ก ก ก FCYP2A6 F F Inhibitory Studies of the Human Cytochrome P450

– – . กกก CYP2A6 Inhibitory studies of the human cytochrome P450, CYP2A6, the nicotine metabolizing enzyme by Thai herbal and fruit juices ก กกก (.) กกก กกก CYP2A6 กก กกกก (.) ก กก Prof. dr. Jung-Ja P.Kim ก Medical College of Wisconsin Assoc. Prof. dr. Emily E. Scott ก University of Kansus ก ก rat CPR CYP2A6 ก Prof. dr. Joyce A. Goldstein ก National Institute of Health (NIH) ก กกกก P450 CYP3A4 CYP2C9 .. ก กกกกก กก-กกกก กก ก กกก CYP2A6 ก Cytochrome P450 2A6 (CYP2A6) ก cytochrome P450 (CYP P450) ก heme กกก กก ก CYP2A6 กกกก Km Vmax กกกก CYP2A6 กกกกกก กก ก CYP2A6 กกก กก CYP2A6 ก กก ก CYP2A6 กกก CYP2A6 กกก coumarin Km 1 uM ก 25 3% v/v ก CYP2A6 50% 95% 40% v/v กกกก CYP2A6 mechanism-based กก CYP3A4 CYP2C9 กก ก CYP2A6 ก กกก Abstract Project Title Inhibitory studies of the human cytochrome P450, CYP2A6, the nicotine metabolizing enzyme by Thai herbal and fruit juices Investigators Songklod Sarapusit, Ph.d, department of Biochemistry, Faculty of Science, Burapha University Pornpimol Rongnoparut, Ph.d, department of Biochemistry, Faculty of Science, Mahidol University Cytochrome P450 2A6 (CYP2A6) is the heme-containing protein that belongs to the cytochrome P450 (CYP or P450) super-family. In human, P450 enzymes play an essential role in metabolism of various endogenous and exogenous compounds such as hormones drugs and many environmental toxicants. The human CYP2A6 enzyme could specifically metabolize nicotine, an additive compound in cigarette, with high efficiency (low Km and high Vmax value). Therefore, nicotine metabolism by the liver-specific human CYP2A6 enzymes has been report as an important route in nicotine detoxification. In addition, some of deficient CYP2A6 allele smokers that drastically decrease in nicotine metabolizing activity of CYP2A6 enzyme tend to smoke few cigarettes per day and are low-risk of nicotine dependence compared to the normal allele smokers. This study aims to screen for Thai herbal juices or fruit juices that could specifically inhibit CYP2A6 enzymatic activity in vitro . As the results, the bacterially expressed and purified human CYP2A6 enzyme is functionally active and is specifically metabolized the coumarin substrate with the K m value approximately 1 uM. Out of 25 herbal and fruit juices tested, an in vitro experiment indicated that only 3% v/v of the Star fruit juice could inhibit CYP2A6-mediated coumarin hydroxylation by 50%. Interestingly, almost 95% of CYP2A6-mediated coumarin hydroxylation was inhibited in the presense of 40% v/v of the Star fruit juice in vitro. Further study indicated Star fruit juice could inhibit CYP2A6 activity by mechanism-based inactivaition process. However, Star fruit juice could also inhibit human CYP3A4 and CYP2C9 activity in vitro . Therefore, care must be taken in cessation smokers who orally take drugs that metabolized by CYP3A4 and CYP2C9 enzyme, a highly distribute human-drug metabolizing enzymes. 1 1.1 กกกก กกก ก กกก 1,100 3,000,000 6 (WHO,2008) กก .. 2550 10.86 ก 20% ก ( 2551) ก กก กก 2 6 10 ก 5-8 กกกก กกก Cytochrome P450 2A6 (CYP2A6) ก Cytochrome P450 (CYP P450) กกกก ก CYP2A6 Km Vmax (Patten et al., 1996) ก CYP2A6 กกกกกก (Hukkanen et al., 2002; Miksys et al., 2002; Su et al., 2000) กกกก CYP2A6 ก CYP2A6 กกกก กกก CYP2A6 ก กก CYP2A6 กกก 8-methoxypsoralrn (8-MOP) ก CYP2A6 ก (Hukkanen et al., 2005) กก 8-MOP กก ก ก CYP2A6 ก กกกก กกก ก กก CYP2A6 กกก กกก CYP2A6 กกก XL-1 blue กก กก ก CYP2A6 ก 50% (IC 50 ) 3% v/v กกกก CYP2A6 ก mechanism based ก ก CYP3A4 CYP2C9 กก ก CYP2A6 ก กกก 1.2ก 1. กกก CYP2A6 2. กกก CYP2A6 1.3กก กก CYP2A6 1.4กก CYP2A6 กก P450 1.5 ก ก (Tranformation) (Competent cell) กก กก CYP2A6 XL-1 blue rat CPR C41 (dE3) SdS-PAGE กกก coumarin กกก CYP2A6 กก กก ( 1-1) กกกกก กกกก dNA CYP2A6 dNA CYPOR กก CYP2A6 CYPOR ก CYP2A6 CYPOR 1-1 กก 2 ก 2.1 ก Cytochrome P450 (CYPs P450s) กกก ก ก ก ก กก P450 ก ก P450s กกก P450 กกกกก ก (Family) กกกก 40 ก (Subfamily) กกกกก 55 ก P450s ก กกกกก P450s กกกกกกก P450s กกกก 1 ก (Bernhardt R, 2006; Mansuy d, 1998; Nelson et al, 1996; Ortiz, 2005) 2-1 ก P450s กกกกก ( 1- 6) กกกก ( 7 8) ก ( 9) Guengerich FG, 2001 กกก P450s ( 2-1) P450s ก ก NAdPH-cytochrome P450 reductase (CPR) กกก (Fe 3+ Fe 2+) ก P450s P450s กก ก กกกกกก กก (iron-hydroxy complex) กกก ก Endoplasmic reticulum กก Squalene monooxygenase, Heme oxygenase ก Cytochrome P450 กกกก (Bernhardt R, 2006; Emre M et al, 2007; Guengerich FG, 2001; Ortiz, 2005) ก cytochrome c ferricyanideก ( 2-2) 2-2 ก NAdPH-cytochrome P450 reducatse ก www.uky.edu/Pharmacy/ps/porter/CPR.htm PLEASE specify reference CPR กกก FAd FMN กก กกก NAdPH ก FAd FMN กก ก CPR ก P450s กก กก CPR CPR , ก (Shen et al., 1989; dohr et al., 2001; Murataliev et al, 1999) กกก CPR CPR ก 4 NH 2-terminal, FMN-binding domain, FAd-binding domain, NAdPH-binding domain (Wang et al., 1997) FMN domain ก ก FAd-binding domain NAdPH-binding domain ก ( 2-3) NAdPH-binding domain FAd -binding domain FMN -binding domain 2-3 Cytochrome P450s () P450s Oxoidoreductase () ก Endoplasmic reticulum 2.2 ก กก ก 2549 กกกก ก 9.86 (Leartsakulpanitch et al , 2007) ก .. 2552 10.90 20.70 กกก 15 52.7 23.04 ( 25-59 ) ก 10.40 0.54 กกกก กก กก กก กก 58.95 6.39 17,059 84.78 ก กกกกกก ก กกก 30.4 4.7 กก 15.6 ( 10.9 ) 1.77 ก กก//ก 38.61 27.42 ก 11.44 กกก ก 20 กกก กกก ( 2551, 2552) กกก กกกก 4,000 กก 40 กก กกก กกกก (WHO, 2008) กกกก ก กก (WHO, 2008) กกก กกกก ก กก กกก ก ก กกกก ก (Hukkanen et al, 2005) กก ก (Benowitz, 2008) กกก (Oral mucosa) 80-90% กก กกกกกก (Beno- witz, 2008; Flammang et al, 1992) ก CYP2A6 ก ก nicotine ∆1(5) - iminium (Hukkanen et al, 2005) กกกก hydroxylation ก trans -3-hydroxycotinine, 5-hydroxycotinine nornicotine ( 2-4) กก ก (glucoronation) ก ก cytochrome P450 (CYP P450) ก heme ก กกกกก ก P450 18 ก (Family) 43 ก (Sub-family) (http://drnelson.utmen.edu/CytochromeP450.html ) CYP2A6 , CYP2A13 , CYP2B6 ก , CYP2d6 CYP2E1 กกก (Hukkanen et al, 2002; Miksys et al, 2002; Su et al, 2000) CYP2A6 Km Vmax (Patten et al, 1996) CYP2A13 กก CYP2B6 CYP2d6 กก (Benowitz et al, 1996) CYP2E1 กกก alcohol กก (McCarver et al, 1998) กก กก ก CYP2A6 กกกกกก 2-4 กกกก (Hukkanen et al, 2005) กกกก CYP2A6 ก coumarin 7-hydroxycoumarin (ก coumarin-7-hydroxylase) coumarin ก CYP2A6 (Miles et al, 1990; Yamano et al, 1990) กก CYP2A6 chlormethiazole กก nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] NNAL [4- methylnitrosamino)-1-(3-pyridyl)-1-butanol] NNN [ N9-nitrosonornicotine] (Patten et al, 1996; Pelkonen et al, 2000;Peterson et al, 1990) ก (Hecht, 1998; Hecht, 1999a, 1999b) กกกกก (Polymorphism) CYP2A6 ก กก 37 กก (http://www.imm.ki.

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