Chem. Res. Toxicol. 1993,6, 880-888
880
Stereoselective Metabolism of (@ -(-) -Nicotine in Humans: Formation of trans-(@-(-)-NicotineN-1’-Oxide Sang B. Park,?Peyton Jacob III,* Neal L. Benowitz,S and John R. Cashman**+ Department of Pharmaceutical Chemistry and Liver Center, School of Pharmacy, University of California, San Francisco, California 94143-0446, and Division of Clinical Pharmacology, Department of Medicine, School of Medicine, University of California, San Francisco, California 94110 Received March 15, 199P The chemical synthesis and chromatographic separation of cis- and trans-(&-nicotine N-1’oxide diastereomers have allowed the development of methods for the quantification of (S)nicotine N-1‘-oxides during in vitro and in vivo metabolic studies. The metabolism of (S)nicotine was investigated in the presence of microsomes, cDNA-expressed and highly purified flavin-containing monooxygenase (FMO) from pig liver, human liver, and rabbit lung. For comparison, the N-1’-oxidation of (5’)-nicotine in the presence of the cytochrome P450 2B1 from rat liver, cytochrome P450 2B10 from mouse liver, and cytochrome P450 4A2 from rabbit lung was examined. The ratio of trans:& @)-nicotine N-1’-oxide formation for pig liver FMOl (form 1)was 57:43. In contrast, cDNA-expressed adult human liver F M 0 3 (form 3) and rabbit lung FM02 formed solely trans-(S)-nicotine N-1’-oxide. Of the cytochrome P450 enzymes examined, formation of (&nicotine N-1’-oxide occurred with a mean trans:& ratio of 82:18. The stereoselectivity of (8-nicotine N-1’-oxide formation was investigated by examining the urine of 13healthy male smokers studied on a protocol which included free-smoking, intravenous infusion of (S)-nicotine-& and dermal patch administration of (S)-nicotine-&. During cigarette smoking or administration of intravenous or transdermal (S)-nicotine,only the trans diastereomer of (S)-nicotine N-1’-oxide was observed in the urine. That the trans-(&nicotine N-1’-oxide metabolite was not appreciably reduced or oxidized further was investigated with infusion studies of (S)-nicotine-dzN-1’-oxide. The mean transxis (5’)-nicotineN-1’-oxide ratio determined from the metabolite isolated from the urine of humans after infusion of the N-1’-oxide was 60:40, which was essentially identical to that of the infusate. Previously, we have observed exclusive trans-(S)-nicotine N-1’-oxide formation in the presence of 14 different adult human liver microsome samples. As described herein, after administration of (S)-nicotine to 13 healthy adult smokers by 3 different routes of administration, we also observed only trans-@)-nicotine N-1’-oxide formation. The excellent agreement between in vitro and in vivo results suggests that human @)-nicotine N-1’-oxygenation is catalyzed predominantly by one monooxygenase. The majority of the data strongly suggests that the adult human liver flavin-containing monooxygenase (form 3) is responsible for trans-@)-nicotine N-1’-oxygenation, and we propose that formation of this metabolite is a selective functional marker for the enzyme.
Introduction Today, about 25 % of adult Americans smoke tobacco. Because many of the pharmacological effects of smoking are due to (5’)-nicotine, this makes @)-nicotineone of the most widely used psychoactive agents in this country. Active smoking is the primary cause of lung cancer ( I ) , but passive smoking also is definitelylinked to lung cancer (2).
@)-Nicotine undergoes extensive metabolism in humans, and presently, 80-90% of a dose can be accounted for in terms of identified urinary metabolites (3). Following smoking or iv infusion, the terminal half-life of @)-nicotineis about 2-3 h. Total clearance averages 1300 mL/min and is highly variable among individuals (4). Plasma protein binding of @)-nicotine(4) or (SI-nicotine N-1’-oxide (5) is not very efficient. An infusion of (SI-
* Address correspondence to this author at the IGEN Research Institute, 130 5th Ave. N.,Seattle, WA 98109;tel: (206) 441-6684, fax: (206) 443-0686. + School of Pharmacy. t School of Medicine. * Abstract published in Advance ACS Abstracts, October 15, 1993.
nicotine can be dosed to achieve levels of (&nicotine similar to those while smoking cigarettes (3). The major excreted human urinary metabolites’ of (S)nicotine are (5’)-cotinine(10-15% ), @)-nicotineN-1’-oxide (4%), trans-3-hydroxy-(S)-cotinine(35%) (61,and (5‘)nicotine glucuronide (7), cotinine glucuronide (B), and trans-3-hydroxy-(S)-cotinineglucuronide (9, 10) (i.e., collectively, approximately 30%) (Scheme I) (11). (SICotinine and @)-nicotine N-1‘-oxide are useful markers of (&nicotine exposure: cotinine has a long half-life (i.e., approximately 16 h), and blood levels are relatively stable with regular tobacco use (12). (SI-Cotinineis extensively metabolized, but @)-nicotine N-1’-oxide is stable and rapidly excreted unchanged (11). In adult human liver preparations, cytochrome P450 2A6 is apparently the primary enzyme that forms (5’)nicotine A1‘*S‘-iminiumion (13) which, in the presence of aldehyde oxidase, is converted to (5’)-cotinine. Although previous studies using expressed human liver cytochrome P450 cDNAs have implicated other cytochrome P450 1 N. L.
Benowitz,P. Jacob, 111, and I. Fong,unpublished observations.
0 1993 American Chemical Society 0893-228~/93/27Q6-088Q$O4.00/Q
Stereoselective (8) - (-1 -Nicotine Metabolism
Chem. Res. Toxicol., Vol. 6,No. 6,1993 881
Scheme I. Overall Metabolism of (@-Nicotine
cis-NICOTINE N'-OXIDE
-2. COTININE
trans-NICOflNE N'-OXIDE
OH
M
N-METHYLENENORNICOTINE IMlNlUM ION
NORNICOTINE
NORCOTININE
COTlNlNE
5-HYDROXYCOTININE
frans-3'-HYDROXYCOTININE
trans-3-HYDROXYCOT1NINE
GLUCURONlDE
enzymes in (5')-nicotine h1'p5'-iminium ion formation (141, the data from adult human liver microsomes clearly shows cytochrome P450 2A6 as the major @)-nicotine oxidase (13). Previously, the major human (&nicotine C-atom oxidase was reported to be cytochrome P450 2B6 on the basis of the oxidation of @)-nicotine with cDNA-expressed enzymes (14). It is possible that, at high substrate concentrations, the rate of @)-nicotine C-oxidation is more a consequence of the nmol of cDNA-expressed cytochrome P450lmgof protein andlor the amount of cytochrome P450 reductase present in the metabolic incubation and not the intrinsic substrate specificity for the monooxygenase. Because little cytochrome P450 2B6 is present in adult human liver preparations thus far analyzed,2we strongly favor a prominent role of cytochrome P450 2A6 in (S)nicotine C-atom oxidation. The adult human liver flavin-containing monooxygenase (FMO)3 FM034 appears to be the major enzyme responsible for (S)-nicotineN-1'-oxide formation (13).However, previous work (15) suggested that adult human urinary (&nicotine N-1'-oxide formation was considerably less stereoselective than the absolute stereoselectivity we observed. The stereoselectivity for (S)-nicotine N-1'-oxide formation (i.e., 100% trans-N-1'-oxide) in the presence of adult human liver microsomes (131,has been observed to be quite distinct from that of FMOl catalyzed (8)-nicotine N-1'-oxide formation [i.e., 49 % :51% trans:& @)-nicotine N-1'-oxide] from pig liver preparations (16).Thus, FMO enzyme structural differences for form 1 and form 3 enzymes are manifested in functional differences as well. The objective of the present study was to examine the stereoselectivity of (&nicotine N-1'-oxygenation in hu2 M. Mirmura,T. Baba,H. Yamazaki, S.Ohmori,Y. Inui, F. J. Gonzalez, F. P. Guengerich,and T. Shimada(1993)Characterizationof cytochrome P450 2B6 in human liver microsomes. Drug. Metab. Dispos. (in press). a Abbreviations: CIMS,chemical ionizationmass spectrometry;FMO, flavin-containing monooxygenase; DETAPAC, diethylenetriaminepentaacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 4 An alternate nomenclature for FMOl (i.e., FMO-A or FMO form I) and FM03 (i.e., FMO-D or FMO form 11) has appeared in our previous publications.FM02 (i.e.,FMO-B)from rabbit lung is yet anotherenzyme form.
Chart I. Deuterated Analogs of (@-Nicotine and (@-Nicotine N-1'-Oxide Used in This Study D
D
mans. In parallel with the in vivo studies and as an in vitro model, various hepatic monooxygenase preparations were employed to determine (&-nicotine N-1'-exygenation stereoselectivity. The regioselectivity of (&-nicotine oxidation (i.e., carbon atom versus nitrogen atom oxidation) was also determined. Thus, (S)-cotinine:(S)-nicotine N-1'-oxide ratios were determined as an index of regioselectivity in adult human (@-nicotine metabolism. Formation of (&nicotine N-1'-oxide was completely stereoselective: only trans-(S)-nicotine N-1'-oxide was observed to be formed. That no evidence for in vitro or in vivo oxidation or reduction of @)-nicotine N-1'-oxide was observed suggests that formation of trans-@)-nicotine N-1'-oxide is a selective functional probe of FM03 activity in adult humans. Materials and Methods Chemicals. (S)-Nicotine was obtained from Aldrich. (5')Nicotine-& cis- and trans-@)-nicotine N-1'-oxide, (S)-nornicotine, and (S)-cotinine were prepared and purified to homogeneity as described previously (13). (S)-Nicotine-S',b-d~and (5')-nicotineN-l'-oxide-S,S-d2were synthesized by oxidation of (S)-nicotine-3',3'-&(5)by the method described before (13). The product was purified by column chromatography on silica gel using ethyl acetate/methanoVconcentrated aqueous ammonia as an eluant (47.5:47.50.5 v/v). The (S)-nicotine N-1'-oxide3',3'-d2 product was shown to be a 62:38 mixture of trans:& diastereomers by lH-NMR and HLPC (Chart I). @)-Nicotine NJV-1,l'-dioxide was synthesized from (@-nicotineas described before (13). The assigned structure of each synthetic material was determined by spectral means (i.e., 'H-NMR, mass spectrometry, and UV-vis) and was essentially identical to that of the authentic samples. All compounds of the NADPH-generating
882 Chem. Res. Toxicol., Vol. 6,No. 6, 1993 system were obtained from Sigma Chemical Co. (Milwaukee, WI). Chemicals, buffers, and other reagents were obtained from Fisher Scientific (Richmond, CA). All other chemicals were of the highest purity and were purchased from commercial sources. Instrumentation. ‘H-NMR spectra were recorded on a General Electric spectrometer operating a t a frequency of 300 MHz. Chemical ionization mass spectra (CIMS) were taken on a VG7OS instrument. Liquid secondary ion mass spectrometry (LSIMS) were performed with a Kratos MS50 a t 6 kV and a source temperature of 50 “C. Gas chromatography-mass spectrometry (GC-MS) was obtained with a Hewlett-Packard 5890 gas chromatograph interfaced with a Hewlett-Packard 5970 B mass-selective detector (17, 18). Liver Preparations. Untreated pig liver microsomes were a generous gift of Professor D. M. Ziegler, (University of Texas, Austin) or were prepared from samples provided by the Department of Surgery (UCSF). Highly purified liver FMOl was isolated and purified from pig liver microsomes by a modified procedure as previously described (20). Pig liver microsomes possessed significant cytochrome P450 activity (21). Microsomes and highly purified FMOl from pig liver possessed N- and S-oxygenase activity characteristic of FMO activity (13, 21,22). Adult human liver microsomes (i.e., J microsomes, see ref 23) were a generous gift of Dr. S. A. Wrighton (Lilly Research, Indianapolis, IN) and were obtained with a protocol from the Medical College of Wisconsin. Use of the human liver microsomes was approved by the UCSF Committee on Human Research. Metabolic Incubation Systems. A typical incubation mixture contained 50 mM potassium phosphate (pH 8.41, 0.5 mM NADP+,2.0 mMglucose 6-phosphate, 1IU of glucose-6-phosphate dehydrogenase, 0.8 mM diethylenetriaminepentaacetic acid (DETAF’AC), 3 mg of rat liver microsome supernatant (asa source of aldehyde oxidase) or 2.0 mg of pig liver microsomes or 45 pg of pig liver FMOl or 0.3-0.5 mg of adult human liver microsomes. When both (S)-nicotine N-1’-oxide and (S)-cotinine formation was studied, rat liver cytosol [Le., the supernatant from the lOOOOOg centrifugation (3 mg of protein)] was added. The components of the incubations were combined and placed on ice. The reaction was initiated by the addition of substrate, and the incubation was continued with constant shaking to maintain adequate oxygen a t 37 “C. At timed intervals (i.e., 10 min), the reaction was stopped by the addition of 3 volumes of 2-propanol/ dichloromethane (1:2 v/v), and after saturating the aqueous phase with approximately 20 mg of anhydrous sodium carbonate, the reaction mixture was mixed thoroughly and centrifuged a t 3000 rpm to separate the aqueous and organic fractions. After filtration of the organic fraction through a 4-pm nylon filter, 50 pL of water was added to the mixture and evaporated to a small volume, and the extract was taken up in methanol for separation and quantitated by HPLC as described previously (13, 24). The recovery of material as judged by HPLC was >go%. The profile of @)-nicotine metabolites was determined by HPLC analysis of 2-propanol/dichloromethaneextracts of the reaction mixture as described before (13). The profile of @)-nicotine N-1’-oxide metabolites was determined by HPLC analysis of 2-propanol/dichloromethaneextracts from microsomes and human urine. The extracts were evaporated to a minimum volume, taken up in methanol, and separated on an IBM 9533 HPLC system with UV detection. The HPLC system utilized a 5-pm AXXIOM (Richard Scientific, Novata, CA) silica-phase column (4.5 pm X 25 cm) with a mobile phase of 70% A and 30% B, where A was methanol/acetonitrile/60% perchloric acid (50:500.03, v/v), a n d B was methanolas previously described (24). The HPLC system efficiently separated the (S)nicotine N,N-1,l’-dioxide, the cis-(S)-nicotine N-1’-oxide, and the trans-@)-nicotine N-1’-oxide, which had retention times of 3.5,4.5, and 4.7 min, respectively, a t a flow rate of 2.0 mL/min. Cytochrome P450-Mediated (@-Nicotine N-1’-Oxygenations. The major phenobarbital-inducible cytochrome P450 from rat (i.e,, P450 2B1) and mouse liver (i.e., P450 2B10) and the major pulmonary cytochrome P450 from pregnant rabbit (Le., P450 4A2) were studied for their ability to form @)-nicotine
Park et al. N-1’-oxide. Rabbit lung cytochrome P450 4A2 was purified from a pregnant rabbit by the method of Williams e t al. (25). The purified enzyme and rabbit lung microsomes were a generous gift of Professor D. Williams (Oregon State University) and exhibited characteristically high benzphetamine N-demethylase activity [30.4 nmol/ (minmmolof protein)] (26),which ww a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25). Cytochrome P450 2B1 was purified from rat liver by the method of Waxman et al. (27). Mouse liver cytochrome P450 2B10 was purified by the method of Bornheim and Correia (28). Both hepatic isozymes had characteristically high pentoxyresorufin 0-dealkylase and 16@testosterone hydroxylase activities 16 and 3 nmol/(minmmol of enzyme)] (29),respectively, and were judged to be homogenous by SDS-PAGE (25). For a typical incubation, cytochrome P450 (0.1 nmol) was reconstituted in the presence of dilaurylphosphatidylcholine (25 pg) and saturating amounts of rat cytochrome P450 reductase [2 nmol, specificactivity 37.6 pmol of cytochrome c reduced/(mg of proteimmin)] and was allowed to stand for 10 min a t 4 “C. Sodium phosphate buffer (50 mM, pH 7.4), the NADPH-generating system (as described above except without NADP+), substrate (500 pM final concentration), and rat liver cytochrome bs (0.1 nmol) were added for a total volume of 1.0 mL. The incubations were initiated by the addition of NADP+ and carried out for 10 min a t 37 “C with constant shaking in air. The reactions were terminated and prepared for HPLC analysis as described above. Clinical Studies. Thirteen healthy adult smokers who had smoked cigarettes for a t least 1year participated in the study. Subjects were confined in the Drug Study Unit a t the University of California,San Francisco, for 13days using a protocol approved by the UCSF Committee on Human Research. The volunteers gave informed consent to participate in the study. Subjects were determined to be in good health based upon a physical examination, medical history, and biochemicaltests of liver and kidney function. Several different studies were conducted as described below. Thirteen male smokers were studied on a 13-day protocol. On days 1 and 2, subjects smoked their chosen brand of cigarettes ad libitum. Subsequently no smoking was permitted. On day 5, a 24-h infusion of deuterium-labeled (SI-nicotine [i.e., (S)nicotine-3’,3’-dzl as the bitartrate salt a t a rate of 0.2 pg/kg/min was initiated. At the same time, a 21-mg (S)-nicotine-do transdermal system (Aha Pharmaceutical Co., Palo Alto, CA) was applied to the skin. For five additional days (from day 7 through day 1l),(SI-nicotine transdermal systems were applied to the skin daily. Urine was collected over 24-h on days 2,5, and 11for analysis of @)-nicotine and (S)-nicotine metabolites. Blood was obtained periodically throughout the study as part of a separate bioavailability study.’ In another study, 7 malesmokers were infused withdeuteriumlabeled (S)-nicotine N-l’-oxide-S’,S’-dz (i.e., 2 pg/kg/min) for 30 min. The infusate consisted of (&nicotine N-l’-oxide-3/,3/-dz in a trans:& ratio of 62:38 as determined by ‘H-NMRand HPLC. Urine samples were collected over the next 4 h after infusion. Urine volume and pH were recorded immediately after each urine collection interval. Human (@-Nicotine Metabolite Analysis. Eachurine and blood sample was split into two groups for separate metabolite quantification and stereoselectivity studies. Urinary and plasma samples were assayed for (5’)-nicotine, (SI-cotinine, and (S)nicotine N-1’-oxide by gas chromatography using nitrogenphosphorous or gas chromatography-mass spectrometry with selected ion monitoring detection as described previously (17, 18). For bioavailability studies,’ plasma samples (1mL) were mixed with 0.5 mL of NaOH (50%) and extracted with 2 mL of CH3CN. For stereoselectivity studies, the urine sample (1mL) was mixed with 0.2 mL of saturated aqueous sodium acetate. The acetonitrile and aqueous sodium acetate fractions for blood and urine, respectively, were loaded onto a 3-mL silica solidphase extraction column (Bakerbond, Fisher Scientific). The column was prewashed with 2 mL of CH3OH/2O% NHdOH ( 8 0
Stereoselective (S)-(-)-Nicotine Metabolism 20 v/v) followed by 2 mL of nicotine-freewater just before the samples were loaded onto the column. The samples were drawn through the solid-phase columns by the use of a homemade vacuum manifold. After the sample was loaded onto the column, the vacuum manifold was adjusted to produce a flow of about 1 mL/min. After the extract was adsorbed onto the column, the column was washed with 2 mL of water and then with 2 mL of 2-propanol. (&Nicotine and (S)-nicotine metabolites were selectively eluted from the column with 4 mL of CHaOH/NHdOH (80:20v/v)by gravity filtration,and the eluantwas evaporated to dryness under a stream of Nz gas. The samples were taken up in CHsOH, and a portion was analyzed by an HPLC method as described previously (13). After HPLC analysis of the urinary metabolites, the unused portion of the sample was pooled together and the (&nicotine N-1’-oxide was purified from the other materials present by preparative TLC (silica gel, 250 pm, 20 X 20 cm, Whatman, Clifton,NJ) with a mobile phase of CHsOH/NH40H (99:1.Ov/v). This system efficiently separated @-nicotine N-1’-oxide from other materials, and the Rfvalue for the N-1’-oxide employing this solvent system was 0.18.
Results The chemical synthesis and purification of cis- and trans-@)-nicotine N-1‘-oxide diastereomers have been previously reported (13). The method we used to separate the N-1’-oxides is more efficient than the traditional procedure involving paper chromatography (15, 30). In addition, quantification by HPLC avoids cumbersome sample handling and possible autoxidation. Highly purified synthetic (SI-nicotine N-1’-oxides were used as standards for in vitro and in vivo characterization of enzymatic and human @)-nicotine N-1’-oxygenation stereoselectivity, respectively. In addition, the formation of (S)-cotinine was also quantified, and (SI-cotinine:(S)nicotine N-1‘-oxide ratios provided information about (S)nicotine metabolism regioselectivity. Efficient methods for the isolation, purification, and chromatographic separation (i.e., TLC, GC, GC-MS, and HPLC) of @)-nicotine, (8)-nornicotine,@)-nicotineN-1’-oxide, (S)-nicotineNJV1,l’-dioxide, and (S)-cotinine have been described and were used in this study to quantify in vitro and in vivo (S)nicotine metabolites (see Materials and Methods). In Vitro (5‘)-Nicotine Metabolism. The metabolism of (S)-nicotine in the presence of various animal and adult human liver preparations was done to determine the stereoselectivityof (S)-nicotineN-1’-oxygenation. Aerobic incubation of hepatic microsomes or highly purified monooxygenases in the presence of NADPH resulted in formation of @)-nicotine N-1’-oxide. Under the in vitro experimental conditions, we did not determine whether (S)-nornicotine or @)-nicotine A1’@-iminium ion was directly formed [the iminium ion was converted to (S)cotinine by aldehyde oxidase present in rat liver cytosol that was added to the incubation]. However, work from our laboratories (11, 13) and others (7)has shown that formation of (S)-nornicotine was only a very minor metabolite in the presence of adult human liver microsomes. We have previously shown that the hepatic preparations used in this study were competent to form the iminium ion that was converted to (S)-cotinine in the presence of aldehyde oxidase. Formation of (SI-nornicotine was not detectable (13). Formation of @)-nicotine N-1’-oxide was dependent on active protein and on NADPH (data not shown). As shown in Table I, a range of enzymatic (&-nicotine N-1’-oxygenation stereoselectivities was observed. In the presence of pig liver mi-
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 883 Table I. Stereoselective Nicotine N-1‘-Oxidation in the Presence of Hepatic Monooxygenases N-l‘-oxide % tram:%cis condition
cotiniie
pig liver microsomes pig liver FMOl pig liver FMOl (exp) rabbit lung microsomes rabbit lung FM02 rat liver cytochrome P460 2BIed m o u e liver cytochrome P450 2BlOCd rabbit lung cytochrome P460 4A2OL monkey liver microsomes human liver micrcaomes human liver FM03 (exp)
2.2 1.2
6.2 11.1 0.8 0.9
formed0
N-l’-oudeb
3.8 194.6 0.1 0.1
60.040.0 67.242.8 64.236.8 84.516.6 100.00 84.216.8 80.219.8 79.021.0 100.00 100.00 100.00
0.2
6.8 4.3 0.1 0.1
0.2 The values are the mean of 2-4 experiments. Values are expressed as nmol/(min-mgof protein). * The percent N-oxide for each diastereomer observed was determined by HPLC. Values expressed are nmol/(min-nmolof enzyme). d The cytochromeP460was isolated from phenobarbital-pretreated animals. e The cytochrome P450was a
isolated from a pregnant rabbit.
crosomes, formation of trans-(SI-nicotine N-1’-oxide was only slightly preferred over formation of cis-@)-nicotine N-1’-oxide. In good agreement with this observation, and what has been reported in the literature (16), is the diastereoselectivity values for (SI-nicotine N-1’-oxide formation by the highly purified pig liver FMOl [i.e., 57.2: 42.8, transxis @)-nicotine N-1’-oxide] and pig liver FMOl from a cDNA expression system in bacteria [i.e., 64.2: 35.8, trans:cis @)-nicotine N-1‘-oxide]. In contrast to highly purified FMOl monooxygenase systems from pig liver, the formation of @)-nicotine N-1’-oxide in the presence of microsomes and highly purified FM02 from rabbit lung was highly stereoselective. Microsomes and highly purified FM02 from rabbit lung formed trans:cis @)-nicotine N-1’-oxide diastereomers in a ratio of 84.5: 15.5 and 100:0, respectively. Next, we examined the stereoselective formation of (8)nicotine N-1‘-oxide in the presence of cytochrome P450 4A2 from pregnant rabbits and the major phenobarbitalinducible form of cytochromes P450 2B1 from rat liver and P450 2B10 from mouse liver. As shown in Table I, for these highly purified monooxygenases, the diastereoselectivityof @)-nicotineN-1’-oxidation was considerably greater than the pig liver FMOl or the cDNA-expressed pig liver FMOl enzymes examined but not as stereoselective as rabbit lung FM02. Thus, rat liver P450 2B1 and mouse liver P450 2B10 and P450 4A2 from pregnant rabbit lung had @)-nicotine N-1’-oxidation formation stereoselectivities of 84.2:15.8; 80.2:19.8, and 79.0:21.0 trans:cis @)-nicotine N-1’-oxide, respectively. The above-described investigations were conducted to serve as animal model systems for @)-nicotine N-1’oxygenation stereoselectivity studies in microsomes from human and nonhuman primates. Adult monkey liver microsomes catalyzed the exclusive formation of trans@)-nicotine N-1‘-oxide. That no cis-@)-nicotine N-1’oxide was observed in adult monkey liver microsomes (n = 8 different preparations were examined) suggested that nonenzymatic autoxidation was not occurring in these studies. The results for monkey liver microsomes were in excellent agreement with the (5’)-nicotine “-oxygenation stereoselectivity (n = 14 different hepatic preparations) observed in the presence of adult human liver microsomes. As reported previously (13), all of the human liver microsomes that we have examined to date showed an absolute stereoselectivity for formation of trans-@)nicotine N-1’-oxide. Because we previously suggested that
Park et al.
884 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
adult human liver FM03 was primarily responsible for (5')-nicotine "-oxygenation in vitro, it was important to examine the (S)-nicotine "-oxygenation stereoselectivity with a preparation containing only adult human liver FM03 activity. We thus expressed the cDNA for adult human liver FM03 in bacteria and confirmed that the preparation was active.5 A number of different preparations ( n = 3) of cDNA-expressed FM03 showed an absolute stereoselectivity for formation of trans-(SInicotine N-1'-oxide (Table I). In summary, the results suggest that, with the exception of rabbit lung FM02, nonprimate animal FMOl or cytochrome P450 monooxygenases examined showed a considerably lower @)-nicotine N-1'-oxide formation stereoselectivity than that observed for monkey or adult human liver microsomes or cDNA-expressed adult human liver FM03. Because a form of FMO has been purified from adult monkey liver microsomes that appears to bear a close identity with adult human liver FM03, it is possible that a nonhuman primate homolog of FM03 exists in adult monkey liver (31) that is capable of producing the same stereoselectivity as that observed for the adult human liver enzyme. We examined the possible oxidative or reductive metabolism of each highly purified cis- or trans-(8)-nicotine N-1'-oxide diastereomers in vitro. In the presence of microsomes from adult human liver, pig liver, or rabbit lung supplemented with NADPH, no detectable oxidation or reduction of either the cis- or trans-(Shnicotine N-1'oxide diastereomers was observed. That no observable (S)-nicotineN,N-1,l'-dioxide was detected suggested that nonenzymatic or autoxidative degradation of (SI-nicotine N-1'-oxide diastereomers was not occurring during our in vitro incubations. To provide further support for a role of a novel @)-nicotine N-1'-oxygenase in adult human liver [Le., FM03, the FMO that is not the dominant (SInicotine N-1'-oxygenase present in nonprimate animal liver tissue], we examined the in vivo metabolism of @)-nicotine in humans. In Vivo (@-Nicotine Metabolism. Thirteen adult human male smokers that were studied on the 13-day protocol had (&nicotine urinary concentrations of 1372 f 797 (SD) ng/mL, range 573-3540 ng/mL after smoking ad libitum. The same smokers infused with (S)-nicotinedz achieved similar mean urinary concentrations of (8)nicotine of 1125 f 593 ng/mL, range 380-2400 ng/mL. Urinary @)-nicotinelevels after wearing the dermal patch for 5 days tended to be less (i.e., 831 f 302 ng/mL range 242-1480 ng/mL), although this was not statistically significant. The reason for this result is that daily intake of (S)-nicotine while smoking was greater than the intake while wearing the transdermal systems.' The concentration of (5')-cotinine in the urine of the 13 male smokers after free smoking was 1867 f 310 ng/mL, range 1360-2450 ng/mL. The same smokers infused with (5')-nicotine-dz achieved a mean concentration of (SIcotinine-dz of 867 f 234 ng/mL, range 255-1136 ng/mL. Urinary (S)-cotinine levels during administration of the dermal patch were observed to be 1246f 400 ng/mL, range 340-1833 ng/mL (Table 11). Levels of @)-nicotine N-1'-oxide concentrations for the 13 men while free smoking averaged 670 f 212 ng/mL, range 330-1110 ng/mL. The same smokers infused with 6
N. Lomri, Z.-C. Yang,and J. R. Caehman, unpublished obeervations.
Table 11. Urinary Nicotine and Nicotine Metabolite Levels after Treatment of Adult Humans by Three Routes of Administration nicotine administrations (ng/mL) subject (day) da do 104 F (2) NDC 1260 634 525 I (5) 951 ND P (11) 642 ND 105 F I 799 594 ND 1150 P ND 1140 106 F I 234 146 P ND 242 107 F ND 970 I 683 467 ND 957 P ND 1860 108 F I 1300 820 P ND 1480 110 F ND 2080 I 1320 1080 899 ND P 112 F ND 797 I 550 435 P ND 617 113 F ND 2020 I 313 312 P ND 955 114 F ND 1350 681 I 882 P ND 783 700 116 F ND I 194 259 P ND 360 117 F ND 906 I 447 402 861 ND P 118 F ND 3540 409 I 586 P ND 766 119 F ND 573 I 288 267 P ND 782
cotinine (ng/mL) da do 21 2130 404 575 ND 1100 ND 1820 346 549 23 1520 ND 1550 126 129 ND 340 ND 1770 484 477 24 1370 ND 2330 385 702 53 1780 ND 1940 407 729 26 1350 23 1630 433 609 35 1640 ND 1910 296 427 28 1220 31 1570 47 657 44 1580 ND 1620 273 626 24 1180 22.5 2090 334 458 ND 1180 ND 2450 256 466 ND 600 ND 1360 232 416 30 1050
nicotine N-1'-oxideb (ng/mL) da do ND 481 187 189 291 ND ND 717 357 401 ND 828 ND 503 52 51.5 117 ND ND 351 287 388 ND 354 ND 636 593 474 ND 680 ND 864 551 530 ND 405 ND 405 101 222 ND 539 740 ND 15 158 ND 698 ND 799 567 586 ND 744 ND 330 159 77 ND 206 763 ND 217 189 427 ND ND 1100 155 185 250 ND 838 ND 151 92 ND 482
a F = free-smoking; I = intravenous; P = dermal patch route of administration. The day of sampling for each protocol is indicated in parentheses. The values were determined by GC-MS as described in the Materials and Methods. Only the trans-nicotine N-1'-oxide diastereomer was observed. Not detectable.
(5')-nicotine-dzor administered (S)-nicotinevia the dermal patch route had similar urinary (8-nicotine N-1'-oxide concentrations which averaged 533 f 356 ng/mL, range 104-1153 ng/mL, and 463 f 214 ng/mL, range 117-828 ng/mL, respectively. The difference in the mean values of the urine volumes for free smokers and transdermal (,%nicotine conditions were not statistically significant. One notable difference observed, however, was that the fractional excretion of (SI-nicotine N-1'-oxide was significantly greater during smoking compared with the transdermal treatment.' Considerable interindividual variability in both the amount of @)-nicotine and type of metabolite [i.e., (S)-cotinine or (S)-nicotine N-1'-oxide1 was observed after administration by the three routes. In general, there was no clear relation between the level of urinary (5')-nicotine and the amount of urinary (S)-cotinine or (5')-nicotine N-1'-oxide observed. This is in agreement with previous studies that showed considerable interindividual variability in both metabolic and renal clearance, as well as volume of distribution of @)-nicotinein humans ( 4 ) . (5')-Nicotine-dzinfusion rate had no influence on any of the pharmacokinetic parameters determined, and there
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 886
Stereoselective @)-(-)-Nicotine Metabolism
was no significant correlation between initial concentration of (23)-cotinineand metabolic clearance of (S)-nicotine (4, 32). Thus, the excretion pattern for the major metabolites for the free smoking and transdermal (S)-nicotine conditions was similar.’ The average (SI-cotinine:(S)-nicotineN-1’-oxide ratio for the 13adult human males examined after free-smoking, intravenous, and dermally administered @)-nicotine was 3.1:l.O f 1.1,2.0:1.0 f 0.9, and 3.0:l.O f 1.0, respectively. The (S)-cotinine:(S)-nicotine N-1’-oxide ratio in the urine was lower during intravenous infusion of (8-nicotine compared with that after smoking or transdermal (SInicotine dosing. This is because (S)-cotinine levels were at steady state during the smoking and transdermal (8nicotine conditions, but because the half-life of (SI-cotinine is long (i.e., 16-20 h), steady-state was not achieved after a single 24-h infusion. In contrast, @)-nicotineN-1’-oxide, which has a much shorter half-life, was at steady state in all three conditions. Also, there is agreat deal of individual variability in urinary (S)-cotinine excretion possibly due to the wide individual variability in glucuronidation of (S)-cotinine (8)or conversion to 3-hydroxy-(S)-cotinine.l To examine whether the stereoselectivity of (S)-nicotine N’-oxygenation was altered as a consequence of route of @)-nicotine administration, the stereochemistry of the @)-nicotine N-1’-oxide metabolite from all three routes of administration was determined by an HPLC chromatographic method that could readily separate cis- and trans-@)-nicotine N-1’-oxide diastereomers. During cigarette smoking or administration of intravenous or transdermal @)-nicotine,only the trans-@)-nicotine N-1’-oxide metabolite was observed. These data support the notion that an absolutely stereoselective monooxygenase is present in humans, exclusivelyforming trans-(5’)-nicotine N-1’-oxide from (5’)-nicotine. That no cis-@)-nicotine N-1’-oxide was observed also suggests that nonenzymatic autoxidative processes leading to even small amounts of diastereomeric (SI-nicotine N-1’-oxide products were not occurring during the workup or sample handling procedure employed in our experiments. Because the formation of trans-(S)-nicotine N-1’-oxide could be a result of exclusive stereoselective reduction of the cis diastereomer (as suggested by others, see refs 3336), we infused a mixture of cis- and trans-(S)-nicotine-dz N-1’-oxide diastereomers into 7 adult human male subjects and analyzed the recovered (S)-nicotine-dzN-1’-oxide from the urine. The stereochemistry of the (S)-nicotine iV-1’oxide recovered in urine was compared with the stereochemistry of the material infused (i.e., the infusate). The infusate had a trans:& (5’)-nicotine N-1’-oxide ratio of 62:38 as determined by a ‘H-NMR and HPLC method previously described (13). The mean trans:cis (8-nicotine N-1’-oxide ratio determined from the metabolite isolated from the urine of humans after infusion of (S)-nicotine-dz N-1’-oxide was 60:40 (Table 111)as determined by HPLC. The result strongly suggested that, within experimental error, the stereochemical integrity of the administered (5’)nicotine N-1’-oxide was unchanged after it was excreted in the urine. In all cases examined, the ratio of trans:cis (&-nicotine N-1’-oxide determined at either the 0-2- or 2-4-h time period was quite similar. The results suggest that diastereoselective enrichment by reduction or oxidation of @)-nicotine N-1’-oxide favoring formation of the trans-@)-nicotine N-1’-oxide was not occurring to a significant extent at least in the first 4 h after N-1‘-oxide
Table 111. Stereochemistry of (S)-Nicotine N-lf-Oxide in Adult Human Urine Samples after (S)-Nicotine N-lf-Oxide Infusion
subject 1
total dose (wg) 5370
2
3690
4
5370
5
5100
6
4380
7
3822
8
4600
time (h) 0-2 2-4 0-2 2-4 0-2 2-4 0-2 2-4 0-2 2-4 0-2 2-4 0-2 2-4
(Sbnicotine N-1’-oxide ratio (trans:cis) 2.551 2.551 2.601 2.25:l 2.65:l 2“ 9
-
2.35:l 2.201
-
2.45:l
-
‘RPHPLC analysis was not possible because the peak correspondingtotrans-nicotine N-1'-oxide was contaminatedwith a minor peak of unknown identity. The value for the infuaatewas determined to be 62:38 (trans:&) nicotine N-1’-oxide by lH-NMR and HPLC.
infusion. It is important to point out that @)-nicotine N,”-1,l’-dioxide was not detected by HPLC as a major urinary metabolite from humans infused with (5’)-nicotine N-1’-oxide. If @)-nicotine N,”-1,l’-dioxide is formed, it is formed in less than 1%yield based on HPLC analyses of organic extracts of urine from humans infused with @)-nicotineN-1’-oxide. Because the trans:cis @)-nicotine N-1‘-oxide ratio is not altered compared with the ratio of the infusate, the results suggest that stereoselective reduction or oxidation of (SI-nicotine N-1’-oxide was not occurring after infusion of @)-nicotine N-1’-oxide to healthy adult human males.
Discussion In humans, both the metabolism (11) and pharmacokinetics (3, 6-10, 15, 34, 37) of (5’)-nicotine have been extensively studied. Cotinine and (5’)-nicotineN-1’-oxide are useful markers of @)-nicotine metabolism in humans because both metabolites are chemicallystable and because they are produced by different enzyme systems. To investigate the molecular basis for (S)-cotinine and (S)nicotine N-1’-oxide formation, we previously showed that, in adult human liver microsomes, @)-nicotine Al’15‘iminium ion and @)-nicotine N-1’-oxide formation was primarily dependent on cytochrome P450 2A6 and the FM03, respectively (13). To extend this analysis and to investigate the possible role of cytochromes P450 in (S)nicotine N-1‘-oxide formation, we examined the N’oxygenation of (SI-nicotine with various hepatic and pulmonary cytochrome P450 monooxygenases (38). In Vitro Studies. As shown in Table I, the phenobarbital-inducible form of cytochrome P450 from rat liver, mouse liver and the pulmonary cytochrome P450 from rabbit catalyzes (SI-nicotine N-1’-oxide formation albeit with modest stereoselectivity (Le., mean trarwcis N-1’oxide ratio 81.1:18.9). From a purely chemical standpoint, it is anticipated that less steric interaction occurs between the N-methyl and pyridyl groups for N-1’-oxidation trans to the pyridine moiety (39). That attack of cytochrome P450 iron-oxo species occurs preferentially cis to the pyridyl ring suggests that the enzyme-substrate complex is “highly ordered and possesses restricted conformational possibilities” as suggested for related @)-nicotine iminium ion formation (40). The data also suggest that selective
886 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
active site-(S)-nicotine pyridyl N atom interactions may be present. For example, H-bonding or ionic interactions with cytochrome P450 active site amino acids may be important to restrict the movement of (S)-nicotine at the active site. The fact that both cis- and trans-@)-nicotine N-1’-oxides are formed suggests either (a) that attack by the cytochrome P450 iron-oxo species occurs from either a top and a bottom direction with respect to the aliphatic tertiary amine lone pair or, more likely, (b) that an intermediate is formed which is capable of equilibrating an oxidized speciessuch that the iron-oxo speciesrebounds preferentially to give the trans-(8-nicotine N-1’-oxide product. Previous work by Castagnoli and co-workers (39, 40) has provided strong evidence that the first step in @)-nicotine A1’v5’-iminiumion formation does not occur via initial nitrogen aminium radical cation formation, but rather, by remarkably stereoselective hydrogen atom removal. It is possible that cytochrome P45O-catalyzed @)-nicotine N-1’-oxide formation follows a completely different mechanism, but it is more likely that the same cytochrome P450 active site steric imperatives present for (SI-nicotine iminium ion formation are at work in the formation of (SI-nicotine N-1’-oxide to give the stereoselectivity observed. Previous studies from this laboratory concerning cytochrome P450-mediated S-oxidation of structurallyrelated aryI-1,3-oxathiolanes(29,411and aryl1,3-dithiolanes (42)have shown stereoselectivity equal to or greater than that observed for (S)-nicotine N-1’oxidation. We conclude that the active site of cytochrome P450 enzymes is rather accomodating to substrates of this class, but once the substrate is committed to catalysis, significant factors affording in some cases a great degree of stereoselectivity are observed for both N - and S-oxide formation. In contrast to the one-electron mechanisms of oxidation employed by cytochromes P450, the mechanism of FMOcatalyzed oxygenation of N- and S-containing substrates does not involve one-electron oxidation (26, 29, 41-43). The mechanism of FMO utilizes a hydroperoxy flavoprotein intermediate, and it has been proposed that, barring steric and ionic limitations, all good nucleophiles that are capable of oxidation by H202 should be oxidized by FMO (44,45). However, different forms of FMO possess distinct substrate binding channel dimensions, and this apparently largely contributes in determining the acceptance of a substrate for, and the stereoselectivity of, N- or S-oxygenation. Thus, FMOl has a broader substrate binding channel than FM03 (46,47) or FM02 (48)based on the ability of each enzyme to N-oxygenate a series of aliphatic tertiary amines. There are undoubtedly other contributions to FMO-mediated substrate oxidation because the N-1’-oxygenation of @)-nicotine by pig liver FMOl gives absolute diastereoselectivity for the trans-(R)-nicotine N-1‘-oxide product, but surprisingly, the N-1‘-oxygenation of (S)-nicotine by pig liver FMOl is done with almost no diastereoselectivity [Le., cis- and trans-(SI-nicotine N-1’oxides are formed in almost equal amounts] (16). This result contrasts the large S-oxygenation stereoselectivity observed for structurally related aryl-1,3-oxathiolanesand aryl-1,3-dithiolanes by pig liver FMOl (29, 41,42). It is possible that some enzyme secondary site interaction involving the pyridine nitrogen atom of (8-nicotine is present in pig liver FMO1-mediated catalysis to facilitate cis-(5’)-nicotineN-1’-oxide formation. Significant binding forces must be at work for FMOl to allow selective binding
Park et al.
of (SI-nicotine with methyl and pyridyl groups in a cis conformation in view of the strong preference for oxygenation of substrates of FMOl a t the least sterically hindered N- or S-atom lone pair ( 16,41,42,49). In marked contrast to FMO1, microsomes and cDNA-expressed FM03 from adult human liver and adult monkey liver microsomes and rabbit lung FM02 catalyzed exclusive formation of trans-(&nicotine N-1’-oxide. This result is in keeping with the notion that FMO generally oxygenates substrates a t the least sterically hindered position and that FM02 and -3 possess a much narrower substrate binding channel. Because the deduced amino acid sequence of FM03 (50)and rabbit lung FM02 (51)are only 55-57% identical to pig liver FMOl (521, it is possible that the putative binding forces present which facilitate cis-@)-nicotine N-1’-oxide formation in the presence of FMOl are absent in both FM03 and rabbit lung FM02. Work from our laboratory (13,53,54)and others (55)has also shown that the stereoselectivity of FM03 from adult human liver microsomes is also quite distinct from that of FMOl from pig or fetal human liver. It was of interest to examine the stereoselectivity of (&-nicotine ”-oxygenation in humans to determine whether an in vitro-in vivo correlation of adult human liver FM03 enzyme activity was present. In Vivo Studies. Infusion of (S)-nicotine-& into healthy male smokers produced plasma levels of (S)nicotine comparable with those observed in smokers (3). Studies to be reported elsewhere’ showed that the pattern of (SI-nicotine metabolism was generally similar when (S)nicotine was inhaled or absorbed transdermally, although considerable individual variability was observed. To examine whether the stereoselectivity of (S)-nicotine N-1’oxide formation was the same as that observed in vitro, the stereochemistry of (SI-nicotine N-1’-oxide was determined for all 13 subjects given @)-nicotine by all three routes of administration. As shown in Table 11,in all cases examined, only trans-(5’)-nicotineN-1’-oxide was observed. The data obtained with human smokers confirm the findings observed in adult human liver microsome studies that showed the metabolism of @)-nicotine to trans-(S)nicotine N-1’-oxide to be absolutely stereoselective. When (SI-nicotine was inhaled as cigarette smoke and administered intravenously or transdermally, of the @)-nicotine N-1‘-oxide diastereomers that could form, only the trans@)-nicotine N-1’-oxide was recovered from the urine. Formation of only the trans-N-1’-oxide diastereomer could be the case if either (a) exclusive trans-@)-nicotine N-1’oxygenation was observed or (b) exclusive reduction of cis-@)-nicotine N-1’-oxide to (S)-nicotine was observed, followed by stereoselective N-1’-oxygenation to the trans diastereomer. Infusion of (S)-nicotine-dz N-1’-oxide diastereomers [i.e., 62 7% :38% trans:cis (S)-nicotine N-1’oxide] to 7 healthy smokers provided no evidence for the latter hypothesis [i.e., (b) above]. This result also suggests that autoxidative or nonenzymatic oxidation of (S)nicotine was not observed during the in vitro or in vivo studies reported here by the metabolite analytical procedures utilized herein. The (S)-nicotine N-1’-oxide stereoselectivity findings described in this work contrast those of previous investigations of @)-nicotine metabolism studies in humans and adult human liver microsomes (34, 56). We attribute the difference in @)-nicotineN-1’-oxide stereoselectivity observed in our studies and those reported by others (15, 34, 56) to be due to differences in the
Stereoselective (S)-(-)-Nicotine Metabolism analytical procedures employed. For example, a previous report (15) utilized overnight paper chromatography separation of human urinary samples concentrated by heating the sample to 70 “Cfor prolonged periods of times. Such harsh and protracted treatment of nicotine N-1’oxide samples likely yielded some autoxidation and necessarily decreased the diastereoselectivity reported previously. Finally, it should be pointed out that, under certain conditions (Le., oral administration), @)-nicotine N-1’oxide could be reduced to @)-nicotine in humans. Presumably, reductases in the gut contents or bacterial flora reduce @)-nicotine N-1’-oxide to @)-nicotine that is then absorbed and subsequently metabolized. That reduction of (S)-nicotine N-1’-oxide was observed in animals (36), but was not observed in humans by the three routes of administration reported here, is in agreement with another report (57)and suggests that (a) disposition of (SI-nicotine and metabolites is highly dependent on the route of administration, (b) different metabolic enzymes (i.e., oxidases and reductases) are present in different species (58),and (c) the results obtained are highly dependent on the analytical methods employed.
Conclusion The studies of the in vitro and in vivo N-1’-oxidative metabolism of @)-nicotine in humans are in remarkably good agreement [i.e., both processes produce exclusively trans-@)-nicotineN-1’-oxide]. We concludethat the same monooxygenase is operating in a highly stereoselective fashion. The data strongly suggest that FM03 is exclusively responsible for @)-nicotine N-1’-oxide formation in humans. Although we cannot rule out the involvement of human cytochromes P450 in @)-nicotineN-1’-oxidation, the majority of the data support the notion that ( S ) nicotine N-1’-oxide formation is a selective functional marker of adult human liver FM03 activity. An important conclusion from the human @)-nicotine N-1’-oxide infusion study is that the (5’)-nicotine N-1’-oxides are not back-converted to nicotine, and in agreement with a previous study (51, (SI-nicotine N-1’-oxide does not represent a reservoir of (8)-nicotine that could play a role in reinforcing the psychoactive properties of (5’)-nicotine.
Acknowledgment. The authors acknowledge the generous help of Professors W. Trager (University of Washington), S. J. Thompson (University of Colorado), D. E. Williams (Oregon State University), and D. M. Ziegler (University of Texas). The financial support of the National Institutes of Health (NIDA DA02277 and DA01696 to N.L.B., and GM36426 to J.R.C.) and the cigarette and tobacco surtax fund of the state of California through the Tobacco-Related Disease Research Program of the University of California (2IT0071 to J.R.C. and 1RT169 to P.J.) is gratefully acknowledged. We acknowledge the generous help of the UCSF BioorganicBiomedical Mass Spectrometry Facility (A. L. Burlingame, Director, supported by NIH Division of Research Resources Grant TT016614).
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