RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate and address(in block letters) /

AMBIKA S,

D/O SHIVA SHANMUGAM K,
#446, FIRST MAIN, FIFTH CROSS,
KUVEMPUNAGAR,
BANGALORE-560016
PH-(080)25657837
2. /

Name of the institution

/

VISVESWARAPURA INSTITUTE OF PHARMACEUTICAL SCIENCES

3. /

Course of study and subject

/

MASTER OF PHARMACY IN

PHARMACOLOGY

4. /

Date of the admission

/ 16th JUNE 2008
5.
6. /

Title of the topic:

“ETORICOXIB AS A BIOLOGICAL PROBE OF COX-2 MECHANISMS IN ECS INDUCED AMNESIA.”
BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY
Electroconvulsive therapy (ECT) is a procedure in which generalized seizures lasting 25 to 150 seconds, induced by the passage of an electrical current through the brain under general anesthesia, and muscle relaxation are used for therapeutic purposes. One of the most effective and safest available treatments for depression, ECT has also been found beneficial in mania, schizophrenia, catatonia, and other neuropsychiatric conditions.1
ECT produces memory and nonmemory cognitive deficits. Memory deficits with ECT include impaired recall (retrograde amnesia) and impaired new learning (anterograde amnesia). Nonsteroidal antiinflammatory drugs (NSAIDs) have been suggested to retard cognitive decrements in patients with Alzhiemer’s disease.2-4Memory protective effect of indomethacin (a nonselective COX inhibitor) against electro-convulsive shock induced retrograde amnesia in rats has been reported.5A negative association between Celecoxib (a selective COX-2 inhibitor) and amnesia in rats has been reported.6As Celecoxib has been withdrawn from market, we sought to experimentally determine the potential ability of Etoricoxib,7(a COX-2 selective inhibitor with the highest selectivity ratio of any coxib for inhibition of COX-2 relative to COX-1) in amnesia. Therefore etoricoxib is selected for the study in ECT induced amnesia.
6.2REVIEW OF LITERATURE
Theory of ECT action.8
The effectiveness of ECT is tied to the production of a series of generalized tonic-clonic type seizures. Evidence to support this contention comes from clinical comparisons of ECT with pharmacoconvulsive therapy and subconvulsive electrical stimulation, as well as from sham ECT studies.
Attempts to elucidate the biological mechanisms by which a therapeutic response has been brought about by ECT have covered three general areas: neurochemical, neuoendocrine, and neurophysiological. Neurochemical basis of ECT in depression is linked to its ability to overcome a hypofunctional central noradrenaline (NA) balance. Also noradrenergic activation hypothesis adds on as a complementary finding to neurochemical mechanism of ECT. Down regulation of 5-HT2 receptors forms the basis of ECT action in mania. The possibility that activation of neurophysiological inhibitory processes may at least in part underlie ECT’s clinical effectiveness in petit-mal status epilepticus. After induced seizures, for example, a period of suppression of neuronal activity is seen that is associated with a generalized hypometabolic state. Also accompanying induced seizures is a transient breakdown of the blood-brain barrier, offering a means by which potential endogenous anticonvulsant substances, once released by the ictus, could spread to other brain regions.
Indications for ECT.8
  1. Major depressive episodes
  2. Schizophrenia
  3. Mania
Complications of ECT.1
  1. Non-memory deficits
  2. Cardiovascular side effects
  3. Treatment-emergent confusion
  4. Treatment-emergent mania
  5. Sore muscles
  6. Postictal and interictal delirium
  7. Memory deficits with ECT
  • Anterograde amnesia
  • Retrograde amnesia
  • Autobiographical amnesia
Nonsteroidal antiinflammatory drugs (NSAIDs) are being explored as putative antidementia agents. Cognitive benefits with NSAIDs were posited as a result of three strands of evidence:
  1. A meta-analysis of 17 epidemilogical studies from nine countries showed a negative association between NSAIDs use (in patients with arthritis) and the incidence of Alzhiemer’s disease.2
  2. Inflammatory changes are demonstrable in brain tissue from patients with Alzhiemer’s disease.3
  3. In a 6-month study of mild to moderately impaired Alzhiemer’s patients, indomethacin in standard anti-inflammatory doses (100-150 mg/day) was found to protect against the cognitive decline that was observed in placebo-treated patients.4
Prostaglandin endoperoxide synthases or Cyclooxygenases (COX-1 and COX-2) play a central role in the inflammatory cascade by converting arachidonic acid, released from membrane phospholipids by a phospholipase A2, into prostaglandin endoperoxide H2 which in turn is converted to bioactive prostanoids by specific terminal synthases. The two COX isoforms COX-1 and COX-2 are key mediators of the inflammatory response in the CNS. Memory protective effect of Indomethacin(a non-selective COX inhibitor) against ECS-induced retrograde amnesia in rats has been reported.5
The importance of COX-2 selectivity in cognition was emphasized following the observation that long-term potentiation (LTP) was normal in COX-1 knockout mice.9LTP is a glutamate-dependent process which is suggestedto be the synaptic representation of learning andmemory in brain structures such as the hippocampus.10Cyclooxygenase-2 (COX-2) mechanisms areinvolved in glutamate-mediated learning and memory as well as in glutamatergic excitotoxicity. Electroconvulsivetherapy (ECT)-induced amnesia may arise from glutamatergicexcitotoxicity; if so, COX-2 inhibition would attenuateretrograde amnesia with ECT.6Etoricoxib, a bipyridine derivative is currently used for the treatment of osteoarthritis, rheumatoid arthritis, gouty arthritis, and acute musculoskeletal pain. Its role in dementia would be an exploratory research to understand COX-2 mechanisms involved in learning and memory.
6.3OBJECTIVE OF THE STUDY
TO STUDY THE EFFECT OF ETORICOXIB ON ECS-INDUCED AMNESIA USING THE FOLLOWING TWO MODELS:
  1. Passive avoidance learning in the step-down apparatus
  2. Spatial learning in water maze
MATERIAL AND METHODS
7.1SOURCES OF DATA
Journals
  1. Journal of Neuroinflammation
  2. Journal of Pharmacology and Experimental Therapeutics
  3. Journal of Biological Psychiatry
  4. Journal of ECT
Text books
  1. Comprehensive Textbook of Psychiatry
  2. Text book of Basic and Clinical Pharmacology
Online sources
  1. Pubmed central
  2. Science direct
  3. Springer link
  4. METHOD OF COLLECTION OF DATA
Data will be generated by performing the experiment using standard animal models.
METHODOLOGY
Rats will be housed 4 per cage with free access to water and standard laboratory diet. The rats will be allowed to acclimatize to the laboratory conditions for a week before the start of experiment.
Animals required
  1. Species : Wistar rat
  2. Age/ weight/ size : About 2-3 months (150- 200g)
  3. Gender : Male
  4. Number to be used : 320
  5. Number of days each animal will be housed : 30 days
MODEL 1
Passive avoidance learning in step-down apparatus
Four groups of animals of 40 male Wistar rats with a body weight of 150-200g will be used. Each group of forty animals would be further randomized into two equal groups to receive either true ECS or sham ECS. The four main groups of animals would receive either vehicle, low dose Etoricoxib, higher dose Etoricoxib or Celecoxib. ECS is admisitered using an electroconvulsiometer through gel-coated ear-clip electrodes. The dose of ECS is 60 mC for 0.4 sec. Sham ECS is a placebo with a dose of 0 mC The ability of Etoricoxib to provide neuroprotection against ECS induce amnesia will be studied.
Group 01 would receive vehicle p.o
Group 02 would receive Celecoxib as standard at a dose of 15 mg/kg/day p.o
Group 03 would receive Etoricoxib at a lower dose of 10 mg/kg/day p.o
Group 04 would receive Etoricoxib at a higher dose of 30 mg/kg/day p.o
EXPERIMENTAL METHODOLOGY.6
Acclimatization
On each of 3 days before the start of the experiment, the animals will be acclimatized briefly in the step down apparatus.
Training in the step – down apparatus
Once a day for three consecutive days, each rat will be trained in the apparatus using a single 60 V, 0.5 s footshock as the aversive stimulus when the animal steps down. On the third day, the time taken by the animal to step down will be recorded as the baseline step down latency.
This apparatus comprises of a 50 cm square box measuring 35 cm in height. The floor of the box formed with 50 steel rods, each 4mm thick and set 6 mm apart. A circular wooden platform, 15 cm across and 5.5 cm high, would be located in the center of the box and enclosed in a hollow, removable cylinder.
During training, each rat will be placed upon the platform within the cylinder. The cylinder will be removed. Immediately after the animal steps down, it would receive a 60 v sinusoidal wave electric shock for 1.2 s. Subsequently the animal will be returned to its home cage. On each occasion the time taken by the animal to step down will be recorded. These times would be designated as the baseline step-down latencies.
Only animals which show adequate or perfect learning will be used in the rest of the study. Adequate and perfect learning refer to baseline step down latencies of 30s and 180s, respectively. To the extent possible, adequate and perfect learners will independently be randomized to receive drug or vehicle. Treatments will be administered once each morning, by slow intra – oral syringing , about 1 – 2 hours pre – ECS on each day that the rats receive true or sham ECS.
Administration of ECS
In each drug group, rats will be sub – randomized to receive true or sham ECS. True ECS will be administered once – daily for 3 consecutive days , through gel – coated ear clip electrodes, using the constant current , brief – impulse ECT device, the stimulus was formed from 0.8 A pulse amplitude, 1.5 ms pulse width, 62.5 Hz pulse frequency bidirectional square waves administered in a stimulus train 0.2 s long,at 30 – 60 mC charge. The seizures will be timed using a stopwatch. Sham ECS will involve an identical procedure sans passage of current.
Assessment of recall
On the day after the last ECS, the animals will be re – exposed to the step down apparatus and the time taken by the animals to step down will be noted as before; this will be designated as the first post –ECS step down latency. In animals which show good recall, the step down latency will be prolonged relative to that at baseline. Therefore, higher final step-down latency scores reflect better recall. Once – daily true and sham ECS will then be resumed for 2 more days, one day after which the second post – ECS step down latency will be recorded as above.
MODEL 2
Spatial learning in water maze
Four groups of animals of 40 male Wistar rats with a body weight of 150-200g will be used. Each group of forty animals would be further randomized into two equal groups to receive either true ECS or sham ECS. The four main groups of animals would receive either vehicle, low dose Etoricoxib, higher dose Etoricoxib or Celecoxib. ECS is admisitered using an electroconvulsiometer through gel-coated ear-clip electrodes. The dose of ECS is 60 mC for 0.4 sec. Sham ECS is a placebo with a dose of 0 mC. The ability of Etoricoxib to provide neuroprotection against ECS induce amnesia will be studied.
Group 01 would receive vehicle p.o
Group 02 would receive Celecoxib as standard at a dose of 15 mg/kg/day p.o
Group 03 would receive Etoricoxib at a lower dose of 10 mg/kg/day p.o
Group 04 would receive Etoricoxib at a higher dose of 30 mg/kg/day p.o
Experimental Methodology.11,12
Acclimatization
Male Wistar rats will be tested ina Morris water maze (180 cm diameter, 60 cm high) filled with water (21°C) made opaque by addition of milk powder. An escape platform will be hidden 2 cm below the surface of the water in a fixed location in one offour quadrants halfway between the wall and the middle of thepool. Before the start of training, animals will be habituated to the pool without a platform 1 min/day for 3 days.
Training in water maze
During training,animals will be required to locate the submerged platformby usingdistal extramaze cues. They will be tested for four trials per day (90s with an intertrial interval of 30 s and beginning from threedifferent start points that varied randomly each day). If an animal did not find the platform, it will be set on it at the end of thetrial. The time to reach the platform (latency in seconds) would be measured. Well trained rats with an escape latency of 10 s would be included into the study. A free-swim trial would generally be performed after the training days where the escape platform is removed and the animal is allowed to swim for 30 s.
To the extent possible, adequate and perfect learners will independently be randomized to receive drug or vehicle. Treatments will be administered once each morning, by slow intra – oral syringing , about 1 – 2 hours pre – ECS on each day that the rats receive true or sham ECS.
Administration of ECS
In each drug group, rats will be sub – randomized to receive true or sham ECS. True ECS will be administered once – daily for 3 consecutive days , through gel – coated ear clip electrodes, using the constant current , brief – impulse ECT device the stimulus, formed from 0.8 A pulse amplitude, 1.5 ms pulse width, 62.5 Hz pulse frequency bidirectional square waves administered in a stimulus train 0.2 s long, at 30 – 60 mC charge. The seizures will be timed using a stopwatch. Sham ECS will involve an identical procedure sans passage of current.
Assessment of recall
On the day after the last ECS, the animals will be re – exposed to the water maze and the time taken by the animals to escape will be noted as before; this will be designated as the first post –ECS escape latency. In animals which show good recall, the escape latency will be shortened relative to that at baseline. Therefore, lower final escape latency scores reflect better recall. Once – daily true and sham ECS will then be resumed for 2 more days, one day after which the second post – ECS escape latency will be recorded as above.
STASTICAL ANALYSIS
The mean values ±SEM will be calculated for each parameter. The data would be analyzed usingtwo-way ANOVA, followed by Dunnet test.
7.3Does the study require any investigations or interventions to be conducted on patients or other human beings or animals? If yes, please describe briefly.
Yes, the study requires investigation on animals. The effect of the drug will be studied on various animal models.
7.4Has ethical clearance been obtained from your institute in case of 7.3?
Yes, institutional ethical clearance certificate is enclosed.
REFERENCES:
  1. Kaplan HI, Sadock BJ. Comprehensive textbook of Psychiatry. 5th ed. Baltimore: Williams & Wilkins. 1989. p. 2129,2136-38.
  2. Mc Geer PL, Schulzer M, Mc Geer EG. Arthritis and anti-inflammatory agents as possible factors for Alzhiemer’s disease. Neurology. 1996;47:425-32.
  3. Aisen PS, Davis KL. Inflammatory mechanisms in Alzhiemer’s disease: Implications for therapy. Am J Psychiatry. 1994;151:1105-13.
  4. Rogers J, Kirby LC, Hempelmen SR, Berry DL, Mc Geer PL, Kaszniak AW, et al. Clinical trial of indomethacin in Alzhiemer’s disease. Neurology. 1999;53:197-201.
  5. Sanjay KR, Chittaranjan A, Kishore R, Maddappa KN, Shivashanmugam T, Suresh C. Memory protective effect of indomethacin against electro-convulsive shock induced retrograde amnesia in rats. J Neural Transm. 2002;51:770-73.
  6. Chittaranjan A, Shivashanmugam T, Nagendra MS, Vinod PS, Sanjay KR, Suresh JC. Celecoxib as an in vivo probe of cyclooxygenase-2 mechanismsunderlying retrograde amnesia in an animal model of ECT.J Neural Transm. 2008;115:941-1085.
  7. Bretram GK. Basic & Clinical Pharmacology. 10th ed. Boston: Mc Graw Hill. 2007. 579.
  8. Kaplan HI, Sadock BJ. Comprehensive textbook of Psychiatry. 5th ed. Baltimore: Williams & Wilkins. 1989. p. 1675-76.
  9. Sang N, Chen C. Lipid signaling and synaptic plasticity.Neuroscientist. 2006;12:425–34.
  10. Stewart C, Reid I. Electroconvulsive stimulation and synaptic plasticity in the rat. Brain Res. 1993;620:139–41.
  11. Vogel GH, Wolfgang HV, Bernward AS, Jurgen S, Gunter M, Wolfgang FV. Drug discovery and evaluation. 2nd ed. Berlin, Germany: Springer; 2002. p. 632-33.
  12. Elodie D, Willy M, Catherine, Michel LM, Pier VP, Djoher NA. Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis.Neurosci. 2003;100:14385-90.

09. / SIGNATURE OF THE CANDIDATE
10. / REMARKS OF THE GUIDE / Project proposal is satisfactory
11. / NAME AND DESIGNATION
11.1 NAME OF THE GUIDE / Mrs. BHARATHI K.N
ASST. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
V.I.P.S, BANGALORE-70.
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY)
11.4SIGNATURE / .
11.5HEAD OF THEDEPARTMENT / Dr. RAJU B KONERI
H.O.D; PROFESSOR
DEPARTMENT OF PHARMACOLOGY
V.I.P.S, BANGALORE-70.
11.6 SIGNATURE
12. / 12.1 REMARKS OF THE
CHAIRMAN & PRINCIPAL
12.2 SIGNATURE

1