Development of Biodegradable Temperature anddbtepromis.gov.in/Documents/...Dr._N._Subramanian.pdf · topical ocular formulation based on of biodegradable hybrid polymer-peptide system

Development of Biodegradable Temperature and pH Responsive Hybrid Polymer-Peptide System for

Efficient Intraocular Delivery of Drugs

Submitted by

Dr. N. Subramanian1, Dr. P. Rajaguru 2, Dr. M. Sivakumar 3 1Department of Pharmaceutical Technology

2 Department of Biotechnology 3 Division of Nanoscience & Technology, Department of Physics

Anna University of Technology Tiruchirappalli Tiruchirappalli – 620 024

Ph: 0431-2407978 Fax: 0431-2407999

E. mail: [email protected]

Submitted to

Department of Biotechnology Block 2, 7th Floor

C.G.O. Complex, Lodi Road New Delhi – 110 003

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PROFORMA – I PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND

DEVELOPMENT, PROGRAMME SUPPORT

PART I: GENERAL INFORMATION 1. Name of the Institute/University/Organization submitting the Project Proposal:

Anna University of Technology Tiruchirappalli, Tiruchirappalli – 620024

2. State: Tamil Nadu 3. Status of the Institute: State Govt. University

4. Name and designation of the Executive Authority of the Institute/University forwarding the

application:

The Registrar, Anna University of Technology Tiruchirappalli,

Tiruchirappalli - 620024

5. Project Title:

Development of Biodegradable Temperature and pH Responsive Hybrid Polymer-Peptide

System for Efficient Intraocular Delivery of Drugs

6. Category of the Project (Please tick): R&D

7. Specific Area : Drug delivery systems

8. Duration : 3 Years

9. Total Cost : 59.664 Lakhs

10. Is the project Single Institutional or Multiple-Institutional (S/M)?: S

11. If the project is multi-institutional, please furnish the following: Not applicable

Name of Project Coordinator

Affiliation:

Address:

12. Scope of application indicating anticipated product and processes

Most of the ocular diseases like glaucoma, dry eye syndrome, conjunctivitis, corneal ulcer

and uveitis etc. require frequent administration of drug to manage/improve the pathological

condition. However, due to various precorneal clearance factors and relative impermeability of

the drugs to corneal epithelial membrane[1], with the currently available ophthalmic drug

delivery systems only a small fraction of the drug, about 1% of the instilled dose is ocularly

absorbed[2]. Although, these issues are addressed in various ophthalmic formulations like

ointments, inserts, hydrogels and suspensions and have offered some advantages over

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conventional liquid formulations, they are not widely accepted because of blurred vision and

patient noncompliance[3]. So, the development of an ideal topical ocular formulation which offers

good ocular bioavailability remains as a challenge to be resolved satisfactorily.

The temperature responsive polymers and pH responsive polymers are extensively used in

stimuli responsive in situ gel forming (phase transition from solution to gel on administration)

ophthalmic formulations. These systems exhibit extended precorneal residence time, enhanced

local ocular bioavailability by sustained release of drug, increased stability of the drug, decreased

dosing frequency and improved patient compliance[4]. But these systems require high

concentration of polymer for efficient gelling and the formed polymeric gel is suffered with weak

mechanical strength, rapid erosion, non-biodegradability, irritation and non-promising manner

of drug release which limit their clinical utility[5,6]. On the other hand, pH responsive peptides are

more biocompatible and less irritant but require high concentration of peptide for effective

retention in the eye.

To overcome these critical issues, we have proposed to develop a novel in situ gel forming

topical ocular formulation based on of biodegradable hybrid polymer-peptide system consisting

of temperature sensitive polymers and pH sensitive peptides as the carriers for therapeutic

drugs. The formulation will be prepared in such a manner that will remain as stable solution at

room temperature and upon topical instillation on the eye, phase transition occurs and become

gel at ocular temperature and pH. This patentable platform formulation containing

biodegradable temperature and pH responsive hybrid polymer-peptide system will be suitable

for encapsulation and delivery of therapeutic drugs to the eyes.

The developed hybrid system is expected to have various advantages over individual

temperature or pH responsive systems like, requiring minimum concentration for effective

gelling, good biocompatibility, biodegradability and good adhesion for a prolonged period of

residence time in eye. Further, the intended controlled and prolonged release of incorporated

drug can be achieved by modifying the composition of polymer and peptide in the hybrid. Due

to its stable crosslinked aggregates, this hybrid polymer-peptide system not only efficiently

delivers the bioactive drug intraocularly and also protects the drug from degradation by

enzymes, oxidation and hydrolysis before released from the system. The released drug from the

system readily permeates through cornea to intraocular, thus it potentially maintains the

therapeutic drug concentration at the diseased site.

13. Project Summary Background Ophthalmic delivery of drugs is limited due to the high extent of constrains such as, blinking reflex, lacrimation, tear turnover, drainage of instilled eye drops, metabolism or degradation of drug in ocular environment, and poor corneal permeability[7], and the drugs administered to eyes are rapidly eliminated and drained into nasopharynx and GI tract[8]. This reduces the contact time of formulation to the cornea and sclera and increases frequency of dosing required to maintain the therapeutic drug concentration. Even though, incorporation of mucoadhesive polymers in the formulations at high concentration extends the ocular contact time, their high viscosity leads to blurred vision, difficulty in instillation of drops and affects the dose precision. The physiological condition responsive in-situ gelling systems are extensively investigated due to their ability to remain in the cornea for an extended period of time and release the drug in a prolonged manner. The temperature responsive PEO-PPO block copolymers or pH responsive polymers in the formulation readily transits the solution to gel phase at ocular temperature or pH, respectively[6]. But these stimuli responsive polymers have also failed to produce clinically acceptable drug delivery due to the use of high concentration of polymers that exhibit weak mechanical strength and rapid erosion of formed gel matrix leading to non-reproducible manner of drug release, variation in biodegradability and irritation[5]. On the other hand, pH responsive peptides are more biocompatible and less irritant but it is also required in high concentration. So, biocompatible, biodegradable hybrid polymer-peptide system is hypothesised as a novel ophthalmic vehicle, which requires low polymer concentration, provides high ocular residence time with prolonged drug release and thus enhance overall ocular bioavailability.

Objective

The aim of the present study is to develop biodegradable hybrid polymer-peptide system, consisting of temperature responsive PEO-PPO block copolymer and pH responsive peptide suitable for encapsulation and delivery of therapeutic drugs to the eyes. The specific objectives of this study are: To synthesis and characterize a biocompatible and biodegradable novel hybrid polymer-peptide

system having both temperature and pH responsive property. To evaluate biocompatibility and biodegradability of synthesized hybrid polymer-peptide system. To incorporate the drugs used for the treatment of glaucoma in the synthesized hybrid polymer-

peptide system and to evaluate their residence time in eye, viscosity, drug release and irritation.

Study Design

In first phase, the hybrid PEO-PPO block copolymer and custom synthesized peptide analogue systems will be developed and characterized for its phase transition under physiological condition. The rheological parameters, biocompatibility and biodegradability of the synthesized hybrid system will be evaluated. In the second phase of the work, ophthalmic formulations with anti-glaucoma drugs using the synthesized hybrid polymer-peptide system will be developed and evaluated. The viscosity, sol-gel transition, isotonicity and drug content, in-vitro drug release and in-vitro transcorneal permeation of the developed formulation will be measured. The corneal resident time, irritation and ocular pharmacokinetics of drug will be studied in rabbit eye. The enhanced therapeutic efficiency of the anti-glaucoma drug with this hybrid polymer-peptide system will be evaluated in glaucoma rat models and compared with the conventional eye drops. The stability of the formulations will also be studied.

Relevance

This hybrid system possess various advantages over individual temperature or pH responsive systems like requiring minimum concentration for effective gelling, good biocompatibility, biodegradability and good adhesion for a prolonged period of residence time in eye. Further, the intended controlled and prolonged release of incorporated drug can be achieved by modifying the composition of polymer and peptide in the hybrid. Due to its stable crosslinked aggregates, this hybrid polymer-peptide system not only efficiently delivers the bioactive drug intraocularly and also protects the drug from degradation by enzymes, oxidation and hydrolysis before released from the system. The released drug from the system readily permeates through cornea to intraocular, thus it potentially maintain the therapeutic drug concentration at the diseased site.

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PART II: PARTICULARS OF INVESTIGATORS Principal Investigator

14. Name : Dr. N. Subramanian

Date of Birth : 15.07.1973 Sex (M/F): M

Designation : Assistant Professor

Department : Pharmaceutical Technology

Institute/University : Anna University of Technology, Tiruchirappalli

Address : Room No. 212, Academic Block

Department of Pharmaceutical Technology

Anna University of Technology, Tiruchirappalli

Tiruchirappalli , Tamil Nadu Pin: 620 024

Telephone: (431)2407978 Fax: (431)2407333 E-mail: [email protected]

Number of research projects being handled at present: One

Co-Investigator

15. Name : Dr. P. Rajaguru


Designation : Professor

Department : Biotechnology


Address : Anna University of Technology, Tiruchirappalli

Tiruchirappalli Pin: 620 024

Telephone: (431)2407993 Fax: (431)2407333 E-mail: [email protected]

Number of research projects being handled at present: Two

Co-Investigator 16. Name : Dr. M. Sivakumar



Department : Division of Nanoscience & Technology,

Department of Physics


Address : Anna University of Technology, Tiruchirappalli

Tiruchirappalli, Pin: 620 024

Telephone: (431) 2407959 Fax: (431)2407333 E-mail: [email protected]

Number of research projects being handled at present: Nil

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PART III: TECHNICAL DETAILS OF PROJECT

17. Introduction

17.1 Origin of the proposal

Even though, a distinct progress is experienced in ophthalmic research and inventions,

delivery of drugs to eye using topical ophthalmic formulations still remains as a challenging

problem. Currently available conventional ocular formulations are failed to produce needed

ocular bioavailability because of eye’s protective mechanism to foreign agents. So, there is a need

for novel drug delivery systems and approaches to circumvent these ocular bioavailability

issues. Among various approaches, the physiological temperature / pH responsive in situ gel

forming ophthalmic formulations are extensively studied to enhance the rate and extent of drug

release to achieve required ocular bioavailability and therapeutic efficacy. But one of the major

drawbacks of these systems is the need of higher concentration of polymers for phase transition

to occur, which creates biodegradability and toxicity issues. Moreover these systems also

suffered with weak mechanical strength and rapid erosion of gel matrix etc. To overcome these

issues, we propose to develop a novel dual responsive hybrid polymer-peptide system consisting

of biodegradable temperature sensitive polymer and pH sensitive peptide, for spontaneous

phase transition at ocular temperature and pH, to provide extended period of corneal adhesion,

to protect the drug from environmental degradation and maintain the therapeutic drug

concentration at disease site.

17.2 (a) Rationale of the study supported by cited literature

The alginate/Pluronic (PEO-PPO block copolymer) polymeric mixture as in situ gelling

vehicle to enhance ocular bioavailability was reported by Sung et al.,[9]. Mishra et al.,[10]

demonstrated the higher drug transport across corneal membrane and increased ocular retention

time of the drug from their developed temperature and ionic responsive in situ gelling

ophthalmic vehicle composed of Pluronic F-127 and chitosan. Likewise Pan et al.,[11] developed

Pluronic F-127-g-poly (acrylic acid) copolymer as ophthalmic in situ gelling vehicle. They

showed 5.0 fold increases in ocular resident time and 2.6 fold increases in resident amount of

drug compared to conventional eye drops. The Pluronic based in situ gelling systems requires

higher concentrations of about 25% (w/v) of polymer to form phase transition on ocular

instillation[9]. But Davidorf et al.,[12] reported the higher concentrations of Pluronic in ophthalmic

formulations produced retinal destruction on chronic use. So, decreasing the Pluronic

concentration is most warranted for the long term ophthalmic use.

Hong et al.,[13] developed physiological pH sensitive peptides, EAK16-II and EAK16-IV and

showed that EAK16-IV forms globular structures at pH 6.5 – 7.5 due to strong intramolecular

electrostatic interaction. But at pH less than 6.5 and above pH 7.5, intramolecular electrostatic

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attractions were weekend and fibrillar structures appeared. Li et al.,[14] investigated the series of

pH sensitive peptides and showed the conformational changes occur in the GALA

(WEAALAEALAEALAEHLAEALAEALEALAA) and KALA (WEAKLAKALAKALAKHLAKA

LAKALKACEA) peptides at physiological pH, which formed self-assembled gelation. Kataoka et

al.,[15] demonstrated that the polymer-peptide conjugates, hybrid PEG-Polypeptide block

copolymer systems, are suitable for drug and gene delivery. These systems also showed

biocompatibility and stimuli sensitivity. Further they have proposed that the system can be used

for targeting of drugs to cancers with minor modification.

Lowe et al.,[16] have developed thermosensitive, biodegradable polymer-peptide conjugates

consisting of Poly(N-isopropylacrylamide)–poly (L-lactic acid) central segment and poly (L-

lysine) dendrons at their ends. These systems tend to deliver bioactive molecules to cells or

across biological barriers under heating. Shi et al.,[17] prepared well-defined block copolymers of

Poly (N-isopropylacrylamide)-b-poly (L-glutamic acid) system that showed pH and temperature

responsive miscellisation behavior in aqueous solution at pH >10 and temperature <33 °C. This

system is developed for the separation of proteins and not suitable for the biological use. These

studies reveal that polymer conjugate with pH responsive peptides might be a promising system

for the development of ocular in situ gelling formulations and which is expected to have more

efficiency and less toxic than individual polymeric system.

17.2 (b) Hypothesis

Our hypothesis to be tested:

To check the effectiveness of proposed novel hybrid polymer – peptide conjugates for its

spontaneous in situ gelling at ocular temperature and pH compared to individual systems

with higher polymeric concentration.

To check the integrity of novel hybrid polymer–peptide conjugates for promising drug

stability, retention time and permeability.

To check the enhanced bioavailability of drug by novel hybrid polymer–peptide conjugates

compared to conventional eye drops.

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Figure: Schematic representation of the hybrid polymer-peptide system.

17.2 (c) Key questions

What are the advantages of dual responsive system over mono temperature/pH responsive

system in ophthalmic drug delivery vehicle?

The phase transition of polymeric solution upon stimuli depends on higher

concentrations of synthetic polymers. This produces biodegradability and toxicity issues.

This can be avoided by designing a dual stimuli responsive system using polymer and

biocompatible peptides which is active in low concentration.

How will the temperature and pH Responsive hybrid polymer-peptide system suitable for

ocular delivery?

Temperature and pH responsive stimuli are present in the ocular surface. The low

concentration of hybrid polymer-peptide system expected to produce good gel with

required mechanical and thermal properties. Further, the system is biocompatible and can

control the release of drugs.

How does the polymer and peptide contribute for spontaneous gelling upon ocular

instillation?

The PEO-PPO block co-polymer part of hybrid system exhibits sol-gel phenomenon

upon heating. They have both hydrophilic and hydrophobic units in their structure. At low

temperature (25 °C), it form micellar subunits in solution but on increase in temperature

(about 37 °C) the gradual desolvation of the polymer increases large micellar aggregation

and creates of cross-linked polymeric networks. On the other hand, the change in pH of the

environment induces conformational modifications from fibrillar to globular structures in

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the peptide part of the system. This is due to the strong intermolecular electrostatic

interactions and formation of hydrogen bonds.

What are the benefits of the phase transition systems over already existing ocular vehicles?

This system can facilitate administration of accurate and reproducible quantities in

contrast to already gelled formulations for promoting precorneal retention. Moreover,

enhanced precorneal residence time and ocular bioavailability, reduced dosing frequency,

dose concentration and total drug and improved patient acceptability are possible by this

novel hybrid system compared to conventional eye drop formulations.

17.5 Current status of research and development in the subject

International Status

Hoffman et al.,[18] developed various polymer-peptide conjugates consisting of

thermoresponsive polymer (PNIPAAm) and various biomolecules/drugs. They demonstrated

various polymer-peptide conjugation methodologies and their uses in diagnostics and affinity

separations. Saiani et al.,[19] designed modified octa-peptide polymer (PNIPAAm-FEFEFKFK)

conjugates and demonstrated its double thermo responsive properties for biomedical

applications. Ghandehari et al.,[20] developed a conjugate of poly (N-(2-hydroxypropyl)-

methacrylamide) (PHPMAAm) and the ανβ3 integrin-targeting peptide RGD4C. This polymer-

peptide conjugate had the ability to deliver therapeutic radioisotopes to angiogenic sites in the

treatment of cancer.

Cui et al.,[21] formulated a controlled polymer-peptide conjugate delivery system for cancer

therapy. The carboxyl group end-functionalized Pluronic triblock copolymers encapsulated the

drug paclitaxel and conjugated with tumor specific peptide ligand, anti-HIF-1α antibody. This

drug delivery system selectively delivered the anticancer drug without cytotoxicity to normal

cells. Hubbell et al., developed hydrogels consisted of star-shape polymers with four PEG arms

functionalized with vinylsulfone and two different peptide sequences: GPQGIWGQ[22] and

RGDSP[23]. The peptide sequences served to mediate cell adhesion and acted as substrates for

matrix metalloproteinase. Klok et al.,[24] designed polymer brushes of poly(2-hydroxyethyl

methacrylate) and poly(poly(ethylene glycol)methacrylate) and functionalized them with RGD

peptide sequence which promoted the endothelialization of blood-contacting biomaterials.

Alexander et al.,[25] developed thermo-responsive copolymers of poly(ethylene glycol)

methacrylate and poly(propylene glycol) methacrylate (PPGMA-co-PEGMA) that tends to form

gel at above 37 °C and dissolves into solution at below 37 °C. However, to the best of our

knowledge, no temperature and pH sensitive polymer-peptide systems have been reported for

ocular drug delivery systems.

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National status

Singh et al.,[26] developed polymer-peptide conjugates by tandem post polymerization

modification. They developed the polymer Poly(pentafluorophenyl methacrylate) obtained by

atom transfer radical polymerization and modified with allylamine, which displaces the active

ester to give well-defined polymers with pendant alkene groups. The produced

poly(allylmethacrylamide) was modified by a second postpolymerization reaction with a thiol-

terminated peptide (CVPGVG) using azobisisobutyronitrile as the radical source. This system

was developed for the separation of proteins or biomolecules. Mishra et al., [10] temperature and

ionic responsive polymeric novel in situ gel system for ocular drug delivery consists of Pluronic

F-127 and chitosan. They showed that the formulation was clear, isotonic solution and converted

into gel at temperatures above 35 ºC and pH 6.9–7.0.

To date, in India, not much research works on development of temperature and pH

responsive polymer-peptide systems for ophthalmic drug delivery is conducted.

17.6 The relevance and expected outcome of the proposed study

The present study is expected to produce the patentable platform formulation of novel

biodegradable temperature and pH responsive hybrid polymer-peptide system suitable for

encapsulation and delivery of biomolecules to the eyes. This hybrid system will possess various

advantages over individual temperature or pH responsive systems like requiring minimum

concentration of polymer and peptide for effective gelling, good biocompatibility,

biodegradability and good adhesion for a prolonged residence time and controlled drug release

in eye. Moreover, the intended controlled and prolonged release of incorporated drug can be

achieved by modifying the composition of polymer and peptide in the hybrid. Due to its stable

crosslinked aggregates, this hybrid polymer-peptide system not only efficiently delivers the

drugs intraocularly and also protects the drug from degradation by enzymes, oxidation and

hydrolysis before released from the system. So, the developed hybrid polymer-peptide system is

expected to be more stable, non-irritant and biocompatible and can be used as a better

formulating agent for prolonged and controlled release of the drug. This will also significantly

reduce the frequency of dosing. Thus this hybrid system may become a viable alternative for the

therapeutically efficacious intraocular delivery of the drugs.

17.7 Preliminary works done so far

We have studied the vitreal pharmacokinetics of dipeptide monoester prodrugs of

ganciclovir (GCV) like Val-Val-GCV and L-glycine-Val-GCV, after intravitreal administration in

conscious rabbit model, using ocular microdialysis and compared the results with published

10

results from anesthetized model. This work has been published in the Journal of Ocular

Pharmacology and Therapeutics[27].

Presently, we are working in the enhancement of corneal permeability of combination

drugs, curcumin and dexamethasone using a novel polymeric nanodispersion for treatment of

age related macular degeneration (AMD). And also, we are developing photosensitizing drug

loaded nanoparticles for the efficient treatment of AMD by photodynamic therapy.

18. Specific objectives

1) To synthesis and characterize a novel hybrid polymer-peptide system having both

temperature and pH responsive property.

The pH sensitive peptides bearing free functional groups COOH or OH will be

obtained by the α-amino acid N-carboxyanhydride ring-opening polymerization or solid

phase peptide synthesis (SPPS) route. The synthesized peptide bearing reactive group of

NH2 / SH will be conjugated to the terminal hydroxyl or carboxyl end group of PEO-PPO

block co-polymer using appropriate chemical reaction. The synthesized polymer-peptide

system will be evaluated for its spontaneous in situ gelling upon ocular pH and

temperature. Its rheological properties and gelling behavior upon physiological condition

will be evaluated.

2) To evaluate biocompatibility and biodegradability of synthesized hybrid polymer-

peptide system.

The biocompatibility of the system will be evaluated by Ocular Irritation Test (HET-

CAM test) [10]. The biodegradation of the system in stimulated ocular environment will be

tested. The cytotoxicity of the biodegradable products will also be evaluated in corneal

epithelial cell lines.

3) To formulate and characterize anti-glaucoma drug incorporated temperature and pH

responsive hybrid polymer-peptide system.

The anti-glaucoma drug will be incorporated in developed hybrid polymer-peptide

system and characterized for its in-vitro characteristics like, viscosity, sol-gel transitions,

isotonicity and drug content. The corneal resident time, irritation and ocular

pharmacokinetics of drug (microdialysis) will be studied in rabbit eye. The enhanced

therapeutic efficiency of the anti-glaucoma drug because of this hybrid polymer-peptide

system will be evaluated in glaucoma rat models and compared with the conventional

ophthalmic formulations. The stability of the formulations will also be studied.

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19. Work Plan

19.1 Methodology

The experiments will be conducted in two phases.

PHASE I

First phase is aimed to develop biodegradable novel hybrid polymer-peptide system

consists of thermo-responsive polymer and pH sensitive peptide capable of forming gel

spontaneously on instillation to the eye.

Preparation of pH sensitive peptide

The peptide will be synthesized in such a way it tends to form aggregates at ocular pH. The

custom designed peptide will be prepared using peptide synthesizer. The terminal functional

group will be modified to SH/NH2 by reacting N-terminus of last amino acid. The custom

synthesized peptide will be cleaned, cleaved and purified. The peptide structure will be

confirmed by proton nuclear magnetic resonance (1H NMR) and its molar mass will be

confirmed using mass spectroscopy (MS). The purity of the synthesized peptide will be

evaluated using HPLC.

Development of hybrid polymer-peptide system

The polymer-peptide system will be synthesized by various chemical reactions.

SCHEME- I

The SH group of the peptide will be reacted with NaOH to get SNa end group (1). The OH

end group of the polymer will be halogenated by treating with PCl3/PBr3 to get OCl/OBr

functional group (2). Then the halogenated polymer will be reacted with SNa end group of

peptide to get polymer-O-S-peptide linkage (3). All the functionalization and polymer-peptide

linkages will be evaluated using 1H NMR and differential scanning calorimetry.

Peptide-SH + NaOH Peptide-S-Na ---------------(1)

Polymer-OH + PCl3 Polymer-OCl ---------------(2)

Peptide-SNa + OCl-Polymer Peptide-S-O-Polymer ---------------(3)

SCHEME-II

The peptide will be synthesized with an end group of NH2. The OH group of the polymer

and NH2 group of the peptide will be conjugated using 2,4,6-trichloro-s-triazine, where the OH

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and NH2 groups will react with chlorine atoms and form the hybrid system. The formation of

linkages will be evaluated by 1H NMR and differential scanning calorimetry.

Polymer OH N

N

N

Cl

Cl

OPolymer

N

N

N

NH

Cl

OPolymer Peptidecyanuric chloride

Na2CO3, benzene, 80 °C

Peptide-NH2

The synthesized hybrid polymer-peptide system will be dialyzed for 4 days to remove the

unconjugated peptides.

Characterization of developed polymer-peptide system

Stimuli responsive gelling behavior of hybrid polymer-peptide system in various pH and

temperature

The known quantity of prepared hybrid system will be dissolved in buffer, vortexes for 2

minutes and will leave for equilibrium for 12 hours. The phase transition from free flowing

liquid to gel will be evaluated based on the concentration of hybrid system, temperature and pH

using inverted tube test and rheometer. These results will be compared with individual polymer

and peptide under same experimental condition.

Rheological properties

The viscoelasticity, intrinsic viscosity of the hybrid polymer-peptide system at normal

storage condition and physiological temperature, ocular pH will be characterized using

rheometer. This evaluation will indicate the tendency of the hybrid systems to get aggregation

due to change in pH and temperature.

Gel morphology and microstructure

The Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM)

techniques will be used to examine the microstructure of the aggregates and networks

uniformity.

NMR studies

The NMR technique will be used to investigate the various chemical bonds occurs in

hybrid system during the phase transition.

Biocompatibility evaluation

The ocular irritation test will be carried out using HET-CAM test and animal models. In

modified HET-CAM test, the fertilized hen eggs will be placed in humidified incubator at 37 ºC

13

for 3 days. Then the egg albumin will be removed using sterile technique and the hole was sealed

by alcohol sterilized paraflim. The eggs will be kept in equatorial position for the development of

chorioallantoic membrane (CAM) away from the shell. The eggs will be candled on the 5th day

of incubation, and every- day thereafter nonviable embryos were removed. On 10th day a

window (2×2 cm) was made on the equator of the eggs through which the polymer-peptide

hybrid will be instilled. A 0.9% NaCl solution will be used as a control nonirritant. Then the score

will be done to evaluate the irritant nature of the system.

The synthesized polymer-peptide system will be instilled on rabbit eyes and the

compatibility will be examined.

Biodegradability evaluation

The biodegradation of the hybrid system will be evaluated in ocular condition stimulated

lacrimal fluid and the toxicity of biodegradable products will be evaluated using corneal

epithelial cell lines.

PHASE II

In second phase, the drug loaded hybrid polymer-peptide system will be prepared.

Formulation of anti-glaucoma drug incorporated hybrid polymer-peptide system

The drug solution will be prepared and incorporated with the hybrid system in isotonic

buffer solution and stored under normal room temperature.

Characterization of formulated drug loaded polymer-peptide system

Physiological stimuli responsive behavior studies will be carried out using rheometer.

Rheological properties like viscoelasticity and intrinsic viscosity will be determined using

rheometer.

Gel morphology and microstructure of the system will be characterized using TEM and

AFM.

The clarity of the formulation at storage condition and after phase transition will be

evaluated by visual observation and light scattering techniques.

The drug content in the formulation will be evaluated by RP- HPLC using suitable

chromatographic conditions.

Sterility of the ophthalmic formulation will be evaluated using membrane filtration and

direct inoculation techniques.

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Evaluation of formulated drug loaded polymer-peptide system

The in-vitro drug release under physiological temperature and pH will be carried out in

stimulated lacrimal fluid at 37 ºC and will be compared with conventional formulations.

In-vitro transcorneal permeation studies will be carried out in corneal diffusion cell using

rabbit cornea, in stimulated lacrimal fluid under 37 ºC. The cumulative amount of drug

permeated will be compared with conventional formulations.

In-vivo transition time and ocular bioavailability of drug from the hybrid polymer-peptide

system will be evaluated using rabbit models. The rate of drug release and concentration of

drug available in the intraocular matrix (aqueous humor) will be evaluated using

microdialysis technique and adopting RP-HPLC with suitable internal standard and

extraction procedures.

The therapeutic efficacy of the hybrid polymer-peptide system in glaucoma condition will be

evaluated using glaucoma induced rat models. The changes in intraocular pressure after the

treatment will be measured using tonometer, and compared with conventional formulations.

The stability studies will be carried out for the optimized formulation as per the ICH

guidelines.

19.2 Connectivity of the participating institutions and investigators (in case of multi-

institutional projects only)

Not applicable.

19.3 Alternate strategies (if the proposed experimental design or method does not work what

is the alternate strategy)

The following alternative approaches may be tried.

i. Using another temperature sensitive polymer Poly(N-isopropylacrylamide) having LCST at

physiological temperature with modification.

ii. The combination of polymer and peptide will be developed instead of conjugation of

polymer and peptide to get the expected thermo, pH responsive system.

iii. The pH sensitive polymers will be investigated instead of pH sensitive peptide, to get an

efficient stimuli responsive polymer system.

15

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13. Hong Y, Legge RL, Zhang S, Chen P. Effect of amino acid sequence and pH on nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV. Biomacromolecules. 2003 Sep-Oct;4(5):1433-42.

14. Li W, Nicol F, Szoka FC Jr. GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv Drug Deliv Rev. 2004 Apr 23;56(7):967-85.

15. Osada K, Kataoka K. Drug and gene delivery based on supramolecular assembly of peg-polypeptide hybrid block copolymers. Adv Polym Sci. 2006;202:113-53.

16. Kim YS, Gil ES, Lowe TL. Synthesis and characterisation of thermoresponsive-co-biodegradable linear-dendritic copolymers. Macromolecules. 2006;39:7805-11.

17. Deng L, Shi K, Zhang Y, Wang H, Zeng J, Guo X, Du Z, Zhang B. Synthesis of well-defined poly(N-isopropylacrylamide)-b-poly(L-glutamic acid) by a versatile approach and micellization. J Colloid Interface Sci. 2008;323(1):169-75.

18. Hoffman AS. Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations. Clin Chem. 2000 Sep;46(9):1478-86.

16

19. Stoica F, Alexander C, Tirelli N, Miller AF, Saiani A. Selective synthesis of double temperature-sensitive polymer-peptide conjugates. Chem Commun (Camb). 2008 Oct 7;(37):4433-5.

20. Mitra A, Nan A, Papadimitriou JC, Ghandehari H, Line BR. Polymer-peptide conjugates for angiogenesis targeted tumor radiotherapy. Nucl Med Biol. 2006 Jan;33(1):43-52.

21. Song H, He R, Wang K, Ruan J, Bao C, Li N, Ji J, Cui D. Anti-HIF-1alpha antibody-conjugated pluronic triblock copolymers encapsulated with Paclitaxel for tumor targeting therapy. Biomaterials. 2010 Mar;31(8):2302-12.

22. Lutolf MP, Raeber GP, Zisch AH, Tirelli N, Hubbell JA. Cell-Responsive Synthetic Hydrogels. Adv Mater. 2003;15(11):888-92.

23. Lutolf MP, Weber FE, Schmoekel HG, Schense JC, Kohler T, Müller R, Hubbell JA. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol. 2003 May;21(5):513-8.

24. Tugulu S, Silacci P, Stergiopulos N, Klok HA. RGD-Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells. Biomaterials. 2007 Jun;28(16):2536-46.

25. Wang W, Liang H, Al Ghanami RC, Hamilton L, Fraylich M, Shakesheff KM, Saunders B, Alexander C. Biodegradable Thermoresponsive Microparticle Dispersions for Injectable Cell Delivery Prepared Using a Single-Step Process. Adv Mater. 2009;21(18):1809-13.

26. Singha NK, Gibson MI, Koiry BP, Danial M, Klok HA. Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem "Ester-Amide/Thiol-Ene" Post-Polymerization Modification of Poly(pentafluorophenyl methacrylate) Obtained Using ATRP. Biomacromolecules. 2011 Aug 8;12(8):2908-13.

27. Janoria KG, Boddu SH, Natesan S, Mitra AK. Vitreal pharmacokinetics of peptide-transporter-targeted prodrugs of ganciclovir in conscious animals. J Ocul Pharmacol Ther. 2010 Jun;26(3):265-71.

17

20. Timeline for year wise plan of the Research work

Period of study Achievable targets

0-6 Months Preparation and characterization of functionalized pH sensitive peptides

7-12 Months Conjugation of pH sensitive peptides with temperature responsive polymer

to get hybrid polymer-peptide system

13-18 Months Characterization and optimization of synthesized hybrid polymer-peptide

system

19-24 Months Formulation of anti-glaucoma drug incorporated hybrid polymer-peptide

system and its in-vitro characterization

25-30 Months Biocompatibility, biodegradability and in-vivo drug release and permeation

studies using rabbit models

31-36 Months Stability studies and documentation of the experimental results,

publication and patenting the completed work will be done

21. Name and address of 5 experts in the field:

Sl.No.

Name Designation Address

1. Dr T. Velpandian Assistant Professor

DRP Centre for Ophthalmic Sciences All India institute for medical sciences New Delhi, India E mail- [email protected]

2.

Dr. V. R. Muthukkaruppan

Director Research

Ophthalmic Research Laboratory Aravind Eye Care System, Madurai E mail- [email protected]

3.

Dr. N. Angayarkanni Head

Department of Biochemistry and Cell Biology Sankara Nethralaya, Nungambakam Chennai- 600 006 Email- [email protected]

4.

Dr. Indupal Kaur Associate Professor

University Institute of Pharmaceutical Sciences Panjab University Chandigarh E mail - [email protected]

5.

Dr. R.K. Khar Professor

Department of Pharmaceutics Faculty of Pharmacy Jamia Hamdard University New Delhi Email- [email protected]

18

PART IV: BUDGET PARTICULARS Budget (In Rupees) A. Non-Recurring (e.g. equipments, accessories, etc.)

S. No.

Item Year 1

Rs. Year 2

Rs. Year 3

Rs. Total Rs.

1. Rheometer 31,60,000/- - - 31,60,000/-

2. Tonometer 2,95,000/- - - 2,95,000/-

3. Multi syringe infusion pump 1,75,000/- - - 1,75,000/- Sub-Total (A) 36,30,000/- - - 36,30,000/-

B. Recurring

B.1 Manpower S. No.

Position Year 1

Rs. Year 2

Rs. Year 3

Rs. Total Rs.

1. JRF (One) @ Rs. 18,000/- pm for 1st and 2nd years and Rs. 20,000/- pm for 3rd year

2,16,000/- 2,16,000/- 2,40,000/- 6,72,000/-

Sub-Total (B.1) 2,16,000/- 2,16,000/- 2,40,000/- 6,72,000/- B.2 Consumables

S.No. Items Quantity Year 1

Rs. Year 2

Rs. Year 3

Rs. Total Rs.

1. 2. 3.

Chemicals Timolol Maleate, Latanoprost, Poloxamer, peptides, 2,4,6-trichloro-s-triazine, Phosphorus trichloride & Phosphorus pentachloride, etc Glass / Plastic ware 96- well plate, Petriplates, Culture flasks, Culture tubes, Pipettes, filters, etc. Minor equipments

Micropipettes

Varying quantity based on

requirements

3,00,000/- 4,00,000/- 3,00,000/- 10,00,000/-

Sub-Total (B.2) 3,00,000/- 4,00,000/- 3,00,000/- 10,00,000/-

S. No.

Other items Year 1

Rs. Year 2

Rs. Year 3

Rs. Total Rs.

1. B.3 Travel 25,000/- 25,000/- 25,000/- 75,000/- 2. B.4 Contingency 60,000/- 70,000/- 70,000/- 2,00,000/- 3. B.5 Overhead @ 20% of B 1,20,200/- 1,42,200/- 1,27,000/- 3,89,400/-

4. Sub-total of B (B.1+B.2+B.3+B.4+B.5) 7,21,200/- 8,53,200/- 7,62,000/- 23,36,400/-

Grand Total (A + B) 59,66,400/-

19

Justification for Non-recurring Expenditure (Equipments) Rheometer

Rheometer is necessary for the evaluation of phase transition behaviour of the proposed

system.

Tonometer

Tonometer is needed to estimate the intraocular pressure and evaluate the therapeutic

efficacy of the proposed polymer-peptide system over currently available conventional

drug delivery systems

Multi syringe infusion pump

Multi syringe infusion pump is essential to carry out the ocular microdaialysis for

continuous determination of drug concentration in aqueous humor.

Justification for Recurring Expenditure

Chemicals

Fine quality chemicals, reagents, serums and glass/plastic wares are essential for the

successful execution of the proposed study.

Manpower

The proposed project involves intensive bench work. Therefore at least one Junior Research

Fellows (JRF) is required for three years to carryout various experiments and successful

completion of the project.

Travel grant

The travel grant may be needed for the PI and other project staff to participate in

conferences/symposia.

Contingency

Contingency is essential for meeting various needs such as publication of the results, postal

charges, photocopying charges, stationeries, purchasing the polymers, experimental

animals and feed etc.

20

PART V: EXISTING FACILITIES IN DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY

Resources and additional information

1. Laboratory:

a. Manpower

Laboratory Assistants – Available

Research Scholars – Seven numbers

b. Equipments

1. HPLC with PDA detector(Shimadzu)

2. UV – VIS Spectrophotometer (Shimadzu)

3. Cooling Centrifuge (Eppendorf)

4. Rotary Mixer

5. Microcentrifuge

6. Vacuum oven (2 nos)

7. Ultra sonicator

8. Tablet Disintegration Test Machine (2 nos)

9. USP Dissolution Apparatus (Veego - 2 nos)

10. Stability Chamber (Bionik)

11. Homogenizer

12. Constant temperature water circulator

13. pH meter (3 nos)

14. Laminar Airflow Chamber (2 nos)

15. Incubator

16. Rotary Shaker

17. Ultrasonic bath Sonicator (3 nos)

18. Infra Red Moisture Balance (Macro Scientific)

19. Sieve Shaker

20. Constant Temperature Bath

21. Deep freezers (-20oC; -80oC)

22. Other minor instruments

2. Other resources such as clinical materials, animal house facility. Experimental garden, pilot

plant facility etc.

Animal house facility - Under construction

21

PART VI: DECLARATION/CERTIFICATION

It is certified that

a) The research work proposed in the scheme/project does not in any way duplicate the work

already done or being carried out elsewhere on the subject.

b) The same project proposal has not been submitted to any other agency for financial support.

c) the emoluments for the manpower proposed are those admissible to persons of

corresponding status employed in the institute/university or as per the Ministry of Science &

Technology guidelines (Annexure-III)

d) Necessary provision for the scheme/project will be made in the Institute/University/State

budget in anticipation of the sanction of the scheme/project.

e) If the project involves the utilization of genetically engineered organisms, we agree to submit

an application through our Institutional Biosafety Committee. We also declare that while

conducting experiments, the Biosafety Guidelines of the Department of Biotechnology would

be followed in Toto.

f) If the project involves field trials/experiments/exchange of specimens, etc. we will ensure

that ethical clearances would be taken from concerned ethical Committees/Competent

authorities and the same would be conveyed to the Department of Biotechnology before

implementing the project.

g) It is agreed that any research outcome or intellectual property right(s) on the invention(s)

arising out of the project shall be taken in accordance with the instructions issued with the

approval of the Ministry of Finance, Department of Expenditure, as contained in Annexure-

V.

h) We agree to accept the terms and conditions as enclosed in Annexure-IV. The same is signed

and enclosed.

i) The institute/university agrees that the equipment, other basic facilities and such other

administrative facilities as per terms and conditions of the grant will be extended to

investigator(s) throughout the duration of the project.

j) The Institute assumes to undertake the financial and other management responsibilities of

the project.

22

PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Principal Investigator

Name : Dr. N. Subramanian


Department/Institute/University : Department of Pharmaceutical Technology

Anna University of Technology

Tiruchirappalli- 620 024.

Date of Birth: 15.07.1973 Sex (M/F): M SC/ST: No

Education (Post-Graduation onwards & Professional Career)

Degree/Position held Institution / University Year of

Passing

M.Pharm. Annamalai University, Chidambaram 1998

Ph.D. Jadavpur University, Kolkata 2005

Post-Doc University of Missouri - Kansas City, USA 2007-08

Assistant Professor Pallavan Pharmacy College, Kancheepuram 1998-2005

Lecturer Bharathidasan University, Tiruchirappalli 2005-2007

Assistant Professor Anna University of Technology, Tiruchirappalli 2007 onwards

Honors/Awards

Sl.No. Year Award Organization

1. 2007-08 BOYSCAST Fellowship DST, Govt. of India

2. 2006 Young Scientist Fellowship Award TNSCST, Govt. of Tamilnadu

3. 2000-04 SRF, Research Fellowship AICTE, Govt. of India

Professional Experience and Training relevant to the Project

Position Institute Duration Supervisor

PDF

Visiting Research Scientist (BOYSCAST Fellow) Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri - Kansas City, USA

2007- 2008

Prof. A.K. Mitra

Visiting Scientist

National Institute of Cholora and Enteric Diseases, Kolkatta

2006 Dr. K. Ramamoorthy

B. Publications (Numbers only):

Books: Chapters - 3 Research Papers: 21 Reports: Nil General articles: 2

Patents: Three Others (Please specify): Conference proceedings - 2

23

Patents

Mitra A.K., Velagaleti P. and Natesan S., Ophthalmic compositions comprising calcineurin or m-TOR inhibitors.

US Patent publication No. 20090092665, Date: 09.04.2009 PCT Publication No. WO/2009/048929 , Date: 16.04.2009 Indian patent Publication No. 1220/KOLNP/2010 Date : 30.06.2010

Subramanian. N, Abimanyu. S, Chandrasekar. P., Method and composition for solublization of hydrophobic compounds. Indian provisional application No. 1423/CHE/2011 Date: 25.04.2011

Selected peer-reviewed publications (Ten best publications in chronological order)

1. N. Subramanian, L. Rameshkumar, K. Venkateshwaran and S. Abimanyu Simultaneous Estimation of Cefixime and Ofloxacin in Tablet Dosage form by RP- HPLC, 2011, Int. J. Pharm. Res. Sci., Vol. 2, 219-224.

2. N. Subramanian, T. Devipriyadharshini, K. Venkateshwaran and P. Chandrasekar Improved RP-HPLC Method for the Simultaneous Estimation of Tranexamic Acid and Mefenamic Acid in Tablet Dosage Form, 2011, Pharm Anal Acta Vol. 2, 115. doi:10.4172/2153-2435.1000115

3. K. G. Janoria, Sai. H. S. Boddu, N. Subramanian, A.K. Mitra. Vitreal Pharmacokinetics of Peptide-Transporter-Targeted Prodrugs of Ganciclovir in Conscious Animals, 2010, Journal of Ocular Pharmacology and Therapeutics, Vol. 26, 265-271

4. S. Gupta, N. Subramanian, A. Acharya, S. Datta, S. P. Moulik. Development and in - vitro Characterization of Cholesterol Nanodispersed Vehicle for the Delivery of Lipophilic Drugs. 2010, Journal Pharmacy Research, Vol. 3, 2808 – 2810.

5. C. Senthil Kumar, B.N. Vedha Hari., S.P. Sharavanan, N. Subramanian, S. Punitha, V. Senthil Kumar. Novel Metronidazole Nanosuspension as a Controlled Drug Delivery System for Anthelmintic Activity. 2010, Journal Pharmacy Research, Vol.3, 2404-2407.

6. N. Subramanian, S. K. Ghosal and S. P. Moulik. Improved Oral Bioavailability of Celecoxib Using Self -Microemulsifying Drug Delivery System, The AAPS Journal Supplement, 2007, 9(S2), 1918.

7. N. Subramanian, S. K. Ghosal and S. P. Moulik. Enhanced in vitro percutaneous absorption and in vivo topical anti-inflammatory effect of a selective cyclooxygenase inhibitor using microemulsions, Drug Development and Industrial Pharmacy, 2005, Vol. 31, 405-416.

8. N. Subramanian, S. K. Ghosal, A. Acharya and S. P. Moulik. Formulation and physicochemical characterization of microemulsion system using isopropyl myristate, medium- chain glyceride, polysorbate 80 and water, Chemical Pharmaceutical Bulletin, 2005, Vol. 53, 1530-1535.

9. N. Subramanian, S. Ray, S. K. Ghosal, R. Bhadra and S. P. Moulik. Formulation design of self-microemulsifying drug delivery systems for improved oral bioavailability of celecoxib. Biological Pharmaceutical Bulletin, 2004,Vol. 27, 1993-99.

10. N. Subramanian, S. K. Ghosal and S. P. Moulik. Topical delivery of celecoxib using microemulsion. Acta Poloniae Pharmaceutica – Drug Research, 2004, Vol. 61, 335-41.

24

List maximum of five recent publications relevant to the proposed area of work.

1. K. G. Janoria, Sai. H. S. Boddu, N. Subramanian, A.K. Mitra. Vitreal Pharmacokinetics of Peptide-Transporter-Targeted Prodrugs of Ganciclovir in Conscious Animals, 2010, Journal of Ocular Pharmacology and Therapeutics, Vol. 26, 265-271

2. S. Gupta, N. Subramanian, A. Acharya, S. Datta, S. P. Moulik. Development and in - vitro Characterization of Cholesterol Nanodispersed Vehicle for the Delivery of Lipophilic Drugs. 2010, Journal Pharmacy Research, Vol. 3, 2808 – 2810.

3. N. Subramanian, S. K. Ghosal and S. P. Moulik. Enhanced in vitro percutaneous absorption and in vivo topical anti-inflammatory effect of a selective cyclooxygenase inhibitor using microemulsions, Drug Development and Industrial Pharmacy, 2005, Vol. 31, 405-416.

4. N. Subramanian, S. K. Ghosal, A. Acharya and S. P. Moulik. Formulation and physicochemical characterization of microemulsion system using isopropyl myristate, medium- chain glyceride, polysorbate 80 and water, Chemical Pharmaceutical Bulletin, 2005, Vol. 53, 1530-1535.

5. N. Subramanian, S. Ray, S. K. Ghosal, R. Bhadra and S. P. Moulik. Formulation design of self-microemulsifying drug delivery systems for improved oral bioavailability of celecoxib. Biological Pharmaceutical Bulletin, 2004, Vol. 27, 1993-99.

C. Research Support Ongoing Research Projects

Sl No.

Title of Project Funding Agency

Amount Date of sanction and Duration

1. Development, characterization and biological evaluation of microemulsion and lipid dispersion for drug delivery and detoxification

DST 20,77,000/- 20–10– 2008 30 Months

2. Simultaneous silencing of multiple pro-angiogenic factors to suppress tumor-induced angiogenesis

DBT, New Delhi

3,90,000/- 14.12.2010 & 3Years

Completed research projects in last 3 years– None

25

Co-Investigator

Name : Dr. P. Rajaguru Designation : Professor Department/Institute/University : Department of Biotechnology

Anna University of Technology, Tiruchirappalli Tiruchirappalli-620 024.

Date of Birth: 20.05.1962 Sex (M/F): M SC/ST : No.


Degree/Position held Institution / University Year of Passing

M.Sc. University of Madras, Chennai 1984

Ph.D. Bharathiyar University, Coimbatore 1998

Post-Doc National Institute of Health Sciences, Tokyo, Japan 2003-04

Lecturer P.S.G. College of Arts and Science, Coimbatore 1986-98

Reader P.S.G. College of Arts and Science, Coimbatore 1998-2005

Assistant Professor Anna University Tiruchirappalli, Tiruchirappalli 2005-2009

Professor Anna University Tiruchirappalli, Tiruchirappalli 2009 onwards

Honors/Awards

Sl.No. Year Award Organization

1. 1998 International Cancer Technology Transfer Award (ICRET)

International Union against Cancer (UICC), Geneva, Switzerland

2. 2002 2nd International Cancer Technology Transfer Award (ICRET)

International Union against Cancer (UICC), Geneva, Switzerland

3. 2003 Postdoctoral Fellowship Japanese Society of Pharmacopia Tokyo, Japan

B. Publications (Numbers only): 21 Books : Nil Research Papers: 21 Reports : 2 General articles : Nil Patents : Nil Others (Please specify):

26

Selected peer-reviewed publications (in last 5 years) 1. Zhan, L., M. Honma, L. Wang, M. Hayashi, D. Wu, L. Zhang, P. Rajaguru, T. Suzuki (2006)

Microcystin-LR is not Mutagenic in vivo in the λ/lacZ Transgenic Mouse (Muta™Mouse). Genes and Environment, 28: 68-73.

2. Luana Y., T. Suzuki, P. Rajaguru, Y. Takashima, H. Sakamoto, M. Sakuraba, T. Koizumi, M. Saito, H. Matsufuji, K. Yamagata, Y. Yamaguchi, M. Hayashi, M. Honma. (2007) Potassium bromate treatment predominantly causes large deletions, but not GC>TA transversion in human cells. Mutat. Res. 619: 113-123.

3. Ramkumar K. M., P. Rajaguru, M. Latha, R. Ananthan (2007) Ethanol extract of Gymnema montanum leaves reduces glycoprotein components in experimental diabetes. Nutrition Research. 27: 97– 103.

4. Ramkumar K.M, P. Rajaguru, R. Ananthan (2007) Antimicrobial properties and phytochemical constituents of an antidiabetic plant Gymnema montanum. Adv. Biol. Res. 1 (1-2): 67-71.

5. Ramkumar K.M, P. Rajaguru, M. Latha, R. Ananthan (2008) Effect of Gymnema montanum leaves on red blood cell resistance to oxidative stress in experimental diabetes. Cell Biol. Toxicol. 24(3): 233-241.

6. Ramkumar K.M, R.S. Vijayakumar, P. Ponmanickam, S. Velayuthaprabhu, G. Archunan, P. Rajaguru (2008) Antihyperlipidemic effect of Gymnema montanum: A study on lipid profile and fatty acid composition in experimental diabetes. Basic Clinical Pharmacol. Toxicol. 103: 538-545.

7. Vijayanand, C., P. Rajaguru, K. Kalaiselvi, K. Panneer Selvam and M. Palanivel (2008) Assessment of heavy metal contents in the ambient air of the Coimbatore city, Tamilnadu, India. J. Hazard. Mater. 160: 548-553.

8. Ramkumar, K.M., C. Manjula, L. Sankar, S. Suriyanarayanan, P. Rajaguru. (2009) Potential in vitro antioxidant and protective effects of Gymnema montanum H. on alloxon-induced oxidative damage in pancreatic β-cells, HIT-T15. Food and Chemical Toxicology. 47: 2246-2256.

9. Latha, M., Pari, L., Ramkumar K.M., Rajaguru, P., Suresh, T., Dhanabal, T., Sitasawad, S., Bhonde, R. (2009) Antidiabetic effects of scoparic acid D isolated from Scoparia dulcis in rats with streptozotocin-induced diabetes. Natural Product Research. 1-13. iFirst

10. Ramkumar, K.M., Ponmanickam, P., Velayuthaprabhu, S., Archunan, G., Rajaguru, P. (2009) Protective effects of Gymnema montanum against renal damage in experimental diabetic rats. Food and Chemical Toxicology. 47: 2516–2521

11. Ramkumar, K.M., K. Krishnamurthi, A.S. Lee, S. Saravana Devi, T. Chakrabarti, K.P. Kang, S. Lee, W. Kim, S.K. Park, P. Rajaguru (2009) Gymnema montanum H. protects against alloxan induced oxidative stress and apoptosis in pancreatic β-cells. Cellular Physiology and Biochemistry. 24:429-440.

12. Ramkumar, K.M., Thayumanavan, B., Palvannan, T., Rajaguru, P. (2009) Inhibitory effect of Gymnema montanum leaves on alpha-glucosidase activity and alpha-amylase activity and their relationship with polyphenolic content. Medicinal Chemistry Research. DOI 10.1007/s00044-009-9241-5.

13. Ramkumar, K.M., L. Sankar, K. Krishnamurthi, S. Saravana Devi, T. Chakrabarti, K. Kalaiselvi, M. Palanivel, P. Rajaguru. (2010). Antigenotoxic potential of Gymnema montanum leaves on DNA damage in human peripheral blood lymphocytes and HL-60. Environmental and Molecular Mutagenesis. 51: 285-293.

27

D. Research Support Ongoing Research Projects

Sl No. Title of Project

Funding Agency Amount

Date of sanction and

Duration

1. Silencing of ER stress response genes using RNA interference to protect hyperglycemia induced pancreatic β-cell death

DST 28,86,000/- (April, 2009)

3 years

2. Simultaneous silencing of multiple pro-angiogenic factors to suppress tumor-induced angiogenesis

DBT 72,66,000/- Dec., 2010 (3 years)

Completed Research Projects – Completed during last 3 years

Sl No. Title of Project

Funding Agency Amount

Date of completion

1 Genotoxicity profiling of ground water in Noyal river basin

UGC 10,57,100/- March, 2010

2 Designing a ‘multitarget RNAi’ effector molecule for simultaneous silencing of multiple genes

DBT 30,71,000/- March, 2010

3

Screening and characterization of active constituents from an antidiabetic plant Gymnema montanum and its pharmacological evaluation using genomic and proteomic approach

DST-JSPS 2,58,000/- May, 2010

28

Co-Investigator

Name : Dr. M. Sivakumar


Department/Institute/University : Division of Nanoscience and Technology

Department of Physics

Anna University of Technology – Tiruchirappalli

Tiruchirappalli - 620 024.

Date of Birth: 24.05.1966 Sex (M/F): M SC/ST: No.


Sl No.

Institution/ Place Degree Awarded

Year Field of Study

1 University of Madras, Chennai M.Sc., 1989 Polymer Chemistry

2 University of Madras, Chennai M.Phil., 1991 Analytical Chemistry

3 Anna University, Chennai Ph.D. 1998 Polymer Technology

A. Position and Honors

Position and Employment (Starting with the most recent employment)

Institution/Place Position From (Date) To (date) Anna University of Technology, Tiruchirappalli

Assistant Professor 02nd Apr 2008 Till date

Nagoya Institute of Technology, Nagoya, Japan

Post-Doctoral Fellow 1st Apr 2005 31st Mar 2008

21st Century Centre of Excellence (COE) fellow

7th May 2002 31st Mar 2005

JSPS Fellow 17 Apr 2000 17 Apr 2002

National University of Singapore Research Engineer 2nd Oct 1998 31st Oct 1998

A.C. Tech, Anna University, Chennai

Research Associate 1st Apr 1998 Sep, 1998

Honors/Awards

Sl. No. Year Honors/Awards Organization

1. 1995-97 Senior Research Fellowship CSIR, Govt. of India

2. 1998 Research Associate CSIR, Govt. of India

3. 2000-02 JSPS (Japan Society for the Promotion of Science) Fellowship for Foreign Researchers awarded

Ministry of Education, Japan

4. 2003-05 Research Associate fellowship awarded by 21st century Centre of Excellence (COE) Program

Nagoya Institute of Technology, Nagoya, Japan

29

Professional Experience and Training relevant to the Project

Position Institute Duration

Research Engineer National University of Singapore 1999 JSPS Fellow Nagoya Institute of Technology, Japan 2000-2002 CO Fellow Nagoya Institute of Technology, Japan 2003-2005 JSPS Fellow Nagoya Institute of Technology, Japan 2005-2007

B. Publications (Numbers only): 33

Books: Nil Research Papers, Reports: 32 General articles: 01

Patents: Nil others (Please specify): Nil

Selected peer-reviewed publications:

1. M.Sivakumar, R.Tominaga, M.Tanaka, T.Kinoshita, Design and fabrication of biosensor for

specific microbe by silicon-based interference colour system, Colloids & surfaces B: Bio

interfaces 2011 (in press) (Elsevier Publications) IF=2.593

2. M.Sivakumar, R.Tominaga, M.Tanaka, T.Kinoshita, Visible Detection of biotin by Thin-film

interference, J. Mater. Chem., 2008,18,976-980(peer reviewed paper).(RSC publication) IF=

4.339

3. M.Sivakumar, R.Tominaga, M.Tanaka, T.Kinoshita, Inorganic-organic thin-layers which

transform bio-molecule binding into visible color change, Adv.Mater.Res(Swiz),2006(11-

12)631-634(Trans Tech Pub)

4. M.Sivakumar, R.Tominaga, T.Koga, T.Kinoshita, M.Sugiyama, K.Yamaguchi, Studies on

visual sensor from self-assembled polypeptides, Sci. Tech. Adv. Mater. 2005, 6, 91-96. (Elsevier

publications)(peer reviewed paper) IF=1.27

5. M.Sivakumar, R.Tominaga, T.Kobayashi, T.Kinoshita, Construction and estimation of

inorganic-organic nanostructured sensing plate. Trans.Mater.Res. Soc.Japan, 2005, 30(2), 349-

352.(MRS-Japan)(peer reviewed)

6. M. Sivakumar, K. P. O. Mahesh, Y. Yamamoto, Y. Tsujita, H. Yoshimizu, Structure and

properties of the delta-form and mesophase of syndiotactic polystyrene membranes prepared

from different organic solvents, J.Polym.Sci, B 2005, 43(14),1873-1880.(Wiley Science) (peer

reviewed paper) IF= 1.524

7. M. Sivakumar, K.P.O. Mahesh, Y. Yamamoto, Y. Tsujita, H. Yoshimizu and S. Okamoto,

Structure and properties of the mesophase of syndiotactic polystyrene IX. Preferential

sorption behaviour of sPS-p- chlorotoluene mesophase membrane in a mixture of solvents,

J.Memb.Sci. 2005, 262(1-2), 11-19 (Elsevier Publications, peer reviewed paper) IF= 3.247

30

8. M.Sivakumar, D.Mohan and R.Rangarajan, Studies on cellulose acetate-polysulfone

Ultrafiltration membranes-I: Effect of additive concentration, J. Memb. Sci., 2006, 268(2), 208-

219.(Elsevier Publications, peer reviewed paper) IF= 3.247

9. M.Sivakumar, L.Susithra, D.Mohan and R.Rangarajan, Preparation and performance of

polysulfone-cellulose acetate blend Ultrafiltration membranes, J. Macromol. Sci, 2006,

A.43(10),1541-1551. (Taylor and Francis group publications, peer reviewed paper) IF= 0.963

10. M.Sivakumar, D.Mohan, R.Rangarajan and Y.Tsujita, Studies on cellulose acetate–

polysulfone ultrafiltration membranes-I: Effect of polymer composition, Polym. Int., 2005.

54(6)956-962. (SCI publications, peer reviewed paper) IF= 2.137List maximum of five recent

publications relevant to the proposed area of work.

C. Research Support

Ongoing Research Projects - NIL

Completed research projects in last 3 years– NIL