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The implications of procainamide metabolism to its induction of lupus.

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994
THE IMPLICATIONS OF PROCAINAMIDE
METABOLISM TO ITS INDUCTION OF LUPUS
JACK P. UETRECHT, RICHARD W. FREEMAN, and RAYMOND L. WOOSLEY
The principal metabolic pathway of procainamide
leads t o formation of the less toxic N-acetylprocainamide and the rapid acetylator phenotype is associated with a lower incidence of procainamide-induced
lupus. Another metabolic pathway forms a reactive metabolite which causes revertants in the Ames test and
covalently binds to microsomal protein. A study of the
metabolism of procainamide revealed three metabolites
that have not been previously described. A comparison
of the metabolites of N-acetylprocainamide with those
of procainamide suggests possibilities for the identity of
the reactive metabolite. The hypotheses to be discussed
explore the relationship between the formation of a reactive metabolite and the induction of lupus.
Procainamide (structure I in Figure 6) produces
the highest incidence of drug-induced lupus of any drug
known. The incidence of clinical lupus in patients receiving prolonged procainamide therapy is about 30%
and the incidence of antinuclear antibodies (ANA) approaches 100% (1-3). The other drug associated with a
high incidence of drug-induced lupus is hydralazine (4).
The major metabolic pathway for both drugs is N-acetylation. The rate of acetylation is a genetically controlled trait and the population can be divided into slow
and rapid acetylator phenotypes (5).
From the Division of Clinical Pharmacology, Departments
of Pharmacology and Medicine, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232.
Supported by grants from the General Clinical Research
Center Program of the Division of Research Resources (5MOlRR-95),
NIH grants GM 07569 and GM 15431, and by a grant from the Kroc
Foundation.
Address reprint requests to Dr. Jack Uetrecht, Department of
Pharmacology, Vanderbilt University Medical Center, Nashville,
Tennessee 37232.
Arthritis and Rheumatism, Vol. 24, No. 8 (August 1981)
N-acetylation of procainamide: a protective
metabolic pathway
It was first recognized by Perry that persons of
the rapid acetylator phenotype have a very low incidence of hydralazine-induced lupus (4). The initial
studies of the relationship between acetylator phenotype
and the incidence of procainamide-induced lupus gave
conflicting results (2,6). In a prospective study of the
time course of induction of ANA in patients taking procainamide we found that, although rapid acetylators did
develop ANA, they did so after a longer period of time
than slow acetylators (Figure 1) (1). A combined retrospective and prospective study of the development of
clinical lupus gave the same results (Figure 2) (1).
Differences in the pharmacokinetics of procainamide and hydralazine probably account for the
lesser degree of protection afforded by the rapid acetylator phenotype to patients on procainamide. Hydralazine has a first-pass hepatic metabolism of 60-8096 and
there is almost no renal elimination of the parent drug
(7). In contrast, the first-pass hepatic metabolism of procainamide is estimated to be only 15%, and 65% of the
parent drug is eliminated by renal excretion (8). The
principal metabolite, N-acetylprocainamide (11), also
has antiarrhythmic efficacy but during prolonged therapy does not cause a significant incidence of ANA or
lupus (9). Thus it appears that the incidence of procainamide and hydralazine-induced ANA and lupus is
a function of the cumulative exposure of unacetylated
drug. This, along with the very high incidence of procainamide-induced ANA and lupus, produces a picture
resembling a dose-response curve rather than a hypersensitivity reaction.
PROCAINAMIDE METABOLISM
995
0SLOW
ACETYLATORS
RAPID A C E T Y L A J O R S
T I M E TO CONVERSION (MONTHS)
Figure 1. Development of procainamide-induced antinuclear antibodies in slow acetylators (open circles) and rapid acetylators (closed
circles) with time. The number of patients followed is listed at each
point.
0
0
E
32
40 48
56
64 72
80
DURATION OF THERAPY (MONTHS)
a
Figure 2. Histogram showing the relationship between acetylator
phenotype and the rate of development of the procainamide-induced
lupus syndrome.
6C
60
50
T
9
50
+A40
h
5
w
v)
a
?! 40
W
E 30
!-31
a
v,
30
!
i
a
a
20
v,
s 20
I-
W
z
c
rY
(L
w
>
k2
10
10
0
I
I
I
I
082
2,o
20
200
& g DRUG PER PLATE
Figure 3. Comparison of the reactivity of procainamide (0)and Nacetyl-procainamide (0)in the Ames test. Data represent the mean f
SE number of revertants above the spontaneous reversion rate.
0.2
2.0
20
Ng DRUG PER PLATE
200
Figure 4. Effect of removal (des-aminoprocainamide A) or replacement by a hydroxyl-group @-hydroxyprocainamide @) of the aromatic amine on the reactivity of procainamide (0)in the Ames test.
Data represent the mean f SE number of revertants above the spontaneous reversion rate.
UETRECHT ET AL
996
Table 1. Comparison of covalent binding of procainamide and Nacetylprocainamide in vitro*
In
W
I
Substrate+
I
1
nmol equivalents per mg
microsomal protein? f SE
0.52 f 0.03
0.05 f O.Ol$
Procainamide
N-acetylprocainamide
~~
* Data represent the mean binding f SE after correction for a small
amount of binding in the absence of NADPH.
t Microsomal suspensions were prepared from Swiss-origin ICRHA male mice. Final concentrations of substrate and microsomal protein were 1.0 mM (1.0 pCi per incubation) and 2 mg/ml, respectively.
Incubation period was 15 minutes.
P 5 0.05 compared to procainamide binding (Student’s t-test).
-
+
I
40 50 60
TIME (minutes)
[PA] mM
I
u
9
PROTEIN I m g )
12
Figure 5. Covalent binding of a metabolite of procainamide in vitro
to microsomal protein as a function of time (A), procainamide concentration (B), and protein concentration (C).
Evidence of metabolic activation of
procainamide
In speculating about the mechanism of an adverse reaction with the aforementioned characteristics
we developed the working hypothesis that pro-
cainamide may form a reactive metabolite that covalently binds to nuclear macromolecules and thereby acts
as a hapten to induce ANA formation. Procainamide, as
well as many other drugs that have been implicated in
the drug-induced lupus syndrome, contains a nitrogen
that could be oxidized to a reactive metabolite (10).
Acetylation of this nitrogen could decrease the amount
of drug available for the oxidative pathway. This would
account for the protective effect seen for the rapid acetylator phenotype.
To test the hypothesis that procainamide forms a
reactive metabolite we used both the Ames test and a
measure of covalent binding. The Ames test measures
the ability of a compound to cause mutations of histidine autotrophs of S fyphrnuriurn which allow them to
grow in a medium free of histidine. Procainamide produced a small but significant increase in the number of
revertants in the Ames test and more than the acetylated metabolite, N-acetyiprocainamide (Figure 3) (1 1).
Addition of microsomal protein and reduced nicotinamide adenine dinucleotide phosphate (NADPH) was
necessary for activity, thus indicating that the activity is
Table 2. Procainamide* covalent binding in vivo: rtJle of mixed function oxidase
Pretreatment
Agent
None
Phenobarbital8
3-methylcholanthreneq
SKF-525A#
Phenobarbital SKF-525A
nmol procainamide
equivalents bound per
Dosage
T i e (days)
gm liver rfr SEt
1.96 f 0.09
50 mg/kg/day; IP
4
2.53 f 0.11
40 mg/kg/day; IP
4
3.06 f 0.19
1.19 f 0.05
1.29 f 0.11
ps
-
< 0.05
c 0.05
< 0.05
< 0.05
* Procainamide was administered intraperitoneally at a dose of 200 mg/kg, 15 pCi per mouse. Specific
activity of injection solution was 0.68 pCi/mol. Animals were killed 2 hours after injection.
f Data represent the mean f SE of 5 male mice per treatment group.
Compared to pretreatment (Student’s t-test).
0 Procainamide was administered 24 hours after the last phenobarbital dose.
1Procainamide was administered 24 hours after the last 3-methylcholanthrenedose.
# SKF-525A (40 mg/kg, ip) was administered 1 hour before procainamide.
+
PROCAINAMIDE METABOLISM
997
0
OJ-N
HC H ~ C H ,N;?"S
YE
Figure 6. Metabolic pathways of procainamide in the perfused rat liver. The dashed arrows on the left side of the figure indicate that since these metabolites were formed when
N-acetylprocainamide was used instead of procainamide it is possible that acetylation is
the first step in their synthesis. The dashed arrow leading to structure IV indicates that very
little, if any, comes from N-acetylprocainamide.
due to a metabolite of procainamide. Other compounds
in which the aromatic amine functional group has been
removed or replaced by a hydroxyl-group also caused
fewer revertants (Figure 4).
Incubation of 14C-procainamidewith mouse microsomes led to covalent binding of a metabolite of procainamide to protein (Figure 5) (12). That this resulted
from a metabolite of procainamide was evidenced by
UETRECHT ET AL
998
Figure 7. Possible intermediates in the pathway from procainamide to the putative 3-hydroxyprocainamide.
the requirement for NADPH, oxygen, and active microsomes. Under the same conditions there was no significant covalent binding of N-acetylprocainamide (Table
1). Injection of ''C-procainamide into mice led to a
demonstration of in vivo covalent binding to hepatic tissue (Table 2). This binding was significantly increased
by induction of mixed function oxidase activity with
phenobarbital and 3-methylcholanthrene and inhibited
by SKF-525A pretreatment.
Metabolites of procainamide and their
possible relationship to a reactive metabolite
With evidence that procainamide forms a reactive metabolite we began a study of the metabolism of
procainamide in search of clues to the identity of this
metabolite.
The metabolism of procainamide has been studied previously in humans, Rhesus monkeys, rats, and
dogs (13-17). With the exception of the dog, the principal metabolite is N-acetylprocainamide (11). DesethylN-acetylprocainamide (VII) has been found in the
Rhesus monkey and humans and small amounts of
desethylprocainamide, p-aminobenzoic acid, and Nacetyl-4-aminobenzoic acid (VIII) have been found in
humans. Other metabolites, including one in which the
aromatic amine appears to have been altered, have been
isolated but not identified.
Using a rat liver perfusion model we found three
metabolites of procainamide which have not been previously described: N-acetyl-3-hydroxyprocainamide
(IV), N-acetylprocainamide-N-oxide(V), and N-acetyl4-aminohippuric acid (VI) (Figure 6) (18).
Because N-acetylprocainamide does not induce
lupus, its metabolism was studied to compare with that
of procainamide. Very little of the phenol metabolite IV
was formed from N-acetylprocainamide, suggesting that
the phenol IV is related to the reactive metabolite. In
addition this implies that, even though it was not observed, the aminophenol I11 is an intermediate in the
formation of the phenol IV. The two most likely intermediates in the formation of the aminophenol I11 are
the epoxide IX and the hydroxylamine X (Figure 7).
Both epoxides and hydroxylamines have been implicated as reactive metabolites of other chemicals and either could represent a reactive metabolite of procainamide. The aminophenol I11 may also be oxidized
to an iminoquinone which could also represent a reactive metabolite of procainamide.
The study of the metabolism of procainamide
continues and it also remains to be determined what
relationship, if any, exists between the formation of a
reactive metabolite and the induction of lupus. If a relationship is demonstrated between the reactive metabolite of procainamide and the induction of lupus, it could
have implications for Reidenberg's hypothesis that
idiopathic lupus is caused by environmental aromatic
amines (19).
PROCAINAMIDE METABOLISM
999
REFERENCES
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