Download (sulpiride) loading on to the PBCA nanoparticles will be calculated

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA,
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
Name of the Candidate
and Address
K.V.ARCHANA
a. Permanent Address
D/O K.V.Srinivas rao ,
15-15/5/1, Plot no.44,rajanagarcolony,
Mirjalguda, Malkajgiri,
Hyderabad, Andhra Pradesh,
Pin code-500047.
b. Postal Address
Al-Ameen College of Pharmacy,
Bengaluru-560027
Karnataka.
2.
Name of the Institute
3.
Course of Study and Subject
4.
Date of Admission to Course
Al-Ameen College of Pharmacy,
Master of Pharmacy in
Pharmacology
6th june 2011
5.
Title of the Topic:
“Pharmacological evaluation of a brain targeted novel nano formulation for the
treatment of negative symptoms of Schizophrenia in rat model”
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6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Schizophrenia is a one of the most common and devastating psychiatric diseases. There is a
continuous increase in the number of the patients and the current morbidity rates are nearly 1 %
of the population worldwide1.Antipsychotic drugs often reduce many symptoms of
Schizophrenia.
Sulpiride
(±)-5-(aminosulfonyl)-N-[(1-ethylpyrrolidin-2-yl)
methyl]-2-
methoxybenzamide is a selective dopamine D2 receptor antagonist and is included in the class
of atypical antipsychotics2. Negative symptoms in Schizophrenia, such as withdrawal, flat
affect, avolition and anhedonia, seem not to be as effectively treated by typical antipsychotics
as are positive symptoms3, 4. Substituted benzamide, such as sulpiride, are thought to have
advantages in treating negative symptoms of Schizophrenia. Sulpiride penetrates the bloodbrain barrier poorly because of its low lipid solubility. With regard to its pharmacokinetic
features, sulpiride is a relatively hydrophilic compound. Gastrointestinal absorption after oral
administration is slow and poor; it is reduced by concomitant food intake and by drugs that
increase the pH of the gastric contents5.As, sulpride poorly penetrates the blood brain barrier, it
is necessary to develop novel approaches to enhance the passing of drug through blood brain
barrier.
Targeting of drugs in the brain is one of the most challenging issues for the pharmaceutical
research, as many hydrophilic drugs and neuropeptides are unable to cross the blood–brain
barrier (BBB) 6. Drug delivery to the brain requires advances in both, drug delivery
technologies and drug discovery7. Drugs that are effective against diseases in the CNS and
reach the brain via the blood compartment must pass the BBB. The BBB has been called ‘‘the
problem behind the problem” of CNS drug development. The BBB prevents the entry of >98%
of small molecules and 100% of large molecules8. The BBB constitutes an insurmountable
barrier for the entry of many drugs and blood-borne substances into the brain9, 10.
The BBB is formed by the capillary endothelial cells lining the micro vessels, which are
coupled by much tighter junctions (zonulae occludentes) than found in peripheral vessels11.
Many strategies have been developed to overcome this problem which includes chemical
delivery systems, magnetic drug targeting, or drug carrier systems such as antibodies,
liposomes, or nanoparticles12-14. Among that polymeric nanoparticles have attracted great
2
attention as potential drug delivery systems in the brain recently. Due to their small size,
nanoparticles penetrate into even small capillaries and are taken up within cells, allowing an
efficient drug accumulation at the targeted sites in the body. The use of biodegradable materials
for nanoparticles preparation, allows sustained drug release at the targeted site over a period of
days or even weeks after injection15.
Many authors have shown that therapeutic agents which normally cannot cross the BBB can be
transported across this barrier into the brain by binding them to poly (n-butylcyanoacrylate)
nanoparticles coated with polysorbate 80. Drugs that have been successfully delivered into the
brain using the carrier poly (n-butylcyanoacrylate) include the hexapeptide dalargin16, the
dipeptide kytorphin17, loperamide18, tubocurarine19 and doxorubicin20. The delivery of drugs
into the brain by using nanoparticles may open a new era for treating diseases such as
Schizophrenia, Alzheimer’s, multiple sclerosis and brain tumors.
Targeting of drugs in the brain with maximum benefits and minimum or no side effects, may be
a breakthrough in the treatment of psychiatric diseases including Schizophrenia as well as other
neurodegenerative disorders. Hence in the present study, the possibility of targeting of an
atypical anti-psychotic drug with polysorbate 80-coated poly (n-butylcyanoacrylate)
nanoparticles to the brain will be investigated.
6.2 Review of Literature:
Schizophrenia (SZ) is a complex, heterogeneous, and disabling psychiatric disorder that impairs
multiple aspects of human cognitive, perceptual, emotional, and behavioral functioning
21
.
Contrasting theories of Schizophrenia propose that the disorder is characterized by a deficit in
phasic changes in dopamine activity in response to ongoing events or, alternatively, by a
weakness in the representation of the value of responses22. Patients with Schizophrenia may
present with positive symptoms (such as conceptual disorganization, delusions or
hallucinations, hyperactivity, stereotypy behavior) or negative symptoms (social withdrawal,
loss of function, anhedonia, decreased emotional expression, impaired concentration and
diminished social engagement). The latter predominate in one third of Schizophrenic
population and are associated with a poor long term outcome and a poor response to drug
treatment23.
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Poly butyl cyanoacrylate (PBCA) is the most widely used drug carrier because it can interact
with different kinds of drugs24. PBCA nano-particles meet ideal requirements for controlled
drug delivery and passive targeting such as biodegradability, low toxicity, ability to alter bio
distribution of drugs25. Polysorbate 80 enhances the central nervous system uptake of drug
nanoparticle in both human and bovine brain capillary endothelial cells, which suggest that it is
a preferred surfactant26.
6.3 Objective of study: The main objective of the present study is targeted delivery of sulpiride
into the brain using polysorbate 80-coated PBCA nanoparticles , for the treatment of negative
symptoms of Schizophrenia in rat model.
SPECIFIC OBJECTIVES:
a. Synthesis and characterization of PBCA- sulpride nanoparticles, a brain targeted novel
nano formulation for the effective treatment of negative symptoms of Schizophrenia.
b. Synthesis and characterization of PBCA-sulpiride nanoparticles coated with 1%
polysorbate 80, a brain targeted novel nano formulation for the effective treatment of
negative symptoms of Schizophrenia.
c. To study the biodistribution of sulpiride in PBCA-sulpride nanoparticles and poly (nbutylcyanoacrylate) sulpiride nanoparticles coated with 1% polysorbate 80 in the brain
homogenate using rat model.
d. To evaluate the therapeutic effects of PBCA-sulpride nanoparticles and PBCAsulpiride nanoparticles coated with 1% polysorbate 80 using experimental rat models for
the negative symptoms of Schizophrenia.
7.
MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from
libraries of Al-Ameen College of Pharmacy, Indian Institute of Sciences, Government College
of Pharmacy etc., and other Research Publications and Journals.
Web sites:
www.sciencedirect.com
www.pubmed.com
www.google.com
www.ijp-online.com
www.elsevier.com.
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Method of Collection of Data: The data collected will be based on animal experimentation as
per the parameters studied under each animal model, which are mentioned under the objectives
of the study.
MATERIAL AND METHODS
Chemical and reagents
Sulpiride, Dextran, D-(+)-glucose and polysorbate 80 (Tween 80), and the monomer nbutylcyanoacrylate. All other materials and reagents used in the study are Analytical/HPLC
grade.
Preparation of PBCA-sulpiride nanoparticles
The PBCA-sulpiride nanoparticles are prepared according to the method described by Kreuter
et al27. Briefly, the monomer n-butyl cyanoacrylate will be added drop by drop under magnetic
stirring to 10 ml of acidic polymerization medium (0.1 N HCl) containing 1% dextran 70,000
and the drug sulpiride. The monomer, n-butyl cyanoacrylate, is a clear liquid with sharp
characteristic odor. It undergoes polymerization in contact with water or in the presence of
moisture. The mixture will be then stirred magnetically at 500 rpm for 4 h to facilitate
nanoparticles formation. The resulting suspension will be neutralized with 0.1 N sodium
hydroxide solution, and filtered through a sintered glass filter (pore size 10 μm) to remove any
agglomerates. Anhydrous glucose (1%) will be added to improve redispersibility of the
nanoparticles after lyophilization. The nanoparticles suspension will be then lyophilized using a
lyophilizer. The drug-free nanoparticles will be prepared in the same manner to that of drug
containing nanoparticles except for omitting the drug.
Coating of PBCA nanoparticles with 1% polysorbate 80
Coating of PBCA nanoparticles formulations will be performed as per the procedure described
by Kreuter et al 27PBCA nanoparticles which contain the drug sulpiride will be resuspended in
phosphate buffered saline at a concentration of 20 mg/ml under constant stirring. Then (relative
to total suspension volume) polysorbate 80 will be added to give a final solution of 1%
polysorbate 80, and the mixture will be incubated for 30 min and finally lyophilized.
5
Determination of drug loading capacity
The drug (sulpiride) loading on to the PBCA nanoparticles will be calculated as the difference
between total amount of drug in suspension and amount of unbound drug. For this purpose, the
unbound drug is separated by filtration through a membrane filter and its concentration in the
filtrate was measured spectrophotometrically at 291 nm28.
Particle Size and zeta potential measurement
Particle size and zeta potential of PBCA-sulpiride nanoparticles coated with 1% polysorbate 80
and without coating will be separately measured using Zetasizer.
In vitro release studies
The PBCA-sulpiride nanoparticles coated with 1% polysorbate 80 and without coating will be
transferred to a 100ml beaker containing 50ml of phosphate buffer saline (PBS) having pH of
7.4 and subjected to continuous stirring at 100rpm. Samples will be withdrawn at various
different time intervals from the beaker and the sulpiride content will be determined
spectrophotometrically at 291 nm wavelength28. The amount of samples withdrawn each time
will be replaced by equal volume of fresh PBS.
Animal testing
The targeting efficacy of PBCA-sulpiride nanoparticles coated with 1% polysorbate 80 and
without coating will be carried out on rats. Healthy adult Wistar rats weighing 180–220 g will
be obtained from the animal house, Al-Ameen College of Pharmacy, Bangalore, India after
getting approval from Institutional Animal Ethics Committee. The experimental animals will be
housed at 25° ± 5°C in a well-ventilated animal house under 12:12 h light and dark cycle. The
rats will have free access to standard rat chow (Amrut Laboratory Animal feed, Maharashtra,
India) containing protein 22.10%, oil 4.13%, fi ber 3.15%, ash 5.15%, sand (silica) 1.12% w/w)
and water ad libitum. There will be no significant difference in the body weight of the treated
rats when compared with control, either at the beginning or at the end of the study period.
6
Experimental protocols
The animals will be divided into four groups and each group contains 6 rats except the control
group which requires only 2 animals.
Group 1: Control.
Group 1: Sulpiride pure drug.
Group 3: PBAC-sulpiride nanoparticles .
Group 4: PBCA-sulpiride nanoparticles of sulpiride coated with 1% polysorbate 80.
For the in vivo experiments the formulations will be re-suspended in phosphate buffered saline.
For surfactant coating 1% polysorbate 80 will be added and the suspension will be incubated
for 30 min under stirring prior to administration. All the formulations will be given in a dose
level equivalent to 10 mg/kg body weight. The formulations will be administered intravenously
in the tail vein of rats. After 1 h of post injection the rats were sacrificed by cervical dislocation.
The brain will be quickly removed and weighed, and stored at 20 0C. The drug content in the
organ will be analyzed by using HPLC30.
Screening of negative symptoms of Schizophrenia using rat model:
The effect of PBCA-sulpiride nanoparticles coated with 1% polysorbate 80 and without coating
will be evaluated using an animal model of negative symptoms of Schizophrenia in rodents.
The proposed model uses NMDA receptor antagonist ketamine for inducing the negative
symptoms of Schizophrenia. It was reported by Chatterjee et al , that forced swim test can be
used as a model for evaluating the negative symptoms of Schizophrenia.The rats will be
pretreated with the drug or the vehicle(saline) 30 mins prior to the administration of ketamine
( 100 mg/kg i.p.)23.
Rats will be divided into five groups and each group contains 6 animals.
Group 1: Vehicle control, the rats will be administered with vehicle.
Group 2: Ketamine 100 mg/kg + vehicle.
Group 3: Ketamine 100 mg/kg + Sulpiride, 10 mg/kg, i.v., single dose.
Group 4: Ketamine 100 mg/kg + PBCA-sulpiride nanoparticles, 10 mg/kg, i.v., single dose.
Group 5: Ketamine 100 mg/kg + PBCA-sulpiride nanoparticles coated with 1% polysorbate80,
10 mg/kg, i.v., single dose.
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7.3 Does the study require any investigation or interventions to be conducted on patients
or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be studied on various
parameters using rats as experimental animal model.
7.4 Has ethical clearance been obtained from your institute
Ethical Committee approved.
8.
List of References:
1. Kilts CD. The changing roles and targets for animal models of Schizophrenia. Biol
Psychiatry 2001; 50: 845-55.
2. Kervin RW. The new atypical antipsychotics. Br J Psychiatry 1999; 164: 141-8
3. Kane JM, Mayerchoff D. Do negative symptoms respond to pharmacological treatment?
Br J Psychiatry 1989; 155: 115-8.
4. Arndt S, Allinger RJ, Andreason NC. The distinction of positive and negative
symptoms: the failure of a two dimensional model. Br J Psychiatry 1991; 158: 317-22.
5. Mauri MC, Bravin S, Bitetto A, Rudelli R and Invernizzi G. Drug safety 1996; 14 (5):
288-298.
6. Tamai I, Tsuji A. Drug delivery through the blood brain barrier. Adv Drug Deliv Rev
1996; 19: 401–424.
7. Vyas SP, Khar RK. Targeted & Controlled Drug Delivery Novel Carrier Systems, CBS
Publishers & Distributors, New Delhi, 2002.
8. Pardridge WM. Drug and gene targeting to the brain via blood–brain barrier receptormediated transport systems. Int Cong Ser; 2005: 49: 49–62.
9. Brightman M. Ultrastructure of the brain endothelium, in: M.W.Bradbury (Ed.),
Physiology and Pharmacology of the Blood–Brain Barrier Handbook of Experimental
Pharmcology, Springer, Berlin, 1992; 1–22.
10. Wolberg H, Lippoldt A. Tight junctions of the blood–brain barrier: development,
composition and regulation. Vasc Pharmacol 2002; 38: 323–337.
11. Bodor N, Prokai L, Wu WM, Farag HH, Jonnalagadda S, Kawamura M, Simpkins J. A
strategy for delivering peptides into the central nervous system by sequential
metabolism. Science 1992; 257: 698–1700.
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12. Devineni D, Klein-Szanto A, Gallo JM. Tissue distribution of methotrexate following
administration as a solution and as a magnetic microsphere conjugate in rats bearing
brain tumors. J Neuro oncol 1995; 24: 43–152.
13. Huwyler J, Wu D, Pardridge WM. Brain delivery of small molecules using immune
liposomes, Proc Natl Acad Sci 1996;L 4164–14169.
14. Vinogradov SV, Bronich TK, Kabanov AV. Nanosized cationic hydrogels for drug
delivery: preparation, properties and interactions with cells. Adv Drug Deliv Rev 2002;
54: 135–147.
15. Kreuter J, Alyautdin RN, Kharkevich DA, Ivanov AA. Passage of peptides through the
blood–brain barrier with colloidal polymer particles (nanoparticles). Brain Res 1995;
674: 171–174.
16. Schroeder U, Sommerfeld P, Ulrich S, Sabel BA. Nanoparticle technology for delivery
of drugs across the blood–brain barrier. J Pharm Sci 1998; 87: 305–1307.
17. Alyautdin RN, Petrov VE, Langer K, Berthold A, Kharkevich DA, Kreuter J. Delivery
of loperamide across the blood–brain barrier with polysorbate 80-coated poly
(butylcyanoacrylate) nanoparticles. Pharm Res 1997; 14: 325–328.
18. Alyautdin RN, Tezikov EB, Ramge P, Kharkevich DA, Begley DJ, Kreuter J.
Significant entry of tubocurarine into the brain of rats with polysorbate 80-coated poly
(butylcyanoacrylate) nanoparticles: an in situ brain perfusion study. J Microencpsul
1998; 15: 67– 74.
19. Gulyaev AE, Gelperina SE, Skidan IN, Antropov AS, Kivman GY, Kreuter J.
Significant transport of doxorubicin into the brain with polysorbate 80-coated
nanoparticles. Pharm Res 1999; 16: 1564–1569.
20. Kreuter J, Alyautdin RN, Kharkevich DA, Ivanov AA. Passage of peptides through the
blood–brain barrier with colloidal polymer particles (nanoparticles). Brain Res 1995;
674: 171–174.
21. Danielyan A., Nasrallah AH. Neurological disorders in schizophrenia. Psychiatric
Clinics of North America2009; 20(4):719-757.
22. Duncan EG, Sheitman BB, Lieberman AJ.An integrated view of pathophysiological
models of Schizophrenia. Brain Research Reviews1999; 29:250-264.
23. Chatterjee M , Jiaswal M , Palit G , Comparative evaluation of Forced swim test and
Tail suspension test as models of negative symptoms of Schizophrenia in rodents;
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www.isrn.com/journals/psychiatry/aip/595141.pdf retrieved on Nov 2011.
24. Mulik R , Mahadik K , Paradkar A.Development of curcuminoids loaded with poly
butyl cyanoacrylate nano particles: Physicochemical
characterization and stability
study. European Journal of pharmaceutical sciences 2009; 37(3-4):395-404.
25. Couvreur et al., Controlled drug delivery with nanoparticles: current possibilities and
future trends. Eur J Pharm Biopharm1995; 41: 2-13.
26. Ramge P, Unger RE, Oltrogge JB, Zenker D, Begley D , Kreuter J, Von Briesen H.
Polysorbate80 coating enhances uptake of PBCA nanoparticles by human and bovine
primary brain capillary endothelial cells, Eur J Neurosci 2000;12(6):1931-40.
27. Kreuter J, Ramge P, Petrov V, Hamm S, Gelperina SE, Engelhardt B, Alyautdin RN,
Briesen HV, Begley DJ. Direct evidence that Polysorbate-80-coated poly (butyl
cyanoacrylate) nanoparticles deliver drugs to the CNS via specific mechanism requiring
prior binding of drugs to the nanoparticles. Pharm Res 2003; 20: 409–416.
28. Spectrophotometric estimation of Levosulpiride in bulk drug and formulations.
International Journal of Pharmacy and Pharmaceutical Sciences. 2011; 3 (2).
29. Preece M, Mukherjee B , Huang CL, Hall LD, Leslie RA, James MF.Detection of
pharmacologically mediated changes in cerebral activity by functional magnetic
resonance imaging:the effects of sulpride in the brain of the anaesthetized rat.Brain Res
2001;916(1-2):107-14.
30. Wilson B, Samanta MK, Santhi K, Kumar KPS, Paramakrishnan N, Suresh B. Targeted
delivery of tacrine into the brain with polysorbate 80-coated poly(n-butylcyanoacrylate)
nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics 2008; 70:7584.
9.
SIGNATURE OF THE CANDIDATE:
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10.
REMARKS OF THE GUIDE:
“Pharmacological evaluation of a brain targeted novel nano formulation for the treatment of
negative symptoms of Schizophrenia in rat model” is to be carried out by K.V.Archana of M.
Pharm. It has been discussed and worked out under my directions and supervision as an official
guide. The project work envisaged is of great importance in the field of pharmacology. The
work can be carried out in pharmacology laboratory of Al-Ameen College of Pharmacy for
which facilities are available. Hence the project is viable and is recommended for clearance and
approval.
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11.1 Name and Designation of Guide
Dr. Noor Zahra
PROFESSOR,
Department of Pharmacology,
Al-Ameen College of Pharmacy,
Bangalore-560027
11.2 Signature
11.3 Head of the Department
Dr. Md. Naseeruddin Inamdar.
HOD
Department of Pharmacology
Al-Ameen College of Pharmacy
Bangalore-560027.
11.4 Signature
12.
12.1 Remarks of the Principal
Recommended for approval.
12.2 Signature
Prof.B.G.Shivananda
Principal,
Al-Ameen College of Pharmacy
Bangalore-560027.
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