טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKizhner Sharona
SubjectModification of L-dopa-induced Increase in Striatal
Extracellular Fluid Dopamine Levels and Metabolism
by the Centrally-acting COMT Inhibitor,
Tolcapone
DepartmentDepartment of Medicine
Supervisor Professor Emeritus John Finberg
Full Thesis textFull thesis text - English Version


Abstract


L-dopa, the most effective therapy in Parkinson's Disease, increases the synthesis and storage of dopamine in remaining axon terminals. With neurodegenerative progression, the appearance of motor complications requires adjunct therapies, such as monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibitors.

Although COMT is a key enzyme in dopamine metabolism, its involvement in the parkinsonian brain is not yet clear. Using tolcapone, a peripherally and centrally acting COMT inhibitor, we investigated the role of CNS COMT in metabolism of L-dopa-derived dopamine in the dopamine-denervated striatum. A rat model of partial dopaminergic striatal lesion was established using intra-cerebroventricular injection of 6-hydroxydopamine (6-OHDA). A preliminary immunohistochemical study showed a linear correlation between the amount of the neurotoxin administered and dopaminergic cell loss, creating a highly reproducible lesion model.

Using a free-moving microdialysis technique, we examined the modification by tolcapone (15 mg/kg) of striatal extracellular dopamine, L-dopa and their metabolites in healthy or partially-lesioned L-dopa-treated rats. Two lesion levels were used, producing 30% and 70% reduction in striatal tissue dopamine (SL and LL rats respectively). L-dopa was administered systemically (50 and 10 mg/kg) together with benserazide (15 mg/kg) for peripheral inhibition of aromatic amino acid decarboxylase (AADC).

Analysis of the dopamine metabolites in microdialysates by HPLC-EC demonstrated a shift from MAO oxidative metabolism to COMT O-methylation only in LL rats. Thus, the role of COMT in dopamine metabolism increases with dopaminergic terminal depletion.

L-dopa pharmacokinetics were improved by tolcapone more effectively in healthy rats than in SL rats. In LL rats, L-dopa pharmacokinetics were not significantly altered by tolcapone, however in these rats a lower dose of L-dopa had to be used because of the extreme excitation caused by the larger dose.

Contrary to previous studies, our microdialysis experiments revealed that tolcapone pre-treatment did not increase extracellular levels of dopamine in healthy rats treated with L-dopa. Interestingly, tolcapone completely inhibited the expected L-dopa-induced increase of extracellular dopamine in SL rats. This intriguing finding could be mediated by inhibition of striatal AADC, thus we conducted an in vitro experiment which demonstrated an inhibitory effect of tolcapone (>1 mM) on AADC. The effect was not seen in LL rats, possibly because they were treated with lower doses of L-dopa, which caused smaller increments in dopamine.

This work sheds more light on the interplay between MAO, COMT and AADC in the partially dopamine-denervated brain, and reveals an additional unsuspected action of tolcapone.