Commonly used L-amino acid decarboxylase inhibitors block monoamine oxidase activity in the rat

Summary

This study examines the effects of peripheral aromatic amino acid decarboxylase (AADC) inhibitors, specifically carbidopa and benserazide, alongside the central AADC inhibitor, 3-hydroxybenzylhydrazine (NSD-1015), on monoamine oxidase (MAO) A and B activity in both peripheral and brain tissues of rats. In vitro experiments revealed that carbidopa, benserazide, and NSD-1015 significantly inhibited hepatic MAO A and B activity, with IC50 values ranging from 10 to 50 µM. In ex vivo studies, systemic administration of NSD-1015 at a dose of 100 mg/kg resulted in 88% and 96% inhibition of hepatic and striatal MAO A and B activity, respectively. In contrast, carbidopa (12.5 mg/kg) and benserazide (50 mg/kg) did not affect striatal MAO A activity or hepatic MAO B activity but inhibited striatal MAO B activity by approximately 45% and 36%, respectively. The findings suggest that carbidopa and benserazide may not only protect L-DOPA from peripheral decarboxylation but also enhance striatal dopamine levels through MAO inhibition. Due to its potent inhibition of rat striatal MAO, NSD-1015 should not be utilized for investigating the neuromodulatory role of L-DOPA.

Introduction

The peripheral L-aromatic amino acid decarboxylase (AADC) inhibitors, carbidopa and benserazide, are commonly used in conjunction with L-DOPA therapy for the treatment of Parkinson’s disease. Their primary function is to prevent peripheral dopaminergic side effects and to maximize the entry of L-DOPA into the brain. The central AADC inhibitor, NSD-1015, is frequently employed as a research tool to investigate the roles of dopamine and serotonin in the brain. It is also used to block the conversion of L-DOPA to dopamine in studies exploring the potential neurotransmitter-like actions of L-DOPA.

Carbidopa, benserazide, and NSD-1015 are irreversible inhibitors of AADC and belong to the hydrazine chemical class, known for inhibiting multiple enzymes. For instance, the antidepressant phenelzine inhibits both MAO A and B while also acting as an AADC inhibitor. This indicates that the AADC inhibitors used to explore brain monoamine functions and to treat Parkinson’s disease may have similar actions. Recent studies suggest that NSD-1015, in addition to inhibiting AADC, may also inhibit MAO. This is evident from observations that it did not block L-DOPA-induced circling in 6-hydroxydopamine (6-OHDA)-lesioned rats but instead delayed the onset and prolonged the duration of the behavioral effects. However, findings regarding NSD-1015′s effects on motor behavior in 6-OHDA-lesioned rats have been inconsistent, with some studies showing both increases and decreases in L-DOPA-induced behaviors.

Further in vivo microdialysis studies indicated that administering NSD-1015 with L-DOPA did not produce the anticipated decrease in striatal dopamine efflux expected from AADC inhibition. Instead, the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was reduced, suggesting inhibition of MAO activity. Earlier investigations noted no significant effects of benserazide on MAO activity despite its strong AADC inhibition, and carbidopa showed only minimal MAO inhibition in the rat brain. However, these studies assessed total MAO activity, leaving the specific effects of these peripheral AADC inhibitors on MAO A and B isoforms largely unexplored.

Consequently, this study aimed to characterize the specific inhibitory effects of the central and peripheral AADC inhibitors, NSD-1015, carbidopa, and benserazide, on MAO A and B activity in vitro and ex vivo in rats. The objective was to determine whether these compounds inhibit striatal and hepatic MAO A and B activities at doses commonly utilized in experimental studies targeting AADC inhibition.

Materials and Methods

Male Wistar rats weighing approximately 190 grams were euthanized through stunning followed by decapitation. The skull was opened, and the brain was extracted. The cortex and underlying corona radiata were carefully removed to expose the striata bilaterally, which were subsequently dissected to the level of the anterior commissure. The abdominal cavity was opened, and a liver sample was collected. The striata or liver samples were homogenized in 20 volumes of ice-cold sodium phosphate buffered saline (PBS; 0.1 M, pH 7.4) at a concentration of 5% w/v. The homogenates were stored at -70°C until they were ready to be assayed for MAO activity. On the day of the assay, samples were defrosted on ice and mixed by vortexing. Liver samples were further diluted 1:10 with ice-cold PBS and vortexed to achieve a final liver homogenate of 0.5% w/v.

Assay conditions for MAO activity were established using a modification of previously described methods. Aliquots of the enzyme preparation (20 µl) from either striatal or liver homogenates were combined with the appropriate substrate dissolved in PBS. The incubation was carried out for 30 minutes at 37°C, after which the reaction was terminated by adding citric acid. All steps leading up to the acid activation were performed at 0°C, except for the incubation periods.

For the extraction of deaminated metabolites, the deaminated product formed from 5-HT (5-hydroxyindoleacetic acid, 5-HIAA) was extracted into a solvent mixture of ethyl acetate and toluene for MAO A activity assessment. For MAO B activity, the deaminated product from PEA (phenylacetic acid, PAA) was extracted into toluene. The solvent was added to the reaction mixture, vortexed, and centrifuged to separate the phases. The solvent phase containing the deaminated metabolite was carefully collected for subsequent radioactivity determination using liquid scintillation spectroscopy.

The effects of NSD-1015, carbidopa, and benserazide on MAO A and B activity in rat liver were assessed in vitro. The assay was conducted following a preincubation of the AADC inhibitors with the liver homogenate at various concentrations.

In ex vivo assays, rats were treated with NSD-1015 or a vehicle control, and after a designated period, they were euthanized to assess MAO A and B activities in striatal and liver tissues. Additional experiments involved treating rats with carbidopa and benserazide or vehicle controls, followed by euthanasia and subsequent assays for MAO activity. Non-enzymatic formation of 14C-5-HIAA or 14C-PAA was evaluated by replacing the enzyme with PBS during the assay process.

Drugs

The radioactive substrates used in this study included 5-hydroxytryptamine-[side chain-2-14C]creatine sulfate (14C-5-HT) and ß-phenylethylamine-(side chain-ethyl-1-14C)hydrochloride (14C-PEA), which were sourced from the Radiochemical Centre in Amersham. All other reagents utilized were standard laboratory reagents of analytical grade whenever possible.

NSD-1015, known chemically as 3-hydroxybenzylhydrazine, was procured from Aldrich, UK. Carbidopa, which is α-methyl-dopa hydrazine, was purchased from Merck, Sharp and Dohme, USA, while benserazide was obtained from Sigma, UK. For the ex vivo studies, NSD-1015 was dissolved in 0.1 M phosphate buffered saline and adjusted to a pH of 7.4 using 5 M sodium hydroxide. Carbidopa was suspended in a solution of 0.1% phosphate buffered saline at pH 7.4. Benserazide was also dissolved in 0.1% phosphate buffered saline at pH 7.4.

Statistics

The IC50 values for the in vitro experiments were calculated using Prism 3 Software from Graphpad Software Inc. Each assay was conducted in triplicate across 4 to 6 separate homogenates. For the ex vivo experiments, the impact of drug treatment on monoamine oxidase (MAO) activity was analyzed using raw data through a one-way ANOVA, followed by either Student’s t-test or Dunnett’s test as appropriate.

Results

Inhibition of Liver MAO In Vitro

NSD-1015, carbidopa, and benserazide were found to be effective inhibitors of both liver MAO A and B in vitro. The analysis was performed using nonlinear regression curve fitting with Graphpad Prism, revealing IC50 values of 14.0 ± 1.2 µM for NSD-1015, 44.9 ± 1.6 µM for carbidopa, and 18.4 ± 1.4 µM for benserazide regarding MAO A. For MAO B, the IC50 values were determined to be 20.2 ± 1.7 µM for NSD-1015, 24.3 ± 2.9 µM for carbidopa, and 42.4 ± 6.0 µM for benserazide.

Inhibition of Hepatic and Striatal MAO Following Intraperitoneal Administration of AADC Inhibitors

The intraperitoneal administration of NSD-1015 at a dose of 100 mg/kg resulted in significant inhibition of both hepatic and striatal MAO A activity. Additionally, both striatal and hepatic MAO B activity were also inhibited following treatment with NSD-1015. In contrast, administration of carbidopa at 12.5 mg/kg or benserazide at 50 mg/kg did not influence striatal MAO A activity one hour post-administration. Similarly, hepatic MAO B activity remained unaffected by either carbidopa or benserazide. However, a noteworthy inhibition of approximately 45% for striatal MAO B was observed following carbidopa administration, while benserazide resulted in a 36% inhibition.

Discussion

The hydrazine derivatives investigated in this study exhibit strong inhibitory effects on aromatic amino acid decarboxylase (AADC) in either peripheral tissues or both peripheral and central nervous systems. The results indicate that in vitro, NSD-1015, carbidopa, and benserazide are potent inhibitors of both MAO A and MAO B. However, the in vivo effects of these compounds on the two isoforms of MAO differ significantly. This discrepancy may be attributed to the varying doses of inhibitors administered during the in vivo studies, which were chosen to reflect commonly used experimental doses aimed at inhibiting AADC activity in rats.

Carbidopa and benserazide did not demonstrate any effect on MAO A or B activity in rat liver when administered intraperitoneally at doses effective for AADC inhibition. This raises questions regarding the central effects of these peripheral AADC inhibitors, which appear puzzling and challenging to interpret. At the doses used in these studies, carbidopa and benserazide are known not to cross the blood-brain barrier or inhibit central AADC activity, and as anticipated, carbidopa and benserazide did not affect MAO A activity in the striatum. The observed inhibitory effect of these compounds on striatal MAO B is therefore unexpected. Since hepatic MAO B activity was not altered in vivo by carbidopa and benserazide, it suggests the involvement of an indirect mechanism, although the precise nature of this mechanism remains unclear and warrants further investigation.

Interestingly, previous studies indicated a small and insignificant inhibition of MAO in rat brain tissue following intraperitoneal administration of carbidopa, which contrasts with the findings of this study. The earlier research did not differentiate between the two isoforms of MAO, which likely led to an underestimation of MAO B inhibition. Additionally, one study reported an increase in MAO B activity without changes in MAO A activity in the rat basal ganglia measured hours after carbidopa administration. It was suggested that melatonin levels, which are reduced by carbidopa, could influence MAO B activity. However, in light of current findings, the increase in MAO B activity may be attributed to an up-regulation of enzyme activity as a compensatory response to prior MAO B inhibition, although the underlying mechanism for this inhibition remains unknown.

In vivo, NSD-1015 was confirmed as a powerful inhibitor of both hepatic and striatal MAO A and MAO B activity. This study represents the first examination of the effects of this widely used inhibitor on the specific isoforms of MAO. Prior investigations have indirectly suggested that NSD-1015 may inhibit MAO activity. The findings of this study indicate that the inhibitory effect of NSD-1015 on both MAO A and MAO B activity has significant implications for research where it has been utilized to assess tyrosine hydroxylase activity or to explore the neurotransmitter or neuromodulatory roles of L-DOPA. The concentrations of NSD-1015 commonly employed in previous studies to block AADC activity may also account for the elevated synaptic dopamine levels observed, even following AADC inhibition. This could partially clarify the findings reported in earlier investigations. The dosage of NSD-1015 used in this study effectively inhibited AADC, and it is routinely employed in rats to block AADC activity for measuring tyrosine hydroxylase activity and examining the neuromodulatory role of L-DOPA on motor activity. The inhibition of MAO activity by NSD-1015 raises questions about whether studies indicating a neuromodulatory role for L-DOPA in motor activity stem from increased dopamine formation or from the inhibition of its metabolism. This inhibition of MAO activity would also explain earlier findings in 6-OHDA-lesioned rats, where pre-treatment with NSD-1015 resulted in a notable increase in the duration of the circling effect produced by L-DOPA. Moreover, microdialysis studies in 6-OHDA-lesioned rats demonstrated that NSD-1015 inhibits the L-DOPA-induced increase in striatal metabolites, which can now be directly associated with the inhibition of both MAO A and MAO B.

Conclusion

The centrally acting aromatic amino acid decarboxylase (AADC) inhibitor, NSD-1015, has been identified as a potent inhibitor of both monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) in both in vitro and in vivo studies conducted on rats. Therefore, its application as a tool for investigating the neuromodulatory role of L-DOPA in the brain is not advisable due to its multiple inhibitory actions. To clarify the role of L-DOPA, it is essential to seek an alternative AADC inhibitor that selectively inhibits only decarboxylase activity.

In vitro studies have shown that the AADC inhibitors, carbidopa and benserazide, are also potent inhibitors of both MAO A and MAO B. However, in vivo, neither carbidopa nor benserazide affects hepatic MAO A or MAO B activity, nor do they have any impact on striatal MAO A activity. Nonetheless, both inhibitors do produce significant inhibition of MAO B activity. The inhibitory effects of carbidopa and benserazide may offer therapeutic advantages when these compounds are co-administered with L-DOPA in the treatment of Parkinson’s Disease, CPI-0610 as they may extend the activity of L-DOPA by inhibiting dopamine metabolism. In humans, MAO B predominates over MAO A in the brain, and the use of MAO B inhibitors such as deprenyl as adjuncts to L-DOPA therapy has been beneficial in prolonging the antiparkinsonian effects of L-DOPA. Conversely, rats exhibit higher levels of MAO A compared to MAO B, and the selective MAO A inhibitor pargyline has been shown to extend L-DOPA-induced circling behavior in 6-OHDA-lesioned rats, indicating that the inhibition of individual isoforms can significantly affect motor behavior. However, the inhibition of MAO B by carbidopa and benserazide in the striatum of rats may not accurately reflect the situation in the human brain. The differences in the distribution of MAO isoforms between rats and humans highlight the existence of species dependency, and it remains unclear whether the inhibitory effects of carbidopa and benserazide on MAO B also occur in the human striatum. Additionally, the doses of carbidopa and benserazide administered to rats are significantly higher than those prescribed to patients with Parkinson’s disease. Therefore, it is uncertain whether these findings can be extrapolated to the treatment of Parkinson’s disease.