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Pharmacokinetics of nicotine in rats after multiple-cigarette smoke exposure
TOXICOL~YANDAPPLIED
69, l-11 (1983)
PHARMACOLOGY
Pharmacokinetics of Nicotine in Rats after Multiple-Cigarette Smoke Exposure’ KEITH S. ROTENBERG' AND JOSEPH ADIR~ Clinical
Pharrnacokinetic
Laboratory,
Received
School
June
of Pharmacy,
28, 1982;
accepted
University
January
of Maryland.
Baltimore,
Maryland
31, 1983
Pharmacokinetics of Nicotine in Rats after Multiple-Cigarette Smoke Exposure. ROTENBERG. Toxicol. Appl. Pharmacol. 69, I- 11. The pharmacokinetics of nicotine and its major metabolites was evaluated in male rats after multiple-cigarette smoke exposure. A smoke-exposure apparatus was used to deliver cigarette smoke to the exposure chamber. The rats were exposed to smoke from a single cigarette every 8 hr for 14 days and to the smoke of a cigarette spiked with radiolabeled nicotine on the 15th day. Blood and urine samples were collected at timed intervals during the lo-min smoke-exposure period of the last cigarette and up to 48 hr thereafter. Nicotine, cotinine. and other polar metabolites were separated by thin-layer chromatography and quantified by liquid scintillation counting. The data were analyzed by computer fitting, and the derived pharmacokinetic parameters were compared to those observed after a single iv injection of nicotine and after a single-cigarette smoke exposure. The results indicated that the amount of nicotine absorbed from multiple-cigarette smoke was approximately lo-fold greater than that absorbed from a single cigarette. Also, unlike the singlecigarette smoke exposure experiment, nicotine plasma levels did not decay monotonically but increased after the 5th hr, and high plasma concentrations persisted for 30 hr. The rate and extent of the formation of cotinine, the major metabolite of nicotine, were decreased as compared with their values following a single-cigarette smoke exposure. It was concluded that nicotine or a constituent of tobacco smoke inhibits the formation of cotinine and may affect the biotransformation of other metabolites. Urinary excretion tended to support the conclusions that the pharmacokinetic parameters of nicotine and its metabolites were altered upon multiple as compared to single dose exposure. K. S., AND ADIR, J. ( 1983).
Numerous investigations have examined the effects of chronic cigarette smoke exposure on induction (Hart et al., 1976; Keeri-Szanto and Pomeroy, 197 1; Jick, 1974; Vestal et al., 1974; Welch et al., 197 1) or inhibition (Ruddon and
Cohen, 1970; Thompson d al., 1974; Weber et al., 1974) of lung or liver metabolizing enzymes. Investigators (McGovern ef al., 1976) have also shown that tobacco smoke inhibits the metabolism of nicotine since the appearance of the major metabolites, cotinine and nicotine- I’-N-oxide, was slower. The pharmacokinetics of nicotine and its metabolites following multiple-cigarette smoke exposure has not been extensively evaluated in the intact animal or man. Previous studies from this laboratory examined pharmacokinetics of nicotine in the rat following single dose iv administration (Adir et al., 1976; Miller et al., 1977) and single-cigarette smoke exposure (Rotenberg et al., 1980). Nicotine was ab-
’ Supported in part by National Cancer Institute Contract NOl-CP-433 12. * Abstracted in part from a dissertation submitted to the University of Maryland by Keith S. Rotenberg in partial fulfillment of the Doctor of Philosophy degree requirements. To whom correspondence should be addressed. Current address: Pennwalt Corporation, Pharmaceutical Division, 755 Jefferson Road, Rochester, N.Y. 14623.
’ Current address: Howard University, College of Pharmacy and Pharmacal Sciences, Washington, D.C.. 20059. I
0041-008X/83
$3.00
Copyright 61 1983 hy Academic Press. Inc All nghtr of reproductmn I” any form reserved.
2
ROTENBERG
sorbed
rapidly
with
concentration cessation
of
The
sure. otine
the
of its were
similar
to
those
of
The purpose of the
of nicotine
in rats
cigarette
position lites observed
kinetics after
elimination
order
kinetics, parameters
observed
after
this
investigation
and
extent
rate
following
smoke
following
first
nicexpo-
iv
ad-
nicotine.
determine to
and
pharmacokinetic
ministration
of
smoke
followed
derived
expo-
elimination
formation
metabolites the
after
smoke
and
as the
plasma
immediately
single-cigarette
as well
and
maximum
single-cigarette
absorption
following
sure
the
occurring
AND
to
multiple single-dose
exposure
compare
nicotine
and
exposure cigarette
to
absorption
multiple
and of
was of
the its
dis-
metabo-
with smoke
those ex-
(Rotenberg et al., 1980) or iv administration (Adir et al., 1976; Miller et al., 1977). posure
METHODS Chemicals. [methyl-‘4C]Nicotine was obtained commerciaIly4 with a specific activity of 6 I .6 rCi/mg. Radiochemical purity was established by TLC and radioscanning techniques. With acetone:henzene:ammonia: ethanol (40:50:5:5) as the solvent, the compound migrated as a single radioactive spot with an R,value corresponding to authentic nicotine. Unlabeled nicotine5 was obtained commerciaIly. and unlabeled cotinine was prepared from nicotine by a previously described method (Bowman and McKennis, 1963). Cigaretfes. Code No. 31 cigarette? were stored upon arrival in a desiccator that provided an atmosphere of 65% humidity. They were reported to contain 9.5 mg of nicotine/g of tobacco and to have a smoke condensate pH of 4.9. Spectrophotometric analysis (El-Sayed and Mohamed, 1975) verified the nicotine content of the cigarettes. One cigarette (the last in the multiple cigarette smoke exposure regimen) was spiked with “C-laheled nicotine by an infusion pump as described earlier (Rotenherg et al., 1980). The final specific activity of the cigarette was 17. I I rCi/mg. To maintain similar nicotine content between the radioactive-labeled cigarette and those used throughout the multiple-exposure regimen (cold), the cold cigarettes were spiked with unlabeled nicotine. Unlabeled nicotine was dissolved in ethanol to a final concentration of 3.65 mg/
4 ICN, Irving, Calif. 92664. 5 Eastman Kodak Company, 6 Enviro Control, Rockville.
Rochester, Md. 20852.
N.Y.
14650
ADIR
ml. One milliliter of the solution was delivered lo each cigarette. Thus. the cigarettes containing either radiolabeled or nonlabeled nicotine had a final nicotine content of 13. I5 mg/cigarette. After spiking. both cigarettes were replaced in the desiccator. -Smoke e.~po.rwe apparatus. The intermittent smoke generation-inhalation system’ has been described earlier (Rote&erg ef al., 1980). AnimuO. Male Fischer-344 rats’ weighing 199 to 228g. were housed in stainless-steel cages and had free access to food’ and water. Three weeks before introduction of radiolabeled cigarette smoke. each animal was anesthetized with ether, and a chronic cannula was implanted into the jugular vein (Upton, 1975). Of the IO rats which entered the study. 4 were removed due to either dislodgement or failure of Ihe cannula. Thus. six rats completed the study. During the multiple-dosing regimen the weight of the rats was recorded each day at 7:30 AM. Throughout the entire dosage regimen. the rats were maintained in a room with a 12-hr light/dark cycle and a constant temperature of 23°C. Protocol. Rats were dosed every 8 hr./i,r 14 consecrctiw dqa with cigarette smoke from nonlabeled cigarettes. They were placed in the smoke-exposure apparatus and administered the smoke of one cigarette at 7 QNI. 3 pm, and 11 pm daily after which they were transfered to individual cages. On the 15th day. the cigarette containing radiolabeled nicotine was administered at 9:30 urn and consumed in IO min (IO puffs), after which the rats were transferred to plastic metabolism cages for the remainder of the experiment and allowed food and water ad lihitum. Blood samples (0.2 ml) were collected into heparinized syringes during the smoking phase at 0.05. 0.08, 0. I I, 0.12, and 0.17 hr and in the post-smoking phase at 0.30. 0.50, 0.75. 1.0, 1.5, 2.0, 3.0, 5.0, 8.0, 10.0. 12.0, 14.0, 18.0, 24.0. and 30.0 hr. The plasma was separated from whole blood by centrifugation and frozen until time of analysis. Urine was collected in containers immersed in ice for up to 48 hr and frozen until analysis. Separation and quantification of nicotine and its metabolites were described earlier (Adir et a/., 1976; Miller e/ al., 1977). Pharmacokinetic unu1vsi.r. The concentration-time profiles of nicotine. cotinine. and other metabolites for each rat were plotted on semilogarithmic graph paper which provided an insight to the operative pharmacokinetic model. These plots were employed to obtain initial estimates for curve fitting of the data by the SAAM 26 digital computer program (Berman and Weiss. 1974). Each datum point was assigned a statistical weight proportional to the reciprocal of the square of a standard deviation representing 10% of the point.
’ Maddox-ORNL, Oak Ridge National Research Laboratory, Oak Ridge. Tenn. 37830. ’ Microbiological Associates, Walkersville. Md. 2 1793. 9 Purina Rodent Laboratory Chow. Ralston Purina Co.. St. Louis, MO. 53 188.
PHARMACOKINETICS
NICOTINE
The puffing profile of the smoke inhalation machine was incorporated into the pharmacokinetic model used to fit the data both during and after cigarette smoke inhalation. The amount of nicotine absorbed was computed (Gibaldi and Perrier, 1975) from the product of the ratio of area under the plasma concentration-time curve (AUC) from 0 to 8 hr (dosage interval) following cigarette smoke inhalation and the AUC from 0 to z, following iv administration from the 0.8 mg/kg iv dose reported earlier (Miller PI al.. 1977).” Alternatively, the amount of nicotine excreted in urine during a dosing interval was compared to the amount of nicotine ultimately excreted in urine following iv administration (expressed as fractions of total radioactivity) to estimate nicotine absorption by inhalation. The dose and the amount of nicotine excreted in the urine from the 0.8 mg/kg dose reported earlier (Miller el a/., 1977) were used in this calculation. The equations used in these calculations have been previously described (Rotenberg ef al., 1980). The plasma nicotine concentration (Cn) versus time data for each rat were fitted to a first order absorption triexponential function which contained the rate constant of absorption (K,) and the other hybrid rate constants. LY and /3. reflecting distributive and terminal phases, respectively. The dose absorbed by each rat (calculated as described above) was included in the data-fitting procedure. The first order rate constants of formation (Kf) and elimination (K,) and the ratio of the fraction of nicotine converted to cotinine (F) to the volume of distribution of the latter ( Vd) were obtained by curve fitting of the plasma cotinine concentration-time data as described earlier (Adir el al., 1976: Miller et al., 1977). Other pharmacokinetic parameters and those of the origin activity were estimated as described previously (Rotenberg ef al.. 1980). Statistical anal.vsis. The t test was used to evaluate differences in the pharmacokinetic parameters of nicotine and its metabolites between the single- and multiple-cigarette smoke exposure or iv administration of nicotine (Steele and Torrie, 1960).
RESULTS Nicotine. Approximately 14% (6.98 X 10’ dpm) of the nicotine present in the mainstream smoke (4.9 X 10’ dpm) was delivered to the exposure chamber. This amount represented the sum of the nicotine absorbed by the rats, retained on the Cambridge filter (4.9 X lo6 dpm), and present in the washings of “In the intravenous study. blood samples were obtained from an arterial cannulation while in the present investigation blood samples were obtained from venous cannulation. A comparative experiment showed that the pharmacokinetic parameters were not affected by the blood sampling technique (Rote&erg, 1978).
CIGARETTE
3
EXPOSURE
the chamber (1.1 X lo6 dpm). The amount of nicotine absorbed (utilizing the weight of the rat on the 15th day) calculated from AUC and urinary recoveries of nicotine are shown in Table 1. Absorption ranged from 3.00 X lo7 to 4.98 X IO7 dpm/kg (0.79 to 1.31 mg/kg) based on plasma AUC and from 1.48 X 10’ to 3.70 X lo7 dpm/kg (0.39 to 0.97 mg/kg) based on urinary excretion. There was a significant difference among the means (p < 0.01) between the average amount calculated from plasma and that from urine. Nicotine was absorbed very rapidly following cigarette smoke inhalation as shown in Fig. 1. During the 10 puffs needed to consume the cigarette, the plasma nicotine levels paralleled the intermittent pattern of smoke exposure (shown in the inset of Fig. 1). Maximum plasma concentration of 9200 dpm/ml (242 rig/ml) occurred at the end of the cigarette smoke exposure. After rapid absorption of nicotine which could be adequately deTABLE NICOTINE
ABSORBED
FOLLOWING
MULTIPLE
SMOKE
EXPOSURES
OF CIGARETTE
Amount absorbed based on AUCb
Rat 1 2 3 4 5 6 R SE
Dose kbm/kg) 4.01 4.89 4.07 4.98 3.00 4.08 4.17 2.93
x x x x x x x x
Dose’ (m&t) 10’ 10’ 10’ 10’ I07 IO’ 10’ lo6
1
1.06 I .29 1.07 1.31 0.79 I .07 1.10 0.08
DOSING
Amount absorbed based b on urinary excretion Dose (&Wmg) 3.31 3.70 2.90 2.49 1.48 2.73 2.77 3.11
x x x x X x
IO’ 10’ 10’ 10’ IO’ 10’ x IO’ x 106
Dose’ (w/kg) 0.87 0.97 0.77 0.66 0.39 0.72 0.73 0.08
a Rats were dosed every 8 hr with cigarette smoke from nonlabeled cigarettes for 14 consecutive days. On the 15th day, a cigarette spiked with radiolabeled nicotine was administered. *Significant differences (p i 0.01) were observed between the dose calculated from plasma versus the dose calculated from urinary excretion of nicotine. ’ Dosage was calculated from specific activity of nicotine in the labeled cigarette, 17. I I &i/mg.
ROTENBERG
AND ADIR
i. I I. TIME
(hr.)
FIG. 1. Semilogarithmic plot of nicotine plasma concentration (x f SE) as a function of time following multiple-cigarette smoke exposure of nicotine. Computer fitting of the data is represented by the solid line. The X-axis of the inset is in minutes.
scribed by first order kinetics, the plasma nicotine concentrations declined biexponentially for the first 2 hr (Fig. I) but increased rapidly in the 5th hr, and high concentrations persisted for 30 hr post-cigarette smoke exposure. This portion of the curve from 3 to 30 hr could not be fitted by an equation which could account for the changes in the pharmacokinetics of nicotine observed. Thus, the AUC during a dosing interval (0 to 8 hrs) was calculated by extrapolation by the observed elimination rate constant (P) to allow comparison with the results from the single-dose studies (Rotenberg ef al., 1980; Miller et al., 1977). The pharmacokinetic parameters of nicotine derived from these data are shown in Table 2. The mean half-life of absorption (t*/2, K’J was 5.3 min. The half-life of the rapidly (P/Z,a) and slowly (f%,8) declining phases were 5.9 and 74.6 min, respectively. There were no significant differences between these half-lives or renal clearance and those ob-
served in the single-cigarette smoke exposure study (Rote&erg et al.. 1980). Cotinine. The concentration vs time profile (Fig. 2) of this major metabolite of nicotine and the pharmacokinetic parameters derived from these data are reported in Table 3. The rate constant of formation (Kr) ranged from 0.6 to 1.12 hr-’ but was not significantly different than that observed in the single-dose iv experiment. Cotinine was eliminated from plasma with a mean (SE) half-life of elimination (P/2, K,) of 4.5 (0.2) hr. There were no significant differences in this parameter between the multiple-dose investigation and the single-dose cigarette smoke exposure or iv administration. Nor were there any significant differences in the time to reach maximum plasma concentration (t,,,) between multiple-dosing and the single-dose experiments. Since the amount of nicotine absorbed upon multiple dosing was greater than that following single-cigarette smoke exposure, the AUC
PHARMACOKINETICS
NICOTINE
CIGARETTE
5
EXPOSURE
TABLE 2 PHARMACOKINETICPARAMETERSOFNICOTINEFOLLOWING MULTIPLE D~SINGOFCIGARETTESMOKE~
Rat I 2 3 4 5 6 R SE
KC3 (mini’)
a (mini’)
P (mini’)
AUC ((dpm . W/W . kg); O-8 hr)
Cl, (ml/hr . kg)
0.0798 0.1381 0.0698 0.2207 0.5079 0.1824 0.1998 0.0660
0.1201 -h 0.1 185 0.1436 0.08 11 0.1617 0.1250 0.0140
0.0101 0.0115 0.008 I 0.0098 0.0097 0.0072 0.0094 0.0006
46.856 57.152 47.532 58,155 35,088 47,610 48,732 3,426
233 101 173 198 132 56 149 27
a See text for definition of parameters. b Although the (Y phase in this rat was discernible by examination of the log concentration-time include sufficient data points to enable computer estimation of this parameter.
of cotinine was expected to be larger and the ratio of AUCcotinlne/AUCnicotine to be significantly smaller (p < 0.05) than that obtained from the single-dose studies of cigarette smoke exposure or iv administration of nicotine (Table 3). However, the renal clearance of cotinine (13 to 45 ml/hr - kg) was not significantly different from that observed in single-dose experiments. Other metabolites. A semilogarithmic plot of the origin activity is presented in Fig. 3. The origin activity declined biexponentially
plot, it did not
following its formation similar to that observed following single-dose cigarette smoke exposure or iv administration of nicotine. There were no significant differences (p < 0.05) in the half-lives of formation, distribution, and elimination of this activity between multiple-dose and single-dose administration of nicotine. The mean (SE) half-life formation was 20.2 (4.8) mitt, while the mean (SE) half-life of the distribution and elimination phases were 31.2 (8.8) min and 23.6 (1.9) hr. respectively (Table 4). There were no
7000
TIME
(hr)
FIG. 2. Semilogarithmic plot of cotinine plasma concentration (J? f SE) as a function of time following multiple-cigarette smoke exposure of nicotine.
6
ROTENBERG
AND ADIR
TABLE 3 PHARMACOKINETKPARAMETERSOFCOTININEFOLLOWING
Rat
I 2 3 4 5 6 ‘I? SE
0.600 0.917 0.729 I.1 I9 0.868 0.785 0.837 0.072
‘See text for definition
0.171 0. I33 0.152 0.141 0. I78 0.155 0.155 0.007
MULTIPLE
4nax W
AU< (@pm . hr)/(ml . kg); O-X hr)
I .92 I .92 1.93 1.71 I.28 I .90 1.77 0.1 I
148.995 215.121 I 13,487 209.40 I 1 18,570 153.103 159.780 17.820
D~SINGOFCIGARETTE
44.85 28.24 12.54 20.10 40.87 24.64 28.54 5.04
SMOKE”
3.18 3.76 2.39 3.60 3.38 3.12 3.26 0.20
0.190 0.194 0.150 0.178 0.206 0.200 0.186 0.008
of parameters.
significant differences (p < 0.05) in the time to reach maximum plasma concentration between multiple- and single-dose administration of nicotine. The values ranged from 0.5 to 1.7 hrs. From the AUC of the origin activity shown in Table 4, it can be seen that the concentrations of origin activity following multiple-dose were higher than those observed in the single-dose cigarette exposure study. The ratio of AUC,h,, actlvity/AUCnicotine ranged from 1.O to 1.6 and were significantly lower (p < 0.05) from the single-dose experiments. Urinary e+xcretion. The 48-hr urinary excretion of nicotine and its metabolites following cigarette smoke inhalation is presented in Table 5. Significantly lower (p < 0.0 1) urinary recoveries of nicotine, cotinine, and origin activities were observed when the 8-hr multiple-dosing interval recoveries were com-
pared to the single-dose cigarette smoke inhalation and iv administration of nicotine studies. However, the 48-hr urinary recoveries were not significantly different from the single-dose studies except when these recoveries were related to the percentage of dose absorbed. Approximately 23, 14, and 39% of the total radioactivity excreted over the 8-hr period consisted of nicotine, cotinine, and origin activity, respectively. It was expected that during a dosing interval approximately 90% of the nicotine would be excreted. In fact, only approximately 60% of the nicotine excreted was found between 0 to 8 hrs. DISCUSSION The purpose of this study was to evaluate the pharmacokinetics of nicotine after mul-
I TIME
1
24
26
28
30
(hr.)
FIG. 3. Semilogarithmic plot of origin activity plasma concentration (d ? SE) as a function of time following multiple-cigarette smoke exposure of nicotine.
PHARMACOKINETICS
NICOTINE
CIGARETTE
TABLE PHARMACOKINETIC
I
OF ORIGIN
I’V2, a (hr)
I%. 0 (hr)
Inm (hr)
0.467 0.172 0.264
20.56
0.50
65,312
281.39
1.39
31.79 26.05 18.83
I .oo I .oo I.71
75.960 48.781 93,232
1.33 1.03 I.61
21.79 22.57
1.00 0.50
184.87 184.03 95.36 281.16 114.51
23.60 I.91
0.96 0.15
.c
0.388 0.249 0.336
0.715 0.352 1.147 0.520
SE
0.080
0.147
5 6
4
PARAMETERS
0.431 0.057 0.258 0.633
2 3 4
‘See text for definition
ACTIVITY
FOLLOWING
MULTIPLE
AK ((dpm . hr)/(mI O-8 hr)
DOSING
TABLE URINARY
EXCRETION
OF NICOTINE
FOLLOWING
kg):
47,560 72.634 67,247 7.100
4
5 6 d SE
kg)
1.36 I.53 1.38
190.16 32.28
0.08
1975). One investigation (Kendrick et al., 1976) further showed that exposure to only five cigarettes within 1 hr was capable of killing 50% of “naive” rats with a cigarette smoke exposure apparatus similar to the one used in the present study. Our earlier pharmacokinetic studies have shown that nicotine had an approximate half-life of about 1 hr while the metabolites had much longer half-lives (e.g., cotinine 4 to 8 hrs) (Adir et al., 1976; Miller 5
AND ITS METABOLITES
CIGARETTE
% of absorbed dose excreted in urine as totaf radioactivity
Time tW
SMOKE
IN RATS 8 AND 48 hr
INHALATION
radioactivity in urine as:
Nicotine
Cotinine
excreted
Orig.
0 0 0 0 0 0
to to to to to to
8 8 8 8 8 8
23.0 12.9 14.4 12.4 22.1 Il.6 16.1 2.1
23.9 18.3 32.0 38.7 14.6 12.8 23.4 4.2
14.7 19.2 5.5 13.7 15.1 17.9 14.4 2.0
40.3 44.4 34.9 29.8 41.8 39.5 38.5 2.1
0 0 0 0 0 0
to to to to to to
48 48 48 48 48 48
44.0 40.3 38.1 26.1 26.4 35.8 35.1 3.0
15.7 11.4 11.4 26.6 13.8 9.0 15.7 2.5
21.2 20.4 13.8 14.4 16.9 17.2 17.3 1.2
35.0 38.5 32.1 31.0 40.2 25.1 33.8 2.2
x SE 1 2 3
SMOKE’
CL (ml/hr.
% of the total
I 2 3 4 5 6
OF CIGARETTE
of parameters
tiple-cigarette smoke inhalation which is the common method of dosing in pharmacologic and toxicologic studies involving this agent. The dosing regimen used in this investigation was derived from toxicologic and pharmacokinetic considerations. Previous cigarette smoke inhalation studies indicated that exposure of two cigarettes during 8 hr each day reduced survival of rats as compared to sham smoking (Davis et al.,
Rat
7
EXPOSURE
act.
8
ROTENBERG
et al., 1977; Rotenberg et al., 1980). Accordingly, rats were exposed to smoke from a single nonradioactive cigarette every 8 hr for 14 consecutive days while a cigarette containing radiolabeled nicotine was used on the 15th day. During the multiple-dose study, the weight of each rat was recorded and a decrease in body weight mean (%CV) of 29.0 (60) g from the first day weight was observed by the 15th day. Similar changes in mean body weight of Fischer-344 rats were reported upon exposure to cigarette smoke (Kendrick et al.. 1976). The weight loss was attributed to stress of confinement in the animal holder and to the exposure of cigarette smoke. Nicotine absorption following multiplecigarette smoke inhalation was determined by comparing the urinary recovery and AUC of nicotine during a dosing interval (8 hrs) with those values obtained following iv injection of a single dose of nicotine. It should be noted, that the AUC during the multiple-dosing regimen is equivalent to the AUC from O-ix, after a single-dose administration (Gibaldi and Perrier, 1975). Also, after multiple dosing, urine collection of a drug during the dosing interval will equal the urine collected to infinity for a single-dose experiment (Gibaldi and Perrier, 1975). Based on these calculations, the total dose absorbed following multiple dosing was approximately 10 times greater than that absorbed following a singledose inhalation, probably because of adaptation by the rats to smoke inhalation following multiple exposure. Furthermore, nicotine plasma concentrations were higher and persisted for an extended period of time following multiple dosing as compared to a single exposure. The mean (SE) half-life of elimination of nicotine following multiple dosing was 1.3 (0.1) hr indicating that seven half-lives later all of the nicotine should have been eliminated from the plasma. However, nicotine levels persisted for 30 hr (Fig. 1). The halflife of build up and elimination of this “nicotine” after 3 hr post-cigarette smoke inhalation were approximately 11.4 and 15.1 hrs,
AND
ADIR
respectively. It should be noted that no nicotine was detected in our single-dose study 5 hrs post-nicotine injection or following cigarette smoke exposure (Adir et al., 1976: Miller et ul.. 1977; Rotenberg et al., 1980). In addition, the average total radioactivity recovered in urine following multiple dosing (Table 5) ranged from 26 to 44.0% of the absorbed dose (0 to 48 hr) while following single-dose exposure, the values ranged from 50 to 79%. These data suggest that in addition to absorption the elimination of nicotine and/or its metabolites was altered upon multiple dosing. It is well known that nicotine is eliminated by urinary excretion and metabolism. One of the metabolites of nicotine is nicotinel’-N-oxide which has an approximate half-life (assuming that it comprises most of the activity at the origin) of 24 hr. It has been reported (Dajani et al.. 1975a) that following ip injection of nicotine- I’-N-oxide (0.80mg/kg) to rats, this metabolite was converted to nicotine. These authors also found (Dajani et al.. 197513) that the liver, small intestine, lung. spleen, kidneys, and heart were capable of reducing nicotine- I’-N-oxide to nicotine in vitro with the liver and small intestine having the greatest activity. Another study (Jenner et al., 1973) showed that following po administration to nicotine-1’-N-oxide (2.0 mg) in man, it was reduced to nicotine in the gastrointestinal tract from which nicotine was subsequentially absorbed and recovered in the urine. However, no reduction of nicotine-l’N-oxide to nicotine occurred when the former compound was injected iv into humans. It is not known whether nicotine- l’-N-oxide is also reduced to nicotine after iv administration to rats: however, only 3% of an injected dose of nicotine was excreted into the bile in 6 hr (Hansson and Schmiterlow. 1962). No data are available on the elimination of nicotineI’-N-oxide in the bile. However, it has been suggested (Smith, 1973) that the increased polarity of a compound may increase its biliary excretion. If the biliary excretion of nicotineI’-N-oxide were greater than nicotine, then the following hypothesis may explain the
PHARMACOKINETICS
NICOTINE
presence and persistence of nicotine at high concentrations 3 hr post-cigarette smoke exposure (Fig. 1). The high tissue levels of nicotine-l’-N-oxide which are likely to occur upon multiplecigarette smoke exposure may result in its increased excretion. Upon reaching the small intestine, this nicotine- I’-N-oxide may be converted to nicotine which is then absorbed. Thus, approximately 3 hr post-nicotine exposure, an increase in nicotine plasma levels would be observed with an elimination rate closely parallel to that of the origin activity from which it is derived. Although this hypothesis seems reasonable, other possibilities such as nicotine removal from deep tissue stores after cessation of cigarette smoke inhalation cannot be ruled out as an explanation for the persistence of nicotine plasma levels. The finding of prolonged exposure of nicotine following multiple dosing may provide additional insight to the possible involvement of nicotine in lung disease, cardiovascular response, and teratogenic and other toxicologic effects of cigarette smoke inhalation. The rate of formation of cotinine, the major metabolite of nicotine, was significantly smaller following multiple exposure than that observed following the single-dose cigarette smoke inhalation. It is possible that following multiple dosing of cigarette smoke an inhibition of the metabolism of nicotine to cotinine may occur. Stalhandske and Slanina ( 1970) have reported that nicotine given ip at 15 mg/kg to mice for 3 days significantly reduced its own metabolism. Nicotine also inhibited the metabolism of benzyprene, a component of cigarette smoke, in vitro and in vivo (Weber et al., 1974). Multiple-cigarette smoke inhalation was also characterized by a decrease in the ratio of AUC,,,inine/AUCnicotine. The possibility that the cotinine volume of distribution was decreased upon multiple dosing is ruled out because any change in the volume of distribution should be reflected in a change in the renal clearance of cotinine. However, this parameter was not significantly different from
CIGARETTE
EXPOSURE
9
that found in the single-dose experiments (Rotenberg el al., 1980; Miller et al., 1977). Data in Table 3 show that the F/L’, ratio for multiple exposure was significantly lower (p < 0.01) than that obtained in the single-dose experiment. These data indicate that F must have decreased upon multiple dosing. It is possible that enzymatic oxidation in the hver microsomal fraction, which was inhibited by SKF 525A (Got-rod et a/., 1971; Jenner et al., 1973; Booth and Boyland, 1970) also was affected by multiple-cigarette smoke inhalation and that the formation of S-hydroxy-nicotine, an intermediate of cotinine formation, has been inhibited. Alternatively it is possible that this intermediate was not further oxidized to cotinine due to inhibition of aldehyde oxidase present in the soluble fraction (Gorrod ef al., 197 1; Jenner et al., 1973; Tjalve et al., 1968). Irrespective of the mechanism of this interaction, nicotine or a constituent product of tobacco smoke appears to inhibit the formation of cotinine. In contrast to cotinine, the time to reach maximum plasma concentration of the origin activity following multiple smoke exposure was not significantly different from that observed following single-dose administration of nicotine. This similarity indicated that the rate constant of formation of this activity had not changed upon multiple-dose cigarette smoke exposure. However, the ratio of AUC onan .. activity/AUC,i,oti”, was decreased. This decrease was not due to an alteration in the volume of distribution of this activity because its renal clearance did not change. It is possible, however, that a constituent of tobacco smoke may inhibit the formation of the origin activity. There is evidence to indicate that the formation of nicotine- I’-N-oxide is microsomal and requires NADPH and oxygen, suggesting the involvement of the hepatic microsomal electron transport chain (Jenner et al.. 1973). However, tertiary amine N-oxidase appears to depend on a flavoprotein other than cytochrome P-450 (Ziegler et al., 1969) and is unaffected by inhibitors of the microsomal mixed-function oxidase system such as SKF
10
ROTENBERG
525A (Bickel, 1969). Also, the formation of nicotine-1’-N-oxide is not inhibited by KCN. Therefore, it is difficult to explain the reduction of the origin activity by inhibition of nicotine-l’-N-oxide upon multiple-cigarette smoke inhalation. Alternatively, the possibility exists that nicotine- l’-N-oxide is excreted in the bile and then reabsorbed from the intestine as nicotine. In conclusion, multiple-cigarette smoke inhalation appears to increase the extent but not the rate of nicotine absorption, decrease the rate and extent of its conversion to cotinine, and enhance the reduction of its polar N-oxide metabolite. These changes are manifested in persistent plasma levels of nicotine. Urinary excretion of nicotine and its metabolites further supports the conclusion that changes in the pharmacokinetics of nicotine occurred following multiple exposure to cigarette smoke.
AND ADIR without prior exposure to 3.4-benzypyrene (BP) given intratracheal instillation. Brit. J. Cancer 31, 469-484. EL-SAYED, M. A.. END MOHAMED. Y. A. (1975). An extractive-spectrophotometric method for the determination of nicotine. Planta Med. 27, 140- 144. GIBALDI, M.. AND PERRIER. D. (1975). Pharmacokinetic,.r. Dekker, New York. GORROD. J. W.. JENNER, P., KEYSELL, G.. AND BECKETT. A. H. (1971). Selective inhibition of alternative oxidative pathways of nicotine metabolism in vifro. Chem.
ADIR. J.. MILLER, R. P., AND ROTENBERG, K. S. (1976). Disposition of nicotine in the rat after intravenous administration. Res. Comm. Chem. Pharmaco. 13, 173183. BERMAN, M. AND WEISS, M. F. (1974). Simulation Analysis and Modeling Manual, U.S. Dept. of Health, Education and Welfare, Public Health Service, N.I.H. Washington, D.C. BICKEL, M. H. (1969). The pharmacology and biochemistry of N-oxides. Pharmacol. Rev. 21, 325-355. BOOTH, J. AND BOYLAND, E. (1970). The metabolism of nicotine into two optically-active stereoisomers of nicotine- I’-N-oxides by animal tissues in vitro and by cigarette smokers. Biochem. Pharmacol. 19, 733-742. BOWMAN, E. R., AND MCKENNIS, H. (1963). (-)Cotinine. Biochem.
Prep.
10, 36-39.
DAJANI, R. M., GORROD. J. W., AND BECKE~T, A. H. ( 1975a). Reduction in vivo of (-)-nicotine- I’-N-oxide by germ-free and conventional rats. Biochem. Pharmacol. 24, 648-650.
DAJANI. R. M., GORROD. J. W., AND BECKETT, A. H. (1975b). In vitro hepatic extra-hepatic reduction of (-)-nicotine- If&oxide in rats. Biochem. Pharmacol. 24, 109-I 17. DAVIS, B. R., WHITEHEAD, J. K., GILL. M. E., LEE, P. N., BUTTERWORTH,A. D., AND ROE. F. J. C. (1975). Response of rat lung to inhaled tobacco smoke with or
Interactions
3, 269-270.
349.
JICK. H. (1974). Smoking and clinical drug effects. Med. C/in. of&orrh /loner. 58, 1 143-l 149. KEERI-SZANTO, M.. AND POMEROY, J. R. (1971). Atmospheric pollution and pentrazocine metabolism. Lancd
REFERENCES
Biol.
HANSSON. E., AND SCHMITERL~W. C. G. (1962). Physiological disposition and fate of 14C-nicotine in mice and rats. J. Pharmacol. Exp. Ther. 137, 91-102. HART, P.. FARRELL, G. C., COOKSLEY, W. G. E., AND POWELL. L. W. (1976). Enhanced drug metabolism in cigarette smokers. Brit. Med. J. 2, 147-149. JENNER,P.. GORROD,J. W., AND BECKETT, A. H. (1973). The absorption of nicotine- If-N-oxide and its reduction in the gastro-intestinal tract in man. Xenohiofica 3,341-
1, 947-950.
KENDRICK, J.. NETTESHEIM,P.. GUERIN, M.. CATON, J., DALBEY, W., GRIESEMER, R., RUBIN, I., AND MADwx. W. (1976). Tobacco smoke inhalation studies in rats. TO-Y. Appl Pharmacol. 37, 557-569. MCGOVERN, J. P.. LUBAWY. W. L., AND KOSTENBAUDER, H. B. ( 1976). Uptake and metabolism of nicotine by the isolated perfused rabbit lung. J. Pharmacol. Exp. Ther.
199, 198-207.
MILLER, R. P., ROTENBERG,K. S., AND ADIR, J. (1977). Effect of dose on the pharmacokinetics of intravenous nicotine in the rat. Drug Mefab. Dispos. 5, 436-443. ROTENBERG,K. S. (1978). The Pharmacokineiics of Nicotine Following Intravenous Administration and Cigaretle Smoke Inhalation in the Rat. Dissertation. Uni-
versity of Maryland. ROTENBERG,K. S.. MILLER, R. P.. AND ADIR, J. (1980). Pharmacokinetics of nicotine in rats after single-cigarette smoke inhalation. J. Pharm. Sci. 69, 1087-1090. RLJDWN, R. W.. AND COHEN, A. M. (1970). Alteration of enzyme activity in rat liver following the acute and chronic admimstration of nicotine. To.x. .Appl. Pharmacol.
16, 6 13-625.
SMITH, R. L. (1973). The E.ucretory Function of Bile. Halstead Press. New York. STALHANDSKE,T. AND SLANINA, P. (1970). Lethal brain concentration of nicotine in mice of different ages.Acta Pharmacol.
To.ricol.
28, 233-240.
STEELE, R. G. D., AND TORRIE, J. H. (1960). Principles and Procedures c?fStafistics. McGraw-Hill, New York. THOMPSON,J. H.. Su, C., SHIH, J. C., AURES, D., CHOI,
PHARMACOKINETICS
NICOTINE
L., BUTCHER, J., LOSKOTA, W. S., SIMON, M.. AND SILVA, D. (1974). Effects of chronic nicotine administration and age on various neurotransmitters and associated enzymes in male Fischer-344 rats. Tax. Appl. Pharmacol. 27, 4 l-59. TJALVE, H., HANSSON, E., AND SCHMITERL~W. C. G. (1968). Passageof 14C-nicotine and its metabolites into mice foetuses and placentae. Acfa Pharmacol. Toxicol. 26, 539.
UPTON, R. A. (1975). Simple and reliable for serial sampling of blood from rats. J. Pharm. Sci. 64, I 12-114. VESTAL, R. E., NORRIS, A. H., TOBIN, J. D.. COHEN. B. H., SHOCK, N. W., AND ANDRO, R. (1974). Antipyrine metabolism in man: Influence of age, alcohol,
CIGARETTE
EXPOSURE
caffeine, and cigarette smoking. C/in. Pharmacol.
11 Ther.
18,425-432.
WEBER, R. P., COON, J. M., AND TRIOLO, A. J. (1974). Nicotine inhibition of the metabolism of 3.4~benzopyrene. a carcinogen in tobacco smoke. Science 184, 1081-1083.
WELCH, R. M.. LOH, A., AND CONNEY. A. H. (1971). Cigarette smoke: Stimulatory effect on metabolism of 3.4-benzyprene by enzymes in rat lung. L[fe Sci. 10, Part I. 215-221. ZIEGLER, D. M., MITCHELL, C. H., AND JOLLOW, D. (1969). Microsomes and Drug 0.xidation.x (J. R. Gillette. A. H. Conney. G. J. Cosmifes, R. W. Eastbrook, J. R. Fouts. and G. J. Mannering. eds.). Academic Press, New York.
69, l-11 (1983)
PHARMACOLOGY
Pharmacokinetics of Nicotine in Rats after Multiple-Cigarette Smoke Exposure’ KEITH S. ROTENBERG' AND JOSEPH ADIR~ Clinical
Pharrnacokinetic
Laboratory,
Received
School
June
of Pharmacy,
28, 1982;
accepted
University
January
of Maryland.
Baltimore,
Maryland
31, 1983
Pharmacokinetics of Nicotine in Rats after Multiple-Cigarette Smoke Exposure. ROTENBERG. Toxicol. Appl. Pharmacol. 69, I- 11. The pharmacokinetics of nicotine and its major metabolites was evaluated in male rats after multiple-cigarette smoke exposure. A smoke-exposure apparatus was used to deliver cigarette smoke to the exposure chamber. The rats were exposed to smoke from a single cigarette every 8 hr for 14 days and to the smoke of a cigarette spiked with radiolabeled nicotine on the 15th day. Blood and urine samples were collected at timed intervals during the lo-min smoke-exposure period of the last cigarette and up to 48 hr thereafter. Nicotine, cotinine. and other polar metabolites were separated by thin-layer chromatography and quantified by liquid scintillation counting. The data were analyzed by computer fitting, and the derived pharmacokinetic parameters were compared to those observed after a single iv injection of nicotine and after a single-cigarette smoke exposure. The results indicated that the amount of nicotine absorbed from multiple-cigarette smoke was approximately lo-fold greater than that absorbed from a single cigarette. Also, unlike the singlecigarette smoke exposure experiment, nicotine plasma levels did not decay monotonically but increased after the 5th hr, and high plasma concentrations persisted for 30 hr. The rate and extent of the formation of cotinine, the major metabolite of nicotine, were decreased as compared with their values following a single-cigarette smoke exposure. It was concluded that nicotine or a constituent of tobacco smoke inhibits the formation of cotinine and may affect the biotransformation of other metabolites. Urinary excretion tended to support the conclusions that the pharmacokinetic parameters of nicotine and its metabolites were altered upon multiple as compared to single dose exposure. K. S., AND ADIR, J. ( 1983).
Numerous investigations have examined the effects of chronic cigarette smoke exposure on induction (Hart et al., 1976; Keeri-Szanto and Pomeroy, 197 1; Jick, 1974; Vestal et al., 1974; Welch et al., 197 1) or inhibition (Ruddon and
Cohen, 1970; Thompson d al., 1974; Weber et al., 1974) of lung or liver metabolizing enzymes. Investigators (McGovern ef al., 1976) have also shown that tobacco smoke inhibits the metabolism of nicotine since the appearance of the major metabolites, cotinine and nicotine- I’-N-oxide, was slower. The pharmacokinetics of nicotine and its metabolites following multiple-cigarette smoke exposure has not been extensively evaluated in the intact animal or man. Previous studies from this laboratory examined pharmacokinetics of nicotine in the rat following single dose iv administration (Adir et al., 1976; Miller et al., 1977) and single-cigarette smoke exposure (Rotenberg et al., 1980). Nicotine was ab-
’ Supported in part by National Cancer Institute Contract NOl-CP-433 12. * Abstracted in part from a dissertation submitted to the University of Maryland by Keith S. Rotenberg in partial fulfillment of the Doctor of Philosophy degree requirements. To whom correspondence should be addressed. Current address: Pennwalt Corporation, Pharmaceutical Division, 755 Jefferson Road, Rochester, N.Y. 14623.
’ Current address: Howard University, College of Pharmacy and Pharmacal Sciences, Washington, D.C.. 20059. I
0041-008X/83
$3.00
Copyright 61 1983 hy Academic Press. Inc All nghtr of reproductmn I” any form reserved.
2
ROTENBERG
sorbed
rapidly
with
concentration cessation
of
The
sure. otine
the
of its were
similar
to
those
of
The purpose of the
of nicotine
in rats
cigarette
position lites observed
kinetics after
elimination
order
kinetics, parameters
observed
after
this
investigation
and
extent
rate
following
smoke
following
first
nicexpo-
iv
ad-
nicotine.
determine to
and
pharmacokinetic
ministration
of
smoke
followed
derived
expo-
elimination
formation
metabolites the
after
smoke
and
as the
plasma
immediately
single-cigarette
as well
and
maximum
single-cigarette
absorption
following
sure
the
occurring
AND
to
multiple single-dose
exposure
compare
nicotine
and
exposure cigarette
to
absorption
multiple
and of
was of
the its
dis-
metabo-
with smoke
those ex-
(Rotenberg et al., 1980) or iv administration (Adir et al., 1976; Miller et al., 1977). posure
METHODS Chemicals. [methyl-‘4C]Nicotine was obtained commerciaIly4 with a specific activity of 6 I .6 rCi/mg. Radiochemical purity was established by TLC and radioscanning techniques. With acetone:henzene:ammonia: ethanol (40:50:5:5) as the solvent, the compound migrated as a single radioactive spot with an R,value corresponding to authentic nicotine. Unlabeled nicotine5 was obtained commerciaIly. and unlabeled cotinine was prepared from nicotine by a previously described method (Bowman and McKennis, 1963). Cigaretfes. Code No. 31 cigarette? were stored upon arrival in a desiccator that provided an atmosphere of 65% humidity. They were reported to contain 9.5 mg of nicotine/g of tobacco and to have a smoke condensate pH of 4.9. Spectrophotometric analysis (El-Sayed and Mohamed, 1975) verified the nicotine content of the cigarettes. One cigarette (the last in the multiple cigarette smoke exposure regimen) was spiked with “C-laheled nicotine by an infusion pump as described earlier (Rotenherg et al., 1980). The final specific activity of the cigarette was 17. I I rCi/mg. To maintain similar nicotine content between the radioactive-labeled cigarette and those used throughout the multiple-exposure regimen (cold), the cold cigarettes were spiked with unlabeled nicotine. Unlabeled nicotine was dissolved in ethanol to a final concentration of 3.65 mg/
4 ICN, Irving, Calif. 92664. 5 Eastman Kodak Company, 6 Enviro Control, Rockville.
Rochester, Md. 20852.
N.Y.
14650
ADIR
ml. One milliliter of the solution was delivered lo each cigarette. Thus. the cigarettes containing either radiolabeled or nonlabeled nicotine had a final nicotine content of 13. I5 mg/cigarette. After spiking. both cigarettes were replaced in the desiccator. -Smoke e.~po.rwe apparatus. The intermittent smoke generation-inhalation system’ has been described earlier (Rote&erg ef al., 1980). AnimuO. Male Fischer-344 rats’ weighing 199 to 228g. were housed in stainless-steel cages and had free access to food’ and water. Three weeks before introduction of radiolabeled cigarette smoke. each animal was anesthetized with ether, and a chronic cannula was implanted into the jugular vein (Upton, 1975). Of the IO rats which entered the study. 4 were removed due to either dislodgement or failure of Ihe cannula. Thus. six rats completed the study. During the multiple-dosing regimen the weight of the rats was recorded each day at 7:30 AM. Throughout the entire dosage regimen. the rats were maintained in a room with a 12-hr light/dark cycle and a constant temperature of 23°C. Protocol. Rats were dosed every 8 hr./i,r 14 consecrctiw dqa with cigarette smoke from nonlabeled cigarettes. They were placed in the smoke-exposure apparatus and administered the smoke of one cigarette at 7 QNI. 3 pm, and 11 pm daily after which they were transfered to individual cages. On the 15th day. the cigarette containing radiolabeled nicotine was administered at 9:30 urn and consumed in IO min (IO puffs), after which the rats were transferred to plastic metabolism cages for the remainder of the experiment and allowed food and water ad lihitum. Blood samples (0.2 ml) were collected into heparinized syringes during the smoking phase at 0.05. 0.08, 0. I I, 0.12, and 0.17 hr and in the post-smoking phase at 0.30. 0.50, 0.75. 1.0, 1.5, 2.0, 3.0, 5.0, 8.0, 10.0. 12.0, 14.0, 18.0, 24.0. and 30.0 hr. The plasma was separated from whole blood by centrifugation and frozen until time of analysis. Urine was collected in containers immersed in ice for up to 48 hr and frozen until analysis. Separation and quantification of nicotine and its metabolites were described earlier (Adir et a/., 1976; Miller e/ al., 1977). Pharmacokinetic unu1vsi.r. The concentration-time profiles of nicotine. cotinine. and other metabolites for each rat were plotted on semilogarithmic graph paper which provided an insight to the operative pharmacokinetic model. These plots were employed to obtain initial estimates for curve fitting of the data by the SAAM 26 digital computer program (Berman and Weiss. 1974). Each datum point was assigned a statistical weight proportional to the reciprocal of the square of a standard deviation representing 10% of the point.
’ Maddox-ORNL, Oak Ridge National Research Laboratory, Oak Ridge. Tenn. 37830. ’ Microbiological Associates, Walkersville. Md. 2 1793. 9 Purina Rodent Laboratory Chow. Ralston Purina Co.. St. Louis, MO. 53 188.
PHARMACOKINETICS
NICOTINE
The puffing profile of the smoke inhalation machine was incorporated into the pharmacokinetic model used to fit the data both during and after cigarette smoke inhalation. The amount of nicotine absorbed was computed (Gibaldi and Perrier, 1975) from the product of the ratio of area under the plasma concentration-time curve (AUC) from 0 to 8 hr (dosage interval) following cigarette smoke inhalation and the AUC from 0 to z, following iv administration from the 0.8 mg/kg iv dose reported earlier (Miller PI al.. 1977).” Alternatively, the amount of nicotine excreted in urine during a dosing interval was compared to the amount of nicotine ultimately excreted in urine following iv administration (expressed as fractions of total radioactivity) to estimate nicotine absorption by inhalation. The dose and the amount of nicotine excreted in the urine from the 0.8 mg/kg dose reported earlier (Miller el a/., 1977) were used in this calculation. The equations used in these calculations have been previously described (Rotenberg ef al., 1980). The plasma nicotine concentration (Cn) versus time data for each rat were fitted to a first order absorption triexponential function which contained the rate constant of absorption (K,) and the other hybrid rate constants. LY and /3. reflecting distributive and terminal phases, respectively. The dose absorbed by each rat (calculated as described above) was included in the data-fitting procedure. The first order rate constants of formation (Kf) and elimination (K,) and the ratio of the fraction of nicotine converted to cotinine (F) to the volume of distribution of the latter ( Vd) were obtained by curve fitting of the plasma cotinine concentration-time data as described earlier (Adir el al., 1976: Miller et al., 1977). Other pharmacokinetic parameters and those of the origin activity were estimated as described previously (Rotenberg ef al.. 1980). Statistical anal.vsis. The t test was used to evaluate differences in the pharmacokinetic parameters of nicotine and its metabolites between the single- and multiple-cigarette smoke exposure or iv administration of nicotine (Steele and Torrie, 1960).
RESULTS Nicotine. Approximately 14% (6.98 X 10’ dpm) of the nicotine present in the mainstream smoke (4.9 X 10’ dpm) was delivered to the exposure chamber. This amount represented the sum of the nicotine absorbed by the rats, retained on the Cambridge filter (4.9 X lo6 dpm), and present in the washings of “In the intravenous study. blood samples were obtained from an arterial cannulation while in the present investigation blood samples were obtained from venous cannulation. A comparative experiment showed that the pharmacokinetic parameters were not affected by the blood sampling technique (Rote&erg, 1978).
CIGARETTE
3
EXPOSURE
the chamber (1.1 X lo6 dpm). The amount of nicotine absorbed (utilizing the weight of the rat on the 15th day) calculated from AUC and urinary recoveries of nicotine are shown in Table 1. Absorption ranged from 3.00 X lo7 to 4.98 X IO7 dpm/kg (0.79 to 1.31 mg/kg) based on plasma AUC and from 1.48 X 10’ to 3.70 X lo7 dpm/kg (0.39 to 0.97 mg/kg) based on urinary excretion. There was a significant difference among the means (p < 0.01) between the average amount calculated from plasma and that from urine. Nicotine was absorbed very rapidly following cigarette smoke inhalation as shown in Fig. 1. During the 10 puffs needed to consume the cigarette, the plasma nicotine levels paralleled the intermittent pattern of smoke exposure (shown in the inset of Fig. 1). Maximum plasma concentration of 9200 dpm/ml (242 rig/ml) occurred at the end of the cigarette smoke exposure. After rapid absorption of nicotine which could be adequately deTABLE NICOTINE
ABSORBED
FOLLOWING
MULTIPLE
SMOKE
EXPOSURES
OF CIGARETTE
Amount absorbed based on AUCb
Rat 1 2 3 4 5 6 R SE
Dose kbm/kg) 4.01 4.89 4.07 4.98 3.00 4.08 4.17 2.93
x x x x x x x x
Dose’ (m&t) 10’ 10’ 10’ 10’ I07 IO’ 10’ lo6
1
1.06 I .29 1.07 1.31 0.79 I .07 1.10 0.08
DOSING
Amount absorbed based b on urinary excretion Dose (&Wmg) 3.31 3.70 2.90 2.49 1.48 2.73 2.77 3.11
x x x x X x
IO’ 10’ 10’ 10’ IO’ 10’ x IO’ x 106
Dose’ (w/kg) 0.87 0.97 0.77 0.66 0.39 0.72 0.73 0.08
a Rats were dosed every 8 hr with cigarette smoke from nonlabeled cigarettes for 14 consecutive days. On the 15th day, a cigarette spiked with radiolabeled nicotine was administered. *Significant differences (p i 0.01) were observed between the dose calculated from plasma versus the dose calculated from urinary excretion of nicotine. ’ Dosage was calculated from specific activity of nicotine in the labeled cigarette, 17. I I &i/mg.
ROTENBERG
AND ADIR
i. I I. TIME
(hr.)
FIG. 1. Semilogarithmic plot of nicotine plasma concentration (x f SE) as a function of time following multiple-cigarette smoke exposure of nicotine. Computer fitting of the data is represented by the solid line. The X-axis of the inset is in minutes.
scribed by first order kinetics, the plasma nicotine concentrations declined biexponentially for the first 2 hr (Fig. I) but increased rapidly in the 5th hr, and high concentrations persisted for 30 hr post-cigarette smoke exposure. This portion of the curve from 3 to 30 hr could not be fitted by an equation which could account for the changes in the pharmacokinetics of nicotine observed. Thus, the AUC during a dosing interval (0 to 8 hrs) was calculated by extrapolation by the observed elimination rate constant (P) to allow comparison with the results from the single-dose studies (Rotenberg ef al., 1980; Miller et al., 1977). The pharmacokinetic parameters of nicotine derived from these data are shown in Table 2. The mean half-life of absorption (t*/2, K’J was 5.3 min. The half-life of the rapidly (P/Z,a) and slowly (f%,8) declining phases were 5.9 and 74.6 min, respectively. There were no significant differences between these half-lives or renal clearance and those ob-
served in the single-cigarette smoke exposure study (Rote&erg et al.. 1980). Cotinine. The concentration vs time profile (Fig. 2) of this major metabolite of nicotine and the pharmacokinetic parameters derived from these data are reported in Table 3. The rate constant of formation (Kr) ranged from 0.6 to 1.12 hr-’ but was not significantly different than that observed in the single-dose iv experiment. Cotinine was eliminated from plasma with a mean (SE) half-life of elimination (P/2, K,) of 4.5 (0.2) hr. There were no significant differences in this parameter between the multiple-dose investigation and the single-dose cigarette smoke exposure or iv administration. Nor were there any significant differences in the time to reach maximum plasma concentration (t,,,) between multiple-dosing and the single-dose experiments. Since the amount of nicotine absorbed upon multiple dosing was greater than that following single-cigarette smoke exposure, the AUC
PHARMACOKINETICS
NICOTINE
CIGARETTE
5
EXPOSURE
TABLE 2 PHARMACOKINETICPARAMETERSOFNICOTINEFOLLOWING MULTIPLE D~SINGOFCIGARETTESMOKE~
Rat I 2 3 4 5 6 R SE
KC3 (mini’)
a (mini’)
P (mini’)
AUC ((dpm . W/W . kg); O-8 hr)
Cl, (ml/hr . kg)
0.0798 0.1381 0.0698 0.2207 0.5079 0.1824 0.1998 0.0660
0.1201 -h 0.1 185 0.1436 0.08 11 0.1617 0.1250 0.0140
0.0101 0.0115 0.008 I 0.0098 0.0097 0.0072 0.0094 0.0006
46.856 57.152 47.532 58,155 35,088 47,610 48,732 3,426
233 101 173 198 132 56 149 27
a See text for definition of parameters. b Although the (Y phase in this rat was discernible by examination of the log concentration-time include sufficient data points to enable computer estimation of this parameter.
of cotinine was expected to be larger and the ratio of AUCcotinlne/AUCnicotine to be significantly smaller (p < 0.05) than that obtained from the single-dose studies of cigarette smoke exposure or iv administration of nicotine (Table 3). However, the renal clearance of cotinine (13 to 45 ml/hr - kg) was not significantly different from that observed in single-dose experiments. Other metabolites. A semilogarithmic plot of the origin activity is presented in Fig. 3. The origin activity declined biexponentially
plot, it did not
following its formation similar to that observed following single-dose cigarette smoke exposure or iv administration of nicotine. There were no significant differences (p < 0.05) in the half-lives of formation, distribution, and elimination of this activity between multiple-dose and single-dose administration of nicotine. The mean (SE) half-life formation was 20.2 (4.8) mitt, while the mean (SE) half-life of the distribution and elimination phases were 31.2 (8.8) min and 23.6 (1.9) hr. respectively (Table 4). There were no
7000
TIME
(hr)
FIG. 2. Semilogarithmic plot of cotinine plasma concentration (J? f SE) as a function of time following multiple-cigarette smoke exposure of nicotine.
6
ROTENBERG
AND ADIR
TABLE 3 PHARMACOKINETKPARAMETERSOFCOTININEFOLLOWING
Rat
I 2 3 4 5 6 ‘I? SE
0.600 0.917 0.729 I.1 I9 0.868 0.785 0.837 0.072
‘See text for definition
0.171 0. I33 0.152 0.141 0. I78 0.155 0.155 0.007
MULTIPLE
4nax W
AU< (@pm . hr)/(ml . kg); O-X hr)
I .92 I .92 1.93 1.71 I.28 I .90 1.77 0.1 I
148.995 215.121 I 13,487 209.40 I 1 18,570 153.103 159.780 17.820
D~SINGOFCIGARETTE
44.85 28.24 12.54 20.10 40.87 24.64 28.54 5.04
SMOKE”
3.18 3.76 2.39 3.60 3.38 3.12 3.26 0.20
0.190 0.194 0.150 0.178 0.206 0.200 0.186 0.008
of parameters.
significant differences (p < 0.05) in the time to reach maximum plasma concentration between multiple- and single-dose administration of nicotine. The values ranged from 0.5 to 1.7 hrs. From the AUC of the origin activity shown in Table 4, it can be seen that the concentrations of origin activity following multiple-dose were higher than those observed in the single-dose cigarette exposure study. The ratio of AUC,h,, actlvity/AUCnicotine ranged from 1.O to 1.6 and were significantly lower (p < 0.05) from the single-dose experiments. Urinary e+xcretion. The 48-hr urinary excretion of nicotine and its metabolites following cigarette smoke inhalation is presented in Table 5. Significantly lower (p < 0.0 1) urinary recoveries of nicotine, cotinine, and origin activities were observed when the 8-hr multiple-dosing interval recoveries were com-
pared to the single-dose cigarette smoke inhalation and iv administration of nicotine studies. However, the 48-hr urinary recoveries were not significantly different from the single-dose studies except when these recoveries were related to the percentage of dose absorbed. Approximately 23, 14, and 39% of the total radioactivity excreted over the 8-hr period consisted of nicotine, cotinine, and origin activity, respectively. It was expected that during a dosing interval approximately 90% of the nicotine would be excreted. In fact, only approximately 60% of the nicotine excreted was found between 0 to 8 hrs. DISCUSSION The purpose of this study was to evaluate the pharmacokinetics of nicotine after mul-
I TIME
1
24
26
28
30
(hr.)
FIG. 3. Semilogarithmic plot of origin activity plasma concentration (d ? SE) as a function of time following multiple-cigarette smoke exposure of nicotine.
PHARMACOKINETICS
NICOTINE
CIGARETTE
TABLE PHARMACOKINETIC
I
OF ORIGIN
I’V2, a (hr)
I%. 0 (hr)
Inm (hr)
0.467 0.172 0.264
20.56
0.50
65,312
281.39
1.39
31.79 26.05 18.83
I .oo I .oo I.71
75.960 48.781 93,232
1.33 1.03 I.61
21.79 22.57
1.00 0.50
184.87 184.03 95.36 281.16 114.51
23.60 I.91
0.96 0.15
.c
0.388 0.249 0.336
0.715 0.352 1.147 0.520
SE
0.080
0.147
5 6
4
PARAMETERS
0.431 0.057 0.258 0.633
2 3 4
‘See text for definition
ACTIVITY
FOLLOWING
MULTIPLE
AK ((dpm . hr)/(mI O-8 hr)
DOSING
TABLE URINARY
EXCRETION
OF NICOTINE
FOLLOWING
kg):
47,560 72.634 67,247 7.100
4
5 6 d SE
kg)
1.36 I.53 1.38
190.16 32.28
0.08
1975). One investigation (Kendrick et al., 1976) further showed that exposure to only five cigarettes within 1 hr was capable of killing 50% of “naive” rats with a cigarette smoke exposure apparatus similar to the one used in the present study. Our earlier pharmacokinetic studies have shown that nicotine had an approximate half-life of about 1 hr while the metabolites had much longer half-lives (e.g., cotinine 4 to 8 hrs) (Adir et al., 1976; Miller 5
AND ITS METABOLITES
CIGARETTE
% of absorbed dose excreted in urine as totaf radioactivity
Time tW
SMOKE
IN RATS 8 AND 48 hr
INHALATION
radioactivity in urine as:
Nicotine
Cotinine
excreted
Orig.
0 0 0 0 0 0
to to to to to to
8 8 8 8 8 8
23.0 12.9 14.4 12.4 22.1 Il.6 16.1 2.1
23.9 18.3 32.0 38.7 14.6 12.8 23.4 4.2
14.7 19.2 5.5 13.7 15.1 17.9 14.4 2.0
40.3 44.4 34.9 29.8 41.8 39.5 38.5 2.1
0 0 0 0 0 0
to to to to to to
48 48 48 48 48 48
44.0 40.3 38.1 26.1 26.4 35.8 35.1 3.0
15.7 11.4 11.4 26.6 13.8 9.0 15.7 2.5
21.2 20.4 13.8 14.4 16.9 17.2 17.3 1.2
35.0 38.5 32.1 31.0 40.2 25.1 33.8 2.2
x SE 1 2 3
SMOKE’
CL (ml/hr.
% of the total
I 2 3 4 5 6
OF CIGARETTE
of parameters
tiple-cigarette smoke inhalation which is the common method of dosing in pharmacologic and toxicologic studies involving this agent. The dosing regimen used in this investigation was derived from toxicologic and pharmacokinetic considerations. Previous cigarette smoke inhalation studies indicated that exposure of two cigarettes during 8 hr each day reduced survival of rats as compared to sham smoking (Davis et al.,
Rat
7
EXPOSURE
act.
8
ROTENBERG
et al., 1977; Rotenberg et al., 1980). Accordingly, rats were exposed to smoke from a single nonradioactive cigarette every 8 hr for 14 consecutive days while a cigarette containing radiolabeled nicotine was used on the 15th day. During the multiple-dose study, the weight of each rat was recorded and a decrease in body weight mean (%CV) of 29.0 (60) g from the first day weight was observed by the 15th day. Similar changes in mean body weight of Fischer-344 rats were reported upon exposure to cigarette smoke (Kendrick et al.. 1976). The weight loss was attributed to stress of confinement in the animal holder and to the exposure of cigarette smoke. Nicotine absorption following multiplecigarette smoke inhalation was determined by comparing the urinary recovery and AUC of nicotine during a dosing interval (8 hrs) with those values obtained following iv injection of a single dose of nicotine. It should be noted, that the AUC during the multiple-dosing regimen is equivalent to the AUC from O-ix, after a single-dose administration (Gibaldi and Perrier, 1975). Also, after multiple dosing, urine collection of a drug during the dosing interval will equal the urine collected to infinity for a single-dose experiment (Gibaldi and Perrier, 1975). Based on these calculations, the total dose absorbed following multiple dosing was approximately 10 times greater than that absorbed following a singledose inhalation, probably because of adaptation by the rats to smoke inhalation following multiple exposure. Furthermore, nicotine plasma concentrations were higher and persisted for an extended period of time following multiple dosing as compared to a single exposure. The mean (SE) half-life of elimination of nicotine following multiple dosing was 1.3 (0.1) hr indicating that seven half-lives later all of the nicotine should have been eliminated from the plasma. However, nicotine levels persisted for 30 hr (Fig. 1). The halflife of build up and elimination of this “nicotine” after 3 hr post-cigarette smoke inhalation were approximately 11.4 and 15.1 hrs,
AND
ADIR
respectively. It should be noted that no nicotine was detected in our single-dose study 5 hrs post-nicotine injection or following cigarette smoke exposure (Adir et al., 1976: Miller et ul.. 1977; Rotenberg et al., 1980). In addition, the average total radioactivity recovered in urine following multiple dosing (Table 5) ranged from 26 to 44.0% of the absorbed dose (0 to 48 hr) while following single-dose exposure, the values ranged from 50 to 79%. These data suggest that in addition to absorption the elimination of nicotine and/or its metabolites was altered upon multiple dosing. It is well known that nicotine is eliminated by urinary excretion and metabolism. One of the metabolites of nicotine is nicotinel’-N-oxide which has an approximate half-life (assuming that it comprises most of the activity at the origin) of 24 hr. It has been reported (Dajani et al.. 1975a) that following ip injection of nicotine- I’-N-oxide (0.80mg/kg) to rats, this metabolite was converted to nicotine. These authors also found (Dajani et al.. 197513) that the liver, small intestine, lung. spleen, kidneys, and heart were capable of reducing nicotine- I’-N-oxide to nicotine in vitro with the liver and small intestine having the greatest activity. Another study (Jenner et al., 1973) showed that following po administration to nicotine-1’-N-oxide (2.0 mg) in man, it was reduced to nicotine in the gastrointestinal tract from which nicotine was subsequentially absorbed and recovered in the urine. However, no reduction of nicotine-l’N-oxide to nicotine occurred when the former compound was injected iv into humans. It is not known whether nicotine- l’-N-oxide is also reduced to nicotine after iv administration to rats: however, only 3% of an injected dose of nicotine was excreted into the bile in 6 hr (Hansson and Schmiterlow. 1962). No data are available on the elimination of nicotineI’-N-oxide in the bile. However, it has been suggested (Smith, 1973) that the increased polarity of a compound may increase its biliary excretion. If the biliary excretion of nicotineI’-N-oxide were greater than nicotine, then the following hypothesis may explain the
PHARMACOKINETICS
NICOTINE
presence and persistence of nicotine at high concentrations 3 hr post-cigarette smoke exposure (Fig. 1). The high tissue levels of nicotine-l’-N-oxide which are likely to occur upon multiplecigarette smoke exposure may result in its increased excretion. Upon reaching the small intestine, this nicotine- I’-N-oxide may be converted to nicotine which is then absorbed. Thus, approximately 3 hr post-nicotine exposure, an increase in nicotine plasma levels would be observed with an elimination rate closely parallel to that of the origin activity from which it is derived. Although this hypothesis seems reasonable, other possibilities such as nicotine removal from deep tissue stores after cessation of cigarette smoke inhalation cannot be ruled out as an explanation for the persistence of nicotine plasma levels. The finding of prolonged exposure of nicotine following multiple dosing may provide additional insight to the possible involvement of nicotine in lung disease, cardiovascular response, and teratogenic and other toxicologic effects of cigarette smoke inhalation. The rate of formation of cotinine, the major metabolite of nicotine, was significantly smaller following multiple exposure than that observed following the single-dose cigarette smoke inhalation. It is possible that following multiple dosing of cigarette smoke an inhibition of the metabolism of nicotine to cotinine may occur. Stalhandske and Slanina ( 1970) have reported that nicotine given ip at 15 mg/kg to mice for 3 days significantly reduced its own metabolism. Nicotine also inhibited the metabolism of benzyprene, a component of cigarette smoke, in vitro and in vivo (Weber et al., 1974). Multiple-cigarette smoke inhalation was also characterized by a decrease in the ratio of AUC,,,inine/AUCnicotine. The possibility that the cotinine volume of distribution was decreased upon multiple dosing is ruled out because any change in the volume of distribution should be reflected in a change in the renal clearance of cotinine. However, this parameter was not significantly different from
CIGARETTE
EXPOSURE
9
that found in the single-dose experiments (Rotenberg el al., 1980; Miller et al., 1977). Data in Table 3 show that the F/L’, ratio for multiple exposure was significantly lower (p < 0.01) than that obtained in the single-dose experiment. These data indicate that F must have decreased upon multiple dosing. It is possible that enzymatic oxidation in the hver microsomal fraction, which was inhibited by SKF 525A (Got-rod et a/., 1971; Jenner et al., 1973; Booth and Boyland, 1970) also was affected by multiple-cigarette smoke inhalation and that the formation of S-hydroxy-nicotine, an intermediate of cotinine formation, has been inhibited. Alternatively it is possible that this intermediate was not further oxidized to cotinine due to inhibition of aldehyde oxidase present in the soluble fraction (Gorrod ef al., 197 1; Jenner et al., 1973; Tjalve et al., 1968). Irrespective of the mechanism of this interaction, nicotine or a constituent product of tobacco smoke appears to inhibit the formation of cotinine. In contrast to cotinine, the time to reach maximum plasma concentration of the origin activity following multiple smoke exposure was not significantly different from that observed following single-dose administration of nicotine. This similarity indicated that the rate constant of formation of this activity had not changed upon multiple-dose cigarette smoke exposure. However, the ratio of AUC onan .. activity/AUC,i,oti”, was decreased. This decrease was not due to an alteration in the volume of distribution of this activity because its renal clearance did not change. It is possible, however, that a constituent of tobacco smoke may inhibit the formation of the origin activity. There is evidence to indicate that the formation of nicotine- I’-N-oxide is microsomal and requires NADPH and oxygen, suggesting the involvement of the hepatic microsomal electron transport chain (Jenner et al.. 1973). However, tertiary amine N-oxidase appears to depend on a flavoprotein other than cytochrome P-450 (Ziegler et al., 1969) and is unaffected by inhibitors of the microsomal mixed-function oxidase system such as SKF
10
ROTENBERG
525A (Bickel, 1969). Also, the formation of nicotine-1’-N-oxide is not inhibited by KCN. Therefore, it is difficult to explain the reduction of the origin activity by inhibition of nicotine-l’-N-oxide upon multiple-cigarette smoke inhalation. Alternatively, the possibility exists that nicotine- l’-N-oxide is excreted in the bile and then reabsorbed from the intestine as nicotine. In conclusion, multiple-cigarette smoke inhalation appears to increase the extent but not the rate of nicotine absorption, decrease the rate and extent of its conversion to cotinine, and enhance the reduction of its polar N-oxide metabolite. These changes are manifested in persistent plasma levels of nicotine. Urinary excretion of nicotine and its metabolites further supports the conclusion that changes in the pharmacokinetics of nicotine occurred following multiple exposure to cigarette smoke.
AND ADIR without prior exposure to 3.4-benzypyrene (BP) given intratracheal instillation. Brit. J. Cancer 31, 469-484. EL-SAYED, M. A.. END MOHAMED. Y. A. (1975). An extractive-spectrophotometric method for the determination of nicotine. Planta Med. 27, 140- 144. GIBALDI, M.. AND PERRIER. D. (1975). Pharmacokinetic,.r. Dekker, New York. GORROD. J. W.. JENNER, P., KEYSELL, G.. AND BECKETT. A. H. (1971). Selective inhibition of alternative oxidative pathways of nicotine metabolism in vifro. Chem.
ADIR. J.. MILLER, R. P., AND ROTENBERG, K. S. (1976). Disposition of nicotine in the rat after intravenous administration. Res. Comm. Chem. Pharmaco. 13, 173183. BERMAN, M. AND WEISS, M. F. (1974). Simulation Analysis and Modeling Manual, U.S. Dept. of Health, Education and Welfare, Public Health Service, N.I.H. Washington, D.C. BICKEL, M. H. (1969). The pharmacology and biochemistry of N-oxides. Pharmacol. Rev. 21, 325-355. BOOTH, J. AND BOYLAND, E. (1970). The metabolism of nicotine into two optically-active stereoisomers of nicotine- I’-N-oxides by animal tissues in vitro and by cigarette smokers. Biochem. Pharmacol. 19, 733-742. BOWMAN, E. R., AND MCKENNIS, H. (1963). (-)Cotinine. Biochem.
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DAJANI. R. M., GORROD. J. W., AND BECKETT, A. H. (1975b). In vitro hepatic extra-hepatic reduction of (-)-nicotine- If&oxide in rats. Biochem. Pharmacol. 24, 109-I 17. DAVIS, B. R., WHITEHEAD, J. K., GILL. M. E., LEE, P. N., BUTTERWORTH,A. D., AND ROE. F. J. C. (1975). Response of rat lung to inhaled tobacco smoke with or
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