interconvertible forms of a single hemoprotein. One view, based on
indirect measurements as cited in Mannering (35), is that cytochrome
P-450 and cytochrome P&50 are similar but separate entities, each of
which can exist in two interconvertible forms.
Direct comparison of cytochrome P-450 and cytochrome P&50 was
made possible through solubilization and partial purification of the
microsomal hemoproteins from phenobarbital and 3-MC treated rats
(unpublished observations of Fujita and Mannering as cited in
Mannering (35)). The absolute spectrum of soluble purified cytochrome
P1-450 is shown in Figure 6, and some properties of cytochromes P-450
and P&O in Table 2. The absolute spectra of the two hemoproteins
are very much alike, but there are differences. The Soret peaks at 443
rnp and 450 rnp (reduced + CO) shown by cytochrome P1-450 and
cytochrome P-450, respectively, accord with what was expected from
spectral studies employing microsomes. The Soret peak at 414 nq.~
rather than at 418 rnp (reduced hemoprotein) also distinguishes
cytochrome P1-450 from cytochrome P-450.
Particularly to be noted is the absence of a peak at about 395 rnp.
Putatively, a peak at 395 rnp characterizes the form of the P-450
hemoprotein that results when PAHs are administered (20, 53). The
most likely explanation for the peak at 395 rnp is that 3,4benzpyrene, a
type I compound (53), or a metabolite, binds with hemoprotein to
produce a type I spectrum. The PAH or its metabolite binds more
avidly than most type I compounds and is not lost during preparation
of the microsomes. However, the loss of 3-MC or its metabolite occurs
when the hemoprotein is solubilized.
Further evidence for the existence of two molecular species of P-450
hemoprotein was obtained by comparing the eytochrome P-420 derived
from cytochromes P-450 and P1-450 (56). When hepatic microsomes
from untreated rats were incubated under nitrogen at 4oC for 24 hours
with 0.0'7% steapsin, about 25 percent of the P-450 hemoprotein was
solubilized as P-420 hemoprotein. After desalting and concentrating
the clear solution to about one-fourth its volume, an aggregate of
cytochrome P-420 was formed consisting of microtubules with globular
substructures (56). Microsomes from rats that had received 3-MC, when
treated in the same manner, also yielded aggregates; but only small
numbers of the tubular structures were seen, their presence possibly
due to the existence of some residual cytochrome P-450 in the
microsomes. Aggregates of cytochrome P-420 showed both type I and
type II binding with drugs, but aggregates of cytochrome P1-420 bound
only with type II compounds. On the basis of heme content, the molar
absorbency of cytochrome P-420 was determined to be 110 mM-`cm-l,
whereas that of cytochrome P&O was 134 mM-`cm-*. Disc electropho-
resis of aggregates solubilized with 8 M urea disclosed differences in
the ionic mobilities of the two P-420 hemoproteins.
12-18

FIGURE C.-Absolute spectra of solubilized microsomal P450
hemoprotein (cytochrome P1-450) from livers of rats treated with 3-
MC (Fujita and Mannering, unpublished results). The hemoprotein
was solubilized by treating microsomes with Triton N-101 and
fractionating the supematant on a DEAE cellulose column. The
preparation was free of cytochrome bsr but contained a small amount
of P-420 hemoprotein. Table 2 summarizes the spectral properties of
solubilized cytochromes P-450 and P1-450
SOURCE: Pamwing. G. (55).
In summary, the preponderance of evidence leads to the following
conclusions:
12-19

I
TABLE 2.-Absorption peaks and molar extinction coefficients of
    absolute sp&tra of soluble cytochromes P-450 and PI-
       45@
C'ondiuons
Cytochrome PW        Cytwhrome PAW
mm (mu)    (mM-km-`)    max (mu)    (mWcm-*)
Oxidized                      360
                     Soret  418
                         537
                         568
Reduced                 soret  418
                         545
Reduced + co                  423
                     soret  450
                         548
49.2
104.2
129
12.3
84i.o
14.9
05.8
89.1
13.9
360          45.7
419         120.3
537          13.5
568          13.4
414          90.1
545          16.4
423          60.0
448         108.0
551          15.4
aThe hemoprotein were aalubilized by treating micmsomea with Triton N-101 and fractionating the aupematant on
s DEAE celluclare column (Fujita and Mannwing, unpublished observations). The preparationa were free of
cytochrome bs, but they contained small amounts of P420. The absolute spectrum of cytochmme PAELI is shown in
Figure 7.
The preparation contsined 3.34 mu moles of P450 hemopmtein/mg of protein. an increase of 4%fo!d over that
contained in the micmwmea from which the preparation was obtained. Recovery of hemoprotein wan 15.6%.
me preparation mntained 4.43 mu moles of PUO hemopmtebumg of protein, an increase of 3.5-fold over that
contained in the micmsomea fmm which the preparation was obtained. Recovery of hemopmtein was 13.9%.
SOURCE, Mannwing. G. (55).
1. The administration of polycyclic aromatic hydrocarbons (PAHs)
causes the biosynthesis of cytochrome P&50, a molecular species of
cytochrome P-450 not normally detectable in appreciable amounts of
microsomes from untreated or phenobarbital-treated animals. This
does not exclude the possibility that small amounts of cytochrome PI-
450 may be found in untreated animals; in fact, this can be expected to
be the case. PAHs or other substances capable of inducing the
synthesis of cytochrome P1-450 may be present in the diet or
atmosphere or may be produced by the intestinal flora. Early
recognition of an exogenous inductive effect on the metabolism of a
foreign substance was made by Brown, et al. (4) and by Beif, et al. (52)
who observed that rancid diets contained oxidized steroids which
stimulated the N-demethylation of aminoazo dyes.
2. Both cytochrome P-450 and cytochrome P1-450 exist in their own
interconvertible forms.
3. Cytochrome P1-450 does not form as a result of the combination of
native cytochrome P-450 with PAHs or their metabolites.
Mechanisms of Induction of Drug Metabolism Enzymes
Gelboin (13) has discussed mechanisms of induction of drug metabolism
enzymes. Significant highlights of this discussion are as follows:
1. The stimulatory effect of PAHs and drugs on certain liver
microsomal enzymes appears not to be mediated through the endocrine
12-20

system, as the stimulation of at least the aryl hydrocarbon hydroxylase
[AHH) is observed in adrenalectomized and hypophysectomized rats.
2. The inducer acts directly on the target tissue.
3. The half-life of induced AHH activity is 3.3 2 1.2 hours.
4. Results of studies in cell culture have suggested the following
sequence of events in microsomal enzyme induction:
a. Upon addition of the inducer to the culture medium, it is rapidly
incorporated, within several minutes, into the cell. This has been
shown by the use of radioactive inducer and fluorescence
microscopy (Miller and Gelboin, unpublished observations cited in
Gelboin (13)). After incorporation, there appears to be a rapid
interaction between inducer and receptor site which is followed by
a period of RNA synthesis. This stage of enzyme induction
involving RNA synthesis is sensitive to actinomycin-D inhibition.
This early RNA synthesis phase is independent of translation,
since it occurs in the presence of inhibitors of protein synthesis.
b. Then follows the protein synthesis stage which is sensitive to
inhibitors of protein synthesis. This stage can proceed in the
absence of the RNA synthesis stage and can occur in the presence
of actinomycin-D. It seems to be a polymerization of amino acid
into polypeptide chains.
c. The next step appears to be an assembly process of the newly-
made polypeptide chains. This is independent of protein synthesis
and may persist for up to two hours. This entire process results in
the appearance of increased levels of AHH. The specific protein,
made and assembled in the microsomes, may be either the
hydroxylase or another protein which may activate by an allosteric
mechanism an inactive form of the hydroxylase. All of these
events appear before there are gross changes in either protein or
RNA synthesis. This suggests that the RNA and protein, which are
required to be synthesized, are very small percentages of total cell
RNA and protein and that many of the gross changes of RNA and
protein synthesis may be subsequent to, and parallel, but not
directly responsible for, the appearance of the early increases of
enzyme level.
Thus, the various studies on the effect of methylcholanthrene (MC)
on nuclear RNA metabolism have shown that: (1) MC causes an
increase in the uptake of erotic acid into nuclear RNA which suggests
increased RNA synthesis; (2) MC increases the amount of RNA in liver
cell nuclei; (3) RNA isolated from the liver cell nuclei of MC treated
rats has greater stimulatory activity in an E. coli phenylalanine-
incorporating system; and (4) the administration of MC in tivo
stimulates RNA polymerase activity of either isolated liver nuclei or
isolated chromatin. These effects of MC suggest an alteration in
genetic transcription.
12-21

TABLE 3.-Summary of effects of methylcholanthrene or
Dhenobarbital on gene-action system
Micmsomes                        NUCIWS
Increases of:
1. Specific enzymes and protein
(MC, PB)
2 Amino acid incorporation (MC.
W
a More mRNA (PB)
  b. More sensitive to added
   mRNA (PB)
3. Effects prevented by:
a. Pummycin (MC, PB)
b. Actinomycin-D (MC, PB)
e. Ethicmine (MC, PB)
Inhibitions of:
1. NADPH cytachmme C
mductase degradation (PB)
2. B degradation (PB)
Changes in:
1. Special pmperties of Pa (MC)
2. Phospholipid metabolism (MC)
3. Kinetic behavior of hydmxylase (MC)
Increases of:
1. Omtic acid-%
incqomtion into RNA
(MC)
2 RNA/DNA ratio (MC)
3. Mwenger RNA content
(MC)
4. Stimulation of RNA
polymeraae (MC, PB)
SOURCE: Gelboin, H. (18)
Table 3 shows a summary of the effects of MC and phenobarbital
(PB) on various aspects of nuclear and microsomal metabolism.
Summary
The pervasiveness of tobacco use in our society and the frequency of
altered disposition and pharmacological effects of many common drugs
in smokers make it apparent that cigarette smoking should be
considered as one of the primary sources of drug interactions in man.
Most of the experimental work in man, animals, and tissues involving
enzyme systems indicates that the dominant effect of smoking is
enhanced drug disposition caused by induction of hepatic microsomal
enzymes. The primary causal agents are probably the polynuclear
aromatic hydrocarbons which are potent and persistent in tissues.
While several of the hepatic microsomal drug-metabolizing enzymes
are stimulated in smokers, the selectivity of this enhancement in
activity is unpredictable. The effects of cigarette smoke on other
potential rate-limiting disposition processes for drugs are largely
unexplored.
12-22

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12-26

Effects on Pharmacokinetics and Pharmacodynamics
The effects of smoking on the action of drugs have become a subject of
an increasing number of investigations. Because the number of
smokers in our population is significant, it is important to determine
whether cigarette smoking alters the pharmacologic effects or the
pharmacokinetics of drugs.
The mechanism of these alterations includes: stimulation or
inhibition of biotransformation of drugs by the various constituents of
tobacco smoke, alteration of physiological processes that control drug
disposition, direct interference in the mechanism of drug action and
modification of psychopharmacological behavior, such as drug con-
sumption and pain threshold. Cigarette smoking may necessitate
modification of drug therapy and alter organ function or responsive-
ness.
Extensive literature is being assembled on the interaction of tobacco
smoke and drugs. Recently, Jusko prepared an excellent review (28) on
the role of tobacco smoke in the pharmacokinetics and pharmacology
of drugs in man and animals. Much of this discussion merely
paraphrases the Jusko review.1 Conney, et al. (14 have previously
reviewed the interaction of smoking and biotransformation of drugs,
and Jick (27) has addressed smoking and clinical drug effects.
Studies of tobacco smoking and nicotine have been closely associated
for many years. Tobacco in the United States yields about 1.2 mg
(range 0.1 to 2.2 mg) of nicotine per cigarette. Chronic nicotine
inhalation produces various types of pharmacological stimulation. The
assimilation of about 0.5 mg/kg/day of nicotine from tobacco smoke
offers the potential for altering drug disposition. The extraction of
nicotine from inhaled smoke by habitual smokers is nearly complete
(25). The half-life of nicotine has been determined to be about one hour
(25). Most studies in animals indicate that nicotine is an enzyme
inducer, which will be described later.
The most common effect of tobacco smoke on drugs in man and
animal is an increase in biotransformation rate consistent with
induction in drug-metabolizing enzymes. The first observation of this
type in man was made by Rottenstein, et al. (65), who found that
intravenous injection of nicotine did not cause nausea in smokers, but
in nonsmokers the same dose produced nausea and vomiting. Beckett
and Triggs (6) subsequently reported that, following intravenous
administration or inhalation of nicotine, the urinary excretion of
nicotine by nonsmokers and smokers was 55 to 70 percent and 25 to 50
percent, respectively. The reduced recovery of nicotine in the smoker
group was explained by an increased biotransformation of the nicotine.
Nicotine had previously been reported to accelerate the biotransforma-
tion of meprobamate in mice (88) and of benzo(a)pyrene (BP) by rat
' Reproduced in part from (08) with permission of William J. Jusko and the Plenum Publishing Company.
12-27

I
Table 4.-Plasma levels of phenacetin in cigarette smokers and
     nonsmokers at various intervals after the oral
    administration of 900 mg of phenacetin
Subjects
     Hours after phenacetin administration
1           2           3.5           5
    Phenacetin concentration in plasma, &ml
Nonsmokers             0.81 + 0.w      2.24 2 0.73     0.39 2 0.13      0.12 z 0.04
Smokers               0.33 + 0.23      0.43 2 0.28      0.09 + 0.04      0.02 r 0.01
*Each value representa the means + SE. for nine subjects
SOURCE: Pantuck, E.J. (55).
liver microsomes (92). Welch, et al. (87) were the first to demonstrate
that inhaled tobacco smoke increased the activity of the enzyme
benzo(a)pyrene hydroxylase in rat lung. This study has stimulated
studies of tobacco smoke as a source of drug interaction.
Phenacetin
Pantuck, et al. (54, 55) first reported that tobacco smoke could induce
the metabolism of a therapeutic agent in man. Oral doses of 900 mg of
phenacetin were administered to nonsmokers and smokers (smoked
more than 15 cigarettes per day). By measuring the concentration of
phenacetin in plasma it was determined that the phenacetin concentra-
tions in the plasma of cigarette smokers were markedly lower than
those in the nonsmokers (Table 4), but the average half-life of
phenacetin (about 50 minutes) in both groups was not different. The
lower plasma levels were not due to altered absorption of phenacetin,
as the urinary excretion of its major metabolite, N-acetyl-p-aminophe-
no1 (APAP), was identical for both groups. The low plasma concentra-
tions of phenacetin in smokers were thus presumed to be caused by
increased metabolism of phenacetin by the enzymes either in the
gastrointestinal tract or during the "first pass" through the liver. On a
theoretical pharmacokinetic basis, an increased degree of "first pass"
metabolism will cause a decrease in the area under the plasma level
curve with little change in half-life (21).
Similar results were reported almost simultaneously by Welch, et al.
(83) on the effect of cigarettes in rats. These workers demonstrated
that the enzyme benzo(a)pyrene (BP) hydroxylase was inducible by 3-
methylcholanthrene (3-MC) and caused lower plasma phenacetin levels
in rats.
Phenacetin has since been extensively studied as a model drug to
investigate various aspects of cigarette smoke-induced changes in
biotransformation rate. Welch, et al. (83, 86) and Pantuck, et al. (53)
exposed rats to cigarette smoke and observed marked increases in the
rate of in z&o metabolism of phenacetin in liver, lung, and intestinal
12-28

homogenates. Similar effects were found when rats were pretreated
with 3-MC or BP. Welch, et al. (86) examined the effects of 3-MC
treatment of rats on the bioavailability of phenacetin and APAP in
portal and peripheral plasma following oral and intravenous adminis-
tration. Comparison of the plasma phenacetin concentration in portal
blood of the control rats and those treated with 3-MC revealed almost
identical plasma concentration of phenacetin. The results indicated
that 3-MC treatment had little effect on the passage of phenacetin into
the portal circulation, but did influence to a very marked extent the
passage of phenacetin from the portal circulation into the general
circulation. These results were interpreted by the authors to mean that
the dominant effect of 3-MC treatment was induction of hepatic rather
than intestinal enzyme activity. On this basis, they concluded that the
reduced plasma phenacetin concentrations in smokers probably
reflected an increased "first pass" metabolism by the liver. However,
Kuntzman, et al. (39) have investigated the stimulation of intestinal
BP hydroxylase in rats following exposure to cigarette smoke or
exposure to BP. Their data showed that rats exposed to cigarette
smoke or to pretreatment with BP enhanced the in tivo metabolism of
phenacetin and stimulated enzymes in the intestinal mucosa to O-
dealkylate phenacetin to APAP. Therefore, the question whether the
stimulatory effect of cigarette smoking on the metabolism of
phenacetin occurs in the gastrointestinal tract or in an additional first-
pass increase in liver metabolism remains unanswered.
Antipyrine
Antipyrine is an analgesic often used as a "marker" for several hepatic
microsomal drug-metabolizing systems in man and animals. Vestal, et
al. (80) studied the effects of aging and cigarette smoking on the
disposition of antipyrine in 307 healthy subjects. Determination of the
half-life and metabolic clearance rate (MCR) of antipyrine revealed
that young and middle-aged smokers metabolized antipyrine more
rapidly than nonsmokers (Table 5). The half-life and the metabolic
clearance rate were defined as: tl/z= 0.693/k, where k, =overall
elimination constant, and MCR = aVd x k, where aVd = apparent
volume of distribution.
The half-life was 16.5 percent longer and the total clearance (Cb)
rate was 18.5 percent less in the older subjects than in the younger. By
old age (66 to 92 years), there was essentially no difference in the Ck
between smokers and nonsmokers, although the CUT diminished with
age in all smoking categories. Similar total clearance values were
reported by Wilson, et al. (89) and found to be 46.0 ml/hr/kg in
smokers and 36.5 ml/hr/kg in nonsmokers following administration of
antipyrine to subjects in the 24- to 45-year age range.
Hart, et al. (23) found enhanced metabolism of antipyrine in
cigarette smokers. These investigators found a mean half-life of 12.5
12-B

TABLE 5.-Effect of age and cigarette smoking on antipyrine
     metabolism. Data are from 307 healthy subjects
  Aice B~OUP        tl'z       Smoking       No. of          MCR
   (yr)        (W         gro"P      subjects      (ml/hr/kg)
   Young       12.7 + 0.50"      Nonsmoker          37        36.6 f 1.34
   (1839)                   MOdWTite          27        37.3 + 239
                    HtWy            9        424 2 4.24
   Middle       13.8 f 0.47      Nonsmoker         102        28.0 f 0.86
   W-59)                  MOde~te          30        37.2 + 221
                     Heavy           18        36.8 + 3.02
Old        14.8 + 0.65      Nonsmoker          67        28.2 2 1.09
(a-92)                   Moderate          14        29.9 2 2%
                  Heavy            3        15, 21. 23
.Nonsmoker: Did not smoke or smoked "once in a while," Moderate: Smoked less than 20 cigarette/day. Heavy:
Smoked more than 20 cigarettes/day.
bMean f SEM
SOURCE: Vestal, R.E. (80).
hours in 17 nonsmokers and 10.8 hours in 25 smokers, a smaller but
significant difference. To determine whether this difference was due
to tobacco consumption, eight smokers were restudied two months
after they stopped smoking. The half-life of antipyrine had increased
in six of the subjects, with an overall increase of about 23 percent.
Welch, et al. (84) reported the mean half-life of antipyrine was 4.2
hours in epileptic patients treated with anti-convulsants for more than
two months; whereas the mean half-life was found to be 12.6 hours in
normal volunteers, three of whom were smokers. These data suggested
that the anti-convulsant, phenytoin, may be a much stronger enzyme
inducer than tobacco smoke. However, Kellermann and Luyten-
Kellermann (31) found that the half-life of antipyrine was decreased 22
percent in normal subjects following 7 days on orally administrated
phenobarbital. This shortening of the antipyrine half-life is almost
identical in the report by Hart, et al. (23).
Kellerman, et al. (31, 32, 33) measured the half-life of antipyrine and
the percent induction of BP hydroxylase by 3-MC in mitogen-
stimulated lymphocytes from normal individuals. Resting lymphocytes
had relatively little BP hydroxylase activity and the capacity to induce
lymphocyte activity in Gtro correlated with hepatic metabolism of
various drugs in the same individual. The antipyrine half-life ranged
from 7.7 to 16.2 hours and showed a high inverse correlation coefficient
(r10.923) with the BP hydroxylase ratio. This indicated that antipy-
rine and BP share one or more common determinants that are
responsible for the observed interindividual variation in the oxidation
rates, and that antipyrine may serve as a useful predictor drug for
12-30

evaluating the drug- and carcinogen-metabolizing capacity of differ-
ent individuals in the human population. The difference in the
antipyrine half-life and the metabolic clearance rate between smokers
and nonsmokers, however, was not large and, therefore, makes
antipyrine an insensitive predicator for smoking effects.
Recently, Ambre, et al. (3) reported the antipyrine total clearance
rate in patients with bronchogenic carcinoma, in patients with chronic
lung disease, and in normal subjects. The mean antipyrine CUT values
were 2.98 -t 0.68,2.02 + 0.67, and 2.14 2 0.69 liters/hour, respectively.
These results could not be reproduced by Tschanz, et al. (74), however.
The latter group examined patients with lung cancer and a malignan-
cy-free control group very well matched for age, sex, drug intake,
smoking, and drinking habits. Their study took more blood samples
than the Ambre study and the mean CUT values were determined to be
47.5 2 0.9 in the cancer group and 55.7 -+ 0.7 mg/kg/hr in the
malignancy-free groups; this was a reversal of the earlier study. This
topic should be investigated further, as an increase in antipyrine Chin
cancer patients would suggest a common factor in the observations of
bronchogenic carcinoma, enhanced drug disposition, and inducibility of
BP hydroxylase. This common factor may be a genetic susceptibility
(33) to the multiple effects of exposure to polycyclic aromatic
hydrocarbons (PAHs, PNAs).
Theophylline and Other Xanthines
Thmphyllim
Theophylline is of primary importance. as a bronchodilator used to treat
acute and chronic asthma or bronchitis. It is generally recognized that
the therapeutic index of theophylline is narrow and the disposition rate
among patients is widely variable. Jenne, et al. (&6), Hunt, et al. (24),
and Powell, et al. (63) have investigated the interaction of cigarette
smoking and theophylline disposition. These investigators have found
that the theophylline half-life ranged from about 4 to 6 hours in
smokers to 7 to 9 hours in nonsmokers. Theophylline appears to be
metabolized mainly in the liver, because only about 10 percent of the
dose is excreted unchanged in the urine. Smokers exhibited a Ck of 100
-+ 44 ml/min/1.73 m2. This value was larger and more variable than 45
,+ 13 ml/min/1.73 m2 found for nonsmokers. A somewhat surprising
finding was that four of the smokers who stopped smoking for three
months had relatively little change in the CIT (24). This suggested that
more than three months is needed for the effects of chronic tobacco use
to dissipate. The average theophylline half-life of smokers who
discontinued their habit for at least 2 months was intermediate
between those of nonsmokers and smoker groups (63). Further studies
by Jusko, et al. (29) showed that increased age offset the increased Ch
of theophylline, as was observed earlier in the case of antipyrine. These
investigators found mean Ck values for theophylline of 55.3
12-31

ml/min/1.73 mzin non/light smokers and 77.5 ml/min/1.73 m2in heavy
smokers. When younger smokers (20 to 40 years) were compared to
older smokers (40 or more years) the mean Ck values were found to be
106 and 61 ml/min/1.73 m2, respectively.
The increased biotransformation rate of theophylline in smokers
appears to be accompanied by a reduced toxicity during clinical use of
this drug. Pfeifer and Greenblatt (62) studied the toxic effects of
theophylline in 2,766 patients, The frequency of adverse reactions
following administration of theophylline correlated negatively with
the daily smoking habit. The data revealed a significant trend, with
nonsmokers exhibiting 12.9 percent, light smokers (20 cigarettes/day)
10.8 percent, and heavy smokers (20 or more cigarettes/day) 7.0
percent incidence of adverse reactions to theophylline.
The dosing of patients on theophylline therapy is important because
of the frequency of adverse reactions of the drug. The rate of
elimination of a drug from the body (total body clearance) can be
ascertained from the plasma half-life and apparent volume of
distribution (aVd) for that drug. The aVd for theophylline does not
appear to be altered in patients with a history of smoking; therefore,
the shorter plasma half-life in smokers indicates that they have more
rapid total body clearance of theophylline. Thus, when a multiple dose
regimen (maintenance dose) is used, the steady-state plasma concen-
tration achieved with a given dose will likely be lower in smokers than
in nonsmokers. Although there appears to be considerable overlap in
the theophylline clearance values, some heavy smokers may require as
much as one and one half to two times the maintenance dose of
nonsmokers. These large maintenance doses required by heavy
smokers could result in toxicity if the patient discontinues smoking.
Because specific information about the recovery of the drug-metaboliz-
ing enzymes following cessation of smoking is not available, clinical
effects should be carefully monitored.
Lohman and Miech (43) have confirmed the inductive effect of 3-MC
on theophylline metabolism by liver slices in rats.
Other Xanthines
Welch, et al. (85) and Parsons and Aldridge (56) reported that the
biotransformation of caffeine in the rat was accelerated by PAHs in
cigarette smoke. Welch, et al. (85) showed that benzpyrene, benzan-
threne, dibenzanthracene, chrysene, and pyrene, which are potent
inducers of the cytochrome P-448 system in liver microsomes, caused a
marked increase in the plasma clearance of caffeine without altering
its volume of distribution. On the other hand, phenanthracene and
anthracene, generally considered very weak inducers of the liver
microsomal cytochrome system, did not change the plasma clearance of
caffeine. Following treatment with BP for three days, the CIT of
caffeine in rats increased from 50.3 to 125.3 ml&. Moreover, the
12-32

subsequent elimination rates in rats of the caffeine metabolites,
theophylline, paraxanthine, and theobromine, were greatly accelerat-
ed. A dose response study with BP indicated that a dose of 1 mg/kg or
more of BP for 3 days was required for the enzyme induction in the rat
and that 0.1 mg/kg had no significant effect. At the higher doses, BP
proved to be a more potent inducer than phenobarbital (equivalent
induction at 75 mg/kg). Thus, increased caffeine biotransformation
may, in part, explain the tendency for smokers to consume more coffee
than nonsmokers.
Other Dtugs
Imipramine
The disposition of the tricyclic antidepressant, imipramine, has been
reported to be affected by smoking. Perei, et al. (60, 61) ggve 29
depressed -patients daily doses of 3.5 mg/kg of imipramine and
determined the mean steady-state plasma concentration of total
imipramine and desmethyl imipramine to be 160 ng/ml in smokers and
290 ng/ml in nonsmokers. A strong correlation-was also found between
these plasma levels and the half-life of phenylbutazone administered to
the same patients. These results implied that the pharmacokinetics of
phenylbutazone may also be affected by smoking, but no direct
evidence is available.
Glutethimide
The metabolism of glutethimide, a hypnotic, has been reported by
Crow, et al. (16) to be altered by smoking. They measured plasma
concentrations of glutethimide given at &hour intervals after attain-
ment of steady-state. The mean area under the curve (0 to 8 hours
after the dose) was determined to be 41 mg/lit.er-hour for four smokers
and 26 mg/litet-hour for four nonsmokers. The half-life of glutethi-
mide was not found to be significantly different between groups.
These results suggested that the bioavailability was changed and that
either the apparent volume of distribution of glutethimide (aVo) was
smaller or the fraction of drug absorbed was larger in smokers. The
latter appeared unlikely because there was no difference in the rate of
excretion of 4-hydroxy-Zethyl-2 phenylglutaramide, an active metabo-
lite, in the urine of smokers and nonsmokers. The presence of other
active metabolites is a possible explanation for these results, since
smokers also performed relatively poorly in a computer-generated
tracking test designed for psychomotor response. The possible
mechanism of this interaction is difficult to assess. Bennett (7) has
pointed out a lack of firm data on the effects. of smoking on most
aspects of gastrointestinal secretion and mobility.
12-33

             I
Vitamin C
Pelletier, et al. (58,59) have reported that the vitamin C levels in serum
and leukocytes were reduced in smokers. It is not clear whether
reduced absorption or enhanced catabolism of the vitamin is the
mechanism for the reduction in vitamin C, as studies to measure the
bioavailability of vitamin C have not been conducted. The studies
carried out by Pelletier, et al. (58) suggest that reduced absorption of
vitamin C by smokers may be involved in reduced levels of vitamin C.
Bilirubin
Nymand (52) recently reported the effects of maternal smoking on
neonatal hyperbilirubinemia. He observed that the biotransformation
of bilirubin was enhanced in newborn infants of smoking mothers. The
incidence of cases with serum bilirubin concentrations below 106
PM/liter was significantly higher in smokers than in nonsmokers. On
the other hand, Conney, et al. (15) reported earlier that the serum
bilirubin levels in newborn of 9 nonsmokers and of 14 smokers showed
no difference in the serum bilirubin levels between the two groups of
newborns. No differences in the serum bilirubin concentration have
been observed between adult smokers and nonsmokers (11).
Subdunces Interfeting with the Assay Procedure
In pharmacokinetic studies, the effect of exogenous chemicals on the
data obtained with nonspecific assays is of particular concern. Beckett,
et al. (5) found that the higher urinary excretion of amphetamine by
smokers was explained by an amine which interfered with the assay.
This interfering substance was subsequently identified as nicotine.
Caution must be used in tobacco-drug studies, because the complex
mixture of chemicals in tobacco smoke could present similar problems
in drug assays carried out on biological samples from smokers.
Biotransformation of Drugs
Jusko (28) has compiled a list of drugs which have clearly been shown
either to have enhanced biotransformation or to have had no effect on
drug disposition in cigarette smokers. This list is given in Table 6. The
majority of the studies of smoking and drug effects have investigated
the drug disposition and clearance, with emphasis on the alterations in
the metabolic rate rather than on the absorption or distribution
process. Except for ethanol, all of the drugs in the list are
biotransformed by microsomal oxidative pathways. Most interesting is
the selectivity in the effects of smoking on drugs which undergo N-
demethylation. This effect may be accounted for by differences in rate-
limiting steps in the overall elimination of the drug. Other rate-
limiting processes are plasma protein binding, metabolism in nonmicro-
somal systems, and metabolism in nonhepatic tissue. Diazepam,
12-34

TABLE O.-Summary of smoking effects on in tiz'o,
     biotransformation of drugs in man
Dw
Major biotransformation pathway
Increased metabolic rate in smokers
Reference
number
Nicotine       Hydmxylation to N of cyclic amine
Phenacetin      0-Dealkylation
Antipyrine      Aliphatie hydmxylation
Theophylline     N&methylation. purine oxidation
lmipramine     N-demethylation
Pentawcine     Allylic hydroxylation
  (6)
(54.55)
(39,80,85)
(z44.iw328,63)
vmw
(30)
Not affected by smoking
Diazepam
Meperidine
Pbenytoin
Nortriptyline
Warfarin
Ethanol
N-demetbylation
Ndemethylation
Aromatic hydmxylation
N-demethylation
Aromatic hydroxylation
Alcohol dehydmgenation
(37)
(48)
(64)
(51)
(50.90
(79)
SOURCE: Jwko. W. (48).
phenytoin, and warfarin, which showed no difference in pharmacoki-
net& in smokers, are highly bound to plasma or protein and, for this
reason, exhibit low total clearance rates. The plasma binding and
diffusion of free drugs may not be altered significantly by tobacco
smoke. Contrarily, meperidine and nortriptyline are drugs which
exhibit very high total clearance rates, and hepatic blood flow may be
the determining factor which is unaffected by smoking. The only
generalization which can be made about these drugs is that the
enhanced metabolism induced by tobacco smoking appears to be a
selective process with several microsomal pathways being induced or
unaffected.
Drug Effects in Man
The uncovering of differences in drug effects related to smoking has
been attributed to the comprehensive in-hospital drug monitoring by
the Boston Collaborative Drug Surveillance Program. Information has
been obtained on drug efficacy and toxicity for all drugs administered
to medical patients in this program. In addition to these data, an array
of basic patient statistics, such as smoking habits, is obtained prior to
admission. Several statistically significant findings that have emerged
from this program are described by Jick (27).
12-35

TABLE `I.-Mean priming dose and maintenance dose of
     pentazocine for supplementation of nitrous oxide
       anesthesia
Group
Smokers
Nonsmoker
No. of
subjecti
15
26
Mean (2 SEM)
priming dose
@Wkg)
0.91 2 0.11 P = o,05
0.57 T 0.13
Mean (+ SEM)
maintenance dose
WWW
SOURCE: Keeri-Szanlo. M. (SO)
Pentazocinc!
A number of clinical reports on the alteration of drug responses in
smokers have been published. One of the first was the examination of
pentazocine dosage requirements for supplementation of nitrous oxide
anesthesia. Keeri-Szanto, et al. (30) found that smokers required larger
priming and maintenance doses of pentazocine than did nonsmokers
(see Table 7).
These results were correlated to plasma concentration of pentazo-
tine, and the increased priming and maintenance doses were attributed
to enhanced drug disposition in smokers. These findings have been
confirmed by Vaughan, et al. (77) by examination of urinary
pentazocine excretion in smokers and nonsmokers. The researchers
determined that smokers metabolize 40 percent more pentazocine than
nonsmokers.
The first drug to be evaluated in detail with respect to smoking in the
Boston Collaborative Drug Surveillance Program was propoxyphene
(10). Propoxyphene was rated ineffective by 10.1 percent of 335
nonsmokers, 15 percent of 347 light smokers, and 20.3 percent of 153
heavy smokers.
A summary of other observations of differences in drug effects in
smokers and nonsmokers made by the Boston Collaborative Drug
Surveillance Program (27) and by Jusko (28) is given in Table 8.
Although the disposition of some drugs (phenacetin, theophylline,
and antipyrine) is known to be increased in smokers, the mechanisms
of other drug/smoking interactions are not well established. An
increased "first pass" metabolism is one possibility. A possible
explanation for the reduced clinical effect of propoxyphene in smokers
is decreased pain threshold. Seltzer, et al. (69) have found that deep
pain tolerance is significantly diminished in white male and female
cigarette smokers as compared to nonsmokers. In addition, two surveys
(one conducted in the United States and the other in Australia) have
12-36

TABLE 8.-Modification of clinical drug effects by smoking:
     observations of the Boston Collaborative Drug
    Surveillawe ?Frn
Incidence relalcvl to smoking
  habit (% p&n!&
Diminished
effwt observed
Non     Light
Heavy     Reference
       number
Pmpoxyphene
~hlorpmmazine
i)iazepam
Chlordiawpoxide
Thenobarbital
WarStin
Theophylline
Pain/headache efficacy
Drowsinepg
CNS depression
None (CNS)
None (CNS)
No modification of
antimagulant needs
various advme
IWCtiOnS
10.1      15.0      20.3      (9)
16       11       3       (78
7.9      7.i      28      WY
9.7      6.1      3.5      (10)
5.9      9.3      4.3      (10)
_ .       _ .      - _      (50)
129      10.8      7.0      W
SOURCE: lick. ?I. (07). Jusko. W. (-988).
found that smokers tend to consume more analgesics than nonsmokers
(19, 68).
other Lkugs
There are a few reported tobacco-drug interactions which do not
involve enzyme induction. Vapaatalo, et al. (76) found that cigarette
smoking somewhat reduced the diuretic effects of furosemide. This
interaction was best explained by an increased secretion of the anti-
diuretic hormone caused by nicotine.
Kershbaum, et al. (35) reported that the stimulating effect of
smoking on adrenocortical secretion could neutralize the suppressive
effect of dexamethasone on plasma corticosteroid concentrations.
Beta-blockers such as propranolol have been used to modify nicotine-
stimulated catecholamine effects such as increased pulse rate, blood
pressure, and ventilator-y function (1.2, 13,20,90,93). Frank1 and Soloff
(20) reported that five subjects who received propranolol, followed by
smoking, experienced significantly decreased cardiac output, signifi-
cantly increased blood pressure, and significantly increased calculated
systemic peripheral resistance compared to smoking without propanol-
01.
Absence of Smoking Effect
Alteration in drug disposition or pharmacological action in smokers
generally received greater attention than those reports demonstrating
no effect of tobacco smoke; it is equally important, however, from a
12-3'7

clinical and pharmacokinetic point of view to identify clearly those
drugs which are not influenced by tobacco smoke.
A Boston Collaborative Drug Surveillance Program report (9) on the
relationship to cigarette smoking of depression of the central nervous
system during chronic diazepam therapy indicated that drug-attrib-
uted drowsiness became less common as the exposure to cigarette
smoke increased. These findings were explained by the stimulation of
diazepam metabolism by one or more of the constituents of cigarette
smoke. Klotz, et al. (37) have reinvestigated the effects of age,
smoking, and liver disease on diazepam disposition. They determined
that an induction of the diazepam disposition would manifest itself by
an increase in the plasma clearance or by a reduction in the tl/z of drug,
yet no obvious differences between these values in smokers and
nonsmokers were seen at any age. The authors concluded that
cigarette smoking did not affect the disposition of diazepam and
suggested that factors other than inferred changes in metabolism were
involved in the greater incidence of side effects of diazepam in
nonsmokers. These results suggest that further study of the effects of
smoking and diazepam disposition is required.
Phenytoin
Phenytoin is subject to highly variable and dosedependent elimination
in patients, and its low therapeutic ratio requires careful patient
monitoring for its use as an anticonvulsant. Rose, et al. (64) found that
the only effect of tobacco smoke on disposition of phenytoin was an
exacerbation of the inherent variability in its elimination, but the
mean total clearance and tl/z values were similar in young, closely
matched smokers and nonsmokers. No difference in the volume of
distribution or the degree of plasma protein binding of phenytoin was
observed between the two groups.
Wurfcwin
The Boston Collaborative Drug Surveillance Program found no
difference in maintenance dosages of warfarin administered to
hospitalized patients who were nonsmokers, light smokers, or heavy
smokers (4.9). Similarly, Yacobi, et al. (91) have determined that
nonsmokers, as well as smokers and patients taking barbiturates, have
similar total clearance and plasma protein binding of warfarin.
Recently Bachmann and Tarloff (4) have uncovered a species
difference in the susceptibility of warfarin disposition to enzyme
induction. They have found that pretreatment with benzo(a)pyrene
decreased the duration of hypoprothrombinemia and shortened the tin
of warfarin rate in rata.
1% 38

Meperidine
Mather, et al. (47) have investigated the effects of cigarette smoking
on meperidine disposition in surgical patients and volunteers. The
mean total clearance value was determined to be 26.9 liters/hr/mz for
smokers and 23.6 liter/hr/mzfor nonsmokers.
Nwtriptyline
Norman, et al. (51) dosed a group of 22 smokers and 31 nonsmokers
with 150 mg/day of nortriptyline and determined steady-state plasma
concentrations. Smokers achieved a mean plasma concentration of
nortriptyline concentration of 191 2 141 ng/ml, but nonsmokers had a
level of 169 2 92 ng/ml. This difference was not determined to be
significant. Age, sex, and number of cigarettes smoked had no effect
on the plasma nortriptyline concentrations achieved.
Ethanol
Smokers tend to consume more coffee, ethanol, and nonnarcotic
analgesics than nonsmokers. Therefore the study by Vestal, et al. (79)
on ethanol disposition and aging is of interest. The mean maximum
biotransformation capacity (Vmax) for five cigarette smokers was
determined to be 75.9 mg/kg/hr while 45 nonsmokers averaged 74.8
mg/kg/hr (79). It should be noted that ethanol metabolism differs
markedly from that of other drug metabolism in that it is primarily
oxidized by the cytosolic hepatic enzyme, alcohol dehydrogenase.
Further studies on the effects of alcohol metabolism and smoking are
needed, because Kopun and Propping (38), in a study using 19 identical
and 22 fraternal sets of male twins, showed that regular alcohol
consumption and heavy smoking correlated with an increased alcohol
elimination rate. The number of individuals used in this study was
somewhat limited.
othm- Drugs
The rate of phenol red excretion was not altered by smoking after
administration of the dye by various routes (42).
Hagedorn and Kostenbauder (22) found that cigarette smoke had no
effect on the metabolism of prostaglandin F-2a in the isolated perfused
rabbit lung, but administration of cigarette smoke was found to have a
pronounced inhibitory effect on the metabolism of both nicotine and
BP in this in vitro system (44,48).
Uotila and Hartiala (75) have reported that the covalent binding of
BP was greatly enhanced by 3-methylcholanthrene pretreatment. The
amount of polar metabolites in the perfusion fluid of 3-MC treated
lung was increased. They suggested that this may indicate induction of
pulmonary BP metabolizing enzyme, but additional studies are needed.
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Mechanism of Tobacco-Drug Interaction
Tobacco smoke is a complex mixture of noxious materials (66). (See the
Chapter on the Constituents of Tobacco Smoke.) The particulate phase
consists of water-soluble materials such as nicotine, other alkaloids,
and a myriad of organic substances. It also contains fat soluble
polycyclic aromatic hydrocarbons (PAHs, PNAs) and more complex
organic compounds. At least 43 major components have been identified
(70) in the PAH fraction. To date only a few of the components of
tobacco smoke have been examined with respect to modifying drug
disposition in man or animal or their effects on tissue or enzyme
systems.
The incomplete combustion of organic materials in tobacco yields
PAH. Akin, et al. (2) separated cigarette smoke into the PAHenriched
fraction which comprised 0.4 percent of the weight of the crude
condensate, but accounted for virtually all the carcinogenic potential.
It has been estimated that a Z&cigarette-per-day smoker of unfiltered
cigarettes would inhale about 0.7 N/day of BP while filtered
cigarettes would yield about 0.4 M/day of BP. It has been reported in a
number of studies that BP induces the microsomal enzyme benzpyrene
hydroxylase (14,39,86). The characteristics of this enzyme system have
been reviewed in the metabolism section of this chapter.
Other Pathophysiological Factors of Smoking
Tobacco smoking is associated with a number of pathophysiological
changes which may not be directly related to any specific drug
interaction, but do offer the potential for contributing to altered drug
disposition. Smoking and nicotine have been shown to increase
corticosteroid secretion (36). It is also known that chronic administra-
tion of steroids will accelerate drug disposition. Nicotine treatment has
been shown to cause catecholamine release; this can result in
mobilization of free fatty acids from adipose tissue (34). The release of
free fatty acids could displace drugs from protein binding sites. Dales,
et al. (17) examined serum chemistry levels in over 65,000 cigarette
smokers and nonsmokers and found slightly lower serum albumin, uric
acid, and creatinine concentration in smokers who were over 30 years
old. This lower serum albumin may relate either to altered hepatic
function or to changes in drug binding. In a similar study, Lellouch, et
al. (40) reported that smokers had lower serum urea and uric acid
concentration than nonsmokers. The lower values for creatinine, urea,
and uric acid may reflect altered renal or hepatic function in smokers.
BP is strongly bound to serum albumin (45) and is therefore capable of
displacing ligands from similar protein binding sites.
There may be other physiological, biochemical, and behavorial
differences in the smoker group. Smokers are a "self-selected" group
which means that the unknown factors that cause individuals to smoke
may be of importance in drug disposition. Studies have examined the
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differences between smokers and nonsmokers. Seltzer, et al. (67) have
reviewed several studies; the consensus was that smokers tend to be
more energetic, restless, and extroverted than nonsmokers. On the
other hand, smokers tend to possess more neurotic traits including
greater psychological tension and more psychosomatic symptoms. In
addition, smokers tend to be hospitalized more often than nonsmokers
and are, as expected, beset with a higher incidence of specific disease
such as hypertension, coronary artery disease, and lung problems. The
self-selection biases are difficult to remove from pharmacokinetic
studies of the effects of smoking.
In the future, it would be helpful if, after cessation of smoking,
careful studies of the reversibility of the smoking effect were
conducted. Present studies indicate that the induction of BP hydroxyl-
ase is not completely reversed following 2 to &month cessation of
smoking (24).
Smoking and Drug Consumption
The relationship of smoking and drug disposition is complicated by the
typical pattern that cigarette smokers tend to consume other drugs
and chemicals more frequently than nonsmokers. Furthermore,
smokers tend to ingest more coffee and alcohol than nonsmokers.
Ferguson (19) found that smokers consumed more alcohol and non-
narcotic analgesics. Weitman, et al. (81) and Seltzer, et al. (69)
examined the incidence of various types of drugs used in relation to
tobacco smoking. In these studies, it was determined that smoking
correlated highly with the use of other drugs. Smokers admitted to
taking more cough medicine, aspirin-containing drugs, pain medica-
tions, prescription analgesics, barbiturates, sleeping pills, tranquilizers,
diuretics, hormones, anemia medicine (iron), amphetamines, antibiot-
ics, stomach medicines, and laxatives than nonsmokers. The only drugs
taken by a larger percentage of nonsmokers were those for allergic
conditions-antihistamines and asthma medicine. Great care must be
used in carrying out pharmacokinetic studies of the effects of smoking.
Because most studies do not or cannot control for many of the
secondary differences between smoker and nonsmokers, care must be
used in the interpretation of the results so that the reported
associations between smoking and pharmacological action of drugs are
not related to psychosomatic differences, drug ingestion patterns, and
therapeutic need (threshold dose) of the two groups.
Studies of the effect of smoking on drug disposition usually attempt
to quantitate smoke intake by vague descriptive categories such as
nonsmokers/smokers, nonsmokers/light smokers/heavy smokers, or
number of cigarettes smoked per day. These measures approximate
only the potential exposure of man to the various chemicals in tobacco
smoke. Factors such as cigarette brand, filters, degree of inhalation,
duration of habit, respiratory rate, pharmacokinetics of the chemical in
12-41

man, and so forth, are unknowns in a study of this type. All of these
factors sometimes make an investigation of the interaction of tobacco
smoking and drugs extremely difficult to assess.
In the future, scientific reports describing the pharmacokinetics or
clinical pharmacology of a drug should list and examine the smoking
status of the subjects employed in the study. Smoking should be
included as a basic characteristic of each subject in the same way as is
age, race, body weight, and presence and type of disease. Monitoring
subjects for intensity of tobacco use might be accomplished by
determining of serum or urine thiocyanate (26). This substance
possesses a long t.l,z (about one week), which allows for an assessment
of chronic smoking at a consumption rate which is most likely to affect
drug disposition. Thiocyanate is relatively easy to assay and serum
concentration has been reported to be proportional to the number of
cigarettes smoked (24).
Marijuana
The subject of tobacco smoking and drug interaction needs to consider
the interaction of drugs and marijuana smoking. It has been estimated
that 13 million people in the United States now smoke marijuana (1).
Animal systems show mixed effects, with marijuana studies
reporting induction and inhibition of the microsomal drug-metabolized
enzymes. Paton and Pertwee (57) reported that cannabis extract
prolonged pentobarbital sleeping time in mice and inhibited the aerobic
metabolism of phenazone in mouse liver microsome preparation. Mitra,
et al. (50) found that chronic treatment with Ag-tetrahydrocannabinol
(THC) for 21 days (10 mg/kg/day) competitively inhibited N- and O-
demethylase activity, but had no inhibitory effect on aniline hydroxyl-
ase activities. Siemens, et al. (71) found a prolonged pentobarbital
sleeping time and a longer tlJz in rats pretreated with various
cannabinoid compositions as well as pure Ag-THC.
Sofia and Barry (7.2) noted both enzyme inhibition and induction in
mice following treatment with As-THC. Pretreatment with a single
high dose of AS-THC (20 mg/kg` increased the duration of the loss of
the righting reflex after a dose of zoxazolamine and hexobarbital, and
enhanced the duration of hr bital sleeping time. Berman and
Bochantin (8) also found that chronic doses of Ag-THC (2.5 or 5.0
mg/kg daily for 4 days) increased liver microsomal dichlorinase
activity (enzymes that metabolize methoxyflurane and halothane) in
rats. Marcotte, et al. (46) have determined that analysis of the smoke
condensate from cigarettes and from marijuana placed in a smoking
machine gave 0.32 and 0.44 ng of BP/mg of PAH condensate, and 0.42
and 0.67 ng of 3-MC/mg of PAH condensate, respectively. These
investigators found that exposure to the smoke of either marijuana or
marijuana placebo (with the cannabinoid removed) maximally stimu-
lated benzpyrene hydroxylase activity in rat lung tissue.
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