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. 2019 Jul 30;24(15):2765.
doi: 10.3390/molecules24152765.

Interspecies Variation in NCMN -O- Demethylation in Liver Microsomes from Various Species

Ziru Dai  1 , Guibo Sun  1 , Jiada Yang  2 , Jie Hou  3 , Ping Zhou  1 , Weijie Xie  1 , Guangbo Ge  4 , Xiaobo Sun  5 , Ling Yang  6
Affiliations

Interspecies Variation in NCMN -O- Demethylation in Liver Microsomes from Various Species

Ziru Dai et al. Molecules. .

Abstract

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.

Keywords: Cytochrome P450 1A (CYP1A); NCMN-O-demethylation; liver microsomes; species differences.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The chemical structure of N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide (NCMN) and its fluorescence response towards mammalian cytochromes 1A from different species.
Figure 2
Figure 2
Representative LC-UV profiles of NCMN and its demethylation metabolite in liver microsomes from human, beagle dog, rat, mouse, minipig and cynomolgus monkey. NCMN (10 µM) was incubated with liver microsomes (0.25 mg protein/mL) from each species at 37 °C for 30 min.
Figure 3
Figure 3
The inhibitory effects of chemical inhibitors of cytochrome P450s (CYPs) on the formation of NCMN in liver microsomes from different species. N.D. means not detected.
Figure 4
Figure 4
The dose-dependent inhibition curves of furafylline on NCMN-O-demethylation in liver microsomes from human (A), cynomolgus monkey (B), beagle dog (C), minipig (D), mouse (E) and rat (F).
Figure 5
Figure 5
Enzymatic kinetic plots of NCMN-O-demethylation in liver microsomes from human (A), cynomolgus monkey (B), beagle dog (C), minipig (D), mouse (E) and rat (F). NCMN (1–150 µM) was incubated with HLM, CyLM, DLM, PLM, MLM or RLM(F) at 37 °C. Please add explanations for (A)–(F)
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