Abstract
Amantadine acetylation was demonstrated to occur both in vivo and in vitro using transgenic male mice overexpressing spermidine/spermineN1-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 ± 1% (mean ± 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 = 267 ± 46 μM and Vmax = 0.009 ± 0.002 nmol/min/mg of protein. Amantadine competitively inhibited spermidine acetylation with an apparent Ki = 738 ± 157 μM. 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 = 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.
Footnotes
-
Send reprint requests to: Daniel S. Sitar, Dept. of Pharmacology and Therapeutics, University of Manitoba, A220-770 Bannatyne Ave., Winnipeg, MB, R3E 0W3, Canada. E-mail:sitar{at}ms.umanitoba.ca
-
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)
- Abbreviations used are::
- SMZ
- sulfamethazine
- DDW
- distilled deionized water
- DTT
- dithiothreitol
- MGBG
- methylglyoxal bis-(guanylhydrazone)
- PABA
- p-aminobenzoic acid
- SSAT
- spermidine/spermineN1-acetyltransferase
- NAT
- N-acetyltransferase
- Received November 11, 2000.
- Accepted January 16, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|