Life Sciences 77 (2005) 1–14

www.elsevier.com/locate/lifescie

Effect of donepezil and lercanidipine on memory impairment

induced by intracerebroventricular streptozotocin in rats

Swapnil Sonkusare, Krishnamoorthy Srinivasan, Chamanlal Kaul, Poduri Ramarao*

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research,

S.A.S. Nagar-160062, Punjab, India

Received 9 March 2004; accepted 23 October 2004

Abstract

Intracerebroventricular (ICV) injection of streptozotocin (STZ) causes cognitive impairment in rats. ICV STZ is

known to impair cholinergic neurotransmission by decreasing choline acetyltransferase (ChAT) levels, glucose and

energy metabolism in brain and synthesis of acetyl CoA. However, no reports are available regarding the

cholinesterase inhibitors in this model. In aging brain, reduced energy metabolism increases glutamate release,

which is blocked by L-type calcium channel blockers. These calcium channel blockers have shown beneficial

effects on learning and memory in various models of cognitive impairment. The present study was designed to

investigate the influence of chronic administration of donepezil (cholinesterase inhibitor, 1 and 3 mg/kg) and

lercanidipine (L-type calcium channel blocker, 0.3 and 1 mg/kg) on cognitive impairment in male Sprague-Dawley

rats injected twice with ICV STZ (3 mg/kg) bilaterally on days 1 and 3. ICV STZ injected rats developed a severe

deficit in learning and memory indicated by deficits in passive avoidance paradigm and elevated plus maze as

compared to control rats. Cholinesterase activity in brain was significantly increased in ICV STZ injected rats.

Donepezil dose-dependently inhibited cholinesterase activity and improved performance in memory tests at both

the doses. Lercanidipine (0.3 mg/kg) showed significant improvement in memory. When administered together,

the effect of combination of these two drugs on memory and cholinesterase activity was higher than that obtained

with either of the drugs when used alone.

D 2005 Published by Elsevier Inc.

Keywords: Intracerebroventricular streptozotocin; Learning and memory; Donepezil; Lercanidipine

0024-3205/$ -

doi:10.1016/j.l

* Correspond

E-mail add

see front matter D 2005 Published by Elsevier Inc.

fs.2004.10.036

ing author. Tel.: +91 172 2214683; fax: +91 172 2214692.

ress: ramaraop@yahoo.com (P. Ramarao).

S. Sonkusare et al. / Life Sciences 77 (2005) 1–142

Introduction

Alzheimer’s disease is a neurodegenerative disorder characterized by progressive loss of

memory followed by complete dementia. It accounts for 50% of dementia cases (Areosa and

Sherriff, 2003). Several pathological mechanisms have been proposed that include g excitotoxicity

as a result of blockade of glutamate uptake into the astrocytes by Ah aggregates, oxidative stress

induced by Ah-aggregates, membrane lipid peroxidation due to C-terminal fragment of APP

(amyloid precursor protein), microglial activation by Ah-aggregates and molecular pathways

activated by Ah-induced stimulation of various kinases including MAP kinases and JNK (Suh and

Checler, 2002). As multiple pathological mechanisms are involved in memory loss, compounds

that inhibit more than one pathological mechanism or a combination of drugs that act by blocking

different pathological mechanisms may be useful.

Intracerebroventricular streptozotocin injection in rats as a model for studying dementia

In all forms of dementia of Alzheimer’s type (DAT), abnormalities in glucose metabolism have

been found. The presence of insulin receptors in the brain is well known (Park, 2001). Insulin plays

an important role in neuronal glucose uptake and memory processing. Insulin receptor desensitization

has been proposed as a cause for reduced energy metabolism and cholinergic deficiency (Biessels et

al., 2002). The levels of energy rich phosphates and intermediates of glycolysis are reduced in AD

brain in presence of arterial normoglycemia. ICV streptozotocin in rats causes desensitization of

insulin receptors and biochemical changes similar to that of AD or ageing brain (Hoyer, 2002a; Hoyer,

2002b; Hoyer et al., 1994). In addition, reduced energy metabolism and synthesis of acetyl CoA

ultimately result into cholinergic deficiency and thereby memory deficit in ICV STZ rats. Apart from

insulin receptor signalling dysfunction, memory impairment in ICV STZ injected rats could occur due

to direct damage to the septohippocampal system. This was supported by reduced choline acetyl

transferase (ChAT) activity in hippocampus (Blokland and Jolles, 1993), reduction in the weight of

septum by more than 40% (Terwel et al., 1995), decrease in the transport of nerve growth factor

(NGF) from hippocampus to septum (Hellweg et al., 1992), microglial activation and specific damage

to myelinated tracts in the fornix through generation of oxidative stress, thereby disrupting

connections between the septum and hippocampus (Shoham et al., 2003). All these pathological

mechanisms play role in dementia of ageing and Alzheimer’s disease. Since ICV STZ causes neuronal

damage without altering blood glucose levels, its action is independent of induction of hyperglycemia.

Bilateral injection of STZ causes more widespread damage and marked impairment in memory as

compared to unilateral damage which causes specific damage to neurons of fornix and corpus

callosum (Weinstock et al., 2001).

Reduced energy metabolism in neurons results into depolarization induced release of glutamate

causing glutamate excitotoxicity, which is blocked by L-type calcium channel blockers (Mason et al.,

1999). L-type calcium channel blockers have shown beneficial effects on learning and memory in AD

patients (Fritze and Walden, 1995) as well as in various animal models of dementia (Ikegaya et al., 2000;

Petkov et al., 2000; Quartermain and Garcia deSoria, 2001). Reduction in blood pressure caused by

calcium channel blockers prevents their use in therapy. It was hypothesized that L-type calcium

channel blockers that exert central effects at much lower doses with minimal lowering of blood

pressure can improve learning and memory.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–14 3

The present study was undertaken to evaluate a cholinesterase inhibitor, donepezil (DON) that is

well known for its activity and is being used therapeutically. Lercanidipine (LER), an L-type

calcium channel blocker has a log P-value (membrane partition coefficient) of 6.88, which is higher

than other commonly used L-type CCB’s (van der Lee et al., 2000). It was found to exert other

central effects at very low doses in our own laboratory (Gullapalli and Ramarao, 2002). We

hypothesized that lercanidipine would have beneficial effect on memory at doses which do not

produce lowering of blood pressure. Plasma half life of lercanidipine is 8–10 hrs and the drug is

not accumulated on repeated dosing. Tmax for lercanidipine is 1.5 to 3 hrs after dosing (Meredith,

1999). Donepezil undergoes extensive metabolism and tissue distribution after i.v. administration to

rats and plasma levels of unchanged donepezil decline biphasically with an apparent T1/2 for the

terminal phase of 3 h. Orally administrated donepezil is absorbed rapidly. The mean plasma levels

of donepezil reach a peak at 30 min after administration, and then decline biphasically (Matsui et

al., 1999). Several reports suggest that 1 to 5 mg/kg, p.o. of donepezil inhibits brain cholinesterase

activity and improves cognition in rats in a dose-dependent manner. The doses of donepezil used

in the present study produce clinically relevant drug levels (Balducci et al., 2003; Kosasa et al.,

1999; Matsui et al., 1999; Tokita et al., 2002). Further beneficial effects of the combination of

these drugs were also evaluated.

Methods

Animals

Adult male Sprague-Dawley rats weighing 320–350 g were procured from Central Animal

Facility (CAF), National Institute of Pharmaceutical Education and Research (NIPER). Animals

were housed three per cage prior to STZ administration and one per cage after STZ administration.

The animals were kept on normal pellet diet and tap water ad libitum in an air conditioned room

(22 F 2). All animal protocols were approved by Institutional Animal Ethics Committee (IAEC),

NIPER.

Intracerebroventricular administration of streptozotocin

Adult male Sprague-Dawley rats weighing 320–350 g were anesthetised with thiopentone

(Neon Laboratories, India, 45 mg/kg, i.p.) The head was placed in position in the stereotaxic

apparatus and a midline saggital incision was made in the scalp. Following coordinates were

used to for ICV injection: 0.8 mm posterior to bregma, 1.5 mm lateral to saggital suture, 3.6

mm ventral from the surface of the brain (Sharma and Gupta, 2002). STZ (Sigma, USA) was

dissolved in citrate buffer (pH 4.4). The STZ group was injected bilaterally with ICV STZ (3

mg/kg) in two divided doses, on the first and the third day. The concentration of STZ in

citrate buffer was adjusted so as to deliver 10 AL of the solution. Rats in the control group

were given ICV injection of same volume of citrate buffer on the first and third day as in

STZ treated. Post operatively, the rats were fed with milk by oral gavage in addition to normal

pellet diet for 4 days after the surgery, followed by normal pellet diet alone. After ICV

injection, povidone-iodine solution was applied and the cut skin was sutured after second

S. Sonkusare et al. / Life Sciences 77 (2005) 1–144

injection followed by daily application of Neosporin.R The body weights were continuously

monitored.

Drug administration

Donepezil HCl (gifted by Hetero Drugs Ltd, Hyderabad, India, 1 and 3 mg/kg/day, p.o.)

treatment was started from 13th day onwards. The drug was dissolved in normal saline and

administered by oral gavage. Control group was treated with normal saline orally. Although

donepezil improves memory on single administration in various models of cholinergic deficiency

that show dementia, multiple administration of donepezil was used to rule out interference by effect

on locomotor activity in memory tests. Chronic treatment with donepezil is used therapeutically in

dementia of AD. Hence effect of multiple administration of donepezil on cholinesterase activity in

brain was determined. Since donepezil was used as a treatment and not as a prophylactic,

administration was started after dementia had been developed i.e. from day 13 onwards.

Lercanidipine (gifted by Recordati Industria Chimica, Italy, 0.3 and 1 mg/kg, i.p.) was administered

for 20 days starting from the first day. Composition of the vehicle for lercanidipine was DMSO: ethanol:

water in a ratio of 20: 20: 60. Control group was injected intraperitoneally with the vehicle alone for all

21 days. To evaluate combination of donepezil (1 mg/kg) and lercanidipine (0.3 mg/kg), the drugs were

administered on the same days as with single drug treatment.

Behavioral tests

Elevated plus maze test

Memory acquisition and retention was tested using elevated plus maze test on days 16 and 17.

The apparatus consisted of two crossed arms, one closed and the other, open. Elevated plus maze

apparatus was placed in a room with a sparse light and the test was carried out only between 10

a.m. and 4 p.m. Each rat was placed on the open arm, facing outwards. The time taken by the rat to

enter the closed arm in the first trial (acquisition trial) on 16th day was noted and was called as

initial transfer latency. Cut-off time was fixed as 300 seconds and in case a rat could not find the

closed arm within this period, it was removed from the maze and rejected. Second trial (retention

trial) was performed 24 hrs after the acquisition trial and retention transfer latency was noted

(Sharma and Gupta, 2002; Veerendra Kumar and Gupta, 2002) similarly. The retention trial latency

was expressed as percentage of initial trial latency.

Step-through passive avoidance task

Step-through passive avoidance task was used on days 18 and 19 to evaluate memory retention

deficit. The apparatus consisted of a chamber illuminated with a 40 W bulb and a dark chamber,

separated by a guillotine door. The floor was made of a metal grid with a shock scrambler. During

acquisition trial, rat was placed in the illuminated chamber. After initial habituation period of 60

seconds, the guillotine door was opened and time taken by the rat to enter the dark chamber was

noted. As soon as the rat entered the dark chamber, it was given a mild footshock of 0.5 mA for 3

seconds through the grid floor. The rat was allowed to remain in the dark compartment for 5

seconds and then was taken out. The latency to step into the dark compartment was recorded as

initial or pre-shock latency (IL). Rats showing initial trial latency of more than 60 seconds were

S. Sonkusare et al. / Life Sciences 77 (2005) 1–14 5

excluded from further experiments. Twenty-four hours later, retention trial was performed and

retention latency (RL) to step into the dark compartment was noted (post-shock latency). Cut-off

time was fixed at 300 seconds (Sharma and Gupta, 2002; Veerendra Kumar and Gupta, 2002). The

retention trial latency was expressed as percentage of initial trial latency.

Closed field activity

Closed field activity was measured to rule out the interference of change in locomotor activity in the

parameters of learning and memory. Spontaneous locomotor activity was measured on day 20th using

animal tracking system (Optoverimex) operated with the help of computer software. The apparatus was

placed in a dark; sound attenuated and ventilated room. Rat was put inside the chamber having infrared

light sensitive photocells and lid was placed in position. After a habituation period of 5 min, ambulatory,

vertical and total activity of the rat was recorded for further 10 minutes (Hartz et al., 2003; Sharma and

Gupta, 2001).

Measurement of blood pressure

Effect of drug treatment on blood pressure was determined using tail-cuff blood pressure monitoring

device (IITC INC., Life-Science Instruments), a non-invasive method to measure blood pressure and it

was measured on 21st day (Dobrian et al., 2001).

Cholinesterase activity estimation

Rats were sacrificed on day 21 after ICV STZ injection. Whole brains were quickly removed,

weighed and homogenized in 25 mM phosphate buffer saline (pH 7.4, 100 mg tissue/mL of PBS) with

polytron homogenisor at 2000 RPM for 30 seconds. For estimation of cholinesterase, the homogenate

was centrifuged at 20,000 g for 30 minutes and supernatant was used.

Cholinesterase activity was determined by modified method of Ellman (Ellman et al., 1961; Han and

Kim, 2003). Briefly, 3 ml of PBS (25mM, pH 7.4), 0.2 ml of 75mM acetylthiocholine iodide and 0.1 ml of

buffered Ellman’s reagent (DTNB 10 mM, NaHCO3 15 mM) were mixed and allowed to incubate for 10

minutes at room temperature. Enzyme sample (20 Al) or buffer (20 Al) was added and optical density wasmeasured at 412 nmwithin 5minutes. Cholinesterase activity was expressed as optical density/ mg protein.

Table 1

Effect of DON and LER on spontaneous locomotor activity in ICV STZ rats

Group Spontaneous locomotor activity

(units activity/10 minutes, n = 5)

Control 882 F 55.1

ICV STZ 1046 F 76.6

ICV STZ + DON (1 mg/kg) 936 F 157.3

ICV STZ + DON, (3 mg/kg) 728 F 126.7

ICV STZ + LER, (0.3 mg/kg) 806 F 106.1

ICV STZ + LER, (1 mg/kg) 320 F 65.3*,a

ICV STZ + DON (1 mg/kg) + LER (0.3 mg/kg) 942 F 72.1

a Control group Vs LER treated ICV STZ group, DON: donepezil, LER: lercanidipine.

* P b 0.05.

(B)

(A)

(C)

0

20

40

60

80

10

120

Control ICV STZ DON 1 mg/kg DON 3 mg/kg

*a

*b **b

0

20

40

60

80

100

120

% ITL

Control ICV STZ LER 0.3 mg/kg" LER 1 mg/kg"

*a

*b

% ITL

0

20

40

60

80

100

120

% ITL

Control ICV STZ DON 1 mg/kg + LER 0.3 mg/kg

*a

** b

Fig. 1. Effect of donepezil (A), lercanidipine (B) and donepezil + lercanidipine (C) on the performance in elevated plus maze

test in ICV STZ injected rats. Data values are RTL, expressed as mean % of ITLF SEM. * P b 0.05, ** P b 0.01 a: ICV STZ Vs

control group, b: Drug treated ICV STZ Vs ICV STZ group (n = 8); DON: donepezil, LER: lercanidipine.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–146

S. Sonkusare et al. / Life Sciences 77 (2005) 1–14 7

Data analysis

All the results are expressed as mean F SEM. Comparisons in the elevated plus maze test, step-

through passive avoidance task and closed field activity were carried out using non-parametric Mann-

0

100

200

300

400

Control ICV STZ DON 1 mg/kg DON 3 mg/kg

**a

**b

**b

0

100

200

300

400

Control ICV STZ LER 0.3 mg/kg LER 1 mg/kg

**b **b

**a

0

100

200

300

400

Control ICV STZ DON I mg/kg + LER 0.3 mg/kg

**a

**b

(B)

(A)

(C)

% ITL

% ITL

% ITL

Fig 2. Effect of treatment with donepezil (A), lercanidipine (B) and DON + LER (C) on passive avoidance paradigm in ICV

STZ rats. Values have been expressed as mean F SEM. ** P b 0.01, a: ICV STZ group Vs control group, b: Drug treated ICV

STZ group Vs ICV STZ group (n = 8); DON: donepezil, LER: lercanidipine.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–148

Whitney rank sum test. Cholinesterase activity and blood pressure were compared using one way

ANOVA.

Results

Behavioral tests

Closed field activity

Spontaneous locomotor activity determined on day 20 (Table 1) did not differ significantly in any of

the groups except the group that received higher dose of LER (1 mg/kg).

Elevated plus maze test

Initial transfer latency (ITL) did not differ significantly in any of the groups. Retention transfer

latency (RTL) of control group was significantly less than that of ICV STZ injected group.

Treatment with DON (1 and 3 mg/kg), LER (0.3 mg/kg) and DON + LER significantly lowered

the RTL in ICV STZ injected rats signifying improvement in learning and memory. The reduction

in RTL with DON + LER was more pronounced than with either of the drug at the doses used

(Fig. 1).

Step-through passive avoidance task

There was no significant difference in the acquisition trial latency between the control, ICV STZ

injected and DON + LER treated ICV STZ injected groups. Retention trial latency (RTL) for the

control group was significantly higher than that of ICV STZ injected group. Treatment with DON (1

mg and 3 mg/kg) and LER (0.3 mg and 1 mg/kg) significantly increased the RTL in ICV STZ

injected rats. Increase in RTL was higher in DON + LER treated rats as compared to that seen with

Table 2

Effect of ICV STZ injection and donepezil treatment on cholinesterase activity in brain in ICV STZ rats

Groups Cholinesterase activity

(Optical density/mg protein)

Control 2.76 F 0.05

ICV STZ injected 3.24 F 0.15*,a

ICV STZ + DON (1 mg/kg) 2.68 F 0.07*,b

ICV STZ + DON (3 mg/kg) 2.01 F 0.06***,b

ICV STZ + LER (0.3 mg/kg) 3.02 F 0.09

ICV STZ + LER (1 mg/kg) 2.98 F 0.16

ICV STZ + DON 1 mg/kg + LER 0.3 mg/kg 2.31 F 0.07**,c

Values have been expressed as mean O.D. (optical density)/ mg protein F SEM.a ICV STZ Vs Control group.b Donepezil treated ICV STZ Vs vehicle treated ICV STZ.c DON 1 mg/kg Vs DON 1 mg/kg + LER 0.3 mg/kg (n = 4); DON: donepezil, LER: lercanidipine.

* P b 0.05.

*** P b 0.001.

Table 3

Effect of lercanidipine and DON + LER treatment on systolic blood pressure

Group Systolic blood pressure

(MM of HG) (mean F SEM)

Diastolic blood pressure

(MM of HG) (mean F SEM)

Control 119.0 F 1.0 80.7 F 1.8

ICV STZ 122.3 F 1.8 81.0 F 2.1

ICV STZ + LER (0.3 mg/kg) 123.3 F 2.9 81.3 F 1.8

ICV STZ + LER (1 mg/kg) 104.3 F 1.2*,b 69.7 F 1.2*,b

ICV STZ +DON (1 mg/kg) + LER (0.3 mg/kg) 122.0 F 2.0 81.3 F 1.8

Values are mean F SEM.a ICV STZ Vs Control.b Lercanidipine treated ICV STZ Vs control (n = 5); DON: donepezil, LER: lercanidipine.

* P b 0.05.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–14 9

either of the drugs when used alone (p b 0.05 for DON + LER Vs DON 1 mg/kg or LER 0.3 mg/

kg) (Fig. 2).

Effect of donepezil treatment on cholinesterase activity in brain

There was a significant increase in cholinesterase activity in ICV STZ injected group as compared to

control group. Donepezil dose-dependently decreased cholinesterase activity in brain in ICV STZ

injected rats. As shown in the Table 2, donepezil (1 mg/kg) reduced the cholinesterase activity almost

back to control levels and a higher dose of DON (3 mg/kg) further reduced the cholinesterase activity.

Lercanidipine alone did not have significant effect on cholinesterase activity. However, inhibition of

cholinesterase activity was more with a combination of donepezil and lercanidipine (1 and 0.3 mg/kg

respectively) than with donepezil alone (1 mg/kg).

Effect of lercanidipine and donepezil + lercanidipine treatment on blood pressure

Chronic administration of 1 mg/kg but not 0.3 mg/kg of lercanidipine reduced the systolic and

diastolic blood pressures significantly as compared to control group as measured with non-invasive tail-

cuff method. Administration of donepezil + lercanidipine did not have any significant effect on systolic

blood pressure at the doses administered (Table 3).

Discussion

Spontaneous locomotor activity was not affected by donepezil (1 and 3 mg/kg), lercanidipine (0.3 mg/

kg) and DON + LER treatment. This rules out any interference by the change of locomotor activity in

performance in the memory tests. The locomotor activity was significantly reduced by treatment with 1

mg/kg of lercanidipine. Moreover, blood pressure was also reduced by higher dose (1 mg/kg) of LER.

Blood pressure was not affected by treatment with DON and DON + LER. These results suggest that the

reduced locomotor activity and blood pressure by LER (1 mg/kg) have interfered in assessment of

memory parameters.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–1410

Donepezil, a second-generation cholinesterase inhibitor (more selective towards AChE), is used

therapeutically for mild to moderate dementia of Alzheimer’s disease. It inhibits acetyl cholinesterase

reversibly and non-competitively (Scarpini et al., 2003). Though a cholinergic deficiency in this model is

known, no cholinesterase inhibitor has been tried as yet. Reduced levels of ChAT in hippocampus has

also been reported in this model. Similar characteristics are observed in an Alzheimer’s brain (Frolich

et al., 1998). Hence, donepezil, an AChE inhibitor that has high bioavailability and relatively lesser side

effects was tested.

Several studies have proved memory-improving action of donepezil in various models of cholinergic

deficiency (Ogura et al., 2000). Donepezil (1 mg and 3 mg/kg; p.o.) treated ICV STZ rats showed

significantly increased retention trial latencies as compared to ICV STZ injected rats in passive

avoidance apparatus and significantly shorter retention transfer latencies in elevated plus maze test as

compared to ICV STZ rats. Improvement in passive avoidance task indicates an improved acquisition

and/or retention of memory and shortened retention transfer latencies in elevated plus maze test signifies

increased ability to learn in rats treated with donepezil.

The results indicate that the cholinesterase activity was increased in ICV STZ injected rats by 15%

as compared to control rats. The activity was reduced by around 15% and 38% by 1 and 3 mg/kg of

donepezil, respectively; as compared to ICV STZ rats i.e. activity was reduced to control levels by 1

mg/kg and to around 23% less than control levels by 3 mg/kg of donepezil. Improvement of

performance in memory tasks and inhibition of cholinesterase activity in rats was in conformity with

several reports on donepezil published previously (Balducci et al., 2003; Carey et al., 2001; Isoma

et al., 2002; Kasa et al., 2000; Tokita et al., 2002). It has also been demonstrated that Ah(25–35)-induced increase in AChE activity in brain is mediated by L-type voltage dependent calcium channels

and was blocked by nifedipine (Sberna et al., 1997). Present study supports this report since the

inhibition of cholinesterase activity was found to be greater with combination of donepezil and

lercanidipine (1 and 0.3 mg/kg, respectively) as compared to that with donepezil alone (1 mg/kg).

However, it is possible that the difference in cholinesterase activity could be higher if activity in

specific regions like hippocampus is measured rather than a whole cerebral homogenate.

Several reports suggest the beneficial effects of L-type calcium channel blockers on memory

processing (Ikegaya et al., 2000; Petkov et al., 2000; Quartermain and Garcia deSoria, 2001). Chronic

administration of amlodipine facilitates learning and memory in mice (Quartermain, 2000).

Nimodipine has been reported to prevent scopolamine induced memory impairment (Norman et al.,

2002) and also prevents the deleterious effects of ethanol on memory (Brooks et al., 2002). It has been

demonstrated that L-type calcium channel blockers prevent cortical neurons from abeta-induced

apoptosis (Yagami et al., 2004). Ah(25–35)-induced depression of long-term potentiation in area CA1

in vivo and in vitro is attenuated by verapamil (Freir et al., 2003). Age-related working memory

impairment is correlated with increases in the L-type calcium channel protein alpha1D (Cav1.3) in

area CA1 of the hippocampus and both are ameliorated by chronic nimodipine treatment (Veng et al.,

2003). L-type Ca2+channel blockers attenuate beta-amyloid peptide toxicity to cortical neurons in

culture (Weiss et al., 1994).

A study has reported dissociation between blood pressure and direct drug effects on the central

nervous system (Denolle et al., 2002). Activation of microglial cells by PrP and beta-amyloid fragments

raises intracellular calcium through L-type voltage sensitive calcium channels (Silei et al., 1999). In

aging brain small, but sustained elevations of cytosolic Ca2+ -concentration might cause damage to the

neurons. Increased calcium flux through voltage dependent calcium channel causes hyperpolarisation

S. Sonkusare et al. / Life Sciences 77 (2005) 1–14 11

that may be responsible for impairment of memory and this process is inhibited by calcium channel

blockers (Brooks et al., 2002). Hence, drugs that reduce the influx of Ca+ into neurons should improve

age-related deficits such as the impairment of memory capability and sensorimotor functions (Izquierdo,

1990). Reduction in blood pressure is the major problem that prevents the use of calcium channel

blockers in therapy for their CNS effects. Studies in our laboratory show that lercanidipine exerts CNS

effects at much lower doses (Gullapalli and Ramarao, 2002). The log P-value (membrane partition

coefficient) of lercanidipine is 6.88, which is higher than other commonly used L-type CCB’s. However,

its potency as a vasodilator does not differ much as compared to other commonly used L-type CCB’s

(van der Lee et al., 2000).

Lercanidipine (0.3 mg/kg) improved performance in elevated plus maze test as evidenced by

significantly reduced retention transfer latency (RTL). This improvement was not observed with 1 mg/kg

of lercanidipine as RTL was even more than that of vehicle treated ICV STZ injected rats. This was

accounted for by the reduced locomotor activity by 1 mg/kg of lercanidipine. Performance in passive

avoidance task was improved by lercanidipine (both the doses, 0.3 mg and 1 mg/kg) as shown by the

retention trial latency. However, improvement by 1 mg/kg was not reliable as this dose also caused

reduction in spontaneous locomotor activity.

In conclusion, lercanidipine (0.3 mg/kg) improved learning and memory in ICV STZ injected rats at a

dose that did not show any effect on systolic blood pressure and locomotor activity.

Combination of donepezil (1 mg/kg) and lercanidipine (0.3 mg/kg) had no significant effect on body

weights, mortality, blood pressure and spontaneous locomotor activity. Increased RTL in passive

avoidance task indicates an improved acquisition and/or retention of memory and shortened retention

transfer latencies in elevated plus maze test signifies increased ability to learn in ICV STZ injected rats

treated with combination of the two drugs at the doses specified. Moreover, results obtained show that

the improvement in memory is even more than that observed with single drug therapy with either of the

drugs.

Conclusions

This is the first study to determine the effects of donepezil, lercanidipine and donepezil +

lercanidipine on memory processing in ICV STZ injected rats. ICV STZ increases cholinesterase activity

in rat brains and this, along with some reports on cholinergic deficiency in this model, provided a

rationale for use of donepezil in this model. Donepezil dose-dependently inhibits cholinesterase activity

and improvement in memory by donepezil correlates well with inhibition of cholinesterase activity. 1

mg/kg of donepezil brings cholinesterase activity down to normal but 3 mg/kg reduces the activity by

around 23% as compared to normal.

Lercanidipine (0.3 mg/kg) has favorable effect on memory processing, but higher dose (1 mg/kg)

reduces blood pressure and locomotor activity. However, it exerts central actions at a dose that does

not have any cardiovascular effects, which is in contrast to many other calcium channel blockers.

This provides a rationale for use of lercanidipine in therapy for dementia. The study clearly

demonstrates that donepezil significantly improves memory in ICV STZ rats and reaffirms that there

is cholinergic deficiency in ICV STZ injected rats. The results give an evidence for involvement of

L-type calcium channel overactivation in memory impairment in ICV STZ injected rats and provide

a rationale for the use of lercanidipine in the treatment of age-related cognitive impairment.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–1412

Lercanidipine when administered along with donepezil, enhances the effect of donepezil on both

memory as well as cholinesterase activity. The effect of combination of donepezil and lercanidipine was

higher than that obtained with either of the drug when administered alone at given dose. The dose of

each drug and therefore its peripheral side effects could be reduced by using combination of reduced

doses of the two drugs, still getting desired effect.

Acknowledgement

The authors acknowledge the gift samples provided by Hetero Drugs Pvt Ltd, Hyderabad and

Recordati Industria Chimica, Italy.

References

Areosa, S.A., Sherriff, F., 2003. Memantine for dementia. The Cochrane Database of Systematic Reviews 3, CD003154.

Balducci, C., Nurra, M., Pietropoli, A., Samanin, R., Carli, M., 2003. Reversal of visual attention dysfunction after AMPA

lesions of the nucleus basalis magnocellularis (NBM) by the cholinesterase inhibitor donepezil and by a 5-HT1A receptor

antagonist WAY 100635. Psychopharmacology (Berl) 167 (1), 28–36.

Biessels, G.J., van der Heide, L.P., Kamal, A., Bleys, R.L., Gispen, W.H., 2002. Ageing and diabetes: implications for brain

function. European Journal of Pharmacology 441 (1–2), 1–14.

Blokland, A., Jolles, J., 1993. Spatial learning deficit and reduced hippocampal ChAT activity in rats after an ICV injection of

streptozotocin. Pharmacology Biochemistry and Behavior 44 (2), 491–494.

Brooks, S.P., Hennebry, G., McAlpin, G.P., Norman, G., Little, H.J., 2002. Nimodipine prevents the effects of ethanol in tests of

memory. Neuropharmacology 42 (4), 577–585.

Carey, G.J., Billard, W., Binch III, H., Cohen-Williams, M., Crosby, G., Grzelak, M., Guzik, H., Kozlowski, J.A., Lowe, D.B.,

Pond, A.J., Tedesco, R.P., Watkins, R.W., Coffin, V.L., 2001. SCH 57790, a selective muscarinic M(2) receptor antagonist,

releases acetylcholine and produces cognitive enhancement in laboratory animals. European Journal of Pharmacology 431

(2), 189–200.

Denolle, T., Sassano, P., Allain, H., Bentue-Ferrer, D., Breton, S., Cimarosti, I., Ouatara, B., Merienne, M., Gandon, J.M., 2002.

Effects of nicardipine and clonidine on cognitive functions and electroencephalography in hypertensive patients.

Fundamental and Clinical Pharmacology 16 (6), 527–535.

Dobrian, A.D., Schriver, S.D., Prewitt, R.L., 2001. Role of angiotensin II and free radicals in blood pressure regulation in a rat

model of renal hypertension. Hypertension 38 (3), 361–366.

Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of

acetylcholinetserase activity. Biochemical Pharmacology 7, 88–95.

Freir, D.B., Costello, D.A., Herron, C.E., 2003. A beta 25–35-induced depression of long-term potentiation in area CA1 in vivo

and in vitro is attenuated by verapamil. Journal of Neurophysiology 89 (6), 3061–3069 (Epub 2003 Feb 3065).

Fritze, J., Walden, J., 1995. Clinical findings with nimodipine in dementia: test of the calcium hypothesis. Journal of Neural

Transmission Suppl 46, 439–453.

Frolich, L., Blum-Degen, D., Bernstein, H.G., Engelsberger, S., Humrich, J., Laufer, S., Muschner, D., Thalheimer, A., Turk,

A., Hoyer, S., Zochling, R., Boissl, K.W., Jellinger, K., Riederer, P., 1998. Brain insulin and insulin receptors in aging and

sporadic Alzheimer’s disease. Journal of Neural Transmission 105 (4–5), 423–438.

Gullapalli, S., Ramarao, P., 2002. Regulation of dihydropyridine-sensitive Ca(2+) channels during naloxone-induced opioid

supersensitivity in rats. European Journal of Pharmacology 451 (3), 271–277.

Han, Y., Kim, S.J., 2003. Memory enhancing actions of Asiasari radix extracts via activation of insulin receptor and

extracellular signal regulated kinase (ERK) I/II in rat hippocampus. Brain Research 974 (1–2), 193–201.

Hartz, B.P., Sohoel, A., Berezin, V., Bock, E., Scheel-Kruger, J., 2003. A synthetic peptide ligand of NCAM affects exploratory

behavior and memory in rodents. Pharmacology Biochemistry and Behavior 75 (4), 861–867.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–14 13

Hellweg, R., Nitsch, R., Hock, C., Jaksch, M., Hoyer, S., 1992. Nerve growth factor and choline acetyltransferase activity levels

in the rat brain following experimental impairment of cerebral glucose and energy metabolism. Journal of Neuroscience

Research 31 (3), 479–486.

Hoyer, S., 2002a. The aging brain. Changes in the neuronal insulin/insulin receptor signal transduction cascade trigger late-

onset sporadic Alzheimer disease (SAD). A mini-review. Journal of Neural Transmission 109 (7–8), 991–1002.

Hoyer, S., 2002b. The brain insulin signal transduction system and sporadic (type II) Alzheimer disease: an update. Journal of

Neural Transmission 109 (3), 341–360.

Hoyer, S., Muller, D., Plaschke, K., 1994. Desensitization of brain insulin receptor. Effect on glucose/energy and related

metabolism. Journal of Neural Transmission Suppl 44, 259–268.

Ikegaya, Y., Nishiyama, N., Matsuki, N., 2000. L-type Ca(2+) channel blocker inhibits mossy fiber sprouting and cognitive

deficits following pilocarpine seizures in immature mice. Neuroscience 98 (4), 647–659.

Isoma, K., Ishikawa, M., Ohta, M., Ogawa, Y., Hasegawa, H., Kohda, T., Kamei, J., 2002. Effects of T-82, a new quinoline

derivative, on cholinesterase activity and extracellular acetylcholine concentration in rat brain. The Japanese Journal of

Pharmacology 88 (2), 206–212.

Izquierdo, I., 1990. Nimodipine and the recovery of memory. Trends in Pharmacological Sciences 11 (8), 309–310.

Kasa, P., Papp, H., Kasa Jr., P., Torok, I., 2000. Donepezil dose-dependently inhibits acetylcholinesterase activity in various

areas and in the presynaptic cholinergic and the postsynaptic cholinoceptive enzyme-positive structures in the human and rat

brain. Neuroscience 101 (1), 89–100.

Kosasa, T., Kuriya, Y., Matsui, K., Yamanishi, Y., 1999. Inhibitory effects of donepezil hydrochloride (E2020) on cholinesterase

activity in brain and peripheral tissues of young and aged rats. European Journal of Pharmacology 386 (1), 7–13.

Mason, R.P., Leeds, P.R., Jacob, R.F., Hough, C.J., Zhang, K.G., Mason, P.E., Chuang, D.M., 1999. Inhibition of excessive

neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells. Journal of

Neurochemistry 72 (4), 1448–1456.

Matsui, K., Mishima, M., Nagai, Y., Yuzuriha, T., Yoshimura, T., 1999. Absorption, distribution, metabolism, and excretion of

donepezil (Aricept) after a single oral administration to Rat. Drug Metabolism and Disposition 27 (12), 1406–1414.

Meredith, P.A., 1999. Lercanidipine: a novel lipophilic dihydropyridine calcium antagonist with long duration of action and

high vascular selectivity. Expert Opinion on Investigational Drugs 8 (7), 1043–1062.

Norman, G., Brooks, S.P., Hennebry, G.M., Eacott, M.J., Little, H.J., 2002. Nimodipine prevents scopolamine-induced

impairments in object recognition. Journal of Psychopharmacology 16 (2), 153–161.

Ogura, H., Kosasa, T., Kuriya, Y., Yamanishi, Y., 2000. Donepezil, a centrally acting acetylcholinesterase inhibitor, alleviates

learning deficits in hypocholinergic models in rats. Methods and Findings in Experimental and Clinical Pharmacology 22

(2), 89–95.

Park, C.R., 2001. Cognitive effects of insulin in the central nervous system. Neuroscience and Biobehavioral Reviews 25 (4),

311–323.

Petkov, V.D., Konstantinova, E., Petkov, V.V., Belcheva, S., Kehayov, R., Vaglenova, J., 2000. Memory effects of the Ca2+ and

5-HT antagonists dotarizine and flunarizine. Acta Physiol Pharmacol Bulg 25 (2), 43–50.

Quartermain, D., 2000. Chronic administration of the Ca(2+) channel blocker amlodipine facilitates learning and memory in

mice. European Journal of Pharmacology 399 (1), 57–63.

Quartermain, D., Garcia deSoria, V., 2001. The effects of calcium channel antagonists on short-and long-term retention in mice

using spontaneous alternation behavior. Neurobiology of Learning and Memory 76 (1), 117–124.

Sberna, G., Saez-Valero, J., Beyreuther, K., Masters, C.L., Small, D.H., 1997. The amyloid beta-protein of Alzheimer’s disease

increases acetylcholinesterase expression by increasing intracellular calcium in embryonal carcinoma P19 cells. Journal of

Neurochemistry 69 (3), 1177–1184.

Scarpini, E., Scheltens, P., Feldman, H., 2003. Treatment of Alzheimer’s disease: current status and new perspectives. Lancet

Neurology 2 (9), 539–547.

Sharma, M., Gupta, Y.K., 2002. Chronic treatment with trans resveratrol prevents intracerebroventricular streptozotocin induced

cognitive impairment and oxidative stress in rats. Life Sciences 71 (21), 2489–2498.

Sharma, M., Gupta, Y.K., 2001. Effect of chronic treatment of melatonin on learning, memory and oxidative deficiencies

induced by intracerebroventricular streptozotocin in rats. Pharmacology Biochemistry and Behavior 70 (2–3), 325–331.

Shoham, S., Bejar, C., Kovalev, E., Weinstock, M., 2003. Intracerebroventricular injection of streptozotocin causes

neurotoxicity to myelin that contributes to spatial memory deficits in rats. Experimental Neurology 184 (2), 1043–1052.

S. Sonkusare et al. / Life Sciences 77 (2005) 1–1414

Silei, V., Fabrizi, C., Venturini, G., Salmona, M., Bugiani, O., Tagliavini, F., Lauro, G.M., 1999. Activation of microglial cells

by PrP and beta-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels. Brain

Research 818 (1), 168–170.

Suh, Y.H., Checler, F., 2002. Amyloid precursor protein, presenilins, and alpha-synuclein: molecular pathogenesis and

pharmacological applications in Alzheimer’s disease. Pharmacological Reviews 54 (3), 469–525.

Terwel, D., Prickaerts, J., Meng, F., Jolles, J., 1995. Brain enzyme activities after intracerebroventricular injection of

streptozotocin in rats receiving acetyl-L-carnitine. European Journal of Pharmacology 287 (1), 65–71.

Tokita, K., Yamazaki, S., Yamazaki, M., Matsuoka, N., Mutoh, S., 2002. Combination of a novel antidementia drug FK960 with

donepezil synergistically improves memory deficits in rats. Pharmacology Biochemistry and Behavior 73 (3), 511–519.

van der Lee, R., Pfaffendorf, M., van Zwieten, P.A., 2000. The differential time courses of the vasodilator effects of various 1,4-

dihydropyridines in isolated human small arteries are correlated to their lipophilicity. Journal of Hypertension 18 (11),

1677–1682.

Veerendra Kumar, M.H., Gupta, Y.K., 2002. Intracerebroventricular administration of colchicine produces cognitive impairment

associated with oxidative stress in rats. Pharmacology Biochemistry and Behavior 73 (3), 565–571.

Veng, L.M., Mesches, M.H., Browning, M.D., 2003. Age-related working memory impairment is correlated with increases in

the L-type calcium channel protein alpha1D (Cav1.3) in area CA1 of the hippocampus and both are ameliorated by chronic

nimodipine treatment. Brain Research Molecular Brain Research 110 (2), 193–202.

Weiss, J.H., Pike, C.J., Cotman, C.W., 1994. Ca2+ channel blockers attenuate beta-amyloid peptide toxicity to cortical neurons

in culture. Journal of Neurochemistry 62 (1), 372–375.

Weinstock, M., Kirschbaum-Slager, N., Lazarovici, P., Bejar, C., Youdim, M.B., Shoham, S., 2001. Neuroprotective effects of

novel cholinesterase inhibitors derived from rasagiline as potential anti-Alzheimer drugs. Annals of New York Academy of

Sciences 939, 148–161.

Yagami, T., Ueda, K., Sakaeda, T., Itoh, N., Sakaguchi, G., Okamura, N., Hori, Y., Fujimoto, M., 2004. Protective effects of a

selective L-type voltage-sensitive calcium channel blocker, S-312-d, on neuronal cell death. Biochemical Pharmacology 67

(6), 1153–1165.