Spermidine/spermine N-acetyltransferase Catalyzes Amantadine Acetylation

Amantadine acetylation was demonstrated to occur both in vivo and in vitro using transgenic male mice overexpressing spermidine/spermine N-acetyltransferase (SSAT). We previously reported that neither NAT1 nor NAT2 was responsible for catalyzing acetylation of the primary amine group of amantadine. We hypothesized that the inducible polyamine-catabolizing enzyme, SSAT, was an alternate pathway for acetylating amantadine. Transgenic mice injected s.c. with 3 mg/kg amantadine excreted 4.5 6 1% (mean 6 S.E.) of the administered dose as acetylamantadine in 24-h urine samples while, by contrast, nontransgenic control mice failed to excrete any detectable acetylamantadine in their urine. In vitro studies with the cytosolic liver fraction from transgenic mice as the source of SSAT demonstrated spermidine acetylation catalytic activity with an apparent Km 5 267 6 46 mM and Vmax 5 0.009 6 0.002 nmol/min/mg of protein. Amantadine competitively inhibited spermidine acetylation with an apparent Ki 5 738 6 157 mM. Incubation of amantadine, SSAT, and an acetyl CoA-regenerating system produced modest amounts of acetylamantadine. The NAT2 substrate, sulfamethazine, inhibited spermidine acetylation with a calculated Ki 5 3.5 mM, suggesting that SSAT may be an alternate pathway for acetylation of NAT2 substrates. The NAT1 substrate, p-aminobenzoic acid, had no inhibitory effect. These results provide evidence that amantadine can be acetylated by SSAT and may be a specific drug substrate for this enzyme. Further investigation of the role of SSAT as a potential drug-metabolizing pathway is warranted. Many drugs are metabolized by acetylation in man, including procainamide, isoniazid, and sulfamethazine (SMZ) (Weber and Hein, 1985). The polymorphically expressed arylamine N-acetyltransferases, NAT1 and NAT2, are the major contributors to this process (Vatsis et al., 1995). The interindividual variation observed in acetylation has been attributed to polymorphism. The ability to rapidly acetylate drugs is inherited as an autosomal dominant trait and is found in different frequencies in different ethnic groups (Kalow, 1982). However, in the presence of alcohol, both fast and slow acetylators increased the amount of acetylated sulfadimidine measured in blood and urine, which was reflected by a decreased serum half-life of sulfadimidine (Olsen and Mørland, 1978). This phenomenon could not be attributed solely to increases in acetate levels, and an alternate pathway not dependent on the NAT2 phenotype was suggested to explain the observed increase in acetylation in the presence of alcohol (Olsen and Mørland, 1982). Amantadine hydrochloride is an achiral polycyclic aliphatic primary amine used in the prophylaxis and treatment of influenza A virus infection and to ameliorate Parkinson’s disease symptoms (Aoki and Sitar, 1988). Preliminary in vivo studies have indicated that 0.1 to 15% of an administered dose of amantadine is acetylated by some humans (Köppel and Tenczer, 1985; Bras et al., 1998). We demonstrated, in vitro, using various sources of enzymes, that amantadine acetylation is not catalyzed by NAT1 or NAT2 (Bras et al., 1998). We hypothesize that the acetyltransferase associated with the conjugation of the naturally occurring polyamine, spermidine/spermine N-acetyltransferase (SSAT), is participating in the acetylation of amantadine. Because substrates for NAT1 or NAT2 are selective and not specific, they may also be conjugated by SSAT and contribute to the overall occurrence of drug acetylation observed in vivo. The use of a transgenic mouse model overexpressing SSAT provided a convenient system to demonstrate not only in vivo but also in vitro activity. It was the purpose of the present study to demonstrate that transgenic mice that overexpress SSAT (Pietilä et al., 1997) could acetylate amantadine in vivo, to evaluate the ability of SSAT to catalyze the acetylation of amantadine in vitro, and to determine whether NAT1or NAT2selective substrates are able to inhibit spermidine acetylation in vitro. Materials and Methods Reagents. Sucrose, potassium chloride, dithiothreitol (DTT), magnesium chloride (MgCl2z6H2O), and hydroxylamine HCl were acquired from Fisher This study was supported in part by a Health Sciences Center Foundation studentship (to A.P.M.B.), the Medical Research Council of Canada (MT-14710), and by the National Cancer Institute, National Institutes of Health (CA 76428) 1 Abbreviations used are: SMZ, sulfamethazine; DDW, distilled deionized water; DTT, dithiothreitol; MGBG, methylglyoxal bis-(guanylhydrazone); PABA, paminobenzoic acid; SSAT, spermidine/spermine N-acetyltransferase; NAT, N-