sandip r final synopsis

SYNOPSIS

For approval of topic of the thesis to be submitted in the partial fulfillment of the

requirement for the degree of Master of Pharmacy in Pharmacology.

“Investigations for pharmacodynamic interaction between agmatine and allopregnanolone in olfactory bulbectomy

induced depression in rats”

By

Sandip R. Rahangdale

B. Pharm.

Guide Co-guide

Prof. C. T. Chopde Dr. R. R. Ugale

M. Pharm., Ph.D., F.I.C. M. Pharm., Ph.D.

s

SMT. KISHORITAI BHOYAR COLLEGE OF PHARMACY,

New Kamptee, Nagpur, Maharashtra, 441 002

Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur.

2010-2011

Investigations for pharmacodynamic interaction between agmatine and allopregnanolone in olfactory bulbectomy induced depression in rats

Introduction:

Agmatine (l-amino-4-guanidinobutane) is an endogenous amine and a novel

neurotransmitter in the central nervous system (Reis and Regunathan, 2000), synthesized by the

enzymatic decarboxylation of L-arginine by arginine decarboxylase, selective distribution in the

CNS, accumulated by uptake, released by depolarization, and its action is terminated by selective

reuptake or enzymatic degradation by agmatinase (Raasch et al., 2001; Reis and Regunathan,

2000). It shows affinity for α2-adrenergic and imidazoline receptors (Raasch et al., 2001; Reis

and Regunathan, 2000), N-methyl-D-aspartate (NMDA) receptors and all isoforms of nitric

oxide synthase (NOS) in the brain (Reis and Regunathan, 2000), opioid receptors (δ and µ)

(Zomkowski et al., 2005), potassium channels (Budni et al., 2007), serotonin receptors

(Zomkowski et al., 2004).

Exogenous administration of agmatine produced several effects including

antinociceptive (Onal et al., 2003), neuroprotective (Olmos et al., 1999), anxiolytic (Lavinsky et

al., 2003), anticonvulsant (Bence et al., 2003), mood elevator (Zomkowski et al., 2002; Halaris et

al., 1999) and antidepressant-like effect in the forced swimming test (FST) and tail suspension

test (TST) in mice, through a mechanism that seems to involve an interaction with NMDA

receptors, the L-arginine–nitric oxide pathway, and/or α2-adrenoreceptors (Zomkowski et al.,

2002). A clinical study has shown an increase in plasma agmatine level in patients with major

depression compared with healthy controls (Halaris et al., 1999). It was also found that brain

agmatine was depleted in depressive patients (Piletz et al., 1994).

Allopregnanolone (3α, 5α THP), dehydroepiandrosterone (DHEA) and its sulfated

moiety DHEA(S) in particular are of significance in depression. Both the indirect genomic and

non-genomic mediated effects of 3α, 5α THP are involved in depression, while DHEA(S) only

exerts non-genomic effect. In patients suffering from major depression, disequilibrium of

neurosteroid concentrations in plasma and cerebrospinal fluid (CSF) from control subjects and

normalize after successful treatment with antidepressants (Romeo et al., 1998; Uzunova et al.,

1998; Strohle et al., 1999, 2000).

The neurosteroid (3α, 5α THP) is synthesized from progesterone in the brain (Baulieu,

1981). It alters rapidly (milliseconds to minutes) neuronal excitability through positive allosteric

interaction with steroid recognition site on gamma-aminobutyric acid type A (GABAA) receptor-

ion channel (GRC) (Paul and Purdy, 1992). At lower (nanomolar) concentrations 3α, 5α THP

increases the frequency or duration of openings, or both of the GRC (Lambert et al., 1995).

However, at very high (micromolar) concentrations, it exerts a certain intrinsic agonistic activity

at GABAA receptor in the absence of GABA (Puia et al., 1990). It also modulate ligand gated ion

channels including the voltage gated calcium ion channels (Ffrench-Mullen et al., 1994),

nicotinic acetylcholine receptors (Bullock et al., 1997) and 5-HT3 receptors (Wetzel et al., 1998).

Neurosteroids regulate the neuronal function through their concurrent influence on

neuronal excitability and gene expression (Rupprecht and Holsboer, 1999). Exogenous

administration of 3α, 5α THP or drugs that increase its endogenous brain content exhibit

antidepressant-like activity in mouse FST (Khisti et al., 2000), behavioral effect such as anti-

stress (Zimmerberg and Blaskey, 1998), anxiolytic (Akwa et al., 1999), anticonvulsant (Frye,

1995), cataleptic (Khisti et al., 1998; Mandhane et al., 1999).

Serotonin (5-HT) system is strongly implicated in the neural regulation of mood.

Dysregulation in 5-HT neurotransmission underlies pathophysiology of depression. Reduced

level of 5-HT and 5-HIAA is reported in CSF of depressed patients (Risch and Nemeroff, 1992).

In addition, enhancement of serotonergic transmission underlies the therapeutic response to

various types of antidepressant treatments (Blier and Montigny, 1994).

The olfactory bulbectomized rat model of depression is a well-characterized animal

model of depression. Bilateral removal of the olfactory bulbs of rats results in a constellation of

behavioral, neurochemical, neuroimmune and neuroendocrine alterations, similar changes

observed in major depressive disorder (Cryan et al., 2002). The most widely studied behavioral

deficit produced by bulbectomy is a response to a novel and brightly lit environment and reduced

gain of body weight (Kelly et al., 1997). It is sensitive almost exclusively to chronic

antidepressant treatment (Cryan et al., 1998). It exhibits ventricular enlargement as well as

altered signal intensity in cortical, caudate, hippocampal and amygdaloid regions as measured by

magnetic resonance imaging (Wrynn et al., 2000).

Considering the fact that i) Agmatine and serotonergic agents produces an antidepressant

effect as well as a well established role of neurosteroids (3α, 5α THP) in FST and TST.

ii)Serotonergic agents modulate antidepressant-like effect of 3α, 5α THP in mice. iii) Fluoxetine

increases the level of neurosteroids (3α, 5α THP) in brain. iv) Agmatine and fluoxetine produce

synergistic effect in sub-effective doses (Zomkowski et al., 2004). Present study sought to

investigate: i) Antidepressant-like effect of agmatine in olfactory bulbectomized rats. ii) Role of

3α, 5α THP in antidepressant-like effect of agmatine by altering endogenous neurosteroids using

neusteroidogenic agents or its biosynthesis blocker in olfactory bulbectomized rats.

Objectives:

1) To investigate the antidepressant-like effect of agmatine in olfactory bulbectomized

rats.

2) To investigate the role of 3α, 5α THP in antidepressant-like effect of agmatine in

olfactory bulbectomized rats.

Plan of work:

1) The rats will undergo surgery for removal of olfactory bulb, after recovery period of not less

than 14 days.

2) Dose dependent study of agmatine and 3α, 5α THP will be carried out by analysing locomotor

activity in open field apparatus and measure body weight. On the basis of this study the final

effective and sub-effective doses will be selected.

3) In another set of experiment the selected sub-effective dose of agmatine will be combined

with sub-effective dose of 3α, 5α THP and effect will be analysed in open field test and measure

body weight.

4) In another set of experiment the positive and negative neurosteroid modulators will be

combined with sub-effective and effective doses of agmatine respectively and effects will be

analysed in open field test and measure body weight.

Materials and method:

Animals:

Male Sprague Dawley rats (200-250 g) will be used. Food and water will be available ad.

Libitum. The experimental procedure will be performed as per the protocol approved by

Institutional animal and ethical committee according to the guidelines of CPCSEA. Every

possible effort will be made to reduce the suffering of animals in all experimental design.

Drugs:

The drugs and other additives used will be: Agmatine, 3α-hydroxy-5α-pregnan-20-one

(3α, 5α THP), progesterone (neurosteroids precursor), (4’-chlorodiazepam (4’CD) (diazepam

binding inhibitor) metyrapone (11β-hydroxylase inhibitor), indomethacin (3α-hydroxysteroid

oxidoreductase inhibitor) (3α-HSOR), trilostane (3β-hydroxysteroid dehydrogenase inhibitor)

(3β-HSD), PK11195 (peripheral benzodiazepine receptors antagonist).

All drugs will be dissolved in 2-hydroxypropyl-β-cyclodextrin (45% w/v) solution and

further diluted with 0.9% saline except agmatine, injected by intraperitonial (i.p.) route in a

volume of 5ml/kg body weight.

Surgery (bilateral bulbectomy):

Procedure: Surgery will be carried out 1 week after the arrival of rats in the animal

house. Rats will be anaesthetized with a ketamine (75 mg/kg i.p.) and xylazine (5 mg/kg i.p.)

combination prepared in sterile water (1 ml/ kg). Bilateral olfactory bulbectomy will be carried

out as follows: rat will be anaesthetized and skull will be shaved with hair remover. Iodine

solution will be applied as an antiseptic solution to exposed skin. The animal will be fixed in

stereotaxic apparatus for bulbactomy. A skin incision will be made to expose the skull overlying

the olfactory bulbs. Two burr holes will be drilled (each 2 mm diameter) over the right and left

olfactory bulbs, respectively, each 1.5 mm from the midline of the frontal bone and 6.7 mm

anterior to bregma. The bulbs will be removed by suction, care being taken to avoid damage to

the frontal cortex. The burr holes will be then filled with haemostatic sponge and the incision

sutured. Control (sham operated) animals will be treated in a similar manner, however the bulbs

will not be aspirated. Postoperative analgesia will be consisted of buprenorphin (Temgesic) (0.3

mg/kg s.c.) once a day for 3 days (Uzunova et al., 2003).

After surgery, the rats will be placed in a group of 5-6 animals per cage and handled daily

throught recovery period to reduce stress and/or aggressive behavior.

Method:

Apparatus:

Open field test:

Bulbectomized and sham control rats will be subjected to an open field test on the 1, 7,

and 14th day of drug administration. Each rat will be placed individually into the center of the

open field apparatus. The open field apparatus consisted of 80-cm diameter arena with 75-cm

high aluminum walls, divided into 10 cm2. A 60 W light bulb will be positioned 90 cm above the

base of the arena, and provided the only source of illumination in the testing room. Each animal

will be placed in the center of the open field apparatus, the ambulation scores (the number of

squares crossed) and the number of rearings (number of times animal stood on its hind limbs)

will be measured during a 3-min period. (Ying et al., 2005).

Experimental design:

The rats will undergo surgery for removal of olfactory bulbs and after recovery different

drug treatment will be started at 10-11 a.m. as described above. Effects on locomotor activity and

body weight will be assessed as given below:

Locomotor activity in open field and measurement of body weight of animal:

After recovery period of two week rats will be given treatment of different drugs once a

day for 14 days and accessed for the acute, sub-chronic and chronic effect of different drug

treatment on 1, 7 and 14th day of initiation of treatment respectively. Several positive and

negative modulators will be given at defferent time interval before administration of sub-

effective and effective dose of agmatine. Effect on locomotor activity will be accessed 30 min

after last dosing of agmatine in open field for ambulations and rearings during a period of 3 min

(as described above) and also measure body weight on same day.

Treatment groups:

1) To study the effect of agmatine:

Bulbectomized animal

A) Control group: Saline (5 ml/kg, i.p.) for 14 days.

B) Treatment group: Agmatine (5, 10 or 20 mg/kg, i.p.) for 14 days.

Sham operated animal

A) Control group: Saline (5 ml/kg, i.p.) for 14 days.

B) Treatment group: Agmatine (5, 10 or 20 mg/kg, i.p.) for 14 days.

Effect on locomotor activity and body weight will be analysed same as described above.

2) To study the effect of 3α, 5α THP:


A) Control group: Vehicle (5 ml/kg, i.p.) for 14 days.

B) Treatment group: 3α, 5α THP (0.5, 1 or 2 mg/kg i.p.) for 14 days.


A) Control group: Vehicle (5 ml/kg, i.p.) for 14 days.

B) Treatment group: 3α, 5α THP (0.5, 1 or 2 mg/kg, i.p.) for 14 days.


3) To study the influence of 3α, 5α THP on antidepressant effect of agmatine:


A) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)

for 14 days.

B) Treatment group: 3α, 5α THP (sub-effective dose, i.p.) 30 min later, agmatine (sub-

effective dose, i.p.) for 14 days.

Sham operated animals


for 14 days.

B) Treatment group: 3α, 5α THP (sub-effective dose, i.p.) 30 min later, agmatine (sub-

effective dose, i.p.) for 14 days


4) To study the influence of neurosteroidogenic drugs (positive modulators of

neurosteroids) on antidepressant effect of agmatine:

i) For observing the effect of progesterone:



for 14 days.

B) Treatment group: Progesterone (5 mg/kg, i.p.) 30 min later, agmatine (sub-effective

dose, i.p.) for 14 days.



for 14 days.

B) Treatment group: Progesterone (5 mg/kg, i.p.) 30 min later, agmatine (sub-effective



ii) For observing the effect of metyrapone:


C) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)

for 14 days.

A) Treatment group: Metyrapone (50 mg/kg, i.p.) 30 min later, agmatine (sub-effective




for 14 days.

B) Treatment group: Metyrapone (50 mg/kg, i.p.) 30 min later, agmatine (sub-effective



iii) For observing the effect of 4’CD:



for 14 days.

B) Treatment group: 4’CD (15 mg/kg, i.p.) 30 min later, agmatine (sub-effective dose,

i.p.) for 14 days.



for 14 days.

B) Treatment group: 4’CD (15 mg/kg, i.p.) 30 min later, agmatine (sub-effective dose,

i.p.) for 14 days.


5) To study the influence of neurosteroid biosynthesis inhibitors (negative modulators of

neurosteroids) on antidepressant effect of agmatine:

i) For observing the effect of indomethacin:


A) Control group: Vehicle (5 ml/kg, i.p.) 20 min later, agmatine (effective dose, i.p.) for

14 days.

B) Treatment group: Indomethacin (5 mg/kg, i.p.) 20 min later, agmatine (effective




14 days.

B) Treatment group: Indomethacin (5 mg/kg, i.p.) 20 min later, agmatine (effective



ii) For observing the effect of trilostane:



14 days.

B) Treatment group: Trilostane (30 mg/kg, i.p.) 120 min later, agmatine (effective




14 days.

B) Treatment group: Trilostane (30 mg/kg, i.p.) 120 min later, agmatine (effective



iii) For observing the effect of PK11195:



14 days.

B) Treatment group: PK11195 (15 mg/kg, i.p.) 30 min later, agmatine (effective dose,

i.p.) for 14 days. Sham operated animal


14 days.

B) Treatment group: PK11195 (15 mg/kg, i.p.) 30 min later, agmatine (effective dose,

i.p.) for 14 days.


Data analysis:

Data will be analysed by two-way analysis of variance (ANOVA). Significant interaction

will be assessed by post hoc Dunnett's or Student–Newman–Keuls test. The criterion for

statistical significance will be P < 0.05.

Possible outcomes:

1) Agmatine may have antidepressant-like effect in olfactory bulbectomized rats.

2) 3α, 5α THP may have role in antidepressant-like effect of agmatine in olfactory

bulbectomized rats.

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Signature of Guide Signature of Co-guide Signature of Candidate

Prof. C. T. Chopde Dr. R. R. Ugale S. R. Rahangdale

Date:

Place:

sandip r final synopsis

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