Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo

Article abstract of DOI:10.1021/jm9606296

S-Methylisothiourea (4), when administered to rats followed by a subsequent dose of ethanol, gave rise to a 119-fold increase in ethanol- derived blood acetaldehyde (AcH) levels-a consequence of the inhibition of hepatic aldehyde dehydrogenase (AIDH)-when compared to control animals not receiving 4. The corresponding O-methylisourea was totally inactive under the same conditions, suggesting that differential metabolism may be a factor in this dramatic difference between the pharmacological effects of O- methylisourea and 4 in vivo. The S-n-butyl- and S-isobutylisothioureas (8 and 9, respectively) at doses equimolar to that of 4 were nearly twice as effective in raising ethanol-derived blood AcH, while S-allylisothiourea (10) was slightly less active. However, blood ethanol levels of all experimental groups were indistinguishable from controls. Pretreatment of the animals with 1-benzylimidazole, a known inhibitor of the hepatic mixed function oxidases, followed sequentially by either 8, 9, or 10 plus ethanol, reduced blood AcH levels by 66-88%, suggesting that the latter compounds were being oxidatively metabolized to a common product that led to the inhibition of AcH metabolism. In support of this, when 8 was incubated in vitro with rat liver microsomes coupled to catalase and yeast AIDH, the requirement for microsomal activation for the inhibition of AIDH activity was clearly indicated. We suggest that S- oxidation is involved and that the S-oxides produced in vivo inhibited AIDH directly, or spontaneously released cyanamide, an inhibitor of AIDH. Indeed, incubation of 8 with rat liver microsomes and NADPH gave rise to cyanamide as metabolite, identified as its dansylated derivative. Cyanamide formation was minimal in the absence of coenzyme. Extending the side chain was detrimental, since S-benzylisothiourea (11) and S-n-hexadecylisothiourea (12) were toxic, the latter producing extensive necrosis of the liver and kidneys when administered to rats.

Full text of DOI:10.1021/jm9606296

1870
J . Med. Chem. 1997, 40, 1870-1875
Novel P r od r u gs of Cya n a m id e Th a t In h ibit Ald eh yd e Deh yd r ogen a se in Vivo†
Frances N. Shirota,‡ J anell M. Stevens-J ohnk,§ Eugene G. DeMaster,‡ and Herbert T. Nagasawa*,‡,§
Medical Research Laboratories, VA Medical Center, and Division of Medicinal Chemistry, College of Pharmacy,
University of Minnesota, Minneapolis, Minnesota 55417
Received September 4, 1996X
S-Methylisothiourea (4), when administered to rats followed by a subsequent dose of ethanol,
gave rise to a 119-fold increase in ethanol-derived blood acetaldehyde (AcH) levelssa
consequence of the inhibition of hepatic aldehyde dehydrogenase (AlDH)swhen compared to
control animals not receiving 4. The corresponding O-methylisourea was totally inactive under
the same conditions, suggesting that differential metabolism may be a factor in this dramatic
difference between the pharmacological effects of O-methylisourea and 4 in vivo. The S-n-
butyl- and S-isobutylisothioureas (8 and 9, respectively) at doses equimolar to that of 4 were
nearly twice as effective in raising ethanol-derived blood AcH, while S-allylisothiourea (10)
was slightly less active. However, blood ethanol levels of all experimental groups were
indistinguishable from controls. Pretreatment of the animals with 1-benzylimidazole, a known
inhibitor of the hepatic mixed function oxidases, followed sequentially by either 8, 9, or 10
plus ethanol, reduced blood AcH levels by 66-88%, suggesting that the latter compounds were
being oxidatively metabolized to a common product that led to the inhibition of AcH metabolism.
In support of this, when 8 was incubated in vitro with rat liver microsomes coupled to catalase
and yeast AlDH, the requirement for microsomal activation for the inhibition of AlDH activity
was clearly indicated. We suggest that S-oxidation is involved and that the S-oxides produced
in vivo inhibited AlDH directly, or spontaneously released cyanamide, an inhibitor of AlDH.
Indeed, incubation of 8 with rat liver microsomes and NADPH gave rise to cyanamide as
metabolite, identified as its dansylated derivative. Cyanamide formation was minimal in the
absence of coenzyme. Extending the side chain was detrimental, since S-benzylisothiourea
(11) and S-n-hexadecylisothiourea (12) were toxic, the latter producing extensive necrosis of
the liver and kidneys when administered to rats.
Sch em e 1
In tr od u ction
Cyanamide, an alcohol deterrent agent used in
Europe, Canada, and J apan, is a potent in vivo inhibitor
of the hepatic class 2 (low Km) aldehyde dehydrogenase
(AlDH2)1 and induces a cyanamide-ethanol reaction
(CER)2 that mimics the disulfiram-ethanol reaction
(DER)3 on consumption of ethanol. This CER is mani-
fested by palpitation, facial flushing, and a general
feeling of malaise that ostensibly serves to deter further
drinking of alcohol. Cyanamide, unlike disulfiram, has
a short duration of action,4 but like disulfiram, must
be bioactivated in vivo since cyanamide itself is inactive
in vitro.5 The enzyme catalase, in the presence of H2O2,
mediates the oxidation of cyanamide6 to N-hydroxy-
cyanamide,7 and the latter spontaneously releases ni-
troxyl,8 the putative inhibitor of AlDH2 (Scheme 1).
Earlier, we had designed and developed a series of
acyl, N-protected R-aminoacyl and peptidyl derivatives
of cyanamide as prodrug forms of this alcohol deterrent
agent.9 Many of these prodrugs of cyanamide were
highly potent, and some were long-acting. For example,
stearoyl and palmitoyl cyanamide retained AlDH in-
hibitory activity for at least 72 h in rats and were
equally effective by the oral route as well as by intra-
peritoneal administration. The activity of these cyan-
amide prodrugs is presumably mediated by enzymatic
cleavage of the acyl cyanamide bond by endogenous
hydrolytic enzymes to liberate cyanamide, which then
inhibits AlDH after further bioactivation according to
Scheme 1.
We now describe a new series of novel, prodrug forms
of cyanamide that require bioactivation by the hepatic
cytochrome P-450 enzymes to release cyanamide in vivo.
These cyanamide prodrugs were found to raise blood
acetaldehyde (AcH) levels in rats following ethanol
administration, a pharmacological consequence of the
inhibition of hepatic AlDH.
Ra tion a le
The report by Hanzlik et al.10 that thiobenzamide (1)
was oxidized by rat liver microsomes to give thiobenz-
amide S-dioxide (2) which spontaneously decomposed
to benzonitrile (3) (Scheme 2) suggested that oxidation
of thiourea itself by the same enzyme system might lead
to the formation of cyanamide by an analogous mech-
anism (not shown). However, thiourea is known to be
toxic and is also a suspect carcinogen and mutagen;11
accordingly, it could not be considered further for
potential drug development. On the other hand, to our
knowledge, the oxidative metabolic fate of S-alkyl-
isothioureas has not yet been described.
† Presented in part at the1994 annual meeting of the Research
Society for Alcoholism, Maui, HI, J une 18-23, 1994.
‡ VA Medical Center.
Thiobenzamide S-oxidation has been shown to be
mediated in large part by the cytochrome P-450 mixed
§ University of Minnesota.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00629-2 CCC: $14.00 © 1997 American Chemical Society

Novel Prodrugs of Cyanamide
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1871
Sch em e 2
Sch em e 3
F igu r e 1. Effect of S-methylisothiourea (4) and O-methyl-
isourea on ethanol-derived blood acetaldehyde levels in male
Sprague-Dawley rats. Results are expressed as means (
SEM; the N’s are indicated in (parentheses) over the bars. The
description of the drug administration protocols can be found
in the Experimental Section. An asterisk (*) means signifi-
cantly different from saline control and the O-methyl com-
pound (P < 0.05).
Ch a r t 1
Sch em e 4
function oxidase system,10b although the participation
of flavin-containing monooxygenases cannot be dis-
counted.12 Enzymatic oxidations on divalent sulfur are
known to yield sulfoxides and, ultimately, S-dioxides (or
sulfones from dialkyl, diaryl, or alkylaryl sulfides).13
Accordingly S-methyisothiourea (4) would be expected
to be oxidized in vivo to its S-oxide (5) or the S-dioxide
(or sulfone equivalent) 6. Intermediates 5 or 6, or both,
should then spontaneously decompose to cyanamide and
methanesulfenic acid or methanesulfinic acid, respec-
tively, according to Scheme 3.
Resu lts
Administration of the commercially available 4 to rats
(0.5 mmol/kg, ip) followed by an ethanol challenge (2.0
g/kg, ip) 5 h later, and measurement of blood AcH 1 h
post ethanol, gave rise to a 119-fold increase in blood
AcH levels compared to saline controls given ethanol
(Figure 1). Blood AcH still persisted above saline
control levels even at 16 h post drug treatment. As
predicted, blood AcH was not elevated above control
levels when O-methylisourea was administered to rats
using the same protocol.
Other S-alkylisothioureas evaluated for their poten-
tial to oxidatively liberate cyanamide in vivo are listed
in Chart 1. With the exception of compound 10, all of
these S-substituted isothioureas are known, and litera-
ture procedures were used or adapted for their prepara-
tion. Compound 13 was prepared by S-alkylation of
thiourea with N-(2-chloroethyl)acetamide.16
Alternatively, the S-oxidized 5 or 6 may react directly
with the active site sulfhydryl group (Cys-302) of AlDH
by carboxamidinating the enzyme (Scheme 4). The
altered enzyme (7) will have a structure identical to that
formed by reaction of cyanamide itself on the enzyme.
This alternative mechanism bears a formal analogy to
the action of the putative reactive metabolite of disul-
firam, viz., methyl N,N-diethylthiolcarbamate S-oxide
(DETC-MeSO), which inactivates AlDH2 by carbam-
oylating the active-site sulfhydryl group.14
S-Methylisothiourea (4) would, therefore, be expected
to behave as a prodrug of cyanamide in vivo in the
manner depicted in Scheme 3 and to inhibit AlDH
following further bioactivation to nitroxyl (Scheme 1)
or, alternatively, may inhibit the enzyme directly fol-
lowing the initial bioactivation step (Scheme 4). Thus,
administration of 4 to rats followed by an ethanol dose
should give rise to elevated blood AcH levels, reflecting
the inhibition of hepatic AlDH (by >70%15). The cor-
responding oxygen analog, O-methylisourea (structure
not shown), which cannot be oxidatively metabolized in
the manner depicted in Scheme 3, should have no effect
on AlDH in vivo, being likely to be O-demethylated to
urea and formaldehyde.
Structural alteration of the S-alkyl group, i.e., re-
placement of the S-methyl group with S-n-butyl (8),
S-isobutyl (9), or S-allyl (10) groups, resulted in in-
creased potency in inhibiting hepatic AlDH, as reflected
by ethanol-derived blood AcH levels that were 40-92%
higher than that observed for 4 (Figure 2). The longer
duration of action of 8 and 9 compared to cyanamide
was evidenced by the persistently elevated blood AcH
levels when measured 1 h following an ethanol chal-
lenge at 15 h post drug treatment. It is noteworthy that
blood ethanol levels were unaffected by these structural
modifications of 4 and were statistically not different
from the blood ethanol of the saline control group
(Figure 3, Supporting Information), suggesting that the

1872 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Shirota et al.
F igu r e 5. Effect of added catalase on the microsomal bio-
activation of 8 to an inhibitor of yeast AlDH in vitro. C.S. )
complete system. The details of the experiment are described
in the Experimental Section. The inhibition observed with 1.0
mM cyanamide (positive control) in the C.S. was set at 1.0.
An askerisk (*) means significantly different from control, i.e.,
without inhibitor (P < 0.05).
F igu r e 2. Ethanol-derived blood AcH in rats following pre-
treatment with the S-substituted n-butyl (8), isobutyl (9), and
allyl (10) isothioureas compared to cyanamide and saline
controls at 6 and 16 h post drug treatment. Statistical
treatment as in Figure 1 legend. All values were significantly
different (P < 0.05) from the saline controls.
NADP + glucose + glucose 6-phosphate dehydrogenase.
The cyanamide produced was extracted into ethyl
acetate and derivatized by dansylation. The dansylated
cyanamide was then separated by thin layer chroma-
tography and the fluorescent product visualized and
compared to an authenic sample of dansylated cyan-
amide (Figure 7, Supporting Information). Cyanamide
formation was minimal in the absence of the NADPH-
generating system, indicating the enzymatic nature of
this reaction. Moveover, microsomes preincubated in
the absence of cofactor still appeared to be fully capable
of producing cyanamide (Figure 7, Supplementary In-
formation), suggesting that flavin monoxygenase was
not involved in the sulfoxidation of these S-alkylisothio-
ureas.
F igu r e 4. Effect of pretreatment with 1-benzylimidazole (0.03
mmol/kg, ip) on ethanol-derived blood AcH levels following the
administration of S-alkylisothioureas (0.5mmol/kg, ip) to rats.
The inhibitor was given 0.5 h prior to the S-alkylisothiourea
and blood AcH levels were measured 1 h following the ethanol
challenge. Statistical treatment as in Figure 1 legend.
Toxicity of High er Hom ologs
On the basis of these favorable in vitro and in vivo
results and the apparent absence of toxicity seen with
the above compounds, we introduced a benzyl side chain
as well as a long aliphatic side chain as the S-substi-
tutent on the isothiourea in anticipation that such
lipophilic groups would greatly prolong activity. These
compounds are represented by the hydrochloride and
hydrobromide salts of S-benzylisothiourea (11) and S-n-
hexadecylisothiourea (12) (Chart 1), respectively. Sur-
prisingly, when the S-benzyl compound (11) was ad-
ministered to rats at a dose of 0.5 mmol/kg, ip, all rats
(n ) 3) died even before ethanol could be administered
at 5 h. Lowering the dose to 0.10 mmol/kg, ip, allowed
survival of the animals, but blood AcH levels were not
statistically different from controls (data not shown).
The S-n-hexadecyl analog (12) at 0.50 mmol/kg, ip, did
give rise to elevated blood AcH when measured at 6 h
post drug and 1 h post ethanol administration (Figure
6), but postmortem examination of the liver and kidneys
at sacrifice showed extensive necrosis. Reducing the
dose to 0.25 mmol/kg, ip, did not appreciably alleviate
the liver and kidney damage observed at the higher
dose. Although the elevated blood AcH levels measured
at 16 h following this lower dose of 12 are not compa-
rable to the results with the higher dose measured at 6
h (Figure 6), their higher levels relative to saline
controls were unequivocal (P < 0.05).
first step in ethanol metabolism, viz., conversion of
ethanol to AcH, was not affected.
That the hepatic microsomal mixed function oxidase
enzyme(s) were involved in the biotransformation of 8,
9, or 10 with subsequent inhibition of AlDH was
adduced by the dramatic reduction in ethanol-derived
blood AcH levels following the administration of 8, 9,
or 10 (82, 66, and 88% reduction, respectively) to rats
pretreated with 1-benzylimidazole (0.03 mmol/kg, ip),
a known inhibitor of cytochrome P-45017 (Figure 4). In
further support, when the n-butyl analog 8 was incu-
bated with rat liver microsomes coupled to catalase and
yeast AlDH, the absolute requirement for microsomes
and the NADPH-generating system was clearly indi-
cated for the inhibition of AlDH (Table 1, Supporting
Information). In contrast to cyanamide itself which
served as positive control in this system, added catalase
only partially enhanced the inhibition of AlDH by 8 (10
mM, Figure 5). However, since isolated liver micro-
somes invariably contain catalase,7 addition of catalase
was not absolutely required for AlDH inhibition by
cyanamide.
The actual formation of cyanamide by cytochrome
P-450 action on 8 was demonstrated by incubation of 8
with liver microsomes isolated from rats pretreated with
3-amino-1,2,4-triazole (to inhibit catalase) in the pres-
ence and absence of the NADPH-generating system,
In order to introduce a biocompatible S-substituted
side chain, the N-acetyl derivative (13) of S-(2-amino-
ethyl)isothiourea (AET) was prepared, the latter, a

Novel Prodrugs of Cyanamide
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1873
release of thiourea following dealkylation of the n-
hexadecyl group by alternative and regioselective side
chain R-hydroxylation, a reaction complementary to
S-oxidation, this being likely enhanced by the lipophilic
long alkyl chain.
Recently, Garvey et al.22 have reported that S-
ethylisothiourea and a wide series of structurally related
compounds were potent inhibitors of human nitric oxide
synthetase (NOS) enzymes in vitro, including the induc-
ible (i), endothelial (e), and neuronal (n) forms of the
enzyme. Interestingly, the series included the S-meth-
yl-(4), S-n-butyl- (8), S-isobutyl- (9), S-2-methylallyl-
(related to our 10), S-benzyl-(11), and S-(2-aminoethyl)-
(related to 13) isothioureas. Although a representative
group including the S-ethyl compound, when tested in
whole cells, were less effective inhibitors than when the
isolated enzymes were used, none were tested in vivo.
On the basis of the present studies, toxicities associated
with compounds that can liberate thiourea by enzymatic
S-dealkylation of the isothioureas may be expected, and
caution must be exercised in extrapolating in vitro data
with isolated enzymes to in vivo efficacy.
In summary, we have shown that short-chain (<C4)
S-alkylisothioureas can be considered to be prodrug
forms of the alcohol deterrent agent, cyanamide, on the
basis that these isothioureas are bioactivated both in
vivo and in vitro by the hepatic mixed function oxidase
enzymes to reactive products that may inactivate AlDH
directly, or may in fact liberate cyanamide, which on
further bioactivation can inhibit this enzyme, thereby
giving rise to elevated ethanol-derived blood AcH.
Larger alkyl-substituted isothioureas are toxic to the
liver and kidneys, presumably by releasing thiourea
itself, a known toxic substance.
F igu r e 6. Ethanol-derived blood AcH in rats following treat-
ment with S-hexadecylisothiourea (12), 0.5 mmol/kg (6 h), and
0.25 mmol/kg (16 h); N ) 3 for 12 and N ) 5 for the saline
controls (P < 0.05).
compound that protects mice against lethal doses of
x-radiation18 possibly by intramolecular rearrangement
to (mercaptoethyl)guanidine.16 Administration of com-
pound 13 to rats elevated blood AcH levels following
ethanol administration significantly above the control
levels without evidence of toxicity; however, potency
(blood AcH ) 67.0 ( 17.4 µM; blood EtOH ) 50.1 ( 1.5
mM; n ) 3) was only approximately one-third that
observed for 4 itself.
Discu ssion
S-Methylisothiourea (4), the lowest member of this
series, has been reported to cause a prompt rise in
arterial pressure and to elicit bradycardia and increased
response to the pressor action of adrenaline on intra-
venous administration to dogs, cats, and rabbits.19a
Compound 4 has also been reported to have therapeutic
efficacy in the treatment of septic shock elicited experi-
mentally in rodents with bacterial lipopolysaccharide
(endotoxin).20 S-Alkylisothioureas also inhibit amide
oxidase activity.21 However, we are unaware of any
report of its disulfiram-like adverse reaction with etha-
nol. The present study constitutes the first observation
that S-alkylisothioureas can be oxidized by the hepatic
mixed function oxidase system both in vivo and in vitro
to (presumably) an S-oxide or an S-dioxide, which
spontaneously releases cyanamide, a potent inhibitor
of AlDH. However, direct inhibition of the enzyme by
the sulfoxidized intermediate by carboxamidination of
the active-site sulfhydryl group is also possible (Scheme
4). The in vitro results (Figure 5 and Table 1, Supple-
mentary Information) where added catalase only slightly
increased the inhibitory potency of microsome-activated
compound 8 suggest that this alternative mechanism
is also viable. However, no distinction between the two
mechanisms can be made at this juncture since cyan-
amide was unequivocally identified as a product of the
microsomal metabolism of 8 (Figure 7, Supplementary
Information).
Exp er im en ta l Section
Melting points were taken either on a Mettler capillary
melting point apparatus or on a Fischer-J ohns hot stage
melting point apparatus and are uncorrected. When melting
point discrepancies with literature values were found, the
compounds were submitted for elemental analysis. IR spectra
were recorded on a Nicollet FT-IR spectrophotometer. 1H
NMR or 13C NMR spectra were recorded on a Varian T-60A
or GE Unity 300 spectrophotometer, respectively. Except
where indicated, thin layer chromatography (TLC) was per-
formed using silica gel plates containing a fluorescent indica-
tor, and the spots corresponding to the compounds were
visualized either by fluorescent quenching observed under UV
light or by exposure to iodine vapors in an iodine chamber.
Rea gen ts a n d Ch em ica ls. The dihydrogen sulfate salts
of O-methylisourea and S-methylisothiourea (4) were pur-
chased from Fluka Chemical Co. (Ronkonkoma, NY) and
Aldrich Chemical Co. (Milwaukee, WI), respectively. All other
S-alkylated isothioureas were prepared according to or by
adaptation of literature procedures. Glucose 6-phosphate,
glucose 6-phosphate dehydrogenase, cyanamide, bovine liver
catalase, and yeast AlDH were purchased from Sigma Chemi-
cal Co. (St. Louis, MO).
S-n -Bu tylisoth iou r ea (8) Hyd r obr om id e; S-Isobu tyl-
isoth iou r ea (9) Hyd r och lor id e; S-Ben zylisoth iou r ea (11)
Hyd r obr om id e. These compounds were prepared according
to literature procedures: 8, mp 77-78 °C (lit.23 mp 80-82 °C);
9, mp 96-97 °C (lit. mp 96 °C,23a 103-105 °C23b); 11, in two
crops, mp 149-151 and 175-176 °C (lit.23a mp 146-148 and
176-177 °C); mp 112-113 °C.23b However, when the melting
points were retaken 8 months later, both crops melted partially
at ∼143 °C and fully at 152-153 °C. The NMR spectra of
either crop in MeOH-d4 were identical to each other, δ 7.39
(m, 5H) 4.46 (s, 2H). Anal. (either crop) (C8H11N2SCl) C, H,
N.
The toxicity of S-benzylisothiourea (11) was unex-
pected, but we suggest that this may be elicited by
liberating thiourea, a known chemical toxin, by prefer-
ential benzylic oxidation as shown in Scheme 5 (Sup-
porting Information) or, alternatively, by para hy-
droxylation followed by elimination. Thus, compound
11 may be considered to be a pro form of thiourea itself
instead of a prodrug of cyanamide. The hepatic and
renal toxicity shown by the hexadecyl compound 12, in
addition to AlDH inhibition, may also reflect the finite

1874 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Shirota et al.
S-Allylisoth iou r ea (10) Hyd r och lor id e. Obtained by
heating allyl chloride (4.1 mL) and thiourea (3.80 g, 5.0 mmol)
in absolute ethanol. Recrystallized from ethanol-ether (67%
yield): mp 87-89 °C; 1H NMR (D2O) δ 5.80 (m, 1H), 5.26 (m,
2H) 3.65 (d, 2H, J ) 6.5 Hz); 13C NMR (D2O) 170.6, 130.4,
119.8, 33.4 ppm. Anal. (C4H9N2SCl) C, H, N.
processed for cyanamide determined essentially as described
by Prunosa et al.,25 except that the dansylation was allowed
to proceed for 20 h at 40 °C. The product was separated by
TLC on Macherney-Nagel silica gel (250 µm) plates without
fluorescent background using EtOAc/MeOH/HOAc (100:5:5) as
solvent, and the plates were viewed under 365 nm UV light.
Sta tistica l An a lysis. Experimental values are expressed
as means ( SEM. Statistical significance was determined
using one-way analysis of variance (ANOVA). Where signifi-
cance was indicated, the Neuman-Keuls criteria were used
to compare the means of multiple groups. Statistical signifi-
cance is indicated by P values of e0.05.
S-n -Hexa d ecylisoth iou r ea (12) Hyd r obr om id e. Recrys-
tallized from ethanol-ether: mp 87-89 °C (lit.24 mp 104-105
°C). Anal. (C17H37NSBr) C, H, N.
S-(Aceta m id oeth yl)isoth iou r ea (13) Hyd r och lor id e.16
Recrystallized from ethanol-ether: mp 196-198 °C (lit.16 mp
193-194 °C).
P h a r m a cologica l Eva lu a tion . These studies were per-
formed in adherence with guidelines established in the Guide
for the Care and Use of Laboratory Animals published by the
U.S. Department of Health and Human Resources (NIH
Publication 85-23, revised 1985). Animals were housed in
facilities accredited by the American Association for the
Accreditation of Laboratory Animal Care (AAALAC), and the
research protocol was approved by the Subcommittee on
Animal Studies of the Minneapolis VA Medical Center. This
committee is vigorous in enforcing its charge of minimizing
the use of animals in research.
Dr u g Ad m in istr a tion P r otocol. Sprague-Dawley male
rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing
170-220 g were fasted ∼24 h prior to the time of sacrifice. All
drugs were dissolved in isotonic saline except 12 which was
suspended in 1% carboxymethylcellulose (CMC). Doses of 0.5
mmol/kg were given ip as 1.0 mL/100 g of body weight except
where noted. Ethanol was administered 1 h before sacrifice
and was given as a 20% (w/v) solution, 1.0 mL/100 g of body
weight. The animals were sacrificed, and blood levels for AcH
and ethanol measurements were determined as described
below.
Ack n ow led gm en t. This work was supported by
Grant 5R01-AA07317, National Institutes of Health,
and by the Department of Veterans Affairs. The
authors thank Dr. David J . W. Goon for helpful discus-
sions and Dr. Terry Conway for the preparation of
dansylated cyanamide.
Su p p or tin g In for m a tion Ava ila ble: Table 1, Figures 3
and 7, and Scheme 5 (4 pages). Ordering information is given
on any current masthead page.
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In Vitr o Stu d ies. Micr osom a l Meta bolism of 8 to a n
In h ibitor of AlDH. Liver microsomes were isolated from
male, Sprague-Dawley rats after an overnight fast as de-
scribed earlier.15 The bioactivation of 8 to reactive metabo-
lite(s) was estimated using a two-step assay system with the
inhibition of yeast AlDH as the end point. The primary
reaction mixture contained 100 mM potassium phosphate
buffer (pH 7.5), 5.0 mM MgCl2, 16 mM KCl, 2.5 mM glucose
6-phosphate, 0.1 µmol of NADP+, and 1.0 unit of glucose
6-phosphate dehydrogenase. After a 3 min preincubation
period, the reaction was initiated by the addition of 8.0 µg of
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removed and added directly to a secondary reaction mixture
containing 0.5 mM NAD+, 1.0 mM EDTA, 30% glycerol, and
90 mM potassium phosphate buffer (pH 8.0) in a final volume
of 1.0 mL. The reaction was initiated by the addition of
benzaldehyde (0.6 µmol), and the remaining yeast AlDH
activity in this secondary mixture was determined spectro-
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ance at 340 nm over time.
Micr osom a l Meta bolism of 8 to Cya n a m id e. For this
experiment, the rats were pretreated with 3-amino-1,2,4-
triazole (12 mmol/kg) 1 h before sacrifice, and the livers were
perfused with 0.15 M KCl to remove residual aminotriazole
before isolation of the microsomes. Each incubation flask
contained 100 mM potassium phosphate buffer, pH 7.4, 16.5
mM KCl, 4.00 mM MgCl2, 50 mM 8, microsomes from 4.0 g
(wet wt) of liver, and, when indicated, the NADPH-generating
system, viz., 2.0 mM NADP, 25 mM glucose 6-phosphate, 312
units of glucose 6-phosphate dehydrogenase, and water to a
final volume of 10.0 mL. After incubation for 60 min at 37
°C, the microsomes were sedimented by centrifugation at
100000g for 60 min, and the supernatant fractions were
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