite. The inhibition of PDGF-mediated signaling and the anti-tumor properties have been ascribed to the parent compound. In spite of its short plasma half-life of the parent molecule, SU101 can be administered intermittently in animal tumor models and retain efficacy. Therefore, the relationship between plasma levels of SU101 and its efficacy in tumor-implanted immuno-compromised mice is not well established. This study was conducted to assess the concentration of SU101 in 3T3/PDGFr alpha and beta cells (NIH3T3 mouse fibroblasts engineered to overexpress human PDGFr alpha or beta) to better understand the cellular levels of SU101 and SU0020. Two strains of 3T3/PDGFr cells (alpha and beta) were incubated with 1, 25, and 100 microM concentrations of SU101 for 1, 6, 24, and 48 hours. Quantitation of SU101 and SU0020 in these cell lines was achieved by a specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Interestingly, in both alpha and beta cell lysates SU101 was much more concentrated than SU0020. The greater concentration of SU101 versus SU0020 that was observed may be due to the preferential partitioning of SU101 into the cells and this shows that significant levels of the parent drug can reach the pharmacological site of action for inhibition of PDGF receptors. The data suggest that the conversion of SU101 to SU0020 is much slower in these cells than in the incubation media.
Zhang
Qingling
Q
SUGEN, Inc. 230 East Grand Avenue, South San Francisco, CA 94080, USA. qingling-zhang@sugen.com
Pang
Wyming Lee
WL
Chen
Hui
H
Cherrington
Julie
J
Lipson
Kenneth
K
Antonian
Lida
L
Shawver
Laura K
LK
D016475:3T3 Cells; D000465:Algorithms; D000814:Aniline Compounds / Q000032:analysis* / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D007555:Isoxazoles / Q000032:analysis / Q000378:metabolism*; D000077339:Leflunomide; D013058:Mass Spectrometry; D051379:Mice; D009570:Nitriles / Q000032:analysis* / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000096:biosynthesis; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet
D000814:Aniline Compounds; D000970:Antineoplastic Agents; D003470:Culture Media; D007555:Isoxazoles; D009570:Nitriles; C460097:SU 0020; D017479:Receptors, Platelet-Derived Growth Factor; D000077339:Leflunomide