Denitrosation as a Determinant of Nitrosocimetidine in Vivo Activity¹

The widely used drug cimetidine (Tagamet) can be nitrosated in the presence of nitrite and under mild acid conditions to form a compound, nitrosocimetidine (NC), which has a chemical structure very similar to those of the mutagens and laboratory carcinogens N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU). NC has given positive indications in several short-term tests for possible carcinogenic activity and is capable of methylating DNA in vitro and in cultured cells in a manner identical to that of MNNG and MNU. Nevertheless, NC has been found to be a weak carcinogen or a noncarcinogen and to be very poor at modifying DNA in vivo when administered p.o. We have found that NC, like MNNG, decomposes very rapidly when incubated with thiol compound in neutral pH buffer. Much of this decomposition is denitrosation. In the presence of excess reduced glutathione, about 35% of the degradation results in denitrosation to produce cimetidine, and in the presence of excess cysteine about 65% results in denitrosation to produce cimetidine. The compound also rapidly decomposes in whole blood isolated from rats; about 70% of this decomposition produces cimetidine. In solution with purified rat hemoglobin, approximately 90% of the NC degradation proceeds via a denitrosation pathway; hemoglobin cysteine residues have been implicated in the denitrosation reaction. In parallel experiments with MNNG, it has been found that, although a fraction of the decomposition of this agent in the presence of thiol compound, in isolated whole blood, and in solution with purified hemoglobin generates the denitrosated derivative, denitrosation is in the range of one-third to one-half of that found for NC. Denitrosation and degradation to form a methylating species appear to be the major NC and MNNG decomposition pathways in vitro. There is no indication that MNU degradation is sensitive to thiols, nor is the compound susceptible to denitrosation at neutral pH. Molarequivalent doses of methyl group-radiolabeled NC, MNNG, and MNU were administered via the tail vein to groups of F344 rats, and the DNA methylation yields in lung, liver, kidney, and brain tissue were assessed. Of the organs considered, DNA methylation was greatest in the lungs of MNNG-treated animals, followed by kidney (25% of the lung value). Methylation of lung tissue DNA in MNU-treated animals was about 50% of that observed in the MNNG experiments; DNA methylation in the other organs was about equivalent to that found in the lung. The DNA alkylation yield found in the lung tissue of NC-treated rats was less than 4% of that found in MNNG-treated animals; DNA alkylation in the other organs was similarly very low. During the 48 hr after i.v. administration of radiolabeled NC to F344 rats, approximately 85% of the radioactivity was recovered in the urine; of this recovered activity, about 70% has been identified as cimetidine. These values are essentially identical to those obtained when radiolabeled cimetidine was dosed i.v. Taken together, these results suggest that NC is rapidly denitrosated when administered i.v., perhaps in the blood mediated by hemoglobin. The partition of NC between aqueous phase and octanol has been determined as a function of aqueous phase pH. At pH values less than 5, NC remains almost entirely in the aqueous phase; the compound has an apparent pK of 6. NC administered p.o. may thus be poorly absorbed from the gastrointestinal tract in those regions of the lumen which are acidic.