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I I I I I I ~ I I I I I I I I I I I Stable isotope studies of nicotine kinetics and bioavailability The stable isotope-labeled compound 3',3'.dideuteronicotine was used to investigate the disposition ki- netics of nicotine in smokers, the systemic absorption of nicotine from cigarette smoke, and the bioavail- ability of nicotine ingested as oral capsules. Blood levels of labeled nicotine could be measured for 9 hours after a 30-minute intnvenous infusion. Analysis of disposition kinetics in 10 healthy men revealed a multiexponential decline after the end of an infusion, with an elimination half-life averaging 203 min- utes. This half-life was longer than that previously reported, indicating the presence of a shallow elimi- nation phase. Plasma clearance averaged 14.6 m1/min/kg. The average intake of nicotine per cigarette was 2.29 mg. A cigarette smoke-monitoring system that directly measured particulate matter in smoke was evaluated in these subjects. Total particulate matter, number of puffs on the cigarette, total puff volume, and time of puffing correlated with the intake of nicotine from smoking. The oral bioavailability of nic- otine averaged 44%. This bioavailability is higher than expected based on the systemic clearance of nic- otine and suggests that there may be significant extrahepatic metabolism of nicotine. (Ci,na Pxxex.Ntncot. THEx 1991;49:270-7.) Neal L. Benowitz, MD, Peyton Jacob III, PhD, Charles Denaro, MBBS,' and Roger Jenkins, PhDt' San Francisco, Calif., and Oak Ridge, Tenn. Cigarette smoking is addicting, and nicotine is the dependence-producing constituent of tobacco.' Phar- maceutical preparations of nicotine are employed as adjuncts to smoking-cessation therapy and may also be of use in treating medical illnesses such as Alzhei- mer's disease.2•3 Central to our understanding of nico- tine dependence and the rational use of nicotine as a medication is an understanding of its disposition kinet- ics and bioavailability from different routes of expo- sure. Because nicotine is a noxious drug in most people From the Division of Clinical Pharmacology and Experimental Therapeutics, Department of Medicine, University of California, San Francisco, and the Analytical Chemistry Division, Oak Ridge National Laboratory. Supported in part by U.S. Public Health Service grants DA02277 and DA01696 and carried out in part in the General Clinical Re- search Center at San Francisco General Hospital Medical Center with support of the Division of Research Resources. National In- stitutes of Health (RR-00083). Received for publication July 9. 1990; accepted Oct. 15. 1990. Reprint requests: Neal L. Benowitz. MD, San Francisco General Hospital Medical Center, Bldg. 30, Fifth Floor, 1001 Potrero Ave., San Francisco, CA 94110. 'Merck International Fellow. bSponsored by the National Cancer Institute under Interagency Agreement No. YOI-CP-30508 under Martin Marietta Energy Systems, Inc.. contract DE-AC05-840R21400 with the U.S. De- partrnent of Energy. 13l1/26117 who do not use tobacco, most studies of the pharma- cokinetics of nicotine have been performed in tobacco users. Such studies are typically performed after a pe- riod of tobacco abstinence, at which time levels of nicotine in the blood have fallen.4'5 However. even after overnight abstinence from tobacco, significant levels of nicotine persist, for which mathematic cor- rection is required in performing pharmacokinetic computations after known doses of nicotine. In addi- tion, there are potential problems with contamination of reagents or glassware with nicotine, which i~ present in significant amounts in the environment be- cause of the widespread use of tobacco. Background levels of nicotine reduce the accuracy of nicotine mea- surements in biologic fluids at very low concentra- tions. The use of stable isotope-labeled drugs allows phar• macokinetic studies to be performed in the presence of unlabeled drug. With a mass spectrometer, the labeled and unlabeled drug can be distinguished from one an- other, and their concentrations can be determined s1- multaneously. In the case of nicotine, the labeled drug is not found in the environment, allowing concentra• tions of the drug administered by infusion to be mc" sured at lower levels. We report here the use of 3',3'-dideuteronicattne (nicotine-d2) to investigate the disposition kinetics of nicotine in smokers and its application in the messurc- 270 I

VoLCMt. 44 ~ ~1~~t8kR 3 Stable isotope studies of nicotine 271 I I I I I I I I I I I I I I I I I 3' (S)-NICOTINE 4' (S)-N I COTI N E-3',3'-D 2 Structures of nicotine and 3',3'-dideuteronicotine. ment of the bioavailability of nicotine inhaled from cigarette smoke and ingested as oral capsules. We also describe the use of an instrumental cigarette smoke monitor and its validation as a method of estimating human smoke exposure. NtETHODS Subjects. Ten healthy men, 24 to 48 years of age, who were regular cigarette smokers were the subjects for the study. They smoked an average of 331/2 ciga- rettes per day (range, 15 to 50), with an average U.S. Federal Trade Commission (FTC) smoking machine ~ield of 1.1 (SD, 0.2) mg nicotine and 17.5 (3.9) mg tar. Subjects were highly dependent on cigarettes based on Fagerstrom score (average 7.3 of a possible .core of 11)6 and admission blood concentration of cotinine (328 ng/ml; SD 144 ng/ml; range 111 to 589 n2 ml). Results of biochemical tests of liver and kid- nn- function were within normal limits for all sub- jects. Experimental protocol. Subjects were hospitalized at the General Clinical Research Center at San Fran- ;isco General Hospital Medical Center for 3 days. The tint day was for acclimatization to the ward and to en- torce no smoking after 10 pm. On the morning of the ,econd day, after overnight abstinence from cigarette •muking and in a fasting state, intravenous catheters uere placed in the antecubital vein of one arm for in- tu,ion of nicotine and into the forearm vein of the uthzr arm for blood sampling. Subjects were asked to •muke one (five subjects) or two (five subjects) of their usual brand of cigarette. The cigarettes were •moked with a cigarette holder attached to the smoke- ,rwnitor system described below. Subjects were in- wucted to smoke the cigarette as naturally as possi- hle, Forty minutes later, after cigarette smoking had bren completed, an intravenous infusion of nicotine- d2, 2 µg base/kg/min, was administered for 30 min- utes. The infusion was administered after completion of smoking so that the exogenously administered nic- otine would not influence smoking behavior, which is determined, at least in part, by the level of nicotine in the body. Frequent blood samples were taken before, during, and after smoking and before, during, and af- ter the infusion as follows: 0, 4, 8, 12, 16, 20, 24, 28, 32, 40, 50, 60, 70, 85, 90, 120, 150, 180, 240, 300, 360, 480, 600, and 720 minutes. Further smoking was not allowed until the time of the last blood sample. On the morning of the third day, again after over- night abstinence from tobacco and food, subjects were given a capsule containing 3 mg (seven subjects), 4 mg (two subjects), or 6 mg (one subject) nicotine base as the bitartrate salt. The 3 mg dose was selected as the one expected to deliver about I mg to the systemic circulation, similar to the dose absorbed from smoking a cigarette. The 4 and 6 mg doses were intended to explore subjects' subjective responses to higher doses. Blood samples were collected at 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300, 360, and 420 min- utes. The intravenous infusion was not repeated. Sub- jects were not permitted to smoke until the completion of blood sampling. Deuterium-labeled nicotine. A nicotine analog in which two deuterium atoms are located on the 3' po- sition of the pyrrolidine ring (structure) was synthe- sized. The site of labeling was chosen because it is re- mote from the two major sites of nicotine metabolism, which include formation of cotinine (oxidized at the 5' position) and nicotine 1'-N-oxide (addition of oxygen to the pyrrolidine nitrogen).7 Previous studies have demonstrated that the disposition kinetics of nicotine-d2 and natural nicotine are similar.8 This deuterium- labeled compound was synthesized as described previ- ously,9 converted to the bitartrate salt, and purifiedby

CL!\ PFikR.'L{COL TtiFF 272 Benowitz et at, I I I I I I I I I I I I I I I recrystallization from aqueous alcohol. A solution of nicotine bitartrate for injection was made up in saline solution, sterilized by autoclaving, and aliquoted into sealed vials under a nitrogen atmosphere. Cigarette smoke monitor. The instrumental ciga- rette smoke monitor system was designed at Oak Ridge National Laboratory to measure directly smoke constituents generated by smokers. The system. a computerized version10 of a system described in detail elsewhere,'' consists of a cigarette holder, a flow measurement system, a smoke concentration detector, and a multiplier/integrator electronics package. Smoke flow and smoke concentration are determined simulta- neously; the signals are multiplied electronically, and the product signal is integrated. The integrator re- sponse was proportional to the mass of smoke particu- lates passing through the holder. The data output of the cigarette smoke monitor system consists of vol- ume, duration, and total particulate matter (TPM) for each puff and time between puffs. Chemical analyses. Plasma concentrations of nico- tine and nicotine-d2 were measured by selected ion monitoring GC/MS, with nicotine-d., used as an inter- nal standard.8 Although the limit of sensitivity of the assay is 0.1 ng/ml, the limit of quantitation (as supported by available quality control data) was 1.0 ng/ml. Therefore values below 1 ng/ml were excluded from pharmacokinetic analysis. Plasma concentrations of cotinine on admission to the study were measured by gas-liquid chromatography,t` modified for use of a capillary column. Data analysis. Plasma nicotine-d, concentrations during and after intravenous infusion were fitted to one-, two-, and three-compartment body models by expended least squares regression (MKMODEL).t3 The two- and three-compartment model fits of the data were markedly superior to the one-compartment model, so the one-compartment data are not pre- sented. The three-compartment model appeared to be superior to the two-compartment model by a combina- tion of visual inspection and the Schwartz criteria.14 in four of the 10 subjects. However, only two subjects had an adequate three-compartment fit based on the confidence intervals of the standard error of the esti- mated parameters. Because the difference in the qual- ity of fit between two- and three-compartment models for these two subjects was marginal, the results of the two-compartment fitting are presented for all subjects. Clearance (CL) and steady-state volume of distribu- tion (Vss) were calculated by two different methods. First, CL was estimated as an unknown parameter in the two-compartment fit, which is analogous to the use of the integral of the equation to the fitted concen- trations to calculate the area under the plasma concentration- time curve (AUC). CL was also calcu- lated as dose/area under the plasma nicotine-d_ concentration-time curve (AUCn,,.d,). AUC„,d, was computed by the linear trapezoidal rule for ascendin; concentrations and the log trapezoidal rule for de- scending concentrations.15 The terminal area of the AUC,,;,_d: was calculated as the last nicotine-d, concentration/k, where k is the terminal slope of the nicotine-d, concentration-time curve, estimated b~ linear regression of the final five concentration-time data pairs. With the parameters estimated for the two-compart- ment fitting, Vss was calculated as follows: uss - Vc(1 t kiz/kz,) in which Vc is the volume of the central compartment and k,, and k,, are the intercompartmental rate con- stants. Vss was also calculated with the area under the moment curve (AUMC), where the terminal area of AUCc-d, and AUMC,,;c_,6, was calculated, with k esti- mated from the last five concentration-time pairs men- tioned above. Computation of the AUMC included correction for the duration of the infusion. The dose of nicotine (D) systemically absorbed from cigarette smoking or oral capsules was deter- mined with the area under the plasma nicotine concentration-time curve for the natural (unlabeled) nicotine (AUC,,,c) and the clearance of labeled nico- tine (CL,,d) as D = AUC,,;, x CL,,;,.d,. The termi- nal portion of the area under the unlabeled plasma nic- otine concentration-time curve was estimated from the last plasma nicotine concentration/k, where k w'a" taken from the terminal portion of the nicotine-d: concentration-time curve. The AUC„ic was corrected for the predosing concentration by subtracting Cc/k • where Co was the plasma level of natural nicotine be- fore smoking or ingesting the capsule. Plasma conccn- trations of nicotine after smoking and oral nicotine were analyzed for up to 300 and 420 minutes. respeC- tively, after which time the concentrations fell belc" the limit of quantitation. The relationship between smoking parameters cOm- puted by the cigarette smoke monitor and absolute availability of nicotine was analyzed by linear regra- sion. RESULTS Plasma concentrations of nicotine-d, c could be ~t1eu- sured accurately for up to 540 minutes after the end c'f nicotine infusion (Fig. 1). The shape of the postinfu• I

I I I I I I I I I I I I I I I I I I ~ctt t'N1F +0 Stable isotope studies of 311fot131e 273, Fig. 1. Plasma nicotine-d, concentration-time curves during and after intravenous infusion of 2 µg/kg/min for 30 minutes in two subjects. The subject shown in the left panel (subject 4) shows a biexponential decline in plasma levels, whereas the subject in the right panel (subject 7) shows an apparent triexponential decline. The solid line indicates the fit based on two- or three-compartment body model equations for subjects 4 and 7, respectively (MKMODEL). Table I. Pharmacokinetics of nicotine-d, Subject No. Body weight (kg) CL (L/min)* CL (Lmin)t Vc. (L)* VSS (L)* VSS (L)t tl,,Q (min)* t1z,, (min)* tt,z0 (min)t 1 81.7 1.07 1.05 62 200 212 9.0 155 185 2 68.3 1.20 1.05 82 202 291 15.2 149 309 3 89.9 1.06 1.07 58 196 206 7.9 151 182 4 71.1 1.21 1.20 67 174 175 8.9 119 124 5 82.8 1.15 1.04 62 202 269 8.8 148 275 6 71.8 1.11 1.05 16 169 211 1.0 116 173 7 68.1 0.96 0.94 34 135 159 6.2 121 170 8 78.2 1.19 1.21 153 242 309 21.3 157 271 9 75.0 0.96 1.01 20 176 213 2.2 148 195 10 84.4 1.46 1.40 27 269 261 0.8 136 149 Mean = SD 77.1 = 7.5 1.14= 0.14 1.10= 0.13 58= 40 196 ± 38 230 - 50 8.1 ± 6.4 140 t 16 203 = 61 CL. Cleannce: Vo, volume of the central compartment: Vu. steady-state volume of distribuuon: t1,;,,. distribution half-life: tt,ZB, elimination half-life. *Paruneter determined by two-comparunent fining procedure. `Parameter determined by noncompartnxntal tnethod, sion plasma concentration-time curve was in all cases multiphasic. In most cases the curve was well de- scribed by a biexponential equation, although in two cases the curve seemed to be described better by a triexponential equation (Fig. 1). Pharmacokinetic parameters are presented for the two-compartment model fit and the noncompartmental analysis, with the half-life (tl,:) determined from the last five data points (spanning the terminal 420 min- utes) (Table 1). CL averaged 1.12 L/min (14.6 ml/min/kg) and was nearly identical as estimated by the two methods. CL values were remarkably similar among subjects, with a coefficient of variation of only 12%. With the two-compartment body model, the tl,, values of the a and (3 phases averaged 8.1 and 140 minutes. respectively. The elimination tl,: derived from the last five concentration points was consis- tently longer, averaging 203 minutes. Vss was consis- tently larger with the noncompartmental method (mean, 203 L or 3.0 LJkg) compared with the two- compartment Vss (140 L or 2.5 L/kg). An example of plasma nicotine concentration- I

, 274 Benowitz et al. `Lm rt~+M~cxni~; Table II. Cigarette smoking, puffing parameters, and bioavailability of inhaled and oral nicotine I FTC nicotine Subject No. yield (mg) FTC tar yield (mg) No. cigarettes smoked Pujjs/eigarette Total puff vol/clgarette (ml) Average puff volume (ml) I 1 0.7 10 2 7.5 460 61.6 2 1.3 23 2 9 436 48.4 3 1.0 16 2 8.5 372 43.6 4 1.1 17 2 10 424 42.1 I 5 1.3 23 2 14.5 588 40.6 6 1.2 17 1 11 721 65.6 7 1.4 21 1 9 329 36.6 8 1.0 16 1 12 630 52.5 I 9 1.1 16 1 16 596 37.3 10 1.0 16 1 14 744 53.2 Overallmean±SD 1.1 -0.2 17.5±3.9 11.1 t 2.9 530 ± 146 48.1 t 10.0 I FTC. U.S. Federal Trade Commission. TPM. total particulate matter. 1 I I I I I : EO 0NE C/GAAETTE Fig. 2. Plasma concentrations of nicotine and nicotine-d2 in a subject showing data for cigarette smoking and simulta- neous infusion of nicotine-d2. Note that nicotine-dZ levels are shown only out to 360 minutes for sake of graphic clar- 20 - 0 __F 100 1 1 200 300 Minutes 400 S00 Fig. 3. Plasma concentrations of nicotine after ingestion of capsules containing nicotine bitartrate. Data represent a mean of seven subjects for the 3 mg nicotine base, two sub- jects for the 4 mg dose, and one subject for the 6 mg dose, I I I I I ity. time curves after cigarette smoking and infusion of nicotine-d2 is shown in Fig. 2. On average, the smok- ers systemically absorbed 2.29 mg nicotinelcigarette, with a range of 0.37 to 3.47 mg. These values were considerably higher than the machine-detetmined nic- otine yields, and there was no correlation between the actual and machine-determined yields. Puffing param- eters and TPM measured by the cigarette smoke do- simeter are shown in Table II. Considering all 10 sub- jects, there was a significant correlation only between nicotine intake per cigarette and TPM (r = 0.72; p <0.01). However, excluding subject 9, who demon- strated an extraordinarily low nicotine intake com- pared to TPM (presumably as a result of puffing with- out inhaling), there were in the remaining group of nine subjects several significant correlations between nicotine intake per cigarette and smoke dosimeter- derived parameters: nicotine intake per cigarette ver- sus number of puffs (r = 0.88; p < 0.01), versus total puff volume (r = 0.76; p < 0.05), versus total puffing time (r = 0.75; p < 0.05), and versus TPM (r = 0.75; p <0.05). It is noteworthy that comparing the average pef- cigarette values for the five subjects who smoked one cigarette with the values for those subjects who smoked two cigarettes, the number of puffs (12.4 ver-

vot.L'ME Ia VL'..1BFR 3 Stable isotope studies of nieotine 275 , I I I I 19.1 ±5.5 39.1 = 11.5 2.29± 1.00 Tvtul puff duratron cigarette (sec) TPM (mglcigarette) Nicotine intakel cigarette (mg) Oral nicotine dose (mg) Oral nicotine absorbed (mg) Oral Bioavailabilitn• 19.4 21.9 1.38 3 1.17 0.39 15.6 46.4 1.78 3 1.18 0.39 9.8 24.0 1.68 3 1.77 0.59 13.6 28.8 2.12 3 1.48 0.49 22.8 43.8 3.26 3 1.27 0.42 26.4 48.1 2.98 3 1.50 0.50 15.1 50.8 2.56 6 2.53 0.42 24.4 47.8 3.47 4 1.60 0.40 18.7 30.1 0.37 3 0.73 0.24 24.9 49.1 3.22 4 1.97 0.49 0.44 ± 0.09 I I I I I I I I 1 I I I sus 9.9), total puff volume (604 versus 456 ml), total puffing time (21.9 versus 16.3 seconds). TPM (45.7 versus 35.0 mg), and nicotine intake (2.52 versus 2.05 mg) were greater when only one cigarette was smoked, although the differences were highly variable and were not statistically significant. Oral nicotine was well tolerated by all subjects. Most were not aware of any pharmacologic effect. The subject who received the highest dose (6 mg) of nicotine complained of nausea and abdominal cramp- ing that began about 30 minutes after ingesting the capsule and lasted for 60 minutes. Nicotine was ab- sorbed quickly, with a peak level occurring at about 90 minutes (Fig. 3). The oral bioavailability averaged 44% (range, 24% to 59%) (Table II). DISCUSSION We demonstrate the use of stable isotope-labeled nicotine for studying the disposition kinetics and bio- availability of the drug. Plasma concentration curves for nicotine-d2 were generally smooth, without the variability and persistent background levels of nicotine commonly seen after administration of natural nico- tine. Blood levels could be followed with confidence for up to 9 hours after the end of the infusion, and no corrections for preinfusion plasma nicotine levels were necessary. The pharmacokinetic parameters derived from infusion of nicotine-d2 are in general similar to those reported previously for natural nicotine.°'s't6't7 However, one difference is noteworthy. It is apparent that. when nicotine levels are monitored for many hours, the elimination phase is multiexponential. With a two-compartment body model, the elimination t1,2 K'as estimated to be 140 minutes, which is similar to those estimated in previous studies after infusion of natural nicotine or after cigarette smoking. However, the curves for most subjects were not perfectly fitted to a biexponential equation. According to model- independent methods, the terminal five plasma con- centration data points, representing the last 7 hours, demonstrated a t1,2 of 203 minutes. Including the ter- minal portion of the elimination phase in the clearance calculation does not appear to be important because the area included under that portion of the curve is small. However, the long elimination t1,2 does indi- cate that the Vss is somewhat larger than that esti- mated by the two-compartment body model. We sug- gest that future pharmacokinetic studies be analyzed by a noncompartmental approach and blood levels be followed for at least 9 hours after the end of adminis- tration for an accurate estimation of elimination t1,2. Stable isotopes are ideal for bioavailability studies in that an intravenous infusion of a known dose can be administered simultaneously with administration of the test drug formulation. In this study the formulation was cigarette smoke. The estimated systemic absorption of nicotine from cigarette smoking in this study averaged 2.3 mg, which is much higher than the average of I mg per cigarette derived from people smoking ad libitum throughout the day.s•18 It is likely that the unusually high level of nicotine intake in our subjects reflects the fact that the subjects (who had not smoked for the previ- ous 10 to 12 hours) knew that they could smoke only one or two cigarettes during the next 12 hours. When access to cigarettes is restricted, cigarette smokersZ1Z can increase their per-cigarette smoke intake by three-© fold or greater.19 Presumably that is what was occur-~ ring in our cigarette-deprived volunteers, despite tn-W structions to smoke naturally. In addition, our subjects~ smoked these cigarettes through a cigarette holder= O O I

CLIN PHARMICOL THER 276 Benowitz et al. MARCH )W: , I I I I I I I I I I I 1 I I (part' of the smoke dosimeter), which is an unnat- ural way to smoke and could have influenced smok- ing behavior and nicotine intake. Effects of tobacco abstinence, either before testing or anticipated after testing, and the use of cigarette holders on smoking behavior should be considered by other investiga- tors. The Oak Ridge National Laboratory cigarette smoke monitor was developed to measure directly the generation of particulate matter from a puff of a ciga- rette. Unlike the smoking machine (FTC method), which measures TPM derived from cigarettes smoked in a mechanical fashion, the smoke monitor can mea- sure particulates generated by a person smoking a cig- arette. This is important because the quantity and composition of tobacco smoke generated during ciga- rette smoking depend on the number of puffs, the size and velocity of the puff, and which part of the ciga- rette is being puffed.20 The smoke monitor had been validated previously with a smoking machine10 but not in people smoking cigarettes. We have confirmed a significant correlation between the number of puffs, total puff volume, and time of puffing with the sys- temic intake of nicotine per cigarette in smokers. Sim- ilar results have been reported by other investigators measuring the increment in plasma nicotine concentra- tion after smoking a cigarette.Z'-23 Our cigarette smoke monitor measures TPM directly, which in gen- eral is correlated with the delivery of nicotine.'`4 We observed a moderate degree of correlation, with only 56% of the variance in nicotine intake accounted for by measurement of TPM. The failure to account for more of the variance is not surprising for two reasons. First, there are differences from brand to brand in tar/nicotine ratios, as seen in Table II. Second, the cigarette smoke monitor measures TPM only in what passes out of the cigarette; it does not measure how much the smoker actually inhales, which varies con- siderably from smoker to smoker and even from ciga- rette to cigarette.25 Because most of the nicotine that is absorbed derives from inhaled smoke, delivery of particulates to the mouth is not expected to be a per- fect indicator of the systemic absorption of nicotine. For example, one of the subjects with a high level of TPM measured by the smoke monitor absorbed very little nicotine systemically, presumably because he was a noninhaler. Of note is that both the systemically absorbed dose of nicotine and the TPM measured by the smoke mon- itor were approximately twice that predicted by the FTC smoking machine (Table II). The similar propor- tional differences from the FTC values further support the validity of the TPM measurement with the smoke monitor. Our subjects generally tolerated oral nicotine well, as has been reported by other investigators.=6 The oral bioavailability was adequate to achieve plasma levels of nicotine similar to those seen after cigarette smok- ing. However, because of the prolonged course of ab- sorption (compared with smoking). the subjective e1- fects were dissimilar to those of smoking. The one subject who had abdominal cramping after a 6 mg dose did not have especially high plasma levels of nic- otine compared with those observed during cigarette smoking, suggesting that the abdominal symptoms represent a direct effect of nicotine on gastrointestinal smooth muscle. That oral nicotine in doses of 3 or 4 mg is well tolerated and achieves blood levels similar to those achieved by chewing nicotine gum suggests that oral nicotine could be employed as a method of nicotine substitution for smoking-cessation therapy. Although there was considerable individual van- ability, the average oral bioavailability of 4417c was higher than expected based on the CL of nicotine. If all of the nonrenal clearance (CLNR) of nicotine were the result of hepatic metabolism. an oral bioavailabil- ity of 33% would be predicted. The CLNR of our sub- jects is estimated to be about 1.0 L/min (based on the CL measurement and published data that indicate that renal clearance is 5% to 10% of CL when urine pH is uncontrolledt'). Assuming that liver blood flow aver- ages 1.5 L/min and all of the CLNR is hepatic. a first• pass extraction of 67% is anticipated, which would correspond to an oral bioavailability of 33%. Our data indicating that oral bioavailability is 44% suggest that one or more of those assumptions is incorrect. Nico- tine is known to be metabolized to some extent by the lung27 and conceivably by other organs; perhaps such extrahepatic metabolism explains the higher than an- ticipated oral bioavailability. In conclusion, we provide data on the use of 3'.3'- dideuteronicotine to study the pharmacokinetic> of nicotine in tobacco users and to determine the absolute availability of nicotine from tobacco smoke or other nicotine-delivery formulations. We suggest that the high degree of analytic sensitivity of the stable isotope method and the specificity of labeled nicotine such that there is no interference by background levels ()f nicotine provides superior-quality pharmacokinetic data compared with measurement of natural nicutine. The results indicate that some of the currently ac- cepted pharmacokinetic parameters for nicotine need to be revised. The application of stable isotuNs I I

%(1l L'MF 41) ~ .I ~taE.R z Stable isotope studies of nicotine 277 I I I I I I I I I I I I 1 I I I should provide a useful tool to examine factors that in- fluence nicotine kinetics and metabolism in tobacco users and to conduct bioavailability studies for newly developed therapeutic nicotine-delivery systems. We thank Beverly Busa and Clarissa Ramstcad for assis- tLincc in clinical studies. Lisa Yu and Chin Savanapridi for performance of nicotine assays. Gunnard Modin for statisti- cal advice, and Kaye Welch for editorial assistance. References 1. Benowitz NL. Pharmacologic aspects of cigarette smoking and nicotine addiction. N Engl J Med 1988:319:1318-30. 2. Hughes JR. Miller SA. Nicotine gum to help stop smoking. JAMA 1984:252:2855-8. :. Newhouse PA, Sunderland T, Tariot PN, et al. Intrave- nous nicotine in Alzheimer's disease: a pilot study. Psy- chopharmacology 1988;95:171-5. 4. Benowitz NL, Jacob P III. Jones RT, Rosenberg J. In- terindividual variability in the metabolism and cardio- vascular effects of nicotine in man. J Pharmacol Exp Ther 1982;221:368-72. 5. Feyerabend C, Ings RMJ, Russell MAH. Nicotine phar- macokinetics and its application to intake from smok- ing. Br J Clin Pharmacol 1985;19:239-47. • 6. Fagerstrom KO. Measuring degree of physical depen- dence to tobacco smoke with reference to individualiza- tion of treatment. Addict Behav 1978;3:235-41. 7. Jacob P III, Benowitz NL, Shulgin AT. Recent studies of nicotine metabolism in humans. Pharmacol Biochem Behav 1988;41:474-9. 8. Jacob P Ill, Benowitz NL, Yu L, Wilson M, Shulgin AT. Selected ion monitoring method for determination of nicotine, cotinine, and deuterium-labeled analogs: absence of an isotope effect in the clearance of (S)-nic- otine-3'-3'd: in humans. Biomed Environ Mass Spec- trom [In press]. 9. Jacob P III, Benowitz NL, Shulgin AT. Synthesis of optically pure deuterium-labelled nicotine, nornicotine and cotinine. J Labelled Comp Radiopharm 1988;25: 1117-28. 10. Jenkins RA, Holmberg RW, Gayle TM. Instrumental cigarette smoke dosimeter designed for human smoking studies. Presented at the Fortieth Tobacco Chemists' Research Conference, Knoxville, Tennessee. Oct. 13-16, 1986. ll. Jenkins RA. Gayle TM. An instrumental cigarette smoke monitor designed for the direct measurement of smoke particulate matter generated in human smoking studies. Behav Res Methods Instrument Comput 1984;16:263-7. 12. Jacob P 111. Wilson M. Benowitz NL. Improved gas chromatographic method for determination of nicotine and cotinine in biologic fluids. J Chromatogr 1981: 222:61-70. 13. Holford N. MKMODEL. Milltown, NJ: Biosoft. 1990. 14. Schwarz G. Estimating the dimension of a model. Ann Stat 1978;6:461-4. 15. Rowland M, Tozer TN. Clinical pharmacokinetics: con- cepts and applications. 2nd ed. Philadelphia: Lea & Fe- biger. 1989:459-63. 16. Rosenberg J, Benowitz NL. Jacob P 111. Wilson KM. Disposition kinetics and effects of intravenous nicotine. Cutti PHARMACOL THER 19H0:28:516-22. 17. Lee BL. Benowitz NL. Jacob P II1. Influence of to- bacco abstinence on the disposition kinetics and effects of nicotine. CuN PHARMACOL THaR 1987:41:474-9. 18. Benowitz NL, Jacob P III. Daily intake of nicotine dur- ing cigarette smoking. CLIN PHARMACOL THF.R 1984; 35:499-504. 19. Benowitz NL, Jacob P III, Kozlowski L. Yu L. Influ- ence of smoking fewer cigarettes on exposure to tar, nicotine, and carbon monoxide exposure. N Engl J Med 1986;314:1310-3. 20. Creighton DE, Lewis PH. The effect of smoking pat- tem on smoke deliveries. In: Thornton RE, ed. Smok- ing behavior: physiological and psychological influ- ences. Edinburgh: Churchill Livingstone. 1978:301-14. 21. Sutton SR, Russell MAH. Iyer R. Feyerabend C. Sa- loojee Y. Relationship between cigarette yields, puffing patterns, and smoke intake: evidence for tar compensa- tion? Br Med J 1982;285:600-3. 22. Herning RI, Jones RT, Benowitz NL. Mines AH. How a cigarette is smoked determines nicotine blood levels. CLIN PHARMACOL THPR 1983;33:84-90. 23. Zacny JP, Stitzer ML, Brown FJ, Yingling JE. Griffiths RR. Human cigarette smoking: effects of puff and inha- lation parameters on smoke exposure. J Pharmacol Exp Ther 1987;240:554-64, 24. Rickert WS, Robinson JC, Young JC. Collishaw NE. Bray DF. A comparison of the yields of tar, nicotine, and carbon monoxide of thirty-six brands of Canadian cigarettes tested under three conditions. Prev Med 1983;12:682-94. 25. Herning RT, Hunt JS. Jones RT. The importance of in- halation volume when measuring smoking behavior. Behav Res Methods Instrument 1983;15:561-8. 26. Jarvik ME, Glick SD, Nakamura RK. Inhibition of cig- arette smoking by orally administered nicotine. Ct-uN PHARMACOL THER 1970;11:5746. 27. Turner DM, Armitage AK, Briant RH, Dollery CT. Metabolism of nicotine by the isolated perfused dog Z~Z lung. Xenobiotica 1975;5:539-51. n ~ ~ W GJ~ ZZ 11Z I