SupervisorDr. Satyanarayana S. V. Padi

Dinesh Kumar DhullB.Pharma.

Co-supervisorMr. Saurabh Sharma

I.S.F. COLLEGE OF PHARMACY, MOGA, PUNJAB

NEUROPROTECTIVE EFFECT OF CYCLOOXYGENASE INHIBITORS IN SPORADIC TYPE OF ALZHEIMER’S

DISEASE

Alzheimer Disease (AD) is aProgressive, irreversible, age-related neurological and psychiatric disorder characterized byProgressive decline in memoryCognitive performanceLoss of acquired skills leading toApraxia, Agnosia, Anomia and Aphasia (Stuchbury and Munch, 2005)

It was named after Alois Alzheimer who was the first to described the disorder in a 51 year old women Auguste D., in 1907 (Maurer et al., 1997)

cont….

Basically, AD is of two types:• Early onset or Familial AD• Late onset or Sporadic AD

The prevalence of AD is rapidly increasing affecting more than 26 million people around the world and this will quadruple by 2050. (Magdalena et al., 2008)

cont….

Familial AD(Early onset)

Genetic abnormalities

Sporadic AD(Late onset)

Insulin signaling and Energy metabolism alterations

• Mutation in certain genes like PS-1,PS-2 or APP gene.

• Neuronal insulin receptor (IR) desensitization• Energy homeostasis imbalance in brain• Decrease in ATP • Decrease in GLUT-1 and GLUT-3 in brain

• Aβ peptide formation• Neurofibrillary tangles

(NFT) formation

cont….

• Oxidative stress• Neurotransmitter abnormalities

• Aβ peptide formation• Neurofibrillary tangles (NFT)

formation

Taskasu et al, 1993; Salkovic et al., 2006; Emilia et al., 2008

CHARACTERISTIC FEATURES

Sporadic Alzheimer’s disease is the most common type of dementia which includes:• Energy metabolism alterations• Oxidative stress• Neuroinflammation• Excitotoxicity• Neurotransmission abnormalities etc.• Senile plaque formation • Neurofibrillary tangles

NEUROPATHOLOGY

Intracerebro-ventricular (I.C.V.) administration of streptozotocin (STZ) resulted in-insulin receptor desensitization behavioral, biochemical, histological and neurochemical alterationsalong with progressive deterioration of cognitive function similar to those seen in sporadic type of alzheimer patients. (Ding et al., 1992; Salkovic et al., 2003)

Animal Model For Experimental Dementia

How I.C.V. STZ induces dementia?ICV STZ

IR desentitization

↓ glucose uptake ↓ GLUT-1 & GLUT-3 ↓ glucose metabolism

↓ ATP

Energy Homeostasis Imbalance

Mitochondrial dysfunction↑ Ca2+

Oxidative Stress

NeurodegenerationHellwag et al., 1992; Miller et al., 2006

• Decreases glucose transporter proteins GLUT-1 and GLUT-3 (Simpson et al., 1994; Harr et al., 1995)

• Decrease in cholineacetyltransferase activity (Hellwag et al., 1992; Blokland et al., 1994)

• Insulin deficiency leads to rapid and large increases in tau phosphorylation (Miller et al., 2006)

• Alters nerve growth factors (NGF) and monoaminergic neurotransmitter (Ding et al., 1992)

• Increase in brain lactate and melondialdehyde and decrease in activities of antioxidative enzymes

(Szkudelski et al., 2001; Sharma et al., 2002; Tahirovic et al., 2007)

• Decreases phospholipids, activates PLA2 and increases free fatty acids (Hoyer et al.,1998)

How I.C.V. STZ induces dementia?

Ramified Microglia Microglial activationROS/RNS

Neuritic Plaques

Chemokines

CyclooxygenaseiNOS

Complement factors

Excitatory amino acids Pro-inflamm.

CytokinesIL-1β,IL-6,TNF-α

Neurodegeneration

NEUROINFLAMMATION

PGD2 PGE2

PGD synthase PGE isomerase

CNS PERIPHERY

PGI2

PGF2 TXA2

PGI synthase PGF synthase TXA synthase

COOH

COOH

COX-1/COX-2/COX-3

COX-1/COX-2/COX-3

Cyclization(2o2)

Peroxidation

PGF1α

PGG2-OOH

PGH2-OH

NADH

NAD.

O2

O2. -

2e.

CYCLOOXYGENASE

• COX-1 expression is present in neurons and microglia (Hirst et al., 1999)

• COX-2 is expressed constitutively and inducible under pathological conditions in the CNS including cortex, hippocampus, hypothalamus and spinal cord. (Breder et al., 1995, 1996)

• COX-3 was first identified as a splice varient of COX-1 by Chandrasekharan in dog brain and is biologically stable. (Chandrasekharan et al., 2002)

• COX-3 is highly expressed in choroid plexus and spinal cord followed by hypothalamus, hippocampus, medulla, cerebellum, cortex, in major brain arteries and brain microvessels but not expressed in neurons.

(Kis et al., 2003,; 2004)

CYCLOOXYGENASE cont….CYCLOOXYGENASE cont….

COX-1 gene

COX-2 gene

COX-1 constitutive

COX-3 constitutive

COX-2b ?? COX-2 constitutive COX-2 inducible

Modulation of sensory process

Pain & FeverNerve

TransmissionNeuroinflammation

Neuronal Death

CNS

Yamagata et al.,1993

Adaptive function

(Normal neurons)

Protective function

(Stressed neurons)

Turini et al., 2002

pCOX1

pCOX1a pCOX1bChandrasekharan et al.,2002

COX-1 gene

COX-2 gene

COX-1 gene CNS

COX-2 gene

COX-1 gene CNS

COX-2 gene

COX-1 gene

CYCLOOXYGENASE cont….CYCLOOXYGENASE cont….

• Non-selective COX inhibitors showed a reduced number of activated microglia (Jin et al., 2008)

• Selective COX-2 and non-selective COX inhibitors prevent the hypoperfusion induced memory impairment but not by protecting hippocampal neurons. (Institoris et al., 2007)

• COX-2 inhibitor protected the brain against Aβ peptide induced memory disturbances. (Cakala et al., 2007)

• Non-selective inhibitors reduced the excitotoxic neurodegeneration.(Silakova et al., 2004)

CYCLOOXYGENASE cont….

• Non-selective COX inhibitor i.e. Mefenamic acid attenuates the I.C.V. STZ induced cognitive deficit in rats.

(Mojarad et al., 2006)

• However, the differential role of COX isoforms-1, -2 and -3 in the pathophysiology of sporadic Alzheimer’s disease is not clearly known.

• Further, the doses employed in the previous studies were selected randomly that may inhibit two or more isoforms.

CYCLOOXYGENASE cont….

HYPOTHESIS ???

To evaluate the neuroprotective effect of cyclooxygenase-1, -2 and -3 inhibitors in pathophysiology of sporadic Alzheimer’s disease.

AIMS AND OBJECTIVES

• AnimalsStrain Wistar ratWeight 200-250gSex MaleFood and Water ad libitum

• MaterialsStreptozotocin (3mg/kg i.c.v.) dissolved in 0.1M citrate buffer pH 4.5 Valeryl salicylate (Selective COX-1 inhibitor)Etoricoxib (Selective COX-2 inhibitor) andPhenacetin (Selective COX-3 inhibitor) dissolved in 0.5% sodium CMC.

:MATERIALS AND METHODS

TREATMENT SCHEDULE

Day 1 Day 4 – 202 3 22

ICVSTZ

ICVSTZ

Administration of COX-1 or -2 or -3 inhibitor

Behavioral analysis

21

Day ofsacrifice

1. Sham control2. ICV STZ control (3 mg/kg, i.c.v.)3. ICV STZ + Etoricoxib (5 mg/kg; i.p.)4. ICV STZ + Etoricoxib (10 mg/kg; i.p.)5. Etoricoxib per se (10 mg/kg; i.p.) 6. ICV STZ + Phenacetin (20 mg/kg; i.p.)7. ICV STZ + Phenacetin (40 mg/kg; i.p.)8. Phenacetin per se (40 mg/kg; i.p.)9. ICV STZ + Valeryl salicylate (5 mg/kg; i.p.)10.ICV STZ + Valeryl salicylate (10 mg/kg; i.p.)11.Valeryl salicylate per se (10 mg/kg; i.p.)

TREATMENT GROUPS

BEHAVIOURAL STUDIES

Assessment of cognitive function:

1. Spatial learning and memory using Morris water maze task(Duckworth et al., 1999)

2. Spatial memory using Modified Elevated plus maze (Gupta et al., 2001)

Assessment of behavioral activity:

1. Motor activity using Actophotometer (Gupta et al., 2001)

Acetylcholine esterase (AChE) (Ellman method, 1961)Lactate dehydrogenase (LDH) (Racon Diagnostic Kit)Superoxide dismutase (SOD) (Mishra and Fridovich, 1972)Nitrite (Green et al, 1982)Lipid peroxidation (LPO) (Will method, 1966)reduced Glutathione (GSH) (Ellman, 1959)Total protein (Biuret method)

OTHER PARAMETERSBiochemical parameters

Histopathological Studies

• Results are expressed as mean ± S.D.• Data for behavioral analysis was analyzed by two way

ANOVA followed by Bonferroni’s posthoc-test for multiple comparisons.

• Data for biochemical analysis was analyzed by one way ANOVA followed by posthoc Tukey’s test for multiple comparisons.

• P < 0.05 considered statistically significant

STATISTICAL ANALYSIS

Intracerebroventricular (ICV) administration of streptozotocin (STZ) induces cognitive impairment, cholinergic deficit, oxidative stress, neuroinflammation and neuronal death.

In the present study, administration of selective COX-1 or -2, but not COX-3, inhibitor attenuated ICV-STZ induced behavioral and biochemical alterations.

DISCUSSION

Accumulating data indicates that central administration of STZ causes• Neuroinflammation

o ATP depletion and uncontrolled leakage of ions causing membrane depolarization

o (Ter Horst and Korf, 1997)o Overproduction of neurotransmitters such as glutamate that lead to

excessive Ca2+ influx (Adibhatla and Hatcher, 2005)o Further activates PLA2, phospholipid hydrolysis and release of AAo -a substrate for COX and is converted into final products i.e PGs o (Lipton, 1999; Phillis and O’Regan, 2003) o Ultimately leading to apoptotic or necrotic cell death. (Ho et al., 1998)

It has been reported that lercanidipine improved the cognitive dysfunction by reducing oxidative stress and AChE activity suggesting the involvement of Ca2+ influx in ICV-STZ induced neurotoxicity and subsequent neuroinflammation. (Sonkusare et al., 2005)

DISCUSSION cont…..

• COX activaton occurs secondary to NMDA receptor activation and increased Ca2+ influx. (Miettinen et al., 1997)

• PGs produced, are neurotoxic in action contributing to neuronal and synaptic loss followed by neurodegeneration. (Kukreja et al., 1986; Bezzi et al., 1998)

• It is known that the induction of PGs formation by central administration of PGE2 or COX inducers such as endotoxin LPS or IL-1β is involved in loss of working and spatial memory. (Jain et al., 2002; Matsumoto et al., 2004; Hein et al., 2007)

• Previous evidence indicates that non-selective COX (COX-1/-2) or selective COX-2 inhibitors improved the memory performances in both EPM and MWM paradigms and also reduce increased AChE activity in various neurotoxin induced models of dementia. (Kumar et al., 2006; Kumari et al., 2007)

• Based on the present data, it is possible that COX-1 and/or COX-2 are activated following ICV-STZ injection.

DISCUSSION cont…..

DISCUSSION cont….. • Oxidative/Nitrosative stress (Shoham et al., 2007; Tiwari et al., 2009)

• During the peroxidase activity of COX, one superoxide free radical is generated that results in ROS production which is an important mediator of neuronal injury. (Pepicelli et al., 2002; Candelario-Jalil et al., 2003; Madrigal et al., 2003)

• ROS formation and subsequent O.S. may cooperate in a series of molecular events that link to memory impairment.

(Beal et al., 1995; Bruce-Keller et al., 1998; Kumar et al., 2006) • Also, COX and iNOS are co-localized in brain cells and COX regulates the

expression of iNOS. (Nogawa et al., 1998)• Induction of COX expression further incresae the expression of iNOS that results

in NO-• free radicals and peroxynitrite generation. Indeed, COX inhibition is reported to decrease iNOS activity. (Goodwin et al., 1999)

• Also, the antioxidant defence mechanism including endogeneous GSH and SOD, is altered.

Previous study indicate that activation of COX is required for execution of oxidative neuronal death. (Lee et al., 2001)

However, NSAIDs administration prevents glutamate mediated excitotoxicity and subsequent O.S. (Fass et al., 2000; Jain et al., 2002; Scali et al., 2003; Silakova et al., 2004)

Thus, it is plausible that ICV-STZ induces activation of COX-1 and/or -2 that results in oxidative and nitrosative stress mediated neurotoxicity.

In the present study, the selective COX-1 and/or -2, but not COX-3, inhibitor significantly attenuated the increased oxidative stress markers i.e. malondialdehyde and nitrite and improved antioxidant defences such as SOD and GSH in the brain of ICV-STZ treated rats.

DISCUSSION cont…..

Prostaglandins such as PGE1 and PGE2 cause neurotoxicity that results in neuronal death. (Kukreja et al., 1986; Hewett et al., 2000; Mana et al., 2002)

LDH – a well known marker of cell death and pathological changes in cortex and hippocampus.

ICV-STZ administration in rats markedly increase the LDH activity and neuronal death in cortex and hippocampus (Hoyer and Lannert, 2007)

Also, COX-2 inhibitor show their cerebroprotection action by reducing infracts in rats following cerebral ischemia (Sugimoto and Iadecola, 2003).

In another study, selective inhibition of COX-1 and/or -2 significantly increases the number of healthy neurons in the CA1 hippocampus. (Sasaki et al. 1988; Nakagomi et al. 1989; Hall et al. 1993 Candelaria-Jalil et al., 2002)

DISCUSSION cont…..

DISCUSSION cont…..

These data along with the present study suggest the neuroprotective effect of COX-1 and/or -2 inhibitors in ICV-STZ-treated rats.

It further suggest that COX-1 and/or -2, but not COX-3, activity is involved in the morphological changes in neuronal cells and the progression of their damage following ICV-STZ administration.

One possibility, that COX-3 inhibitor fail to show protection, is that COX-3 might be sparsely distributed and/or COX-3 might not be induced in the brain areas that are prone to neuronal damage following central STZ injection.

Selective COX-1 or -2, but not COX-3, inhibitor showed the neuroprotective effect in ICV-STZ induced experimental model of SAD.

They significantly-• improved impaired cognitive performance,• attenuated oxidative stress,• improved altered cholinergic function and • reduced neuronal damage

CONCLUSION

“A theory is something nobody believes, except the person who made it.An experiment is something everybody believes, except the person who made it”

Albert Einstein