148

Neuroprotective Effects of Novel Cholinesterase Inhibitors Derived from Rasagiline as Potential Anti-Alzheimer Drugs

MARTA WEINSTOCK,

a

NATANJA KIRSCHBAUM-SLAGER,

a

PHILIP LAZAROVICI,

a

CORINA BEJAR,

a

MOUSSA B.H. YOUDIM,

b

AND SHAI SHOHAM

c

a

Departments of Pharmacology, Hebrew University Faculty of Medicine, Jerusalem, Israel

b

Technion-Faculty of Medicine, Eve Topf and NPF Centers, Haifa, Israel

c

Herzog Hospital, Jerusalem, Israel

A

BSTRACT

: TV3326, (

N

-propargyl-(3R)-aminoindan-5-yl-ethyl,methyl car-bamate) was prepared in order to combine the neuroprotective effects of rasa-giline, a selective inhibitor of monoamine oxidase (MAO)-B with thecholinesterase (ChE) inhibitory activity of rivastigmine as a potential treat-ment for Alzheimer’s disease. The study reported here examined the neuropo-tective effects of TV3326 against various insults

in vitro

and

in vivo.

TV3326caused a dose related (10–500

�

M) reduction in death induced in NGF differ-entiated rat pheochromocytoma (PC12) cells by 3–4 hour exposure to oxygen–glucose deprivation. A single sc injection of TV3326 given five minutes afterclosed head injury in mice significantly reduced the cerebral edema, and accel-erated the recovery of motor function and spatial memory several days later.Unilateral icv injection of streptozotocin (STZ) 1.5 mg in rats, caused specificdamage to myelinated neurones in the fornix and corpus callosum accompa-nied by microgliosis. Three bilateral injections of STZ, 0.25 mg each, causedmore widespread damage, and a marked impairment in spatial memory.Chronic oral treatment with TV3326 (75

�

mols/kg) reduced the neuronaldamage and microgliosis and almost completely prevented the memoryimpairment. The neuroprotective effect in PC12 cells may be due to a combi-nation of ChE inhibition and antiapoptotic activity. The latter does not resultfrom ChE inhibition. It is associated with the presence of the propargyl group,since it occurs with other propargylamines that do not inhibit MAO, but notwith drugs that inhibit only ChE.

K

EYWORDS

: PC12 cells; Glucose–oxygen deprivation; Closed head injury;Streptozotocin memory deficits.

Address for correspondence: Professor Marta Weinstock, Leon & Mina Deutsch Professorof Psychopharmacology, Department of Pharmacology, School of Pharmacy, Hebrew Univer-sity Medical Centre, Ein Kerem, Jerusalem 91120, Israel. Voice: 972-2-6758731; fax: 972-2-6758741.

martar@cc.huji.ac.il

149WEINSTOCK

et al.

: NEUROPROTECTION BY TV3326

INTRODUCTION

Although the cause of Alzheimer’s disease (AD) has not yet been fully elucidat-ed, indirect evidence has implicated oxidative stress in the etiology of the neurode-generation by increasing glutamate release and inducing apoptosis.

1,2

Theproduction of oxidative free radicals (ROS) is either a cause or a consequence ofmitochondrial dysfunction. Complex IV activity has been reported to be decreasedin the brains of patients with AD.

3,4

The initial neurodegeneration, whatever itssource, is further exacerbated by the release of cytokines and the reactive oxygenspecies from activated microglia.

5,6

In AD, nerve cell loss occurs principally in larger neurons of the superficial cortexand in terminal synapses of neurons arising in the nucleus basalis of Meynert. Thedegree of memory impairment correlates well with the loss of cholinergic transmis-sion in the temporal lobe and other cortical brain regions.

7

Cholinesterase (ChE)inhibitors induce a modest improvement in memory and/or delay the rate of itsdecline.

8

However, there is little or no evidence that they can significantly reduceneuronal damage by interfering with the production or action of ROS, or by reducingmicrogliosis.

The monoamine oxidase (MAO)-B inhibitor, selegiline was reported to delay theprogression of AD without significantly improving cognitive function.

9,10

In rats,selegiline suppresses the formation of the cytotoxic

�

OH radical and protectednigrostriatal neurons from oxidative damage induced by MPTP.

11

It also decreasesbrain damage induced by hypoxia-ischemia, probably by increasing the activities ofthe protective enzymes, superoxide dismutase and catalase.

12

In vitro,

selegiline pro-tected cultured cells from the toxic effects of serum deprivation.

13

Selegiline ismetabolized to desmethylselegiline, methamphetamine, and amphetamine.Although desmethylselegiline is responsible for the neuroprotective effect of theparent compound, the other metabolites appear to interfere with this activity

14

there-by compromising the effect of selegiline.

Rasagiline,

N

-propargyl-(3R)-aminoindan, is another selective inhibitor ofMAO-B

15

that exerts neuroprotective actions against a variety of insults

in vitro

and

in vivo.

These actions are similar to those of selegiline,

16–19

but rasagiline is notmetabolized to amphetamine or methamphetamine. TV3326, (

N

-propargyl-(3R)-aminoindan-5-yl-ethyl,methyl carbamate) was synthesized in an attempt to combinethe neuroprotective properties of rasagiline with the ChE inhibiting activity ofrivastigmine, a drug with proven efficacy in AD patients.

8

The introduction of a car-bamate in the 6 position of rasagiline reduced by more than 1,000-fold, MAO-Binhibitory activity

in vitro

or after acute administration in mice and rats. However,after chronic oral administration to rats the compound inhibited MAO-A and B andChE in the brain at similar doses, but showed very little effect on MAO in the intes-tine and liver.

20

This reduced the likelihood of tyramine potentiation and adversecardiovascular reactions.

The rat pheochromocytoma cell line (PC12) has been widely used as a model ofneuronal cell death induced by serum and nerve growth factor (NGF) deprivation,

21

toxins,

22

and ischemia.

17

Inhibitors of both ChE

23,24

and MAO-B

17,25

have beenshown to protect PC12 cells against some of these insults. Rasagiline and rivastigmine

150 ANNALS NEW YORK ACADEMY OF SCIENCES

significantly reduced brain edema and speeded the recovery of motor function andspatial memory after brain injury in mice.

18,26

Unilateral intracerebroventricular (icv) injection of streptozotocin (STZ 1.5 mg)into middle-aged rats decreased glucose utilization in those brain areas that showedextensive morphological changes in AD patients.

27

Icv STZ also reduced cholineacetyltransferase activity in these brain areas and altered levels of NGF in a mannerconsistent with cholinergic deafferentation.

28,29

Although it was suggested that STZmay act by interfering with insulin mediated glucose uptake,

30

it is possible that itproduced its effects by inducing ROS and lipid peroxidation, as it does in peripheraltissues.

31,32

Parenteral administration of STZ also causes damage to myelin

33

asso-ciated with diabetes.

In this article, we report the neuroprotective effects of TV3326 against oxygen–glucose deprivation (OGD) in PC12 cell cultures and

in vivo

as seen by its reductionof the sequelæ of closed head injury in mice and neuronal damage caused in rats byicv injection of STZ.

MATERIALS AND METHODS

Drugs

TV3326 hemitartrate, TV3279 mesylate and TV3262 hydrochloride were sup-plied by Teva Pharmaceuticals Israel, Ltd. Rivastigmine hemitartrate was suppliedby Novartis, Switzerland. Because of its chemical instability, streptozotocin (Sigma,St. Louis, MO, U.S.A.) was prepared freshly as a solution in artificial cerebrospinalfluid (CSF) immediately before injection into each rat. All doses or concentrationsare expressed as

µ

moles/kg or

µ

M of the appropriate salt.

Neuroprotective Effect of TV3326 Against Oxygen–Glucose Deprivation in Nerve Growth Factor Differentiated PC12 Cells

PC12 cells (1.5–2

×

10

5

) per dish were grown in Dulbeco’s modified Eagle’smedium supplemented with 7

%

fetal calf and horse serum containing NGF(50 ng/ml) for 8–10 days and then subjected to OGD for 3–4 h in a specially con-structed ischemic device as described elsewhere.

17

At the end of this period, glucose(4.5 mg/ml) was added and the cultures reoxygenated for an additional 18 h. Underthese conditions, cell death determined by the leakage of lactic acid dehydrogenase(LDH) into the medium ranged between 30 and 50

%

of the total content. TV3326,its S-isomer, TV3279, or rivastigmine was added to the cultures 15 min before appli-cation of OGD in concentrations in the range 1–500

µ

M.

Cholinesterase Inhibition by Putative Neuroprotective Agents in PC12 Cells

ChE activity was measured by the method of Ellman

et al.

,

34

in PC12 cells grownin 25-ml culture bottles at a density of 100,000 cells/ml. The various experimentalgroups were incubated with the drugs (1–500 M) for 4 or 18 hours. They were thencentrifuged at 1,000 rpm for 10 minutes and solubilized in 100

µ

l 0.1M phosphatebuffer pH 8.0 containing 1

%

Triton. ChE inhibition was calculated by comparing theenzyme activity in cells taken from cultures with and without added drugs.

151WEINSTOCK

et al.

: NEUROPROTECTION BY TV3326

Cholinesterase Inhibition by TV3326 and TV3279 in Mouse Brain

Mice were injected sc. with saline, (1 ml/kg), TV3326 or TV3279 (50, 75, or 200

µ

moles/kg). The mice were decapitated 60 and 120 min later, and the whole brainremoved, rapidly weighed, and homogenized (100 mg/ml) in 0.1M phosphate bufferpH 8.0 containing 1

%

Triton. ChE activity was measured in 25

µ

l aliquots of enzymehomogenates.

34

Inhibition of ChE (percentage) induced by TV3326 and TV3279was calculated by comparison with enzyme activity obtained from mice injectedwith saline under the same conditions.

Protective Effects of TV3326 Against the Sequelæ of Brain Trauma in Mice

The following experiments were performed in mice and rats according to theguidelines of the University Committee for Institutional Animal Care, based on thoseof the National Institutes of Health, U.S.A. Male mice of (40) Sabra Hebrew Univer-sity strain, weighing 35–40 gm were trained in three daily trials for five days to findthe escape platform in a Morris water maze (1 m diameter) that remained in the sameplace throughout the test. All the mice reached escape latencies ranging from 22–30sec at the end of this period. They were then separated randomly into four equalgroups. Moderate to severe closed head injury to the left hemisphere was induced inthe mice under ether anesthesia by a weight-drop device as described elsewhere.

35

Saline (1 ml/kg), TV3326 or TV3279 (75

µ

moles/kg or 200

µ

moles/kg) were inject-ed sc, five minutes after injury. Motor dysfunction and impairment of spatial mem-ory was assessed for up to 14 days after injury, as described by Huang

et al.

18

Themaximal possible score for motor dysfunction one hour after injury was 14.

Protective Effect of TV3326 Against Neuronal Damage and Spatial Memory Deficits Induced in Rats by icv Injection of Streptozotocin (STZ)

The experiments were performed in order to determine the nature and extent ofany morphological changes that are produced by icv STZ in rats, and to see if thesechanges could be prevented by TV3326. Ten 3–4-month old male Sprague-Dawleyrats, weighing 250–300 g were given TV3326 (75

µ

moles/kg) by gastric gavage, twohour before they were anesthetized with equithesin (1 ml/kg) and another 10received 1 ml/kg water. The treatment with TV3326 or water was continued oncedaily for three weeks after injection of STZ. STZ 1.5 mg (prepared in artificial CSFimmediately before injection) or CSF alone was injected once, unilaterally in a vol-ume of 4

µ

l. The stereotaxic coordinates were

−

0.9 mm anterior, 1.8 mm lateral and

−

3.7 mm ventral from bregma. Forty days after the icv injection of STZ, the spatialmemory of the rats was tested in the Morris water maze (1.4 m diam.) as describedelsewhere.

36

Briefly, the rats were given two daily trials for five days in the mazewith an intertrial interval of 15 min. The point of entry of the rat into the maze andthe position of the escape platform were changed daily, but not between the firstand second trials each day. The decrease in escape latency from day to day in trial 1represents reference or long term memory, whereas that from trial 1 to trial 2, is con-sistent with working, or short term memory.

37

On the following day, the rats weredeeply anesthetized with sodium pentobarbitone (60 mg/kg). The brains were fixedby transcardial perfusion, with phosphate buffered saline (0.02 M PBS, pH 7.4)

152 ANNALS NEW YORK ACADEMY OF SCIENCES

containing heparin (5 U/ml) and 4

%

paraformaldehyde in 0.1 M PBS, pH 7.5 con-taining sucrose 4

%

. Brain sections, 30-

µ

m thick, were cut and stained for potentialaxonal degeneration by means of silver impregnation.

38

In addition, reactive micro-glia were stained by means of an antibody to complement receptor 3.

39

Rating scaleswere constructed to evaluate the degree of pathology; one scale was based on the cre-syl violet stain and depicted the damage to the hippocampus, fornix, and corpus cal-losum. The second scale was based on immunohistochemical staining of microgliaand depicted the extent of microglial reactions in these structures. The scale for dam-age ranged from 0 for no damage to 5 for total loss of fornix, anterior hippocampus,and dorsal hippocampus. The rating scale for microglial reaction in the dorsal branchof fornix was also 0 for no change (microglia are evenly distributed with radialbranching of dendrites) to 3 for a condition in which orientation of microglial fibersparallels orientation of axons together with an increase in the number of fibers overthe entire dorsal fornix on that side (ipsilateral or contralateral).

In another experiment, the same total amount of STZ was given but injected bilat-erally on day 1, 5, and 21 in three doses each of 0.25 mg/side. Control rats were givenartificial CSF in the same way. TV3326 or water was given as described abovefor three weeks, starting two hour before the first STZ injection. Spatial memorywas tested in the Morris water maze as described above, 40 days after the first STZinjection.

RESULTS

Neuroprotective Effect of Drugs Against Oxygen–Glucose Deprivation in Nerve Growth Factor (NGF) Differentiated PC12 Cells

Exposure of NGF differentiated PC12 cells to OGD for 3.5 h and reoxygenationresulted in death of about 45

%

of the cells compared to 8

%

under control conditionsat 37

°

C (see T

ABLE

1). Rivastigmine reduced cytotoxicity by a similar amount (about33

%

) at all concentrations tested, ranging from 1–500

µ

M. By contrast, TV3326 andTV3279 caused a dose related reduction in cell death that reached levels of 58

%

ata concentration of 100

µ

M, and more than 80

%

at 500

µ

M.

T

ABLE

1. Cell death (percent of total LDH) induced by OGD (3–4 h) in PC12 cells

Note: Significantly different from OGD alone,

*

p

<

0.01,

**

p

<

0.001.s

Concentration (

µ

M)

Drug 0 1 10 100 500

No insult 8.5

±

1.5

OGD 45.3

±

3.5 — — — —

Rivastigmine

+

OGD 29.0

±

1.9

*

30.2

±

2.5

*

27.2

±

2.2

*

26.8

±

2.9

*

TV3326

+

OGD 43.9

±

3.0 37.2

± 4.9 18.9 ± 4.7* 9.4 ± 1.0**

TV3279 + OGD 40.0 ± 5.4 34.9 ± 4.8 19.1 ± 3.8* 8.9 ± 0.7**

153WEINSTOCK et al.: NEUROPROTECTION BY TV3326

Inhibition by Drugs of ChE in PC12 Cells

The inhibition (percent of control) of ChE in PC12 cells incubated for 4 or 18 hwith the drugs during their exposure to OGD is shown in TABLE 2. Although rivastig-mine caused a significant inhibition of ChE after four hour incubation at a concen-tration of 1 µM, TV3326 and TV3279 were only effective at this concentration after18 h incubation.

Inhibition by Drugs of ChE in Mouse Brain

The effect of TV3326 and TV3279 on brain ChE reached its peak two hour aftersc injection. For TV3326 this was 33% at 75 µmoles/kg and 54% at 200 µmoles/kg.TV3279 (75 µmoles/kg) inhibited the enzyme by 49%. Neither isomer caused any

TABLE 2. Inhibition of cholinesterase by drugs (percent of control) after OGD (fourhour) followed by reoxygenation (18 h)

Concentration (µM)

Drug Time of incub (h) 1 10 100

Rivastigmine 4 34.8 ± 4.9 77.7 ± 2.2 96.0 ± 0.6

TV3326 4 0 9.5 ± 2.1 44.3 ± 4.0

18 20.9 ± 8.1 38.0 ± 0.6 49.0 ± 5.6

TV3279 4 2.0 ± 3.9 12.5 ± 5.5 70.6 ± 8.7

18 38.8 ± 6.0 52.9 ± 12.5 —

FIGURE 1. Effect of acute administration of TV3326 and TV3279 on the disturbancein motor function induced by closed head injury in mice. Significantly different fromsaline, * p < 0.05. Doses in µmoles/kg. , saline; , TV3326 (75); , TV3326(200); , TV3279 (75).

154 ANNALS NEW YORK ACADEMY OF SCIENCES

inhibition of MAO-A or -B in the brain of mice after acute sc injection of 75µmoles/kg (Gross, unpublished observations). TV3326 inhibited MAO-A by 17%and MAO-B by almost 50% at a dose of 200 µmoles/kg, whereas TV3279 was inef-fective in doses below 500 µmoles/kg

Protective Effects of TV3326 Against the Sequelæ of Brain Trauma in Mice

All the mice showed a motor disability score of about 12 points, one hour afterinjury (see FIGURE 1). This gradually declined by about 50% in the saline treated ani-mals over the next two weeks. TV3326 (200 µmoles/kg) reduced the motor disabilityscore, even one day after injury, whereas 75 µmoles/kg of each isomer reduced thissignificantly from the seventh day onwards. Both isomers also significantly reducedescape latencies from the second day after injury at a dose of 75 µmoles/kg. Theescape latencies of mice given the larger dose of TV3326 were significantly lowerthan those of saline-treated mice on day 4 (see FIGURE 2).

Effect of TV3326 Against Pathological ProcessesInduced in Rats by icv Injection of STZ

Two major types of pathological processes were observed after unilateral injec-tion of STZ 1.5 mg: shrinkage of the dorsal hippocampus and adjacent fornix andgliosis in myelinated periventricular brain structures, including the corpus callosum

FIGURE 2. Effect of acute administration of TV3326 and TV3279 on spatial memoryafter closed head injury in mice. �, saline; �, TV3279 (75 µmoles/kg); �, TV3326 (75µmoles/kg); �, TV3326, (200 µmoles/kg). Closed head injury was administered at the timeindicated by arrow. Escape latencies of mice treated with TV3279 and TV3326, 75µmoles/kg, were significantly lower than those of saline-treated mice from day two andthose treated with TV3326, 200 µmoles/kg, from day four.

155WEINSTOCK et al.: NEUROPROTECTION BY TV3326

FIGURE 3. Effect of TV3326 75 µmoles/kg on neuronal damage and microgliosisinduced in the dorsal fornix induced by a single unilateral icv injection of STZ in rats. Signif-icantly different from water-treated rats, *p < 0.05. , STZ + water ipsilateral; , STZ+ water contralateral; , STZ + TV 3326 ipsilateral; , STZ + TV 3326 contralateral.

FIGURE 4. Effect of three bilateral icv injections of STZ on escape latencies of ratsin the Morris water maze. CSF trial 1, �; trial 2, �; STZ trial 1, �; trial 2, �. Significantlydifferent from artificial CSF, # p < 0.05. Significantly different from trial 1, * p < 0.05.

156 ANNALS NEW YORK ACADEMY OF SCIENCES

and fornix. Damage to the fornix and anterior hippocampus was more severe ipsilat-eral than contralateral to the site of injection, both at the dorsal and hypothalamicbranch of fornix. A change in the orientation of microglial fibers and an increase intheir number occurred in the dorsal fornix, corpus callosum, and hypothalamicbranch of the fornix. Chronic treatment with TV3326 reduced the degree of damageand microglial reaction in the dorsal fornix (see FIGURE 3) but not in the hypotha-lamic branch.

Effect of STZ and TV3326 Treatment on Spatial Memory in Rats

A single icv injection of STZ (1.5 mg) did not significantly affect spatial learningand memory in the Morris water maze compared to that in rats treated with icv CSF.However, when the same amount of STZ was given in three divided doses over aperiod of three weeks, both reference and working memory were significantlyimpaired 40 days after the first injection of STZ (see FIGURE 4). Chronic treatmentwith TV3326 (75 µmoles/kg) prevented the learning deficits and changes in spatialmemory, restoring escape latencies to those seen in control rats injected with artifi-cial CSF, bilaterally (see FIGURE 5).

DISCUSSION

A growing body of evidence has indicated that oxidative stress plays an importantrole in the progressive neuronal degeneration that occurs in AD.40,41 Oxidative stress

FIGURE 5. Reduction by TV3326 of the spatial memory impairment induced by threebilateral icv injections of STZ. STZ trial 1, �; trial 2, �; STZ + TV3326 (75 µmoles/kg)trial 1, �; trial 2, . Significantly different from STZ, # p < 0.05. Significantly differentfrom trial 1, * p < 0.05.

157WEINSTOCK et al.: NEUROPROTECTION BY TV3326

arises when the production of ROS exceeds the rate of consumption by cellular anti-oxidants, leading to peroxidation of lipids and cell damage.42 The brain is particu-larly vulnerable to ROS because it contains a relatively high concentration of easilyperoxidizable fatty acids43 and a relatively low concentration of protective antioxi-dant enzymes.44 In AD the damage occurs initially in cholinergic neurones in thebasal forebrain and results in characteristic memory and cognitive impairment. It is,therefore, reasonable to suggest that a drug that can prevent the formation of ROS oraccelerate their consumption by activating protective enzymes and also increasecholinergic transmission, should improve memory and delay the progression of thedisease. When developing such a drug it is essential to show that it has protectiveeffects against neuronal damage caused to cholinergic neurones in the brain, as wellas in neuronal cultures.

In our study, TV3326, an aminoindan derivative with both carbamate and propar-gylamino groups, induced dose related protection against cell death caused by OGDin NGF differentiated PC12 cells. This could have resulted from blockade of ChE,since it also occurred with rivastigmine in concentrations that inhibited ChE by atleast 40% in these cells. However, rivastigmine that lacks a propargyl amino groupwas only able to reduce cell death by about 33% at any concentration in the range1−500 µM, whereas TV3326 and TV3279 reduced the number of dead cells by morethan 80% at the higher concentrations. Such levels of neuroprotection against OGDwere also seen with higher concentrations (1 µM or more) of rasagiline,17 which hasno ChE inhibitory activity. The mechanism of neuroprotection by ChE inhibitors andpropargylamines is not yet established. However, other ChE inhibitors, tacrine,donepezil, and huperazine protected undifferentiated PC12 cells against oxidativestress resulting from exposure to β-amyloid fractions23,45 or H2O2.24 In this model,the effect of ChE inhibitors appeared to be mediated by activation of nicotinic recep-tors and involved an increase in the activity of catalase and glutathione peroxidase.45

Propargylamines may also prevent the reduction in the activity of theseenzymes.12 Furthermore, they have been shown to reduce apoptosis in response tooxidative stress or serum deprivation by increasing the synthesis of BCL-2 and byinhibiting activation of caspase 3.19,46 These activities are shared by other propargy-lamine derivatives like the S-isomer of rasagiline, which does not block MAO in theconcentrations used in the experiment (Naoi, personal communication).

The reduction by a single injection of TV3326 and TV3279 of the cerebral ede-ma, induced by brain trauma and acceleration of the recovery of motor function andspatial memory are similar to that seen with another ChE inhibitor, rivastigmine.26

It is likely that the drugs prevented the reduction in cholinergic transmission that hasbeen shown to occur after brain trauma47,48 since all the effects of rivastigmine weresignificantly reduced by cholinergic receptor blockade.20,26

In addition to the reported effect of icv STZ on cerebral glucose metabolism, ourstudies show that it causes ventricular enlargement and distinct white matter lesionsin the fornix and corpus callosum, accompanied by activation of microglia. All thesepathological changes are seen in the brains of AD patients and were reduced byTV3326. To our knowledge this is the first time that a detailed histological examina-tion has been performed and the nature and extent of the morphological damageinduced by icv STZ described. Damage to myelinated nerves in the periphery has alsobeen reported following parenteral administration of STZ, in association with an

158 ANNALS NEW YORK ACADEMY OF SCIENCES

increase in ROS, although this could have resulted from the ensuing hyperglycemia.31

It is not known whether the damage to the fornix and corpus callosum results fromoxidative stress or apoptosis, in a similar manner to that induced in pancreatic cells.49

Examination of brain phospholipid metabolism three weeks after icv injection of STZrevealed significant losses in phosphatidyl ethanolamine, but none that are indicativeof peroxidative damage.50 If apoptosis is involved in the specific lesions induced bySTZ and if it acts in vivo as it does in neuronal cell cultures, then this could explainhow TV3326 causes protection.

In contrast to studies in older animals in which about 50% showed a deficit in spa-tial memory,51 the limited damage caused by a single injection of STZ in young ratsdid not produce any memory impairment in the Morris water maze. In a more recentstudy, STZ was given in the same total amount bilaterally in three injections over aperiod of three weeks in rats aged one year. This produced memory deficits that weredetected after 40 days.52 We also obtained a marked impairment in working and ref-erence memory after icv injection of STZ in young rats. Chronic treatment withTV3326 throughout the three weeks during which STZ was given almost completelyprevented the occurrence of memory deficits when tested 19 days after the lastadministration.

The neuroprotective effects of TV3326 in cell cultures and animal models inwhich neurodegeneration occurs, together with its ability to inhibit ChE and MAOselectively in the brain, make it a potentially useful therapeutic agent for the treat-ment of AD.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the support of Teva Pharmaceuticals Ltd.,Israel for funding and for supplying the test compounds, and thank its personnel forhelpful discussions during the course of this research.

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