Inhibition of kynurenine hydrolase by benserazide, carbidopa and other aromatic hydrazine derivatives: evidence for sub-clinical iatrogenic niacin deficiency [proceedings].

It has been shown previously that kynurenine hydrolase (L-kynurenine hydrolase, EC 3.7.1.3) is inhibited by the hydrazide Benserazide {R,, 4-4602, DL-serine 2-[(2,3,4trihydroxyphenyl)methyl] hydrazide), a compound that is used clinically in the treatment of Parkinson’s disease (Bender et al., 1977). Benserazide is a potent inhibitor of aromatic amino acid decarboxylase (L-aromatic amino acid carboxy-lyase, EC 4.1.1.28) and is used in conjunction with dopa (3,4-dihydroxyphenylalanine) therapy. Since Benserazide does not cross the blood-brain barrier, it will inhibit only extra-cerebral aromatic amino acid decarboxylase, and hence allow a greater proportion of the administered dose of dopa to enter the brain for cerebral dopamine (3,4-dihydroxyphenethylamine) synthesis. This allows use of smaller doses of dopa, and hence decreases the incidence of dopa-induced side effects in Parkinsonian patients. Since Benserazide is a hydrazide, it is not surprising that it inhibits kynurenine hydrolase, which is a pyridoxal phosphate-dependent enzyme. It is well established that many hydrazine derivatives inhibit pyridoxal phosphate-dependent enzymes, by formation of biologically inactive hydrazones of the cofactor (Vilter, 1964). In the present study the pattern of inhibition of kynurenine hydrolase by Benserazide has been studied. At the same time another hydrazine inhibitor of aromatic amino acid decarboxylase that is used in treatment of Parkinsonism together with dopa, Carbidopa (MK-486, S-a-hydrazino-3,4-dihydroxy-a-methylbenzenepropionic a id monohydrate) has also been studied. For comparison, four other aromatic hydrazine derivatives have also been used: isonicotinic acid hydrazide, an anti-tuberculosis agent known t o inhibit kynurenine hydrolase in vivo under clinical conditions (Roe, 1971), phenylhydrazine, Girard’s reagent T (2-hydrazino-NNN-trimethyl-2-oxoethanaminium chloride) and Girard’s reagent P (2-hydrazino-2-oxoethane-N-pyridinium chloride), Liver from freshly killed rats was homogenized in 2ml of 0 . 1 5 ~ N a C l / g of tissue; 0.5ml of this homogenate and 0.5ml of 0 .2~-sodium phosphate buffer, pH7.0, were incubated together for 5min at 30°C. For inhibition studies the inhibitor was dissolved in the buffer, so that it was present with the enzyme throughout this preincubation period. The reaction was initiated by addition of 0.2ml of a solution of DL-kynurenine; five concentrations of substrate were used, over the range 0.5-4.0m~. After incubation for a further 30min the reaction was stopped by addition of 1 ml of 1 M-trichloroacetic acid. Denatured protein was removed by centrifugation and the supernatant was made approximately neutral by the addition of 0.5ml of 1 M-NaOH. The volume of the supernatant was then adjusted to 5 ml by the addition of 1 M-sodium phosphate buffer, pH 5.5, and the fluorescence due to anthranilic acid measured (activation 310nm,