possible Mechanism of
Tolerance to Ntircotic Drugs

study was begun were used throughout
the study. The average gain in weight in
all groups of rats was approximately the
same.

Other pathways in the in vitro meta-
bolism of narcotic drugs, such as O-de-
methylation of codeine (7)) hydrolysis
of diacetyl morphine (8)) and conjuga-
tion of morphine (9)) were also exam-
ined. No differences in the enzymic
0-demethylation, hydrolysis, and con-
jugation of narcotic drugs in the control-
and morphine-treated rats were found.

Although many hypotheses have been
offered to explain the development of
tolerance to narcotic drugs (I ), adequate
experimental data have not been pre-
sented to elucidate this phenomenon. In
recent studies at this laboratory, we have
observed several striking similarities be-
tween the receptors for narcotic -drugs
and the enzymes that N-demethylate
these drugs. The enzymes and receptors
have been found to be alike with respect
to substrates with which they interact,
stereospecificity,  and antagonism by
N-allylnormorphine (2). Since the en-
zymes that N-demethylate narcotic drugs
were similar in several ways to narcotic
drug receptors, it appeared likely that
these enzymes might serve as a model for
the receptors. Thus, any changes occur-
ring in enzyme activity during the devel-
opment  of tolerance might reflect
changes taking place on the drug recep-
tor. With this in mind, an examination of
the effect of repeated administration of
morphine to rats on the enzymic N-de-
methylation of morphine and other nar-
cotic drugs was undertaken. The effect of
the administration of morphine, together
with its antagonist, N-allylnormorphiner
on enzymic N-demethylation was also in-
vestigated, since it has been shown that
this combination reduces the develop-
ment of tolerance (3).

Twenty-four hours after the test period
the animals were sacrificed, and the liv-
ers were examined for their ability to
N-demethylate morphine, dilaudid, mep-
eridine (Demerol), and cocaine. The
livers were prepared for enzyme assay by
a procedure described previously (2))
and the degree of. enzymic N-demethyla-
tion was determined by estimating the
amount of formaldehyde liberated (4).

The changes in the enzymic N-de-
methylation in the various groups of rats
are shown in Fig. 1. In the case of mor-
phine-treated animals (group M) , a
profound reduction in the ability to
N-demethylate morphine occurred. In
addition, the enzymic N-demethylation
of dilaudid, a compound that shows
cross-tolerance to morphine (5)) was re-
duced to about the same degree as that
of morphine, while the demethylation of
meperidine, a drug that exhibits limited
cross-tolerance to morphine (6)) was
only partially reduced. Enzymic N-de-
methylation of cocaine, for which no
cross-tolerance to morphine occurs (5))
was unaffected by chronic morphize ad-
ministration. In the group of animals
that was treated with- both' N-allylnor-
morphine and morphine (group NM),
the reduction in the enzymic demethyla-

From the results described here, a
striking parallelism between the enzymic
N-demethylation of narcotic drugs and
the development of tolerance to these
drugs was found. The repeated adminis-
tration of morphine reduced both en-
zymic demethylation and pharmacologi-
cal response. In addition, there was a
correlation between demethylation of
substrates and cross-tolerance to mor-
phine. Furthermore, N-allylnormorphine,
which blocks development of tolerance
to morphine, also blocks reduction of
enzyme activity. It appears that N-allyl-
nor-morphine not only antagonizes the
pharmacological action and the enxymic
demethylation of narcotic drugs but also
protects the enzyme and perhaps the re-
ceptor sites. Animals that are withdrawn
from narcotic drugs recover their phar-
macological responses to these drugs;
similarly, the demethylating-enzyme ac-
tivity in rats withdrawn from morphine
returns to normal.    :
The changes in enzyme activity in mor-

phine-treated rats suggest a mechanism
for the development of tolerance, if one
assumes that enzymes which N-demethyl-
ate narcotic drugs and the receptors for
these drugs are probably closely related.
The continuous interaction of narcotic
drugs with the demethylating enzymes
inactivates the enzymes. Likewise, the

Twelve rats were made tolerant to
morphine by a daily intraperitoneal in-
jection of morphine sulfate. The animals
were given an initial dose of 20 mg/kg
of morphine sulfate, and the amount of
drug administered was then progressively
increased during a period of 35 days until
daily injection of 150 mg/kg was reached
(group M). Another group of eight rats
was given N-allylnormorphine and mor-
phine in a ratio of l/4 for 35 days
(group NM). A group of 12 rats was
given the same dosage regimen of mor-
phine as described in group M for 35
days, following which the drug was
abruptly withdrawn for 12 days (group
W). Fourteen rats receiving a daily in-
jection of isotonic saline served as con-
trols (group C) . Fisher-strain male rats
that were 120 to 130 days old when the

Fig. 1. Effect of morphine
treatment, N-allylnormor-
phine, and withdrawal on
the enzymic N-demethyla-
tion of narcotic drugs. Ver-
tical bracketed lines on
bars are standard devia-
tion of the mean. (group
M) morphine-treated rats ;
L7p -NM) morphine-
   N-allylnormorphine-

MORPHINE

DILAUDID

MEPERIDINE   COCAINE

treated rats ; (group W)
rats treated with morphine
and   then  withdrawn;
(group C) normal rats.

lo AUGUST 1956                                                                           263

Reports

tion of narcotic drugs was significantly
less than in those that received morphine
only. The enzyme activity with respect to
all substrates had returned to the control
level or above in withdrawn animals
(grow WI.


continuous interaction of narcotic drugs
with their receptors may inactivate the
receptors. Thus, a decreased response to
the narcotic drugs may develop as a re-
sult of unavailability of receptor sites
(10).

JULIUS AXELROD

National Institute of Mental Health,
National Znstitutes of Health,
U.S. Public Health Service,
Bethesda, Maryland

1.

2.

3.

4.
5.

6.

7.
Z:

10.

M. H. Sewers and L. A. Woods. Am. J. Med.
14, 546 (1953) ; N. B. Eddy, Origin of Re-
sirlance to Toxic Agcntr (Academic, New
York, 1955) p. 223.
J. Axelrod, J. Pharmacol. Expll. Therop., in
press; J. Axelrod and J. Co&in, Federation
Proc. 15, 395 (1956).
P. D. Orahovats, C. A. Winters, E. G. Leh-
man, J. Pharmacol. Exptl. Themfi. 109, 413
(1953).
J. Ax&&, ibid. 114, 430 (1955).
E. Joi+l and A. Ettinger, Arch. Expfl. Pathol.
Phcnmakof. 115, 334 (1926).
F. E. Shideman and H. T. Johnson, J. Phor-
mcrcol. Exptl. The@. 92, 414 (1948).
J. Axelrod, ibid. 115, 259 (1955).
C. I. Wright, ibid. 75, 328 (1942).
F. Bemheim and M. L. C. Bcmheim, ibid.
83, b (1945).
I wish to thank N. B. Eddy and J. Co&in
for many helpful discussions.

4 June 1956