Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2002-03-01
2004-11-02
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007210
Reexamination Certificate
active
06811967
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for assaying activity of the enzyme spermidine/spermine N
1
-acetyltransferase (SSAT) using SSAT substrates by detecting acetylated forms of the SSAT substrates. SSAT substrates may include amantadine wherein metabolism of amantadine occurs in part by the action of the inducible enzyme SSAT to produce the acetylated metabolite N-acetylamantadine.
BACKGROUND OF THE INVENTION
Amantadine was first synthesized at the Du Pont laboratories in the 1960s (Davies et at., 1964) and has a unique polycyclic aliphatic structure, with an achiral primary amine that makes it a weak base (pKa=10.1). At physiological pH, it exists mainly in the cationic form. Amantadine has been used as an adjunct in the symptomatic relief of Parkinson's disease and for the prophylaxis and treatment of influenza A virus infection the two approved clinical indications for amantadine in Canada.
The first report that addressed amantadine metabolism was by Bleidner et al. They stated that there was no evidence of acetylated or methylated forms of amantadine in human urine or other extraneous peaks that could be attributed to metabolites of the drug (Bleidner et al., 1965). This view was accepted and formed the basis of opinion concerning amantadine metabolism and the assumption that incomplete oral absorption could account for some of the unrecovered dose. More recently evidence indicates that amantadine is acetylated by a specific acetyltransferase.
Amantadine Metabolism
Koppel and Tenczer (1985) provided the first evidence for metabolic disposition of amantadine in humans. These researchers reported that 5 to 15% of the administered dose (200 mg) was recovered in the urine as acetylamantadine in three healthy young male volunteers. Furthermore, they reported the existence of other minor metabolites, which were not quantified. They suggested that other minor metabolic pathways may be involved in N-methylation, formation of Schiff bases and N-formiates. There was no evidence for oxidation of the adamantane ring (Koppel & Tenczer, 1985). This observation was extended by Sitar et al., when they reported that acetylation of amantadine was not correlated with the N-acetyltransferase 2(NAT2) acetylator phenotype This observation suggested that NAT2 was not the acetyltransferase enzyme that catalyzed this conjugation reaction (Sitar et al., 1991). Further still, Bras et al. (1998) reported amantadine acetylation may be effected by acetyltransferases other than N-acetyltransferase 1 (NAT1) or NAT 2.
Spermidine/Spermine Acetyltransferase (SSAT)
Spermidine/spermine N
1
-acetyltransferase (SSAT), is ubiquitously distributed in mammalian issues and plays a role in catabolism and elimination of polyamines from cells (Cohen; 1998; (Morgan, 1998). However, in normal or uninduced mammalian tissues SSAT is present at very low levels (Casero & Pegg, 1993; Cohen, 1998). SSAT is an inducible enzyme that catalyzes the transfer of an acetyl group from acetyl-coenzyme A to the aminopropyl moiety of polyamines. This action by SSAT facilitates polyamine degradation, excretion, cycling and/or intracellular cycling (Casero & Pegg, 1993). In this manner SSAT participates in the maintenance of polyamine homeostasis in mammalian cells.
Regulation of SSAT
Induction of SSAT can be caused by different drugs, growth factors, polyamines, polyamine analogues, toxic substances, hormones, and physiological stimuli (Casero & Pegg, 1993). All could cause induction, but the induction occurs at different times for each individual compound. The regulation of SSAT expression occurs at the levels of transcription, mRNA stability, mRNA translation and protein stability (Fogel-Petrovic; et al., 1997).
The SSAT gene contains a polyamine responsive element located in a region that occurs at −1522 to −1492 with respect to the SSAT transcriptional start site (Wang et al., 1998). Within this 31 base pair sequence, the polyamine response element was identified as a 9 base pair sequence. The polyamine response element mediates transcriptional induction of SSAT by the polyamine analogue N
1
,N
12
-bis(ethyl)spermine or natural polyamines (Wang et al., 1998).
SSAT, the rate-limiting enzyme in the catabolic pathway plays a regulatory role in maintaining spermidine and spermine homeostasis. It has been estimated that less than 1000 molecules of SSAT are present in a rat hepatocyte, compared to 60,000 molecules in an induced cell (Matsui & Pegg, 1981; Pegg et al., 1982). The induction of mammalian tissues by the various inducers can result in increased levels of SSAT that in turn may serve as a potential acetylator of primary amine-containing compounds.
Induction or over-expression of SSAT is usually required for there to be sufficient SSAT enzyme present in cells or 100,000×g supernatant before in vitro experiments can be successfully undertaken (Casero & Pegg, 1993; Fogel-Petrovic et al., 1997; Matsui & Pegg, 1980; Pietila et al., 1997).
Thus, while the literature teaches that SSAT is an acetylating enzyme specifically for substrates including spermine and spermidine or its analogues, SSAT activity, SSAT enzyme kinetics and assay methodology for non-spermine/spermidine substrates of SSAT has not been understood. Current methods exist to quantitate SSAT activity however these techniques are dependent on highly skilled personnel and involve complicated experimental methods. More specifically, there has been a need for assay methodology which quantifies the activity of SSAT through detection of acetylated forms of non-spermine/spermidine substrates of SSAT, including amantadine which may be used to detect various pathological conditions.
SUMMARY OF THE INVENTION
In accordance with the invention, there is provided a method for determining the activity of spermine/spermidine N
1
-acetyltransferase (SSAT) in a mammal comprising the step of assaying a sample derived from the mammal for the level of an acetylated form of a non-spermine/spermidine, (or analogues thereof), SSAT substrate in the sample.
In one embodiment, the SSAT substrate is amantadine and the acetylated form of an SSAT substrate is acetylamantadine. The method may include incubating the SSAT substrate with the mammal or mammalian tissue or cells at a specific SSAT substrate dosage level, preferably in the range of 1-4 mg/kg and more preferably at 3 mg/kg. Samples to be assayed may be urine and/or blood samples from the mammal which may be collected at 2-24 following substrate incubation and preferably at 8 hours following incubation.
The relative level of the non-spermine/spermidine SSAT substrate in the sample is preferably correlated to a standard curve representing known activity levels and may be assayed by a variety of techniques including but not limited to gas chromatography, radio-labelling, mass spectrometry, high performance liquid chromatography (HPLC) and thin-layer chromotography.
In another embodiment of the invention, the assay method is used to correlate SSAT activity to pathological conditions in the mammal including gastric carcinoma, ovarian cancer, acute myelocytic leukemia, lymphoma, breast cancer, renal cancer, colorectal cancer, prostate cancer or alcohol consumption.
REFERENCES:
Diane H. Russell, Nature New Biology, vol. 233, (Sep. 29, 1971), pp. 144-145.
Martin J. Pine et al., The Journal of Urology, vol. 141, (1989), pp. 651-655.
Marko Pietila et al., The Journal of Biological Chemistry, vol. 272, No. 30, (Jul. 25, 1997), pp. 18746-18751.
Anthony E. Pegg et al., Biochimica et Biophysica Acta, vol. 1171, (1992), pp. 106-108.
Anthony E. Pegg et al., Federation Proceedings, vol. 41, (1982), pp. 3065-3072.
Lisa Parry et al., Biochem. Journal, vol. 305, (1995), pp. 451-458.
Yanlin Wang et al., The Journal of Biological Chemistry, vol. 273, No. 51, (Dec. 18, 1998), pp. 34623-34630.
Yanlin Wang et al., The Journal of Biological Chemistry, vol. 274, No. 31, (Jul. 30, 1999), pp. 22095-22101.
Yanlin Wang et al., Biochemical Journal, vol. 355, No. 1, (2001), pp. 45-59.
Robert H. Tannen et al., Drug Metaboli
Bras Alvaro P. M.
Sitar Daneil S.
Birch & Stewart Kolasch & Birch, LLP
Srivastava Kailash C.
The University of Manitoba
Weber Jon P.
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