Ethanol-derived Blood Acetaldehyde Research Articles

Prodrugs of nitroxyl and nitrosobenzene as cascade latentiated inhibitors of aldehyde dehydrogenase.

The prototypic aromatic C-nitroso compound, nitrosobenzene (NB), was shown previously to mimic the effect of nitroxyl (HN=O), the putative active metabolite of cyanamide, in inhibiting aldehyde dehydrogenase (AlDH). To minimize the toxicity of NB in vivo, pro-prodrug forms of NB, which were designed to be bioactivated either by an esterase intrinsic to AlDH or the mixed function oxidase enzymes of liver microsomes, were prepared. Accordingly, the prodrug N-benzenesulfonyl-N-phenylhydroxylamine (3) was further latentiated by conversion to its O-acetyl (1a), O-methoxycarbonyl (1b), O-ethoxycarbonyl (1c), and O-methyl (2) derivatives. Similarly, pro-prodrug forms of nitroxyl were prepared by derivatization of the hydroxylamino moiety of methanesulfohydroxamic acid with N, O-bis-acetyl (7a), N,O-bis-methoxycarbonyl (7b), N, O-bis-ethoxycarbonyl (7c), and N-methoxycarbonyl-O-methyl (7d) groups. It was expected that the bioactivation of these prodrugs would initiate a cascade of nonenzymatic reactions leading to the ultimate liberation of NB or nitroxyl, thereby inhibiting AlDH. Indeed, the ester pro-prodrugs of both series were highly active in inhibiting yeast AlDH in vitro with IC50 values ranging from 21 to 64 microM. However, only 7d significantly raised ethanol-derived blood acetaldehyde levels when administered to rats, a reflection of the inhibition of hepatic mitochondrial AlDH-2.

Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo.

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 (A1DH)-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 A1DH, the requirement for microsomal activation for the inhibition of A1DH activity was clearly indicated. We suggest that S-oxidation is involved and that the S-oxides produced in vivo inhibited A1DH directly, or spontaneously released cyanamide, an inhibitor of A1DH. 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.

Latent alkyl isocyanates as inhibitors of aldehyde dehydrogenase in vivo.

On the basis of our previous observation that N1-alkyl substituted chlorpropamide derivatives when administered to rats nonenzymatically eliminated n-propyl isocyanate, a known inhibitor of aldehyde dehydrogenase (AlDH), we have synthesized other latentiated n-propyl isocyanates as in vivo inhibitors of AlDH. N1-Allylchlorpropamide 3 was, as expected, a potent inhibitor of hepatic AlDH in rats, as indicated by the 4-fold increase in the levels of ethanol-derived blood acetaldehyde relative to that elicited by chlorpropamide itself. Closely following in activity in decreasing order were N3-(n-propylcarbamoyl)uracil (7),N-(n-propylcarbamoyl)saccharin (6), and the S-(n-propylcarbamoyl) derivative (9) of benzyl mercaptan. However, two hydantoin derivatives, 5 and 8, were totally inactive in inhibiting AlDH in vivo. A prodrug of N1-ethylchlorpropamide, viz., its N3-trifluoroacetyl derivative (4b), was a good in vivo inhibitor of AlDH, mimicking the activity of the parent N1-ethylchlorpropamide. These results suggest that latent alkyl isocyanates are inhibitors of AlDH, giving further support to the hypothesis that the inhibition of AlDH in vivo by the hypoglycemic agent chlorpropamide may be due to the release of n-propyl isocyanate following metabolic bioactivation.

A Nonhypoglycemic Chlorpropamide Analog that Inhibits Aldehyde Dehydrogenase

Chlorpropamide (CP), a sulfonylurea-type oral hypoglycemic agent, is known to provoke a flushing reaction reminiscent of the disulfiram-ethanol reaction in certain individuals. This is manifested in rodents by an increase in blood acetaldehyde levels after ethanol administration. When the sulfonamide N1-nitrogen of CP was substituted with an ethyl group, the product, N1-ethylchlorpropamide, was found to be three times as active as CP in raising ethanol-derived blood acetaldehyde. However, whereas CP lowered fasting blood glucose in rats measured over 6 h, N1-ethylchlorpropamide was devoid of hypoglycemic activity, suggesting that the latter might be potentially useful as an alcohol deterrent agent.

Acyl, N-protected alpha-aminoacyl, and peptidyl derivatives as prodrug forms of the alcohol deterrent agent cyanamide.

Cyanamide (H2NC identical to N), a potent aldehyde dehydrogenase (AlDH) inhibitor that is used therapeutically as an alcohol deterrent agent, is known to be rapidly metabolized and excreted in the urine as acetylcyanamide (1). On the basis of our observation that 1 is deacetylated to cyanamide in vivo, albeit very slightly, thereby serving as a precursor of prodrug form of the latter, several acyl derivatives of cyanamide were synthesized specifically as prodrugs, including benzoylcyanamide (2), pivaloylcyanamide (3), and 1-adamantoylcyanamide (4), as well as long- and medium-chain fatty acyl derivatives such as palmitoyl- (6), stearoyl- (7), and n-butyrylcyanamide (5). N-Protected alpha-aminoacyl and peptidyl derivatives of cyanamide were also synthesized, and these include N-carbobenzoxyglycyl- (10), hippuryl- (13), N-benzoyl-L-leucyl- (14), N-carbobenzoxyglycyl-L-leucyl- (18), N-carbobenzoxy-L-pyroglutamyl- (22), L-pyroglutamyl-L-leucyl- (19), and L-pyroglutamyl-L-phenylalanylcyanamide (20). All of these prodrugs of cyanamide raised ethanol-derived blood acetaldehyde levels in rats significantly over controls 3 h after ip drug administration, and some of these were still capable of elevating blood acetaldehyde 16 h post drug administration. A selected group of cyanamide prodrugs were also evaluated by the oral route of administration and showed nearly equivalent activity as the ip route in elevating ethanol-derived blood acetaldehyde. These results suggest potential utility of these prodrugs as deterrent agents for the treatment of alcoholism.

ChemInform Abstract: LATENT INHIBITORS OF ALDEHYDE DEHYDROGENASE AS ALCOHOL DETERRENT AGENTS

AbstractDargestellt und pharmakologisch untersucht werden die Analoga (II) von Pargylin (I).

Disulfiram and erythrocyte aldehyde dehydrogenase inhibition.

During disulfiram therapy erythrocyte aldehyde dehydrogenase (ALDH) was fully inhibited. The time for total loss of erythrocyte ALDH activity ranged from 36 to 120 hr. In contrast to the 85% recovery of in vitro disulfiram-inhibited ALDH activity, this in vivo disulfiram-ALDH inhibition could not be reversed by mercaptoethanol. It is proposed that the in vivo and in vitro mechanisms of ALDH inhibition by disulfiram differ. Erythrocyte ALDH activity can be readily monitored to determine patient compliance and is an accessible model for investigations of in vivo mechanisms of drug inhibition. Because the disulfiram-inhibited erythrocyte ALDH is not regenerated until new erythrocytes are made (120 days), a significant portion of the extrahepatic acetaldehyde metabolic capacity remains inhibited for long periods after disulfiram is discontinued. Thus, the recidivistic patient who discontinues disulfiram and waits several days (to regenerate liver ALDH activity) before drinking will be exposed to even higher ethanol-derived blood acetaldehyde levels than usual, which may induce further alcohol-associated organ damage and alcohol dependence.

Metabolic activation of cyanamide to an inhibitor of aldehyde dehydrogenase in vitro

The inhibition of aldehyde dehydrogenase (AlDH) by cyanamide is dependent on the conversion of the latter to an active metabolite. This accounts for the in vivo activity of cyanamide in raising ethanol-derived blood acetaldehyde levels to the mM range in the rat (ED 50 for cyanamide=0.11 mmole/kg) and its lasc of inhibitory activity in vitro with purified AlDH enzymes. Liver mitochondria were shown to catalyze this activation. The Low K m mitochondrial AIDH isozyme was strongly inhibited by cyanamide when measured in intact rat liver mitochondria (I 50=2.0 μM). Cyanamide also inhibited yeast AlDH when incubated in the presence, but not in the absence, of rat liver mitochondria (I 50=7.8 μM). Using yeast AIDH activity as a measure of cyanamide activation, the subcellular distribution of the cyanamide-activating system was assessed. Microsomes plus an NADPH generating system were equally active as mitochondria in activating cyanamide. In the absence of NADPH, microsomal activity was about half that of mitochondria. Little or no activity was found in the cytosolic fraction. A series of cyanamide analogs and derivatives were screened for their ability to inhibit the low K m AlDH isozyme measured in intact mitochondria. Only monoalkylcyanamides exemplified by n-butylcyanamide showed significatn inhibition. Other cyanamide analogs and derivatives including N-acetylcyanamide, the major urinary metabolite of cyanamide, were inactive in this system.

Studies on the cyanamide-ethanol interaction: Dimethylcyanamide as an inhibitor of aldehyde dehydrogenase in vivo

Administration of dimethylcyanamide (DMC) to rats caused a marked elevation in ethanol-derived blood acetaldehyde (AcH) and depressed the specific activity of the low K m mitochondrial aldehyde dehydrogenase (AlDH) by 90% at 12–24 hr, coincident with depletion of hepatic glutathione levels. Comparison of the relative efficacy of DMC and cyanamide in elevating blood AcH measured at 2hr and 1 hr post-drug treatment, respectively, indicated that DMC was at least one-fifth as active as cyanamide. However, since the comparison was not made at optimal times for DMC (12–24 hr), it is likely that its activity in vivo approaches that of cyanamide itself. DMC was essentially inactive in vitro as an inhibitor of the low K m AlDH isozyme in intact rat liver mitochondria. Although methylcyanamide, the product of N-demethylation of DMC, was too unstable to be prepared for this evaluation, the higher monoalkyl cyanamide, n-propylcyanamide, was synthesized chemically and was shown to be a good inhibitor of the mitochondrial enzyme in vitro. These results suggest that DMC must be N-demethylated before being converted to a reactive species that inhibits AlDH activity.