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Keywords:

  • dopamine;
  • enzyme inhibition;
  • monoamine oxidase inhibitor;
  • MPTP;
  • neuroprotection;
  • novel cholinesterase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

(R)-[(N-propargyl-(3R) aminoindan-5-yl) ethyl methyl carbamate] (TV3326) is a novel cholinesterase and brain-selective monoamine oxidase (MAO)-A/-B inhibitor. It was developed for the treatment of dementia co-morbid with extra pyramidal disorders (parkinsonism), and depression. On chronic treatment in mice it attenuated striatal dopamine depletion induced by MPTP and prevented the reduction in striatal tyrosine hydroxylase activity, like selective B and non-selective MAO inhibitors. TV3326 preferentially inhibits MAO-B in the striatum and hippocampus, and the degree of MAO-B inhibition correlates with the prevention of MPTP-induced dopamine depletion. Complete inhibition of MAO-B is not necessary for full protection from MPTP neurotoxicity. Unlike that seen after treatment with other MAO-A and -B inhibitors, recovery of striatal and hippocampal MAO-A and -B activities from inhibition by TV3326 did not show first-order kinetics. This has been attributed to the generation of a number of metabolites by TV3326 that cause differential inhibition of these enzymes. Inhibition of brain MAO-A and -B by TV3326 resulted in significant elevations of dopamine, noradrenaline and serotonin in the striatum and hippocampus. This may explain its antidepressant-like activity, resembling that of moclobemide in the forced-swim test in rats.

Abbreviations used
AD

Alzheimer disease

ChE

cholinesterase

DA

dopamine

DHPG

3,4-dihydroxyphenylglycol

DOPAC

3,4-dihydroxyphenylacetic acid

5-HIAA

5-hydroxyindole-3 acetic acid

5-HT

serotonin

HVA

homovanilic acid

MAO

monoamine oxidase

NE

noradrenaline

PD

Parkinson's disease

PEA

phenyletheylamine

TH

tyrosine hydroxylase

A considerable proportion of patients with Alzheimer's disease (AD) and Parkinson's disease (PD) have co-morbidity with each other and many of these also have symptoms of depression (Newman 1999; Honig and Mayeux 2001). The latter are not alleviated by cholinesterase (ChE) (EC 3.1.1.7, EC 3.1.1.8) inhibitors used to reduce memory impairments or by selective monoamine oxidase (MAO)-B (EC 1.4.3.4) inhibitors for their extrapyramidal disorder. In an attempt to address these problems, we have synthesized (R)-[(N-propargyl-(3R) aminoindan-5-yl) ethyl methyl carbamate] (TV3326), by introducing a carbamate moiety into selective neuroprotective MAO-B inhibitor rasagiline, to add ChE inhibitory activity (Weinstock et al. 2000a,b; Youdim et al. 2001; Maruyama et al. 2002; Youdim and Weinstock 2002) TV3326 antagonizes scopolamine-induced impairments in spatial memory, indicating that it can cause significant increases in brain cholinergic activity (Weinstock et al. 2000a). We found that chronic treatment of rats and rabbits with TV3326 produced an irreversible brain-selective inhibition of MAO-A and -B, with little or no effect on these enzymes in the liver and small intestine (Weinstock et al. 2000a). This resulted in a relatively small degree of potentiation of the pressor response to oral tyramine (Weinstock et al. 2002b). TV3326 also significantly reduced immobility in the forced-swim test, an action consistent with that of known antidepressants, including MAO-A inhibitors (Weinstock et al. 2002a). In contrast, TV3279, the S-isomer of TV3326, [(N-propargyl-(3S) aminoindan-5-yl) ethyl methyl carbamate], that inhibits ChE but not MAO (Weinstock et al. 2000a), was devoid of antidepressant-like activity.

The aim of the present study was to examine the ability of TV3326 and TV3279 to prevent striatal dopamine depletion by the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We also determined the degree of MAO inhibition by TV3326 required for protection against the effects of MPTP and its effects on striatal dopamine, serotonin and noradrenaline.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

MPTP-HCl, 1-methyl-4-phenylpyridinium-HCl (MPP), Bradford reagent, dopamine (DA), homovanilic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), noradrenaline (NA), 3,4-dihydroxyphenylglycol (DHPG), serotonin (5-HT), 5-hydroxyindole-3 acetic acid (5HIAA), 6-methyl-5,6,7,8-tetrahydropterin, catalase, l-tyrosine, d-tyrosine, l-3,4-dihydroxyphenylalanine (l-DOPA), 3,4-dihydroxybenzylamine (DHBA), were purchased from Sigma Chemical Co. (St Louis, MO, USA). The protease inhibitor mixture (Complete™, EDTA free) was purchased from Boehringer-Mannheim (Mannheim, Germany). 4-phenylpyridine was purchased from Fluka Chemie, Stenheim, Germany. [14C]phenylethylamine HCl (specific activity 44.13 mC/mmol) and [14C]5-HT binoxalate (specific activity 44.9 mC/mmol) were purchased from PerkinElmer (Boston, MA, USA).

TV3326 and (N-propargyl-(3S) aminoindan-5-yl) ethyl methyl carbamate hemitartarate (TV3279), were generously supplied by Teva (Netanya, Israel).

Animals and treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

All procedures were carried out in accordance with the National Institutes of Health Guide for care and use of laboratory animals, and were approved by the Animal Ethics Committee of the Technion, Haifa, Israel. Male C57/BL mice (20–22 g, Harlan, Rehovot, Israel) were housed under a 12-h light/dark cycle. Mice were handled daily and allowed at least 3 days to acclimatize before any treatment. Mice were injected i.p. with TV3326 (150 μmol/kg i.p.), or TV3279 (150 μmol/kg) once daily for 18 days. After 14 days of treatment the mice received MPTP (24 mg/kg i.p.) for 4 consecutive days. Control mice received saline (0.1 mL/100 g i.p.) For the correlation study, mice were treated with TV3326 (150 μmol/kg/once daily) for 1, 2, 4, 8 days, after which MPTP (24 mg/kg/day for 4 days) was also given for 4 days. The animals were killed by decapitation 4 days after the last injections, and the striatum was removed and frozen in liquid nitrogen.

Preparation of striatal homogenates

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

Each striatum was homogenized separately in 20 vol of 0.25 mol/L ice-cold sucrose containing a mixture of protease inhibitors (Complete™, Boehringer-Mannheim) followed by the addition of TritonX-100 to a final concentration of 1%. After 20-min incubation on ice, the homogenates were centrifuged at 12 000 g for 10 min at 4°C, and the supernatants were taken for further analysis. Protein concentration was determined with Bradford reagent at 595 nm, using bovine serum albumin as a standard.

Determination of MAO activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

MAO-A and -B activity was measured according to Tipton and Youdim (1976) with the following modifications: triplicates of 70 μg protein homogenate were incubated with [14C]5-HT (100 μm), for 30 min (final concentration 100 μm), as a substrate for MAO-A, or [14C]phenylethylamine for 20 min (final concentration 10 μm), as a substrate for MAO-B. For determination of MAO-A or -B, brain homogenates were pre-incubated with 75 nm l-deprenyl or 75 nm clorgyline, respectively, for 1 h in 37°C prior to the addition of the substrates. Reaction was stopped with (2 m) ice-cold citric acid, and the metabolites were extracted and determined by liquid-scintillation counting in c.p.m. units.

Measurement of striatal MPP+ levels

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

HPLC with UV detection (wavelength 295 nm) was used to measure striatal MPP+ levels according to Przedborski et al. (1992) with the following changes: mice were killed 90 min after an MPTP injection (24 mg/kg i.p.). Striata were dissected out as above, weighed and placed into 1.5-mL microcentrifuge tubes. One hundred and fifty microlitres of 5% ice-cold trichloroacetic acid containing 4 μg/mL of 4-phenylpyridine as internal standard was added to tissue samples, followed by homogenization as above on ice. Tubes were placed on ice for 10 min, then centrifuged twice at 16 000 g for 15 min. Supernatant was quickly removed, placed into a 250-μL vials. One hundred and 30 microlitres of supernatant were injected onto a cation-exchange Altima-Partisil- SCX 10-μ column (Alltech, Nicolasville, USA). The mobile phase consisted of 90% 50 mm phosphoric acid (pH 3.0) solution and 10% acetonitrile. The flow rate was 1.4 mL/min. Detector used was L-4200 UV-VIS detector (Merck-Hitachi).

Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

We first characterized the inhibitory effect of chronic treatment with TV3326 and its enantiomer TV3279 (75 and 150 μmol/kg/day for 14 days) on MAO-A and -B activities in mouse brain. While TV3326 caused a significant inhibition of MAO-A and -B in the cortex and brainstem (Table 1), TV3279 had no effect on either of the iso-enzymes. TV3326 (150 μmol/kg once daily for up to 21 days) induced a similar degree of inhibition of MAO-A and -B in the two brain regions (Fig. 1). The time course of MAO inhibition by chronic TV3326 showed that enzymes inactivation is time dependent and that MAO-B is more sensitive to inhibition than MAO-A. Inhibition of MAO-B and -A reached values of 90 and 65%, respectively, at 14 days. The values for MAO inhibition were no greater at 21 days of treatment (data not given). This suggests that TV3326 has a greater selectivity for MAO-B than -A, as has been shown for rat and rabbit brain enzymes. Treatment with a lower dose of TV3326 (75 μmol/kg) for 14 days resulted in a MAO-A and -B inhibition similar to that of the higher-dose treatment for the same duration (data not shown). In order to achieve maximal inhibitory effect on MAO-A and -B, we continued experiments with chronic treatment for 14 days.

Table 1.  Effect of treatment with TV3326 on MAO activity in mice
EnzymeTV3326 doseBrainstem MAO inhibition (% of control)Cortex MAO inhibition (% of control)
  1. C57/BL mice received either TV3326 (75/150 μmol/kg/day i.p.), or saline for 14 days. Mice were killed 4 days after last injection. MAO-A and -B activity were measured in brainstem and cortex. Results represent the mean (% inhibition of control activity) ±SEM (n = 5), each determined in triplicate.

MAO-A 75 μmol/kg38 ± 3.961 ± 3.2
150 μmol/kg45 ± 3.655 ± 2.3
MAO-B 75 μmol/kg53 ± 2.047 ± 4.5
150 μmol/kg60 ± 4.349 ± 2.1
image

Figure 1. Inhibition of striatal MAO activities after treatment with TV3326 (▵, MAO-A inhibition; □, MAO-B inhibition). Mice were injected with TV3326 (150 μmol/kg/day i.p.) as indicted, and were killed 5 min after the last injection. Results represent the mean percentage of activity ± SEM (n = 5). Two-way anova: ap < 0.05 versus MAO-A; bp < 0.05 versus control.

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Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

To determine the rate of recovery of MAO-A and -B activities after maximal inhibition by TV3326 (150 μmol/kg i.p.), we administered the drug chronically for 14 days. Mice were killed after the last dose at time indicated (Fig. 2). It was found that the recovery from their inhibition by TV3326 of MAO-A and -B activities in both regions from their inhibition by TV3326 are different and do not follow a first order kinetics, as has been shown to occur when MAO-A and -B are selectively inhibited with clorgyline and pargyline, respectively (Corte and Tipton 1980). In both brain regions, the recovery of the MAO-A and -B activities were biphasic, consisting of a rapid and a slow phase, and their time course differed. However, in both regions, the early recovery of MAO-B activity was slower than that of MA- A (Fig. 3). The half-lives of striatal MAO-A and -B after inhibition by TV3326 were calculated as 20.6 and 14.4 days, respectively, according to Corte and Tipton (1980).

image

Figure 2. The recovery of MAO activity after inhibition by chronic treatment with TV3326 (▵, MAO-A striatum; ▴, MAO-A hippocampus; □, MAO-B striatum; ▪, MAO-B hippocampus). Mice were treated for 14 days with TV3326 (150 μmol/kg i.p.), and were killed at the times indicated. Results represents the mean percentage of activity ± SEM (n = 5).

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image

Figure 3. The recovery of MAO activity. The first 12 h showed a faster rate (a1; □, MAO-A; ▵, MAO-B) and (a2; •, MAO-A; ▴, MAO-B), both in MAO-A as well as MAO-B recovery, than after 12 h, and a faster rate for MAO-A over MAO-B. Half-life for recovery plotted from data in (b1; □, MAO-A; ▵, MAO-B) and (b2; •, MAO-A; ▴, MAO-B), was 14.4 and 20.6 days for MAO-B and -A, respectively. The results represent percentage inhibition ± SEM (n = 5).

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Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

MPTP treatment resulted in more than a 60% reduction in striatal DA levels (Fig. 4), as well as in a parallel reduction in the concentrations of the metabolites DOPAC and HVA, as previously shown (Levites et al. 2001). Pre-treatment with TV3326 (150 μmol/kg) prevented catecholamine depletion by MPTP, resulting in a preservation of more than 90% of the DA content compared with that of saline treated controls. Similarly, the lower dose of TV3326 (75 μmol/kg) also reduced the fall in DA maintaining almost 70% of control levels. However TV3279, which lacks MAO inhibitory activity, did not prevent the fall in DA.

image

Figure 4. Protection of TV3326 against MPTP-induced catecholamine depletion TV3326 (75 or 150 μmol/kg/day i.p.), TV3279 (75 or 150 μmol/kg/day i.p.) or saline were given for 18 days. The last 4 days, mice received MPTP (24 mg/kg/day i.p.) or saline. Mice were killed 4 days after the last MPTP injection. Results represent the mean striatal catecholamines concentration ± SEM (n = 5). Two-way anova; ap < 0.05 versus MPTP, bp < 0.05 versus control.

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As expected, striatal DA turnover (DOPAC + HVA/DA) was increased, by almost 50% in MPTP-treated mice (Fig. 5), while pre-treatment with TV3326 restored turnover to control values. TV3279, 75 but not 150 μmol/kg, given prior to MPTP had no significant effect on the higher striatal DA turnover rate induced by MPTP neurotoxicity.

image

Figure 5. Protection of TV3326 against MPTP-induced catecholamine turnover increment in mice. C57/BL mice received TV3326 (75 or 150 μmol/kg/day i.p.), TV3279 (75 or 150 μmol/kg/day i.p.) or saline for 18 days. For 4 days, 14 days from beginning mice received MPTP (24 mg/kg/day i.p) or saline. Mice were killed 4 days after last MPTP injection. Striatal catecholamines were measured by EC-HPLC. Results represent the mean ± SEM (n = 5). Two-way anova; ap < 0.05 versus MPTP, bp < 0.05 versus control.

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Following MPTP treatment, striatal TH activity was reduced by 70% (from 15 ± 2.4 to 6 ± 1.5 Pmol/min/mg protein) (Fig. 6). TV3326 (150 μmol/kg/) alone did not influence TH activity (14 ± 1.0 Pmol/min/mg protein). However, it increased TH activity by nearly 200% (30 ± 2.1 Pmol/min/mg protein) when given with MPTP. The protective effect of TV3326 on striatal DA, correlated with its prevention of the fall in TH activity. TV3279 had no influence on TH activity (data not shown).

image

Figure 6. Protection of TV3326 against striatal TH activity depletion induced by MPTP in mice. C57/BL mice received TV3326 (150 μmol/kg/day i.p.), or saline for 18 days. For 4 days, 14 days from beginning mice received MPTP (24 mg/kg/day i.p) or saline. Mice were killed 4 days after last MPTP injection. Results represent the mean activity ± SEM (n = 8). One-way anova; ap < 0.01 versus MPTP, bp < 0.01 versus control.

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Effect of TV3326 and TV3279 on striatal neurotransmitters

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

The extensive inhibition of MAO-A and -B by chronic treatment with TV3326 in control animals, resulted in significant increases in striatal DA, NA and 5-HT levels in untreated mice (Table 2). Chronic treatment with TV3326 resulted in a 50% increase of control DA (Fig. 4). This effect was detected at both doses (75 and 150 μmol/kg), as well as a reduction of nearly 75% of DOPAC. However, lower dose of TV3326 (75 μmol/kg) did not reduce the concentrations of DOPAC. In contrast, chronic treatment with TV3279 (75 μmol/kg) did not alter the levels of striatal neurotransmitters or their metabolites. TV3279 150 μmol/kg decreased DOPAC levels by nearly 50%. TV3326, but not TV3279, also increased both NA and 5-HT levels (Table 2).

Table 2.  Effect of treatment with TV3326 on striatal amines levels in MPTP-treated mice
 ControlTV3326MPTPTV-3326/MPTP
  1. Mice received either TV3326 (150 μmol/kg/day i.p.), or saline for 18 days. For 4 days, 14 days from beginning mice received MPTP (24 mg/kg/day i.p.) or saline. Mice were killed 4 days after last MPTP injection. Results represent the mean amine content ± SEM (n = 8). anova; ap < 0.05 versus control.

DHPG (Pmol/mg tissue)2.9 ± 0.603.6 ± 0.601.0 ± 0.503.7 ± 0.90
NE (Pmol/mg tissue)0.5 ± 0.101.4 ± 0.1a0.4 ± 0.21.5 ± 0.09a
5-HIAA (Pmol/mg tissue)1 ± 0.51 ± 0.92 ± 11 ± 0.7
5-HT (Pmol/mg tissue)7 ± 0.310 ± 0.5a6 ± 0.59 ± 0.2a

Effect of TV3326 on MPP+ levels following MPTP treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

To determine whether or not TV3326 interferes with brain uptake of MPTP, we measured the levels of the MAO-B-derived MPTP metabolite, MPP+, in the striata of mice treated chronically with TV3326 (150 μmol/kg for 14 days) plus MPTP as described above in the neurotoxicity studies. MPP+ concentrations in mice treated with TV3326 and MPTP, was reduced by 90% as compared with that in saline and MPTP-treated mice (Table 3).

Table 3.  Effect of TV3326 on striatal MPP+ content
TreatmentSaline + MPTP (24 mg/kg)TV3326 (150 μmol/kg/day)/ MPTP (24 mg/kg)
  1. Mice were injected chronically with TV3326 (150 μmol/kg i.p.) or saline for 14 days. A single MPTP injection (24 mg/kg i.p.) was given 5 min after last injection. Mice were killed 90 min after MPTP administration. Striatal MPP+ was measured by UV-HPLC. Results represent the mean MPP+ content ± SEM (n = 5). Two-tail student t-test; ap < 0.001 versus MPTP group.

MPP+ concentration37 ± 8.54 ± 2.6a
(Pmol/mg tissue)

The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

Previous studies on the prevention by MAO-B inhibitors of MPTP neurotoxicity employed doses that completely inhibited the enzyme selectively. While MPTP treatment alone reduced striatal DA content to 30 ± 1.6% of control prior to MPTP, 1, 2, 4 and 8 days of treatment with TV3326 resulted in 37 ± 2.9, 44 ± 2.2, 82 ± 4.6, 113 ± 4.2% of DA content as compared with non-MPTP-treated controls, respectively (Fig. 7). Even 2 days of pre-treatment with TV3326, which resulted in an inhibition of MAO-A and -B by 10 and 50%, respectively, produced a significant protection against DA depletion by MPTP, while 4 days of pre-treatment with TV3326 almost completely protected against MPTP-induced DA depletion (85 ± 5.0%) (Fig. 7), even though striatal MAO-B was inhibited by only 70% (Fig. 1). Eight days treatment with TV3326 resulted in an almost complete inhibition of MAO-A and -B and an increase of striatal DA to 115 ± 5.0% of control (Fig. 4). A significant correlation was found between the degree of MAO-B inhibition (Fig. 1) and the prevention of MPTP-induced striatal DA depletion (Fig. 6). (Spearman correlation, two-tail, p < 0.005) suggesting that total inhibition of MAO is not required to prevent MPTP neurotoxicity.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References

A significant percentage of subjects with Alzheimer's disease have co-morbidity with dementia and depression. A similar co-morbidity is reported in patients with Parkinson's disease. Such subjects may benefit from therapy with a cholinesterase inhibitor and an antidepressant (Barber et al. 2001; Robert 2002). TV3326 is a bifunctional cholinesterase-monoamine oxidase inhibitor that possesses pharmacological activities that are relevant for the treatment of this disease (Weinstock et al. 2002a). As TV3326 is a propargylamine derivative, it was assumed to inhibit MAO irreversibly, like rasagiline, from which it is derived.

In this study we have shown that, on chronic treatment, TV3326 time-dependently inhibited MAO-A and -B in the cerebral cortex, brainstem and striatum of mice. However, TV3326 showed a significantly greater inhibition of MAO-B. Unlike the selective MAO-A (clorgyline) or -B (selegiline, rasagiline) inhibitors, that do not affect brain levels of DA, its activities were similar to that of non-selective inhibitors like tranylcypromine (Green and Youdim 1975); Green et al. 1977). TV3326 increased levels of striatal dopamine and of 5-HT and NA.

This study also shows that MAO inhibition by TV3326, like other propargylamines, such as clorgyline, l-deprenyl and rasagiline, is irreversible and slow to recover. However, the recovery of the activity of each enzyme in the striatum and hippocampus differ and it is biphasic. This is in contrast to what has consistently been reported for the first order recovery of brain MAO-A and -B activities from inhibition by clorgyline, selegeline and rasagiline (Corte and Tipton 1980; Youdim and Tipton 2002). The latter studies also showed that the half life for recovery of enzyme activity is significantly shorter for MAO-A than for MAO-B (Goridis and Neff 1971; Carlo et al. 1996). In the present study, the terminal half life of enzyme activity was faster for MAO-A than for MAO-B in both the hippocampus and striatum. The biphasic nature of the recovery of activity of both MAO enzymes from the inhibition by TV3326 may be explained by the fact that TV3326, gives rise to a number MAO inhibitory metabolites (Sterling et al. 2002; Youdim and Weinstock 2002). These are generated as a consequence of the dealkylation and hydrolysis of the carbamate moiety on the 6 position of the aminoindan structure. Each of these metabolites has a different profile of MAO-A and -B inhibitory activity. This also explains the slow rate at which chronic in vivo treatment with TV3326 inhibits MAO-A and -B. It is worth noting that rasagiline from which TV3326 is derived is a highly irreversible inhibitor of MAO-B, about 5–10 times more potent than selegeline (Youdim et al. 2001). The introduction of carbamate ChE inhibitory moiety into rasagiline results in a considerable reduction in MAO-B inhibitory activity because of the bulkier molecule which is more difficult to attach to the MAO isoenzymes, as suggested by structure analysis (Binda et al. 2002). Thus, the biphasic nature of the recovery of the brain from MAO-A and -B inhibition may be related to the generation of metabolites and the faster rate at which they interact with the active sites of these enzymes and inhibit them. Existence of multiple active metabolites probably also explains why the recovery of MAO-B following chronic treatment with TV3326 was longer than after other MAO-B inhibitors, such as rasagiline and l-deprenyl (Youdim and Tipton 2002).

Chronic treatment for 14 days with TV3326 resulted in maximum inhibition of both isoenzymes, which, in turn, completely attenuated MPTP-induced striatal dopamine depletion and its metabolites and the loss of TH. However, its optical isomer TV3279 that does not inhibit MAO was ineffective. TV3326 also increased striatal concentration of 5-HT and NA in control and MPTP-treated mice, further confirming that TV3326 inhibits both MAO-A and -B in the brain. In preventing MPTP-induced neurotoxicity by MAO-B or non-selective inhibitors, the doses that have been employed were those that completely inactivated the enzyme (Heikkila et al. 1985; Kindt and Heikkila 1986). The present study shows that even a limited MAO-B inhibition, as seen after 2 and 4 days of chronic TV3326 treatment (Fig. 6), is sufficient to partially prevent MPTP neurotoxicity as seen by the degree of DA depletion. Almost total protection is observed when the enzyme is inhibited by about 70%. Thus we may assume that partial inhibition of MAO-A by this inhibitor also contributes to its neuroprotective activity against MPTP, even though this tertiary amine is a substrate for MAO-B (Tipton et al. 1986). It is now well documented that MAO-A or -B selectivity metabolize amines such as 5-HT and phenylethylamine (PEA), respectively, but this separation seems to be less clear-cut. A number of studies have clearly shown that when either MAO-A or -B is completely inhibited and the concentration of amine that is not a substrate for the particular MAO rises sufficiently to satisfy the Km of the enzyme, the substrate will be metabolized by the other isoenzyme (Green and Youdim 1975; Schoepp and Azzaro 1981). This has been shown to be true for 5-HT, NA and PEA and can not be ruled out for MPTP, that has a similar structure to PEA.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and treatment
  6. Catecholamine analysis
  7. Preparation of striatal homogenates
  8. Tyrosine hydroxylase activity
  9. Determination of MAO activity
  10. Measurement of striatal MPP+ levels
  11. Statistics
  12. Results
  13. Effect of chronic treatment with TV3326 on mouse brain MAO-A and -B activities
  14. Turnover of striatal MAO-A and -B activities in response to chronic treatment with TV3326
  15. Effect of TV3326 and TV3279 on MPTP induced neurotoxicity in mice
  16. Effect of TV3326 and TV3279 on striatal neurotransmitters
  17. Effect of TV3326 on MPP+ levels following MPTP treatment
  18. The degree of striatal MAO inhibition versus MPTP-induced dopaminergic neurotoxicity
  19. Discussion
  20. Acknowledgements
  21. References
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