Benserazide and carbidopa as substrates of catechol-O-methyltransferase: new mechanism of action in Parkinson's disease.

-The DOPA-decarboxylase (aromatic-L-amino acid decarboxylase) inhibitors benserazide, its active metabolite 2,3,4-trihydroxybenzylhydrazine and carbidopa are substrates of highly purified catechol-O-methyltransferase. The affinity and maximal velocity of both benserazide and trihydroxybenzyl hydrazine are extremely favourable implying that these compounds may compete as substrates for catechol-O-methyltransferase with L-DOPA. These results are discussed in the light of the ability of benserazide to cause further elevation of plasma L-DOPA than carbidopa and the use of these compounds in Parkinson's disease. Parkinsonism, a disease predominantly of the elderly characterized by chronic motor dysfunction, is caused by a degeneration of dopaminergic neurons of the nigro-striatal pathway. The degree of dopaminergic deficiency corresponds with the loss of melanin-containing cells of the pars compacta of the substantia nigra. Replacement therapy utilizes L-DOPA, the normal precursor of dopamine, in doses of up to 8 g/day, which is transported across the blood-brain barrier and subsequently replenishes the depleted dopamine stores. L-DOPA is often combined with an inhibitor of peripheral DOPA-decarboxylase (aromatic-Lamino acid decarboxylase, EC 4.1.1.28) to maintain elevated plasma levels [1]. The two clinically available inhibitors are benserazide [NL(oL-seryl)-N 2(2,3,4-trihydroxybenzyl)hydrazine] (Preparation: M A D O P A R 4:1 w/w L-DOPA: benserazide) and carbidopa (tr-methyldopahydrazine) (Preparation: S I N E M E T 10:1 w/w L-DOPA: carbidopa). Since L-DOPA is effectively catabolized by catechol-O-methyltransferase (COMT, EC 2.1.1.6) [2], O-methylation becomes the exclusive route of peripheral D O P A metabolism if DOPA-decarboxylase is inhibited. Inhibition of COMT potentiates the effect of L-DOPA in man [3]; and Marx [4] suggested that COMT inhibitors could be used to overcome the decreasing response to chronic L-DOPA therapy and to combat side-effects, which may be due to high concentrations of O-methylated DOPA metabolffes [3]. Benserazide has been shown to be a potent competitive COMT inhibitor [5-7] and O-methylated metabolites have been detected in the urine of rat and man [8] which has led to the present study of benserazide and carbidopa as substrates for COMT. The active benserazide metabolite (2,3,4-trihydroxybenzylhydrazine) and several other COMT substrates were compared to further our understanding * To whom correspondence should be sent. of the potential effects of these drugs on peripheral L-DOPA metabolism. COMT is an enzyme of wide specificity concentrated mainly in the liver [9] from which source it can be purified to homogeneity by affinity chromatography [10]. This highly purified enzyme was used in our investigations. MATERIALS AND METHODS All reagents used were of analytical quality (BDH, Poole, U.K.) and dissolved in glass distilled water. All pH measurements are relative to 20 °. Catechol-O-methyltransferase was purified from fresh pig liver by the method of Gulliver and Tipton [10]. Briefly, the COMT activity from the supernarant was precipitated by ammonium sulphate, redissolved, subjected to chromatography on Sephadex G75 (Pharmacia, Uppsala, Sweden) and affinity chromatography on 2,6-dimethoxyphenol-azophenyl-methylene-anilino-agarose. The purification scheme is given in Table 1. The magnesium chloride concentration in all buffers was raised to 2.6 mM, as originally used in the affinity chromatography step only. During purification, COMT activity was assayed by the direct extraction radiochemical method [11] using 3,4-dihydroxyphenylacetic acid as the methyl acceptor and methyl-tritiated S-adenosylmethionine as the methyl donor in 0.2 M pH 7.20 triethanolamine hydrochloride buffer, pH 7.20. One unit (U) of activity represents the formation of one micromole of product in one minute. Adenosine deaminase was partially purified from Takadiastase (Koch-Light, Colnbrook, U.K.) using the method of Sharpless and Wolfenden [12] up to and including the dialysis stage. Kinetic determinations were carried out by the coupled assay method of Coward and Wu [13] as modified by Gulliver and Tipton [11]. The assay contained in a volume of 500 #1 : 1.6 mM magnesium