1

January 2015

Dabur Research Foundation

22, Site IV, Sahibabad

Ghaziabad – 201010

Uttar Pradesh, INDIA

www.daburresearch.in

2

Dabur Research Foundation (DRF)

Indian Contract Research Organization focused on Preclinical drug discovery &

Development

Led the R & D programs of one of the largest Indian Healthcare groups (1979 -

2008)

Strategic spin off of the parent group in year 2008 to become a Contract Research

Organization in the niche area of Preclinical development

Positioned as a Biology specialist CRO with services in several therapeutic areas viz

Oncology, Inflammation, Metabolic diseases & others

Strength of 80 scientists with close to 40 % being Ph.Ds recruited from the top 5

Universities of India

Contract services for end to end preclinical development of Cytotoxics, Botanicals,

Phytochemicals, generics & differentiated formulations in multiple therapeutic areas

GLP compliant and non GLP studies

Located near New Delhi, well connected to the International Airport

3

The DRF Advantage

Over 20 years of experience in preclinical development of Cytotoxics, biologically

targeted molecules, Phytochemicals, generics & differentiated formulations in multiple

therapeutic areas

No conflict of interest with client projects. No internal R &D programs

Comprehensive Services in Cell Biology, Pharmacology, Toxicology, DMPK, Bioanalytical,

Analytical & formulation development to enable lead identification as well as lead

development

Availability of stand alone service modules & complete service packages to meet varied

client requirements

Availability of guideline driven services as well services customized for the clients

A dedicated Central Innovation Research Team for customized model development

GLP compliant studies managed by Project coordinators, Technical Coordinators & QAU

teams

Experience & knowledge of regulatory requirements for submission of data packages

Technical consulting for development of road map for client pipeline

4

About Parent Company

• Established in 1884, Dabur India Ltd is among the oldest and largest healthcare company in

India

• Has over 5000 employees working in more than 20 countries

• Market Cap of over 4 bn USD, DIL recently achieved sales of 1 bn USD

• More than 600 herbal products in market

• 17 ultra-modern manufacturing units spread around the globe

• Products marketed in over 60 countries

• More than 5000 distributors and over 2.8 million retail outlets all over India

5

Location

Delhi (NCR,India)

R & D center, Sahibabad, India

New Jersey, USA

Malmo, Sweden

6

Management Team

CSO- Dr. Manu Jaggi Post Graduate in Pharmaceutical Sciences with Doctorate in Cancer Biology. More than 22 years of experience in Drug Discovery, advanced preclinical development & regulatory submissions for NCEs & Botanicals. Expertise in Pharmacology, Safety assessment & Pharmaceutical development

Vice President (R&D) – Dr. Anu T. Singh Post Graduate in Biotechnology with Doctorate in Cancer Biology. More than 22 years of research experience in drug discovery, Cell Biology & early preclinical development of NCEs & Botanicals. Expertise in Cell Biology, target identification and development of disease models in Oncology & allied areas

Director, Business Development (US) Dr. William Heilman has more than 30 years of experience in business development. He has previously worked in large pharma / biotech companies in the US including Wyeth, Comgenex, AMRI & Morphotek. Dr. Heilman has done is Ph.D in Medicinal Chemistry from University of Kansas.

Advisor Dr. Ashok Mukherjee has more than 35 years of experience in human and animal

pathology. Ex-Director, of Institute of Pathology (ICMR), Dr. Mukherjee has carried out

extensive research in communicable diseases. He has been associated with DRF since 1999.

CHAIRMAN - Dr. Burman holds a Ph.D in Pharmaceutical Chemistry from the University of Kansas. Dr. Burman set up the pharmaceutical division for Dabur India in 1989 and he is a prominent and respected figure within the international oncology industry.

7

1985

SIRO

recognition

by DST

1994

Set Up state-

of-art labs

for Discovery

& Preclinical

division

1995

First US

Patent

granted

1999

Registered

with

CPCSEA

2003

Strategic

Expansion of

capabilities

in non

Oncology

areas

2004

First Polymer

based

nanoparticle

delivery

system

enters

clinical

development

2008

Filed 400th

patent

application

2008 April

Started

development

of botanicals

for Cancer &

CVS

2009 March

Dev &

regulatory

submission

of liposomal

Docetaxel

2010 Expansion

of facility at

SBD & initiation

of GLP

compliant

studies

2013- SBD

Facility II

initiated

The Story

continues…

2008 May

Partnered

with Oxford

University

for drug

development

in oncology

for a new

target

1979

DRF

incorporated

2000

First

Anticancer

NCE goes

into clinical

development

2007

Nanoxel

launched in

India

2008 May

Expansion of

contract

Research

capabilities

2009 April

Partnered

with a large

health care

Finnish

company for

development

of molecules

for alopecia

Key Milestones

20013

Completed

1000 studies

8

Experience from 1 to 19 years

Scientific Personnel

Pharmacists

Biotechnologists

Toxicologists

Pharmacologists

Biochemists

Life Science

graduates

9

Research Laboratories

10

Infrastructure

FACILITY DETAILS

Facility - Spread on a single floor

Area - Approx. 30,000 sq/ft

Labs - in vitro / in vivo Pharmacology, Cell Biology, Toxicology, DMPK lab, Small Animal Facility(SAF), Tissue Culture Facility (TCF)

Water - Sufficient with separate lines of Normal and DM Water Milli Q Water Purification System.

Temperature controls - Area controlled by AHUs/ACs

Temperature/Humidity data loggers in each labs Safety - Safety devices at each location.

Fire Extinguishers, Fire Alarms First Aid

CCTV Surveillance in all labs

Access Control System in all labs

Fire Proof Archival Room for Wet Archives

Fire Proof Archival Room for Dry Archives

Provision for Ante-rooms wherever necessary

11

Infrastructure

TOXICOLOGY LAB

Equipments :

Hematology Analyzer

Biochemistry Analyzer

Manual Rotary Microtome

Tissue Embedder (Paraffin Dispenser)

+ Water bath + Hotplate

Water Warming Table Digital

Experienced Study Director (6 to 17 years)

Expert Pathologist (> 30 years experience)

PHARMACOLOGY LAB

Equipments :

CNS pharmacology

Pressure Application measurement (PAM)

Von Frey

Small animal anesthesia system

RA-50 Chemistry system

Inverted and Phase contrast microscope

Homogenizer

OHM meter, Histamine chamber

Digital Plethysmometer

Digital Caliper

Organ bath

Hot and cold plate method

Rectal thermometer

Refrigerated centrifuge

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Infrastructure

CELL BIOLOGY LAB

Equipments:

Flow cytometer (Guava Technologies)

Multiwell multimode reader (Biotek)

Fluorescence microscope (Nikon)

Phase contrast microscopes (Nikon)

Dissecting microscope (Motic)

Spectrophotometer (Shimazdu)

CO2 incubators

Laminar hood cabinets

Liquid nitrogen cans

UV irradiation chamber

Centrifuges, water bath, shakers

Analytical Balance

Micropipettes, Dispensettes

Vortexer mixers, homogenizer

Fridges, Freezer, Deep Freezer.

DMPK LAB

Equipments :

Analytical HPLCs

Preparative HPLCs

LC/MS-MS

Nitrogen and Vacuum Evaporator

SPE Vacuum Manifold, Temp. & humidity data loggers

Analytical Balance

Dispensette/ Micropipettes

Centrifuge/Homogenizer/Sonicators

Water Bath with Shaker

Fridges, Freezer,(-20°C) Deep Freezer(-80°C)

Lyophilizer

Buchi Rotavapour

Franz diffusion cell system

Nitrogen generator

13

Infrastructure

TISSUE CULTURE FACILITY

Equipments:

Laminar air Flow

CO2 incubators

Biosafety cabinet

Refrigerated centrifuge

Fridges and Deep freezers

Liquid nitrogen container

Inverted & Phase contrast Microscope

ANIMAL FACILITY

Equipments:

Isolators, Data loggers

Small Animal Anesthesia System

Individually Ventilated Caging System (Techniplast, Italy/Citizen

Industries, India)

Animal Cage Changing Station (Citizen Industries, India)

Horizontal Autoclave (P. L. Tandon & Co., India)

Lux Meter (HTC, China)

Sound Level Meter (CENTER, Taiwan)

Laminar Flow Hood (Klenzaids, India)

Freezer (Vest Frost)

Water Purifier (Eureka Forbes)

Digital Hygro-thermometer (MEXTECH)

Facilities: Small laboratory animals breeding and experimental

facility

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Mammalian cell

culture

Small Animal

Facility

Microscopy

Histopathology

Archival

Labs & support facilities

Preclinical

Safety

Preclinical

Efficacy

Bioanalytical

Experimental

Models

Cellular &

Molecular Biol

ADME-PK

Quality

Assurance

Study Directors

Project

Management

15

Publications

1. Evaluation of 5-hydroxy-2,3-diaryl (substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anti angiogenic and anticancer agents, Vinod Kumar

Sanna,et.al, Investigational New Drugs, Volume 28, Number 4 (2010), 363-380

2. Anticancer and immunomodulatory activities of novel 1,8-naphthyridine derivatives”,Kumar V, et.al, J Enzyme Inhib Med Chem. 2009 Oct; 24(5):1169-78.

3. “1,8-Naphthyridine-3-carboxamide derivatives with anticancer and anti-inflammatory activity”. Kumar V, et.al, Eur J Med Chem. 2009 Aug;44(8):3356-62.

4. “Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives, Kumar V, et.al, Bioorg Med Chem Lett. 2008 Sep 15;18(18):5058-62.

5. “Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents”. Kumar V, et.al, J Enzyme Inhib Med Chem. 2008 Aug 11:1.

6. Eclipta alba extract with potential for hair growth promoting activity. Kakali Datta, et.al, J Ethnopharmacol 124(3): 450-6 (2009)

7. Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats, Bansal T, et.al, Eur J Pharm Sci. 2008 Dec 24.

8. Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives, Kumar V, et.al, Bioorganic & Medicinal Chemistry Letters18 (2008) 5058–5062

9. Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents, Kumar V, et.al, Journal of Enzyme Inhibition and Medicinal

Chemistry2008 Aug 11:1

10. Development and validation of reversed phase liquid chromatographic method utilizing ultraviolet detection for quantification of irinotecan (CPT-11) and its active metabolite, SN-38,

in rat plasma and bile samples, Bansal T, et.al, Talanta 2008 Sep 15;76(5):1015-21. Epub 2008 May 4.

11. Pre-clinical evidence for altered absorption and biliary excretion of irinotecan (CPT-11) in combination with quercetin: possible contribution of P-glycoprotein, Bansal T, et.al, Life

Sci.2008 Aug 15;83(7-8):250-9

12. Protective effects of Terminalia arjuna against Doxorubicin-induced cardiotoxicity, Gurvinder Singh, et.al, Journal of Ethnopharmacology, 2008 Apr 17;117(1):123-9. Epub 2008 Feb

3.

13. Anticancer activity of a peptide combination in gastrointestinal cancers targeting multiple neuropeptide receptors, Manu Jaggi, et.al, Investigational New Drugs, Volume 26, Number

6 (2008), 489-504

14. Modulation of key signal transduction molecules by a novel peptide combination effective for the treatment of gastrointestinal carcinomas, Anu T Singh, et.al, Investigational New

drugs, Volume 26, Number 6 (2008), 505-516,

15. Synthesis of 1-(2,6-dichlorophenyl)-3-methylene-1,3-dihydro-indol-2-one derivatives and in vitro anticancer evaluation against SW620 colon cancer cell line, Virsodia V, et.al, Eur J

Med Chem, Volume 44, Issue 3, March 2009, Pages 1355-1362.

16. Anticancer and anti-inflammatory activities of 1,8-naphthyridine-3-carboxamide derivatives, Srivastava SK, et.al, Bioorg Med Chem Lett, Dec 1;17(23):6660-4. Epub 2007 Aug 11.

17. Bombesin analogs containing alpha-amino-isobutyric acid with potent anticancer activity, Prasad S, et.al, J Pept Sci,13(1): 54-62, (2007)

18. Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography; Utility in Caco-2

intestinal absorption studies, Bansal T, et.al, Biomed Life Sci.,Oct 10; (2007)

19. Development of Dendritic-cells based assay to screen molecules for potential anti-inflammatory activity, Alka Madaan, et.al, 33rd Indian Immunological Society Conference, 2007 in

Indian Journal of Biochemistry and Biophysics,

20. Delivering multiple anticancer peptides as a single prodrug using lysyl-lysine as a facile linker, Prasad S, et.al, J Pept Sci,13(7): 458-67, (2007)

21. Pharmacological evaluation of C-3 modified Betulinic acid derivatives with potent anticancer activity, Rajendran P, et.al, Invest New Drugs,2008, Vol 26, No 1, Pg 25-34

22. Substance P analogs containing alpha,alpha-dialkylated amino acids with potent anticancer activity, Prasad S, et.al, J Pept Sci,13(8):544-8, (2007)

23. Synthesis and evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anticancer agents, Gurjar MK, et.al, J

Med Chem,19; 50(8): 1744-53, (2007)

24. “Synthesis and structure-activity relationships of potent antitumor active quinoline and naphthyridine derivatives, Sanjay K, et.al, Medicinal Chemistry. Page no. 685-709, Volume 7,

Number 6,2007

25. “Anticancer & Antiinflammatory activities of 1,8 Naphthyridine –3-carboxamide derivatives, Sanjay K, et.al, Bioorg Med Chem Lett. 2007 Dec 1;17(23):6660-4. Epub 2007 Aug 11

26. “Sesquiterepene lactone derivatives: synthesis and their cytotoxicity”. Sanjay K, et.al, Bioorganic & Medicinal Chemistry Letters, Volume 16, Issue 16, 15 August 2006, Pages

4195–4199

27. Betulinic Acid Derivatives as Anticancer Agents: Structure Activity Relationship., R. Mukherjee, et.al,Anti-Cancer Agents in Medicinal Chemistry, 6(3), 271-279 (2006).

16

Publications

28. Synthesis of 13-Amino Costunolide derivatives as Anticancer Agents, Sanjay K, et.al, Boiorg. Med. Chem Lett, 16, 4195-4199 (2006) 29. Conformational studies of 3,4-dideoxy furanoid sugar amino acid containing analogs of the receptor binding inhibitor of vasoactive intestinal peptide., Chakraborty TK, et.al,

Tetrahedron (2004) 60: 8329-8339. Tetrahedron, Volume 60, Issue 38, 13 September 2004, Pages 8329-8339 30. Furanoid sugar amino acids as dipeptide mimics in design of analogs of vasoactive intestinal peptide receptor binding inhibitor , Prasad S, et.al, J. Peptide Res. (2005) 66: 75-84. 31. Octapeptide analogs of somatostatin containing ,- dialkylated amino acids with potent anticancer activity., Prasad, S, et.al, Int. Journal of Peptide Res. & Therapeutics (2006) 12:

179-185. 32. Bombesin analogs containing -aminoisobutyric acid with potent anticancer activity, Prasad S. et.al, J.Peptide Science (2006). 33. Octapeptide analogs of somatostatin containing ,- di-alkylated amino acids with potent anticancer activity, Prasad S, et.al, Int J of Peptide Research & Therapeutics, 2006, Vol 12 (2),

179-185. 34. A simple method of isolation of chloramphenicol in honey and its estimation by liquid chromatography coupled to electrospray ionization tandem mass spectrometry, K. Vivekanandan,

et.al, Rapid Commun. Mass Spectrom. (2005) 19: 3025-3030. 35. Furanoid Sugar Amino Acids in Design of Analogs of VIP Receptor Binding Inhibitor, Prasad S, et.al, American Peptide Symposium, 2005: 661-662. 36. Identification of degradation products from aqueous carboplatin injection samples by electrospray tandem mass spectrometry, K. Vivekanandan, et.al, Int. Journal of Pharmaceutics

(2006) 313: 214-221 37. Identification of Isocephalomannine in presence of cephalomannine isomers and alkali metal ion adducts in paclitaxel active pharmaceutical ingredient using electrospray tandem mass

spectrometry, K. Vivekanandan, et.al, Rapid Commun. Mass Spectrom. (2006) 20: 1731-1735. 38. DRF7295: A novel peptide based signal transcluction modulator for the treatment of gastrointestinal carcinomas, Singh AT, et.al, R. Clin. Cancer Research (2005) 11: 9010S. 39. Anticancer activity of DRF7295: A peptide combination targeting multiple neuropeptide receptors in gastrointestinal cancers, Jaggi M, et.al, R. Clin.Cancer Research. (2005) 11:

9081S-9082S. 40. DRF7295: a novel peptide based signal transduction modulator for the Treatment of Gastrointestinal carcinomas.,Singh AT, et.al, Clin. Cancer Res. Dec 15,200511,24 (Suppl) Abstract

No. A186 41. Anticancer Activity of DRF7295: A Peptide Combination Targeting Multiple Neuropeptide Receptors for the Treatment of Gastrointestinal carcinomas, Jaggi M, et.al, Clin. Cancer

Res; Dec 15,2005 , 11,24 (Suppl) 42. Synthesis of 13-amino costunolide derivatives as anticancer agents, Srivastava SK, et.al, Bioorg Med Chem Lett. 16, 2006 Jun 9; 4195 – 4199. 43. Octapeptide analogs of Somatostatin containing , - Dialkylated amino acids with potent anticancer activity, Prasad S, et.al, Int. J of Peptide research & therapeutics 2006 Vol 12,

No.2, June 2006 pp179 -185 44. Bombesin Analogs Containing -Amino-isobutyric acid with Potent Anticancer Activity. Prasad S, et.al J Pept Sci, 2007 Jan;13(1):54-62 45. Synthesis and cytotoxic evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4, Mukund K, et.al, J Med Chem. 2007

Apr 19;50(8); 1744-53. 46. Furanoid sugar amino acids as dipeptide mimics in design of analogs of vasoactive intestinal peptide receptor binding inhibitor, Prasad S, et.al, Journal of Peptide Research (2005),

66(2), 75-84. 47. Synthesis and cytotoxic activity of 3-O-acyl/3-hydrazine /2-bromo/20,29-dibromo betulinic acid derivatives, Mukherjee R, et.al, Bioorganic & Medicinal Chemistry Letters (2004),

14(15), 4087-4091. 48. Synthesis of 3-O-acyl/3-benzylidene/3-hydrazone/3-hydrazine/17-carboxyacryloyl ester derivatives of betulinic acid as anti-angiogenic agents., Mukherjee R, et.al, Bioorganic &

Medicinal Chemistry Letters (2004), 14(12), 3169-3172. 49. Betulinic acid and its derivatives as anti-angiogenic agents, Mukherjee R, et.al, Bioorganic & Medicinal Chemistry Letters (2004), 14(9), 2181-2184. 50. Manuscript entitled “Synthesis and cytotoxic evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4, Gurjar MK,,

et.al, J Med Chem. 2007 Apr 19;50(8):1744-53 51. Octapeptide analogs of somatostatin containing ,- dialkylated amino acids with potent anticancer activity, Prasad, S, et.al, Ameri Pept.Symposia, 2006, Vol 9, Part 8, 639-640, 52. LC–UV Detection of 5′-Chloro-2,3-didehydroindolo(2′,3′:2,3)betulinic Acid in Rat Plasma and its Application to a Pharmacokinetic Study, Gautam M, et.al, Chromatographia , (2011).

Volume 73 (3):281-289. 53. "Efficiency and Mechanism of Intracellular Paclitaxel Delivery by Novel Nanopolymer based Tumor Targeted Delivery System, Nanoxeltm, Hrishikesh K, et.al, Clinical and

Translational Oncology,

17

OECD Organization for Economic Co-operation

and Development

Schedule Y

Drugs and Cosmetic Act 1947, Government of India

EU European Union

EPA Environmental Protection Agency

ICH International Conference on Harmonization

Regulatory Guidelines Followed

18

Registrations / Approvals

Registered under the Indian Companies Act, 1956

Registered by CPCSEA, Govt. of India

IAEC-Institutional Animal Ethics Committee-

Animal studies are approved as per CPCSEA norms

SAC – Scientific Advisory Committee

Recognition by two premier Indian Universities for Ph.D program

Funding from DST, DBT

19

Regulatory Experience

FDA, US Pre IND & IND experience

MHRA, UK F2F meetings seeking CT approval

BfArM, Germany Received preclinical go-ahead for an

anticancer drug

DCGI, India Participated in several meetings / discussions

For preclinical & clinical development plans

SFDA, China Customization of preclinical studies as per

SFDA requirements

Swedish & Dutch Compliance of preclinical requirements

20

Phase I Phase II Phase III Clinical

Support

Drug

Discovery

Early

Preclinical

Advanced

Preclinical

API Synthesis

& Form. Dev

Drug

Manufacture

CLINICAL

Biochemical &

Cell based screens

Target based screens

Signal transduction

Molecular modeling

in silico

Computational designing

Chemistry

• Medicinal

• Combichem

• Computational

• Natural Product

• Analytical

Efficacy

• Oncology

• Diabetes

• Pain

• Inflammation

• Dermatology

• Hair

ADME

Bioanalytical

Characterization

Pharmacokinetics

Toxicology

Special Toxicity

Safety Pharmacology

Process Development

Scale up

Characterization

GMP Synthesis

GMP

• Manufacture

• Solid oral

• Injectible

Bioavailability

Bioequivalence

Tissue banking

• Data Management Plan

• Database Design

• CRF Management

• Double Data Entry

• Central Lab Data Import

• Medical / AE Coding

• Query Management

• Manual Data Quality Control

Our Focus Areas

CRAM DRUG DISCOVERY & PRECLINICAL

21

Preclinical Services

ONCOLOGY IMMUNOMODULATION

HEPATOPROTECTIVES

DIGESTION

PAIN METABOLIC DISEASES

DERMATOLOGY

HAIR BIOLOGY

IMMUNOGENICITY

INFLAMMATION

INNOVATION RESEARCH TEAM

Therapeutic Areas

22

Services

EFFICACY

SAFETY

DMPK

PRODUCT DEVELOPMENT

MECHANISTIC PROFILING

SERVICES OFFERED

BIO ANALYTICAL

23

Service Modules

Mechanistic Profiling

Vascularization

Target Based Screens In vitro Screens Efficacy in Animals

ADME and PK Bioanalytical Techniques

Pain and Inflammation

Skin and Hair Biology

Immunomodulation Diabetes Technical Consultation

Systemic Safety Dermal safety

Hypersensitivity

Clinical Toxicities

24

What Do We Offer

M R T Package

Screening

Module for

Accelerating

Research and

Therapy

A comprehensive package from Discovery to Pre-IND selection

S A

in vitro screens Tumor Models ADMET Studies Lead Selection Pre formulation

Dev Toxicity Special Studies Mechanism

25

Rapid ADME Profiling of Investigational New Drugs (RAPID Screen)

R A P I D Package

Rapid

ADME

Profiling of

Investigational new

Drugs

A comprehensive package for ADME profiling

Solubility Metabolic stability Plasma protein

binding Permeability

CYP 450 phenotyping

CYP 450 Inhibition

26

Screens Available For Frequently Encountered ToxicitY (SAFETY Screen)

S A F E T Package

Screens

Available for

Frequently

Encountered

ToxicitY

Y

A comprehensive package for clinical toxicity screening

Normal cell toxicity

Neutropenia Alopecia Neuropathy GI Toxicity Cardiotoxicity

27

Cellular screens for Mechanism of Action Profiling (CellMAP Screen)

Cell M A Package

Cellular screens for

Mechanism of

P

Action

Profiling

A comprehensive screening platform for Mechanism of action profiling

Angiogenesis Signal

Transduction Target

Expression Cell Cycle Intracellular

Tracking Apoptosis Drug Uptake

28

Our National & Overseas Partners

U.K. U.S.

Germany

Finland

Korea

Thailand

China

India Hong Kong

Australia

Taiwan

Egypt

Pakistan

Israel Japan

Denmark

France

Switzerland

Russia

Malaysia

29

Our National & Overseas clients

30

Our National & Overseas clients

Lee’s Pharmaceutical Holdings Ltd

Sirbal Ltd.

Innoveda Biological Solutions Pvt Ltd

31

Our National & Overseas clients

Invictus Oncology

32

Case Studies

33

DISCOVERY AND DEVELOPMENT OF BETULINIC ACID DERIVATIVES

FOR THE TREATMENT OF CANCER

Manu Jaggi, MJA Siddiqui, Praveen R, Anand Vardhan, Rama Mukherjee, Anand C.Burman

Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad. Uttar Pradesh. INDIA

www.daburpharma.com

In-vitro Anti-cancer activity

S.No Cell line Cytotoxicity of Betulinic acid

ED50 (µg/ml)

1 HL 60 (Human myelogenous leukemia) 2.80 0.32

2 K 562 (Human myelogenous leukemia) 3.25 0.49

3 MOLT-4 (Human lymphoblastic leukemia) 1.23 0.70

4 Jurkat E6.1(Human lymphoblastic leukemia) 0.65 0.04

5 CEM.CM3 (Human lymphoblastic leukemia) 0.98 0.03

6 U937 (Human histiocytic lymphoma) 0.69 0.01

7 BRISTOL-8 (Human B-cell lymphoma) 0.84 0.05

8 MiaPaCa2 (Human pancreas) > 10

9 HeLa (Human cervical) > 10

10 PA-1 (Human ovary) > 10

11 U87MG (Human glioblastoma) > 10

12 U373MG (Glioblastoma) > 10

13 MDA.MB.453 (Breast) > 10

14 T47D (Breast) > 10

15 HT29 (Colon) 1.8 0.0

16 SW 620 (colon) > 10

17 CoLo 205 (colon) > 10

18 A549 (lung) > 10

19 L132 (lung) 1.30 0.55

20 KB (Oral) > 10

21 DU145 (Prostate) 1.13 0.35

22 Malme 3M (Melanoma) 2.20 0.70

23 RPMI 8226 (Myeloma) >10

S.No. Cell line Cytotoxicity of lead molecules

ED50 (µg/ml)

LEAD1 LEAD5 LEAD2 LEAD3

1 HBL100 2.82 3.94 2.87 6.98

2 DU145 0.82 3.24 2.37 8.92

3 KB 16.43 >20 13.53 16.3

4 SW620 3.4 5.9 9.57 4.77

5 Hs294T 3.09 >20 11.59 7.87

6 MiaPaCa-2 3.17 3.11 3.95 7.88

7 HuTu-80 16.27 10.50 10.99 12.56

8 U87MG >20 >20 >20 18.91

9 Hep-2 13.39 7.65 9.59 7.11

10 PA-1 3.63 3.4 7.53 6.81

11 CHO 13.95 7.2 >20 14.84

S.No. Cell line Specificity of lead molecules to tumor cells

(ED50 normal cell line[CHO] / ED50 tumor cell line)

LEAD1 LEAD5 LEAD2 LEAD3

1 HBL100 4.94 1.82 >6.96 2.15

2 DU145 17.01 2.22 >8.43 1.66

3 KB 0.85 0.36 >1.48 0.91

4 SW620 4.1 1.22 >2.08 3.11

5 Hs294T 4.5 <0.36 >1.7 1.88

6 MiaPaCa-2 4.4 2.31 >5.12 1.88

7 HuTu-80 0.86 0.68 >1.81 1.18

8 U87MG 0.70 <0.36 1.0 0.78

9 Hep-2 1.04 0.93 >2.1 2.08

10 PA-1 13.84 2.1 >2.65 2.17

5.5

18.5

49.221.7

33.4

46.710

11.7

0 10 20 30 40 50

% reduction in area

Betulinic acid

LEAD3

LEAD4

LEAD2

LEAD6

LEAD5

LEAD1

LEAD7

Effect of Betulinic acid and

lead molecules on

tube formation of ECV304 cells

Representative image analy sis photograph of tube

formation (left panel) and its inhibition by incubation

with LEAD 4 after 48 hrs (right panel)

Lead selection

Physico-chemical and ADME studies

Physico-chemical & ADME characteristics of lead molecules

Pharmacokinetics in RatSolubility Permeability

(PAMPA)

Metabolic

stability

Plasma

protein

binding

CYP450

(1A2,2C9,2D6,3A4)

inhibition

Intravenous Oral

Poor Poor Good High Does not inhibit key

enzyme isoforms

2 compartment,

1st order elimination

model

Poor oral

availability

Toxicity / Safety

S.No Derivative Safe Dose (mg/Kg. B.Wt) Lethal Dose (mg/Kg. B.Wt)

1 LEAD2 150 200

2 LEAD6 25 37.5

3 LEAD1 25 37.5

4 LEAD5 300 ND

5 Betulinic Acid 150 ND

6 Vehicle Equivalent to 200 ND

ND=Not determined (above highest dose tested)

Acknowledgements

We would like to acknowledge the contributions made in this work by scientists of Molecular Oncology and Analytical Development divisions of

Dabur Research Foundation.

Reference

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2;14(15):4087-91.

Abstract

Betulinic acid is a naturally occurring pentacyclic triterpenoid that has demonstrated

selective cytotoxicity against melanoma and glioblastomas. It’s application in treatment

of cancer has been limited due to its poor solubility. Betulinic acid is currently

undergoing advanced preclinical development for treatment of melanoma where it is

applied topically as a cream. We have for the first time established betulinic acid as a

potential anti-cancer drug with broad-spectrum anti-cancer activity in leukemia,

lymphoma, prostate, ovary, lung, melanoma & colon human tumor cell lines and

xenografts.

In continuation and in line with development of more potent derivatives with improved

physicochemical properties we have synthesized more than 1000 novel betulinic acid or

dihydro-betulinic acid derivatives with modifications in C2, C3, C20, C28 and C29 positions

and identified more than 50 novel molecules with better activity profile as compared to

the parent molecule. We further elucidated the mechanism of action of Lead compounds

wherein it was demonstrated that the compounds have anti-apoptotic and anti-angiogenic

potential as well as significant PKC inhibitory activity in cancer cells. These molecules

are being tested for metabolic stability, potential for drug interactions,

permeability/absorption, pharmacokinetics and toxicity. Being a natural product derived

molecule with ready availability of starting material and high yield of synthesis coupled

with low toxicity in animals these molecules are promising anticancer agents.

Introduction

Betulinic acid is a pentacyclic lupane-type triterpene. One of the most widely reported

sources of betulinic acid is the birch tree where both betulinic acid and betulin can be

obtained in substantial quantities. (See Photograph)

Betulinic acid was reported to be a melanoma-specific cytotoxic compound [1].

However, recent evidence indicates a broader spectrum of activity against other cancer

cell types [2-7]. It was shown to act through induction of apoptosis [1] independent of

the cell’s p53 status [4,8,9] by causing changes in mitochondrial membrane potential,

production of reactive oxygen species, and permeability transition pore openings [3].

This leads to the release of mitochondrial apoptogenic factors, activation of caspases,

and DNA fragmentation [8,10,11].

Betulinic acid also inhibited the in vitro activity of aminopeptidase N, an endogenous

angiogenic factor [12] and inhibited the mitochondrial function in endothelial cells

[13]. It is active in-vivo against TPA-induced tumors [14,15], ovarian [4] and

melanoma [1] xenografts in mice. Remarkably, betulinic acid exhibited no toxic effects

in mice even at a concentration of 500 mg/kg [1]. However, doses as low as 5 mg/kg

were determined to significantly impede tumor development [1]. Recently, we have

reported the broad-spectrum anti-cancer and anti-angiogenic activity of several

promising derivatives of betulinic acid [16-18]. These findings have made betulinic

acid and its derivatives attractive candidates for the clinical treatment of various forms

of cancer.

We have synthesized more than 1000 novel betulinic acid or dihydro-betulinic acid

derivatives with modifications in C2, C3, C20, C28 and C29 positions and identified more

than 50 novel molecules with better activity profile as compared to the parent molecule.

We further short-listed the derivatives based on potency and specificity to tumor cells

and were able to select 3-O-Acyl, 3-Hydroxyloxime, 3-Hydrazone, 3-Hydrazine and 3-

Benzylidene derivatives for further LEAD development.

Betulinic acid (R = COOH)

Betulin (R = CH2OH)

Selection of LEADS

Day 10 Day 18 Day 25 Day 35

An illustrative photograph showing stages of tumor regression of

(PTC) colon xenograft following treatment with LEAD1

Materials and Methods

Cell culture

ECV304 cell line was generously gifted by Dr. Takahashi (Tokyo University, Tokyo, Japan). All other cell

lines were procured from NCCS, Pune, India. Cell lines were grown in DMEM, containing L-glutamine

and 25mM HEPES and supplemented with 10% fetal bovine serum, penicillin (100 units/mL),

streptomycin (100 lg/mL), and amphotericin B (0.25 lg/mL) and maintained at 37 0C , 5% CO2, 100%

humidity.

Cytotoxicity assay

Cells (1.5 x 104) were incubated with the molecules dissolved in DMSO, in triplicate wells of 96-well

tissue culture plate to obtain drug concentrations of 0.5 to 20 g/mL (final DMSO < 1%). Cytotoxicity was

measured after 72 h by tetrazolium-based MTT assay. Each experiment was repeated thrice and mean ED50

values (half-maximal cytotoxicity) as calculated using Prsim® software has been reported.

Tube formation assay

104 ECV304 cells in growth medium (DMEM containing 10% FBS) were seeded on Matrigel

TM (70 L).

Compounds were solubilized in DMSO and were added in duplicate wells at non-cytotoxic concentration

and incubated at 4 µg/ml ((final DMSO < 1%) overnight after which the control cells start to form an

intense network of tube-like structures. The total tube area was measured by Image analysis (VideoPro®,

Australia) and percentage inhibition of tube formation was calculated as compared to controls.

Tumor xenograft assay

Human tumor xenografts were initiated in athymic nude mice by subcutaneous inoculation of a single cell

suspension (containing 107 cells) of PTC (Primary tumor cells of colon adenocarcinoma) or L132 (Lung

adenocarcinoma) tumor cells. The test compound was formulated in nanoparticles When tumors were

around 100-300 cu.mm mice were dosed intravenously between 10 - 40 mg/kg B.wt. on alternate days for

about 2-3 weeks. Tumor growth was monitored by measuring tumor dimensions using vernier caliper once

every week and calculating tumor volumes using the formula 0.4 xW2xL (W = smaller dia, L = larger dia,).

Measurement of VEGF, bFGF, Endostatin levels

K562 (Chronic myelogenous leukemia), cells (1x106) were incubated with test compounds at 1 µg/ml in a

6-well tissue culture plate. After 6 hrs incubation the culture supernatant was analyzed for the levels of

different pro-angiogenic molecules VEGF, bFGF and Endostatin using commercially available ELISA kits

by following kit instructions. Quantikine human VEGF kit, Quantikine human bFGF kit (both from

R&D systems), Human Endostatin Protein Accucyte EIA (from Oncogene, USA).

Measurement of levels of Bcl-2, Nucleosome, and Protein Kinase (PKC) activity

Briefly 1 x 10 6 human ovarian cancer cells (PA1) suspended in culture medium (DMEM) were incubated

with test compound dissolved in DMSO (2.5%) at concentrations between 5 - 200 µg/ml in 6-well tissue

culture plates. After incubation cell lysates were prepared and analyzed using commercially available

ELISA kits. The level of free nucleosomes was measured after 6 hours of incubation using Nucleosome

ELISA kit, Oncogene Research Products,USA. The levels of Bcl-2 were measured after 20 hours using

Bcl-2 EIA, Oncogene Research products, USA,Cat no. QIA23. PKC activity was measured after 20

minutes using Protein kinase non-radioactive kit, Calbiochem, USA.

ADME studies

Solubility of the molecules was determined using the shake flask method. After 17 hours of shaking in

phosphate buffer (pH7.4) the soluble portion was filtered out and analyzed using HPLC.

Permeability was determined using the Parallel Artificial Membrane Permeability Assay (PAMPA,

Millipore, USA). Molecules dissolved in DMSO were added to donor wells at 100µM (final DMSO=5%).

The transport across a lipid layer was determined by analyzing the contents of the acceptor well after 16 hrs

by HPLC.

Metabolic stability of the molecules was determined by HPLC by calculating the amount of the compound

remaining un-metabolized following incubation for 60 min in pooled human liver microsomes(BD Gentest,

USA).

Plasma protein binding was determined in Rat plasma. Test compounds were spiked in Plasma at 20µM

and incubated at 370C for 1 hr followed by centrifugation across 10 KD YMC membrane (Millipore, USA)

at 2000xg for 30-45 min. The amount of the test compound in the filtrate and retentate was measured by

HPLC.

Toxicity/Safety studies

Adult Balb/c mice, age 6-8 wks, weighing between 20-25gms were selected for the study. 3 animals per group

were administered a single intravenous dose of the test compound (dissolved in co-solvents) at doses ranging

from 10 to 300 mg/kg. Mortality, body weight and apparent toxic signs/symptoms were recorded over a

period of 14 days. The maximum tolerated dose at which no toxic signs were seen was designated as ‘Safe

dose’ and the dose at which atleast one mortality was seen was designated as ‘Lethal dose’.

Pharmacokinetics

Male Wistar Rats, age 6 -10 weeks, weighing between 100-150 gms were selected for the study. Test

compounds were dissolved in co-solvents at a concentration of 5 mg/ml (intravenous dose) or in 0.5% CMC

suspension at a concentration of 15 mg/ml (oral dose). 3 animals per group were given a single dose,

approximately 8-10 mg/kg (intravenous) or 150 mg/kg (oral). Blood samples were collected at different time

points (3 min, 10 min, 30 min, 1 hr, 2hr, 4hr, 6hr, 8hr, and 24 hr). The plasma layer was separated, extracted

using organic solvents, centrifuged and supernatant evaporated to dryness. It was reconstituted with 200 l of

10% DMSO in Methanol and analyzed using HPLC. The pharmacokinetic parameters were determined using

WinNonlin 4.0 software

In vivo anti-tumor activity

Effect of LEAD1 formulation on

colon (PTC) xenograft

0

2000

4000

6000

8000

10000

0 10 20 30 40

Days post inoculum

Tu

mo

r v

olu

me

(c

u.m

m)

Control

LEAD1

Effect of LEAD1 formulation on

lung (L132) xenografts

0

100

200

300

400

500

600

0 10 20 30

Days post inoculum

Tu

mo

r v

olu

me

(c

u.m

m)

Control

LEAD1

In vitro Anti-angiogenic acitivity

Cytotoxicity in ECV304 cells

IC50 (ug/ml)

LEAD1 LEAD2 LEAD4 LEAD5 LEAD3

3.49 3.64 2.26 12.87 0.98

S.No. Cell line Endothelial cell specificity (ECS)

(ED50 Tumor cell / ED50 Endothelial cell)

LEAD1 LEAD5 LEAD2 LEAD3

1 HBL100 0.8 0.3 0.8 7.1

2 DU145 0.2 0.3 0.7 9.1

3 KB 4.7 >1.55 3.7 16.6

4 SW620 1.0 0.5 2.6 4.9

5 Hs294T 0.9 >1.55 3.2 8.0

6 MiaPaCa-2 0.9 0.2 1.1 8.0

7 HuTu-80 4.7 0.8 3.0 12.8

8 U87MG >5.73 >1.55 >5.49 19.3

9 Hep-2 3.8 0.6 2.6 7.3

10 PA-1 1.0 0.3 2.1 6.9

11 A549 0.3 >1.55 >5.49 >20.4

12 HT29 4.1 >1.55 >5.49 >20.4

13 CHO 4.0 0.6 >5.49 15.1

14 ECV304 1.0 1.0 1.0 1.0

ECS less than 10 = Low ECS

ECS between 10 and 20 = Moderate ECS

ECS greater than 20 = High ECS

Mechanism of action

Effect of Betulinic acid on levels of

Bcl-2 in PA-1 (ovarian) cell line

0

50

100

150

5 20 100

ug/ml

% o

f c

on

tro

l

Bcl-2

Effect of Betulinic acid on

Nucleosome release in

PA-1(ovarian) cell line

0

2

4

6

8

5 20 100

ug/ml

Fo

ld in

cre

as

e

vs c

on

tro

l

Nucleosome

Effect of betulinic acid on

pro-angiogenic factors in

K562 (leukemia) cell line at 1ug/ml

0

10

20

VEGF bFGF Endostatin

% in

hib

itio

n

Effect of Betulinic acid on Protein

Kinase(PKC) activity in PA-1 (ovarian)

cell line

0

50

100

150

5 20 100

ug/ml

% o

f contr

ol

• Betulinic acid has broad-spectrum anti-cancer activity. The derivatives have

better potency and varying degree of specificity to cancer cells.

• Betulinic acid inhibits endothelial cell growth, tube formation, and pro-

angiogenic factors viz. VEGF, bFGF and Endostatin. The derivatives have

better potency and varying degree of specificity to endothelial cells.

• Betulinic acid causes cell death by Apoptosis as demonstrated by inhibition of

bcl-2 and induction of nucleosome release. The apoptotic cell death induced

may be mediated by inhibition of PKC activity

• One of the derivatives (LEAD1) was shown to inhibit and cause regression of

human tumor (colon and lung) xenografts in nude mice.

• The derivatives had poor solubility and permeability with high protein binding

and poor oral bioavailability (as shown in PK studies). However they were

shown to have good metabolic stability and did not inhibit key CYP enzymes

capable of causing drug interactions.

• The derivatives show varying levels of toxicity and safety profiles as

compared to Betulinic acid.

Conclusions

34

35

Anthracycline antibiotics like Doxorubicin & Daunorubicin are among the most effective antineoplastics

with broad-spectrum efficacy. However their clinical application is limited by cumulative dose related

cardiotoxicity that presents itself in acute or chronic forms in 2 –20 % of patients. Of several

cardioprotectors that have been evaluated, only Dexrazoxane (ICRF –187), an iron chelator has been

approved for clinical use.

A panel of potential cardioprotectives were evaluated by us in an in vivo model representing acute

Doxorubicin induced cardiotoxicity (Circulation 2000,102:2105 – 2110). Male Wistar rats were treated

with Doxorubicin ranging from 15 –30 mg/kg given intraperitoneally with or without the potential

cardioprotectives. Hearts excised 24 – 48 hours after the treatment were evaluated for superoxide

Dismutase & catalase activity, reduced Glutathione & lipid peroxidation. Serum levels of Creatine

Kinase MB (CKMB) & Lactate Dehydrogenase (LDH) were evaluated as an index of myocardial injury.

The cardiac tissues were also scored electron microscopically.

A novel synthetic free radical scavenging molecule (NDR/NCE25) was identified which significantly

reduced the Doxorubicin induced CKMB & LDH levels. Further it induced Superoxide Dismutase

activity & reduced the lipid peroxidation. This correlated with histopathological evidence of reduction in

the severity of myocardial tissue damage.

Anthracycline antineoplastics are amongst the most active anticancer drugs that are effective against

malignancies like leukemias, lymphomas and many solid cancers. These include Doxorubicin ( sold as

ADRIAMYCIN, NSC 123127, From Adria Laboratories, Columbus, Ohio), Daunorubicin, Epirubicin,

THP- Adriamycin and Idarubicin. Doxorubicin is the drug of choice, alone or in combination with other

chemotherapeutic agents, in the treatment of metastatic adenocarcinoma of the breast, carcinoma of the

bladder, bronchogenic carcinoma, neuroblastoma, and metastatic thyroid carcinoma. It exerts its

antitumour effects due to inhibition of DNA replication by intercalating between base pairs and/or steric

inhibition of RNA activity.

Cardiotoxicity is the major limitation in the use of doxorubicin (Weiss, R.B., Semin. Oncol. 19, 670 –

686, 1992). The risk of developing cardiomyopathy becomes unacceptably high beyond the cumulative

dose of 550 mg/m2 (Lefrak et al., Cancer 1973, 32, 302- 314). In addition to clinical heart failure,

cardiotoxicity encompasses clinical cardiotoxicity such as congestive heart failure and / or cardiac

arrhythmias, and subclinical cardiotoxicity such as that detected by pathologic changes in cardiac biopsy

or decrease in ventricular ejection fractions.

Thus, it has been found that doxorubicin treatment often must be terminated before the maximum

effective cumulative dose has been administered to a patient bearing a neoplasm, because of the

development of life-threatening cardiomyopathy. Hence while doxorubicin is considered a highly

effective anti-tumor agent, this effectiveness is significantly reduced by the concomitant cardiotoxicity

encountered with the use of the drug. Apart from doxorubicin, cardiotoxicity is a major setback

associated with other chemotherapeutics agents also like Mitoxantrone at doses >100-140mg/m2

,Cyclophosphamide at doses >100-120mg/m2 and at conventional doses of Ifosphamide, Cisplatin and

Flourouracil.

Doxorubicin induced cardiotoxicity is mediated through several different mechanisms including lipid

peroxidation ( Bordoni, A. et al., Biochim. Biophys. Acta 1999, 1440: 100- 106), free radical formation

( Yin, X. et.al., Biochem. Pharmacol. , 1998, 56: 87- 93, Hershko, C. et al, Leuk. Lymphoma 1993, 11

: 207 –214 ), mitochondrial damage (Cini Neri, G. et al., Oncology 1991, 48: 327- 333), and iron

dependent oxidative damage to biological macromolecules (Thomas, C.E. and Aust, S.D., Arch.

Biochem. Biophys. 1986, 248: 684 – 689).

The complete mechanisms for doxorubicin and other anthracycline-induced cardiotoxicity are not

completely understood. Three intracellular mechanisms are ascribed to Anthracyclines : interactions

with DNA synthesis, binding to cell membranes and altering membrane functions, and intracellular Na

& Ca2 concentrations and stimulation of lipid peroxidation to form oxygen radicals (Young, R.C et. al,

N. Engl. J. Med. 1981 , 305: 139-153). Further Doxorubicin administration is associated with a

decrease in the presence of the endogenous antioxidants. Doxorubicin directly depresses cardiac

glutathione peroxidase activity, the major defense against free-radical damage.

It also may induce apoptosis in cardiomyocytes ( Arola, O.J. et al., Cancer Res., 2000 Apr 1, 60 (7) :

1789- 1792). The membrane interaction of Doxorubicin appears to be an integral part of the

biochemical mechanisms of its toxicity. Chronic administration of Doxorubicin modulates the

membrane bound adenylate cyclase and cAMP levels (Robison , T.W. and Giri, S.N. , Virchows Arch.

B cell Pathol. Incl. Mol. Pathol. 1987, 54( 3): 182- 189 ).

Pharmacological methods for development of novel cardioprotectives has involved the exploration of

diverse classes of molecules.

At present, Dexrazoxane (ICRF –187, Zinecard), an iron chelator, is the only drug available for human

clinical use to reduce Doxorubicin induced cardiotoxicity (Swain, S.M. et al., J. Clin. Oncol., 1997 : 15

: 1333 – 1340 , and Swain, S.M. et. al., J. Clin. Oncol., 1997, 15: 1318 – 1332).

Diverse classes of molecules or active principles of plants, have shown cardioprotective activities for

Doxorubicin induced cardiotoxicity in animal models. These include lipid lowering drugs like

Lovastatin (Feleszko, W. et al., Clin. Cancer. Res. Vol 6, 2044 – 2052, May 2000) and probucol

(Li, T. and Singal, P., Circulation, 2000, 102: 2105 - 2110), cytoprotective drugs like Amifostine

(Jahnukainen, K. et al., Cancer Research, 61, 6423 – 6427, September 1, 2001), free radical scavengers

like Vitamin E or N-acetylcysteine, calcium channel antagonists like Amlodipine (Yamanaka, S. et al.,

J. Am. Coll. Cardiol. 2003 , Mar. 5 ,41(5): 870- 878 ) , non selective adrenoceptor blocker and

vasodilator like Carvedilol ( Santos, D.L. et al., Toxicol. Appl. Pharmacol., 2002 Dec 15, 185(3): 218 -

227 ), Angiotensin converting Enzyme inhibitors like Captopril and Enalapril ( El Aziz, M.A. et al., J.

Appl. Toxicol., 21, 469 – 473, 2001 ) and plant extracts like curcumin (Venkatesan, N., Br. J.

Pharmacol. 1998, Jun, 124 (3); 425 – 427). These molecules & extracts have not shown acceptable

success in clinical trials, thus underscoring the need for the development of new cardioprotectives . This

remains the essential premise of the current work.

IDENTIFICATION OF NOVEL CARDIOPROTECTIVES FOR CHEMOTHERAPY

INDUCED CARDIOTOXICITY Anu T. Singh, Gurvinder Singh , Ashok Mukherjee , Rama Mukherjee

Dabur Research Foundation, 22, Site IV, Sahibabad. Ghaziabad. Uttar Pradesh. INDIA

www.daburpharma.com

Quantitation of free radical scavenging activity by DPPH (1,1, Diphenyl-2-picrylhydrazyl)

Method

DPPH is a 1° stable free radical that in the presence of free radical quencher undergoes a one-molecule

reduction, which can be detected spectrophotometrically at 517nm as its color changes from violet to yellow.

In its radical form (DPPH·) it absorbs at 517nm, but upon reduction by an antioxidant or radical scavenger

(AH) the extent of absorption decreases.

Dilutions of NDR/NCE-25 were prepared in the concentrations ranging from 0.45 g/ml to 1mg/ml and radical

scavenging activity was measured at 517nm with DPPH. Vitamin C & Melatonin was taken as a reference

standard and The ECR 50 (ratio of the concentration of the molecule to the concentration of DPPH that

reduces the optical density of DPPH by fifty percent at five minutes) values for radical scavenging were

calculated.

Super-oxide dismutase (SOD) activity assays:

Superoxide anions are generated in a system comprising of NADH and Phenazine methosulphate (PMS). This

anion reduces the Nitro blue tetrazolium (NBT) and forms a blue formazan, which is measured at 560nm.

Superoxide dismutase inhibits the reduction of NBT and thus the enzyme activity is measured by monitoring

the rate of decrease in optical density at 560 nm. The assay was carried out as described by Kakkar etal., 1984.

Briefly frozen myocardial tissue were taken and homogenized in Tris-Sucrose buffer at 40C . The homogenate

was centrifuged at 10,000 rpm for 15 minutes at 40C. SOD activity was measured in the supernatant by adding

0.6 mL of supernatant to a tube containing 1.2mL of Sodium Pyrophosphate (0.052 M, pH 8.3), 0.1mL of

Phenazine methosulphate (186 µM) and 0.6 mL of water.. Reaction was initiated by adding 0.2 mL of NADH

(780 µM) solution and was terminated after 90 seconds by the addition of 1 mL of glacial acetic acid to the

tubes. Absorbance was measured at 560nm. SOD activity was expressed as U/mg protein.

Catalase activity assays:

Catalase causes the decomposition of H2O2 to H2O in phosphate bufferthat can be followed directly by the

decrease in absorbance at 240nm. The assay was carried out as described by Aebi H. 1974.

Briefly frozen myocardium tissue was taken and homogenized in phosphate buffer at 40C.The homogenate was

centrifuged at 5,000rpm for 10 minutes at 40C. To the supernatant, 0.01mL of ethanol and 0.1mL of 10%

Triton 100X was added and incubated in ice for 30 min. Catalase activity was measured in the 0.05mL of

prepared samples by adding to the 1.95mL of phosphate buffer and then by adding 1mL of 30% H2O2 at

240nm. Catalase activity was expressed as U/mg protein.

Reduced Glutathione (GSH) Assays:

Bis-P-nitrophenol disulphide reacts with GSH at pH 8.0 to produce one mole of highly colored p-nitrophenol

anion per mole of thiol that can be measured at 412nm. The assay was carried out as described by Ellman etal.

1959.

Briefly frozen tissue was homogenized in cold TCA buffer at 40C and centrifuge at 3,000 rpm for 10 minutes

at 40C. To the glass test tube added 0.1mL of supernatant/Standard, 0.4mL of H2O, 2mL of GSH buffer and

0.5mL of DTNB solution, vortexed and incubated for 10 minutes at room temperature. Absorbance was taken

at 412nm. GSH was expressed as g/mg tissue.

Lipid peroxidation assays:

The assay for quantitation of lipid peroxidation was carried out as described by Okhawa H. etal. 1979.

Briefly Malonaldehyde (MDA) reacts with 2-thiobarbituric acid to give pink colored complex which can be

read at 532nm.

MDA + TBA Pink color complex.

(Read at 532nm)

Briefly frozen tissue was homogenized in cold TCA buffer at 40C. To the glass test tube added 0.2mL of

Tissue homogenate, 0.2mL of 8.1% SDS, 1.5mL of 20% acetic acid (pH 3.5), 1.5mL of 0.8% Thiobarbituric

acid (TBA) solution and 0.6mL of water. Mixed and heated at 950C for 60 minutes. Cool it and 5mL of

mixture of n-Butanol & pyridine (15:1) was added. Test tube was centrifuged at 5,000rpm for 10 minutes.

From junction of two layers 200µL of liquid was taken and absorbance was taken at 540nm. Lipid

peroxidatipn was expressed as M of MDA/mg of tissue.

Quantitation of serum CK-MB levels:

Serum CK MB levels were estimated using commercially available reagents from Bayer Diagnostics as per the

manufacturer’s instructions

Briefly rats were anaesthetized and blood was collected from retro-orbital vein and serum was separated.

Serum was diluted five times with saline and CK-MB values were estimated as described by the manufacturer

Quantitation of serum LDH levels:

Serum LDH levels were estimated using commercially available reagents from Bayer Diagnostics as per the

manufacturer’s instructions.

Briefly rats were anaesthetized and blood was collected from retro-orbital vein and serum was separated.

Serum was diluted five times with saline and LDH values were estimated as described by the manufacturer.

Electron Microscopy of cardiac tissues:

Cardiac muscle was fixed in 2.5% Glutaraldehyde in Phosphate buffer. After necropsy the heart was immersed

in the fixative solution and diced into 2mm cubes. The cubes of Tissue were kept in the fixative for 24hrs at

40C and then transferred and stored in Sucrose-cacodylate buffer till processing.

Tissues were then post fixed in osmium tetra oxide and processed through alcohol and

propylene oxide for final embedding in resin. After polymerization of the resin blocks, semithin sections at 1-2

u thickness were cut and stained with toluidine blue for selection of final areas. Ultrathin sections were cut at

60-90nm thickness from selected areas, contrasted with uranium and lead salts for final viewing under

PHILIPS Transmission Electron microscope at an accelerating voltage of 120KV.

STANDARD CURVES

ELECTRON MICROSCOPIC IMAGES OF CARDIAC TISSUES FROM

WISTAR RATS TREATED WITH ADRIAMYCIN OR ADRIMYCIN &

NDR/NCE25

Aebi H. 1974. In: Bergmeyer HU Ed. Methods of enzymatic analysis. Verlag Chem Acad.

Press Inc. 2: 673-685

Arola, O.J. et al. 2000. Cancer Res., Apr 1, 60 (7) : 1789- 1792

Bordoni, A. et al. 1999. Biochim. Biophys. Acta. 1440: 100- 106

Cini Neri, G. et al. 1991. Oncology. 48: 327- 333

El Aziz, M.A. et al. 2001. J. Appl. Toxicol. 21, 469 – 473

Ellman etal. 1959. Tissue sulphydryl groups. Arch Biochem Biophy. 82: 70-77

Feleszko, W. et al. 2000. Clin. Cancer. Res. Vol 6, 2044 – 2052

Hershko, C. et al 1993. Leuk. Lymphoma, 11 : 207 –214

Jahnukainen, K. et al. 2001. Cancer Research. 61, 6423 – 6427

Kakkar P.etal. 1984. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem

Biophys. 21: 130-132

Lefrak EA, Pitha J, Rosenheim S, Gottlieb JA. 1973. A clinicopathologic analysis of adriamycin

cardiotoxicity. Cancer 32(2): 302-14.

Li, T. and Singal, P. 2000. Circulation. 102: 2105 – 2110

Okhawa H. etal. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.

Anal Biochem. 95: 351-358

Robison , T.W. and Giri, S.N. , Virchows. 1987. Arch. B cell Pathol. Incl. Mol. Pathol. 54( 3): 182-

189

Santos, D.L. et al. 2002. Toxicol. Appl. Pharmacol.185(3): 218 - 227

Swain, S.M. et. al. 1997. J. Clin. Oncol. 15: 1318 – 1332

Swain, S.M. et al. 1997. J. Clin. Oncol. 15 : 1333 – 1340

Thomas, C.E. and Aust, S.D. 1986. Arch. Biochem. Biophys. 248: 684 – 689.

RESULTS

Comparative free radical scavenging

activity of Vitamin C, NDR/NCE-25 and

Melatonin

0

20

40

60

80

100

0 500 1000 1500 2000

Conc. (µg/mL)

% R

adic

al

scav

eng

ing

Vitamin C NDR/NCE-25 Melatonin

EFFECT OF NDR/NCE-25 ON SUPEROXIDE DISMUTASE (SOD)

ACTIVITY OF ADR TREATED RAT MYOCARDIUM

0

10

20

30

40

50

CO

NT

RO

L

AD

R-3

0mg

/kg

ND

R/N

CE

25-

8mg

/kg

+AD

R

30m

g/k

g

ND

R/N

CE

25-

17.5

mg

/kg

+AD

R

30m

g/k

g

SO

D (

U/m

g P

rote

in)

EFFECT OF ADRIAMYCIN ON RAT

MYOCARDIAL SUPEROXIDE

DISMUTASE(SOD) ACTIVITY

0

40

80

120

Control ADR(15mg/kg) ADR(30mg/kg)

SO

D A

ctiv

ity(

% o

f C

on

tro

l)

EFFECT OF NDR/NCE-25 ON LIPID

PEROXIDATION IN RAT MYOCARDIAL TISSUE

0

40

80

ADR-30mg/kg NDR/NCE-25-

8mg/kg AND

ADR-30mg/kg

NDR/NCE-25-

17.5mg/kg AND

ADR -30mg/kg

Lip

id P

er-o

xida

tion

(% o

f co

ntro

l)

EFFECT OF NDR/NCE-25 ON

REDUCED GLUTATHIONE (GSH)

ACTIVITY OF ADR TREATED RAT

MYOCARDIUM

0

40

80

120

NDR/NCE-2

5

ADR(10m

g/kg)

NDR/NCE-1

5+ADR(10m

g/kg)

GS

H (%

of C

ontr

ol)

Effect of NDR/NCE-25 on Catalase activity

in Rat hepatic tissue.

0

50

100

150

200

250

AD

R

30m

g/kg

AD

R30

mg/

kg

and

ND

R/N

CE-

25-

17.5

mg/

kg

AD

R30

mg/

kg

and

ND

R/N

CE-

25-

40.4

mg/

kg

Ca

tala

se a

ctiv

ity

( %

of

con

tro

l)

Time kinetics of release of CK-MB

in the serum of Adriamycin treated

Wistar Rats

0

400

800

1200

1600

0 20 40 60 80

Time (Hrs)

CK

-MB

(U

/L)

Control Adriamycin Treated (15mg/kg)

Effect of NDR/NCE-25 on CK-MB

levels in an Acute in vivo model for

Adriamycin (ADR) induced

Cardiotoxicity (24hr)

0

200

400

600

800

1000

Control NCE-19 ADR

15mg/kg

NCE19 +

ADR

15mg/kg

CK

-MB

(U

/L)

Effect of NDR/NCE-25 on LDH levels

in an Acute in vivo model for

Adriamycin (ADR) induced

Cardiotoxicity (24hr)

0

400

800

1200

Con

trol

NDR/N

CE-

25

ADR 1

5mg/

kg

NDR/N

CE-

25 +

ADR 1

5mg/

kg

LD

H (

U/L

)

Standard Curve for Super-oxide

Dismutase activity

0

0.04

0.08

0.12

0.16

0.2

0 5 10 15 20

SOD (In Units)

Ab

so

rba

nce

(560n

m)

Standard Curve for

Catalase activity

0

0.02

0.04

0.06

0.08

0.1

0 20 40 60 80 100Concentration (In units)

Fa

ll i

n A

bso

rba

nce

(240n

m)

Standard Curve for Reduced

Glutathione levels

0

0.04

0.08

0.12

0.16

0 2.5 5 7.5 10

Concentration (µg/mL)

Absorb

ance (

412nm

)

Standard Curve for

Lipid Per-oxidation

0

0.1

0.2

0.3

0 20 40 60

MDA (µM)

Ab

sorb

an

ce

(540n

m)

ABSTRACT

INTRODUCTION

CONCLUSIONS

REFERENCES

NDR/NCE-25 is a potent free radical scavenging molecule in vitro.

NDR/NCE-25 increases SOD enzyme activity in animals treated with Adriamycin in

acute study model.

NDR/NCE-25 decreases Lipid peroxidation in animals treated with Adriamycin in

acute study model.

NDR/NCE-25 decreases CK-MB and LDH levels in animals treated with Adriamycin

in acute study model.

No effect of NDR/NCE-25 on myocardial catalase in acute study model was

observed.

NDR/NCE-25 increases liver catalase activity in animals treated with adriamycin in

acute study model.

Adriamycin treated animals showed significant electronmicroscopical changes in

cardiac muscle fibres in the form of nuclear chromatin margination, cytoplasmic

Z-line disarray, swelling of Smooth Endoplasmic Reticulum (SER), mitochondrial

swelling and rarefaction of the cytoplasm with lifting up of plasma membranes.

The study group treated with NDR/NCE-25 and Adriamycin displayed fewer clusters

of Z band disarray compared to that seen in the animals treated with Adriamycin

alone. None of the other changes as seen the Adriamycin group were seen when the

animals were pretreated with NDR/NCE-25. The data was suggestive of the potential

of NDR/NCE-25 for reducing the severity of cardiac tissue damage caused by

Adriamycin

MATERIALS & METHODS:

Venkatesan, N. 1998. Br. J. Pharmacol., Jun, 124 (3); 425 – 427

Weiss, R.B. 1992. Semin. Oncol. 19, 670 – 686.

Yamanaka, S. et al. 2003. J. Am. Coll. Cardiol. 41(5): 870- 878

Yin, X. et.al., 1998. Biochem. Pharmacol. 56: 87- 93,

Young, R.C et. al, 1981. N. Engl. J. Med. 305: 139-153.

MECHANISM OF DOXORUBICIN INDUCED

CARDIO TOXICITY

Inhibit Na+/K+ ATPase

Block the exchange

between Na+ and K+ ions

DOXORUBICIN

Inhibit Ca2+ATPase Decreased Adenylate

Cyclase

Increase the release of Ca2+

from Intracellular Storage

Sites

Decreases cAMP

Causes the Alteration in

Contraction and

Relaxation Cycle of

Myocardium

Decreases the β1 receptor

mediated signaling

response

Increases the

Depolarization of

Myocardial Cells

Decreases myocardial

contraction and heart rate

Increases the Contraction

of Myocardium

HEART FAILURE

36

ANTICANCER ACTIVITY OF DRF7295:

A PEPTIDE COMBINATION TARGETING MULTIPLE NEUROPEPTIDE RECEPTORS IN COLORECTAL CANCER Manu Jaggi, Anu T. Singh, Sudhanand Prasad, Praveen Rajendran, Sarjana Dutt, Anand C. Burman, Rama Mukherjee

Dabur Research Foundation, 22, Site 4, Sahibabad, Ghaziabad-201010, Uttar Pradesh, India www.daburpharma.com

Figure 1: Structures of the component peptides of DRF7295

Figure 6

PTC (colon) cells probed with a polyclonal

antibody to Vasoactive Intestinal Peptide

(x400).

Table 1

Receptor affinity [KD(M) and number [R(M/L)] of

inidividual peptides on 8 primary tumor cultures

(PTC-1 to PTC-8) of human adenocarcinoma.

Table 2

Percent inhibition of the binding of the

native neuropeptides on PTC (colon) by

DRF7295

Fold excess of cold Peptide combination

Neuropeptide 400 fold

(1.2 µM)

1000 fold

(3 µM)

30,000 fold

(90 µM)

VIP 2.6% 35.87% 94.01%

Somatostatin 20.31% 42.91% 96.6%

Bombesin 7.89% 39.08% 93.13%

Substance P 5.06% 27.71% 97.52%

TGF Nil Nil 4.5 %

Cell line Tumor type % Cytotoxicity

Colon

PTC 94.2 3.1

HT29 41.4 2.7

SW620 33.2 4.7

Pancreas MiaPaCa.2 85.4 2.9

Duodenum HuTu80 92.1 2.2

Lung L132 36.2 4.3

Breast MCF-7 34.6 5.8

Leukemia

MOLT-4 81.3 4.2

K562 41.9 4.8

Ovary PA-1 28.4 4.1

Oral KB 70.0 2.7

Neuropeptides function peripherally as paracrine and endocrine factors to regulate diverse physiological processes and act as

neurotransmitters and neuro-modulators. In a majority of cases, the receptors, which mediate signaling by neuropeptides, are

members of the superfamily of the G protein coupled seven membrane-spanning receptors [1]. Neuropeptides have been

documented to play important roles as autocrine /paracrine growth factors for human cancers [2]. The interruption of autocrine

and paracrine neuropeptide signaling with specific antagonists or broad spectrum biased antagonists offer new therapeutic

approaches to the treatment of cancer [3]. Neuropeptides and their analogs bind to specific high affinity transmembrane

receptors on target cells to initiate a cascade of cytoplasmic signaling events [4]. The role of neuropeptides in cancer and cancer

associated angiogenesis has been extensively reviewed [5-7]. Recently, extensive reviews have also been appeared describing

the neuropeptide receptors as target for cancer treatment as well as diagnosis of cancer [8-10]. Earlier studies have demonstrated

the presence of several different receptors for gastrointestinal hormones or neurotransmitters on human colon cancer cell lines,

including bombesin-related peptides, VIP, somatostatin, substance P, beta-adrenergic agents, calcitonin gene-related peptide,

gastrin, muscarinic cholinergic agents, and opiates (11). We hypothesized that analogs/antagonists to gastrointestinal peptides

would block cell proliferation and lead to cancer cell death. In order to test our hypothesis, we chose to work on colon cancer, as

it is the second most common cause of cancer death in the Western world, resulting in 56,730 deaths in the US alone according

to a recent report [12].

Neuropeptides function in an autocrine/paracrine manner and

possess specific cell surface receptors in colon cancer cells

DRF7295 displaces the binding of neuropeptides to

receptors on colon cancer cells

DRF7295 inhibits growth of human tumor cell lines

while sparing normal Cells

Table 3

Percentage cytotoxicity caused by

DRF7295 on human tumor cell lines

Background Neuropeptides play an important role as growth factors for human cancers. The interruption of autocrine and paracrine neuropeptide signaling with specific analogs offer a therapeutic approach for the treatment of cancer. Purpose To identify neuropeptides that act as growth factors for adenocarcinomas including colorectal cancer and to develop structurally designed synthetic peptide analogs with anticancer activity. Methods We have developed an ELISA capable of detecting secretion of neuropeptides in the culture supernatants of human colon adenocarcinoma with a sensitivity of 0.5 - 5.0 ng/ml (Jaggi M., Mukherjee R. J Immunoassay 15 (2), 1994). Further, we characterized these cells for receptor number and affinity for neuropeptides. A panel of novel analogs of peptides were designed, synthesized and characterized. (US 6,316,414, US 6,489,297, US 6,596,692). These peptides were screened for cytotoxicity using the MTT assay and in vivo efficacy determined on tumor xenograft models. Toxicity and Pharmacology studies were conducted as per regulatory guidelines. Results Vasoactive Intestinal Peptide, Bombesin, Substance P and Somatostatin were found to be secreted by colon adenocarcinoma cells. Moderate to high affinity receptors for the respective peptides were detected on cell surface. The in vitro screening of peptides for cytotoxicity led to the identification of four analogs, the combination of which was code-named DRF7295 (US 6,828,304). DRF7295 competed with the binding of native peptides to their membrane bound receptors on colon adenocarcinoma cells without interfering with the binding of specific growth factors of the EGF - TGF family. In vitro anticancer activity of DRF7295 in a large panel of human adenocarcinomas showed cytotoxicity ranging from 60 - 95% with colon cancer cell lines being most sensitive. Efficacy studies for individual peptides and DRF7295 were conducted in vivo on colon xenografts. While individual peptides showed a mean T/C% in the range of 2.0-54.8% in primary tumor cells of colon adenocarcinoma (PTC), DRF7295 showed significant tumor regressing activity at 320g/kg injected twice daily for 14 days. The mean T/C % was < 1.0 % and 19.1% for PTC and HT29 (colon) xenografts respectively. No tumor recurrence was observed in the normal life span of the treated animal. Further, DRF7295 demonstrated better tumor regression when used in combination with standard cytotoxics for treatment of colon cancer. Acute and long-term toxicity studies as well as safety pharmacology studies indicate the safety of the drug upon systemic administration with no significant adverse pharmacological effects. Conclusion Preclinical studies demonstrated DRF7295 to have potent in vitro and in vivo anticancer activity with a potential for use as monotherapy or in combination for treatment of colon cancer. Phase I dose escalation study of DRF 7295 presented at the ASCO Meetings (Abstract No: 948, 2003 ASCO Annual Meeting, Abstract No: 3094, 2004 ASCO Annual Meeting) have shown it to be a well-tolerated anticancer drug devoid of toxicities associated with cytotoxics. DRF7295 is presently in Phase II clinical trials and is being evaluated in patients with colorectal cancer.

DRF7295 causes tumor regression in GI cancer xenografts in nude mice

0

2000

4000

6000

8000

10000

12000

14000

0 5 10 15 20 25 30 35 40

Days post inoculum

Tu

mo

r v

olu

me (

cu

.mm

) Unt reat ed

Pept ide 1

Pept ide 2

Pept ide 3

Pept ide 4

Figure 8

Antitumor activity of Peptide 1,2,3 & 4 on

PTC (colon) xenograft

0

2000

4000

6000

8000

0 5 10 15 20 25 30

Days post inoculum

Tu

mo

r v

olu

me

(c

u. m

m)

Treated

Control

Figure 9

Antitumor activity of DRF7295 on

PTC (colon) xenograft.

Figure 10

Antitumor activity of DRF7295 on

HT-29 (colon) xenograft.

ABSTRACT

INTRODUCTION

MATERIALS & METHODS

RESULTS & DISCUSSION

0

1000

2000

3000

4000

5000

0 5 10 15 20 25 30

Days post inoculum

Tu

mo

r vo

lum

e (

cu

.mm

) Untreated

DRF7295

Figure 11

Antitumor activity of DRF7295 on

HuTu80 (Duodenum) xenograft.

Figure 2

HPLC chromatogram of DRF7295

2.00 6.00 10.00 14.00 18.00 22.00 26.00 30.00 34.00 Time 0

100

%

3.55

5.17

16.61 10.13

DRF 7295

Figure 3

LC-MS profile of DRF7295

DRF7295 is safe for systemic administration

CONCLUSIONS

REFERENCES

Peptide KD(1) KD(2) R1 R2

VIP 5.886.4 E-09 2.232.6 E-06

1.841.6 E-

10 3.062.7 E-08

Somatostatin 5.914.2 E-10 7.924.1 E-08

8.704.0 E-

11 3.421.9 E-09

Bombesin 1.357.4 E-08 -

1.540.7 E-

09 -

Substance P 5.841.4 E-10 -

2.790.4 E-

11 -

Adenocarcinoma is a heterogeneous population of cancer cells. They autonomously synthesize multiple pro-proliferative

growth factors and express high affinity receptors for these factors on their plasma membrane.

Specific high affinity receptors were found for Vasoactive intestinal peptide, Somatostatin, Bombesin and Substance P.

They were found to be secreted by the tumor cells. The native peptide hormones act as growth factors for tumor cells and

this effect is inhibited by their antagonists/analogs.

DRF7295 (combination of four peptide analogs) was developed which could kill greater than 80% of gastrointestinal tumor

cells in vitro.

DRF7295 competitively inhibits the binding of physiologically relevant concentrations of the native peptides.

Two-week administration of DRF7295 with two 12-hourly injections given by intravenous route caused significant tumor

regression of GI cancer xenografts.

DRF7295 is devoid of any acute toxicity/mortality and observable untoward effects. The cardiovascular system showed a

species varied reversible mild to moderate hypotensive response, not associated with any increase in the heart rate or

changes in the ECG. It also possessed some degree of acute anti-inflammatory effect.

In acute toxicity studies, no treatment related toxic signs or symptoms or mortality were observed at any dose level studied

in mice and rats. In chronic toxicity studies in rabbits and mice had NOEL at 5x dose level by i.v. and s.c. routes.

The support of Dabur Pharma Ltd, Dabur India Ltd., Department of Science & Technology ,

Ministry of Science & Technology , India during this work is gratefully acknowledged..

A representative illustration of stages of tumor regression

on treatment with DRF7295

1. Burbach J.P., Meijer O.C. Eur J Pharmacol, 227, 1-18, 1992.

2. Rozengurt E. In : Pusztai, L.L etal ( editors)., Cell proliferation in cancer : Regulatory mechanisms of Neoplastic cell growth, Oxford, Oxford University press, page

247- 259, 1996.

3. Favoni RE, de Cupis A. Pharmacol Rev. 2000 Jun;52(2):179-206.

4. Pimentel, In Growth factors and neoplasia, In Handbook of Growth factors, CRC Press, U.S.A, Vol 1, 329-337, 1994.

5. Matsumoto Y, Kawatani M, Simizu S, Tanaka T, Takada M, Imoto M. Anticancer Res 2000 Sep-Oct; 20(5A):3123-9.

6. Danesi R, Del Tacca M, Metabolism 1996 Aug ; 45(8 Suppl 1) : 49-50.

7. Woltering EA, Barrie R, O'Dorisio TM, Arce D, Ure T, Cramer A, Holmes D, Robertson J, Fassler J. J Surg Res 1991 Mar; 50(3): 245-251.

8. Reubi,J.C. Endocrine Reviews 24(4),389-427.

9. Janin,Y. Amino Acids. 2003 Jul;25(1):1-40.

10. Schally AV, Szepeshazi K, Nagy A, Comaru-Schally AM, Halmos G. Cell Mol Life Sci. 2004 May;61(9):1042-68.

11. Frucht H, Gazdar AF, Park JA, Oie H, Jensen RT. Cancer Res. 1992 Mar 1;52(5):1114-22.

12. Cancer Facts and Figures 2004, American Cancer Society, Inc., ©2004.

13. Mukherjee R. Jaggi M. US Patent 5744363.

14. Jaggi M., Mukherjee R. Anticancer Research 12 (6A), 1992.

15. Jaggi M., Mukherjee R. Anticancer Research 12 (6B) : 2340, 1992.

16. Jaggi M, Mukherjee R. New, sensitive and specific ELISA for the detection of neuropeptides in culture supernatants.et al. J Immunoassay. 1994 May;15(2):129-46).

17. Quin JA, Sgambati SA, Goldenring JR, Basson MD, Fielding LP, Modlin IM, Ballantyne GH. J Surg Res. 1995 Jan;58(1):111-5.

• LD50 in mice and rat by i.v. and s.c. routes > 50 times the therapeutic dose tested.

• In 3-month toxicity studies in mice and rabbits - body weight, food/water consumption, hematological, blood biochemistry

& urine parameters were within limits. Histopathological examination was found normal.

• Does not cause irritation at the site of administration when injected by i.v. or s.c. route.

• In safety pharmacology studies in rats, no gross behavioural effects were observed. The compound had a mild CNS

depressant action and partial anticonvulsant effect at 15x while it was devoid of any neurotoxicity or muscle relaxant effect

at the same dose.

• The CVS effects in rats show mild reversible hypotension at 15x, while in cats, the CVS, respiratory and autonomic

ganglionic transmission were not effected at a dose of 1x.

• Effects on isolated tissues were studied wherein DRF7295 produced spasmogenic effect on guinea pig ileum without

interfering with cholinergic or histaminergic responses. It also seems to posses oxytocic activity in the estrogen primed rat

uterus.

• Found to posses some degree of acute anti-inflammatory effect in the carageenan-induced paw oedema method.

• 15x dose produced moderate diuretic effect without potassium sparing action in fasted rats (SD strain).

• No effects were seen on liver function and the compound was devoid of any hypoglycemic effect.

Peptide synthesis

The peptides were synthesized by standard solid phase peptide chemistry methods using Fmoc chemistry on peptide synthesizer CS536 (CS Bio, San Carlos, CA, USA).

All the amino acids were protected by Fmoc group at the N-terminal. All the Fmoc protected amino acids and reagents were procured from Advanced Chemtech,

Louisville, KY, USA. The peptide was purified by Preparative HPLC system and characterized by mass spectra, amino acid analysis and mass sequencing.

Cell Culture

Human tumor cells K562 (leukemia), MOLT-4 (lymphoma), L132, (lung carcinoma), MCF-7, (breast), SW620, HT29, (colon), Mia.PaCa.2 (pancreas), HuTu80

(duodenum), KB (oral), PA-1 (ovary) were obtained from NCCS, Pune, India. PTC (colon) is a primary tumor cell line developed by us [13,14]. Cell lines were cultured

in Dulbecco’s modified Eagles medium- DMEM (GibcoBRL, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, USA) and 100U/mL penicillin and

100µg/mL streptomycin (Hyclone USA) in a humidified atmosphere of 5% CO2 and 95 % air at 37oC.

Cytotoxicity assay

A modified MTT method [15] was followed. Briefly, 10,000 cells were incubated with growth medium (control) or medium containing DRF7295 (1 µM – 0.1nM) for 3

days by dosing at 0, 24 and 48 hrs. The assay was terminated after 3 days using MTT and IC50 values were calculated by non-linear regression.

ELISA for detection of neuropeptides in culture supernatants

A sandwich ELISA method was developed by us [16] was used for the detection and identification of each of the four neuropeptides, namely, Vasoactive intestinal

peptide, Somatostatin, bombesin and Substance P. 100 µL of Amicon concentrated culture supernatant of PTC (colon cancer) cells was added to round bottomed wells

coated with 1 g of purified anti-peptide antibody and incubated for 1 hour at 37oC. For color development, 25 ul of substrate (1 mg/ml ortho phenyl diamine + 1 ul

H2O2) in Citrate Phosphate Buffer, pH 5.5 was added to each well and incubated in dark for 5 minutes at 37oC. The absorbance in each well was determined at 490 nm.

T/C% = 2%

T/C% =54.1%

T/C% =10.8%

T/C% =32.7

T/C% = < 1%

T/C% = 19.1%

T/C% = 2.8%

0

20

40

60

80

100

120

140

0 1 2 3 4 5

Days

Per

cen

t via

bil

ity

Splenocytes Endothelium L. intestine S. intestine Brain PTC

Figure 7

DRF7295 is cytotoxic to colon tumor cells

(PTC) while sparing the normal cells

Figure 5

Detection of peptides in culture supernatant of tumor cells.

Four peptides, namely VIP, somatostatin, bombesin and

Substance P were detected in supernatants of colon cancer

cell line (PTC) by a highly sensitive and specific sandwich

ELISA.

0

1

2

3

4

5

6

1 2 3 4 5 6Days

co

nc (

ng

/ml) VIP

Somatostatin

Bombesin

Substance P

Indirect Immunofluorescence

PTC (colon cancer) cells (103) cultured on coverslips were incubated at 37oC for 1 hour with 1:50 dilution of anti-peptide polyclonal antibody . The cover slips were washed and tumor cells incubated under same conditions with

1:200 dilution of anti-rabbit Immunoglobulin-Fluorescein Isothiocyanate (IgG-FITC) conjugate. The tumor cells were scanned under UV light on a Microphot FX microscope (Nikon).

Determination of receptor affinity and number

PTC cells (0.5 x 106 cells/50 µL) were suspended in binding buffer comprising of 5% Bovine Serum Albumin (BSA) in RPMI 1640. Radioactive counts were measured on a gamma counter initially and after incubation at 37oC for 1

hour. The tubes were centrifuged at 2500 rpm for 10 minutes at 4oC. The optimum cell number and tracer counts per tube were determined from the standard curve. Cold competition experiments were performed at these saturation

conditions. A fixed cell concentration and tracer counts, as optimized earlier, were added to assay tubes. This was followed by the addition of increasing concentrations of cold VIP, Somatostatin, Substance P & Bombesin in

duplicates to the tubes.

Receptor binding assay 125I labeled VIP, Bombesin, Somatostatin, Substance P and TGF each of specific activity 2000Ci/mmol were obtained from DuPont NEN, USA. The assay was carried out on intact PTC cells as described [17]. The cells were

washed twice with ice-cold Binding buffer (10mM MgCl2, 1% BSA, 1mM EGTA, 0.25mM phenyl methyl sulfonyl fluoride (PMSF) and 10 µM/ml aprotinin in RPMI 1640). Cells were incubated with 3nM of either VIP, Bombesin

or Substance P or Tranforming Growth Factor – alpha (TGF) or with 2nM of Somatostatin in the presence or absence of various concentrations of cold peptide combination and allowed to incubate for 2 hrs. at 4C. The cells were

subsequently washed thrice with ice cold binding buffer to remove unbound radioligand and lysed with 20mM Tris-HCl buffer, pH 7.4, containing 1% SDS. The radioactive counts in the cell lysate were measured using a gamma

counter (LKB Wallace, Finland). Nonspecific binding was determined in the presence of 1nM of the native peptide. The counts were processed using the EBDA Biosoft program to obtain Kd and Bmax (pmol/ mg cellular protein)

values

Animals

Athymic nude mice (Nu/Nu, Balb C background), 20-25 g of either sex, bred in National Centre For Laboratory Animal Sciences, Hyderabad were used in the xenograft study. Colony bred adult albino Swiss mice; adult Sprague

Dawley or Charles Foster rats; Guinea Pigs and trapped, quarantined cats of either sex maintained at 24 1 C were obtained from the Central Drug Research Institute, Lucknow, India and used for Toxicology and Safety

pharmacology studies. Due permission was taken by the Institutional Animal Ethics Committee (IAEC) to perform experimentation on the animals

Tumor xenograft assay

Human tumor xenografts of colon cancer were initiated in athymic nude mice by subcutaneous inoculation of a single cell suspension, containing approximately 10-15 million tumor cells. When tumors were between 400-800

cu.mm mice in the treatment group were dosed intravenously with individual peptide or DRF7295 at 0.32 mg/kg in two divided doses continuously for a period of 2 weeks. Control group of animals was not treated and the tumors

were allowed to grow. Tumor volumes were calculated using the formula 0.4 xW2xL (W = smaller dia, L = larger dia,). Tumor growth inhibition was calculated at the end of treatment using the formula (1- tumor volume treated /

tumor volume control) x 100.

Toxicity studies

Single dose : Acute toxicity study was conducted on swiss albino mice and Wistar rats of both sexes by injecting the test substance by two routes viz. intravenous and subcutaneous at 5 dose levels of 2.5 x, 5.0 x, 10.0 x, 25.0 x and

50.0 x. ( “x” is 0.32 mg/kg as determined from efficacy studies conducted on tumor bearing nude mice). A single injection (i.v. or s.c.) was administered and animals were observed daily for 2 weeks for mortality, body weight and

for any toxic signs. The control animals received vehicle only.

Long term Toxicity: Long term toxicity study was conducted on Swiss albino mice and New zealand strain rabbits of both sexes by injecting DRF 7295 by i.v. or s.c. route at 3 dose levels for 90 days at doses of 5x, 10x and 15x.

Mice were divided into groups consisting of 10 male and 10 female mice and rabbits were divided into groups consisting of 3 male and 3 female rabbits. Three groups of animals received DRF7295 intravenously and the other three

received DRF7295 subcutaneously. A fourth group treated in a similar manner with comparable volume of distilled water served as control. Weekly charting of body weights and consumption of food/water of all the animals were

done. Initial and final recordings of hematological parameters and urinalysis parameters were done. All the animals were sacrificed at the end of study, and terminal blood biochemistry and histopathology of all the important organs

and tissues was studied.

Figure 4

Detection of peptides in culture supernatant of

tumor cells. Four peptides, namely VIP,

somatostatin, bombesin and Substance P were

detected in supernatants of colon cancer cell

line (PTC) by HPLC

37

DEVELOPMENT OF A DENDRITIC-CELL BASED ASSAY TO SCREEN MOLECULES FOR POTENTIAL

ANTI-INFLAMMATORY ACTIVITY

Alka Madaan, Manu Jaggi, Rama Mukherjee

Dabur Research Foundation, 22, Site-IV, Sahibabad, Ghaziabad, Uttar Pradesh - 201010

Pro-inflammatory cytokines, such as Tumor Necrosis Factor (TNF)-

and Interleukin (IL)-1- have been explored as potential targets in

therapeutic interventions for various inflammatory disorders (1).

Currently available TNF-inhibitors include monoclonal antibodies

(Infliximab, D2E7) to neutralize TNF- α activity , TNF- α specific

recombinant receptor construct (Etanercept), and CTLA4 fusion

protein (Abatacept) to block TNF- α activity by inhibiting T cells

activation (2,3). Dendritic cells (DC), which recognize invading pathogens and

translocate the processed antigens to secondary lymphoid organs for

T cells activation, have been qualified as the target cells to investigate

pharmacological role of various Immunomodulatory agents (4-8).

An in vitro septic shock assay was developed with murine bone

marrow-DCs to screen new molecules for potential anti-inflammatory

activity. Extent of modulation in pro-inflammatory cytokines and

chemokines was taken as an indicator of anti-inflammatory activity.

OBJECTIVE

To develop an in vitro Dendritic-cell based assay for screening of

New Chemical Entities for potential anti-inflammatory activity.

Immature DCs

Mature DCs

Increased level of pro-inflammatory cytokines

Downregulation of pro-inflammatory cytokines

Identification of potential anti-inflammatory molecules

LPS

INTRODUCTION

Generation of BMDC cultures : Primary DC cultures

were generated from mouse bone marrow. Bone marrow progenitor

cells were cultured in presence of rmGMCSF, at 37C, 5%CO2.

Immature DCs were preincubated with LPS, resulting in elevated

levels of pro-inflammatory cytokines and chemokines.

Activity Screening of new molecules: 1-8 naphthyridine

derivatives were screened from an in-house library of NCEs

synthesized at DRF. Primary screening of new molecules was done

at two specific concentrations 0.1g/ml and 1 g/ml. Molecules with

potential anti-inflammatory activity were subjected to screening over

a multiple dose concentration range of 0.001 g/ml to 10 g/ml.

Supernatants were collected for analyses.

Cytokine and chemokine analyses: The panel of pro-

inflammatory cytokines and chemokines analyzed using specific

enzyme linked immunosorbent assay kits for modulatory activity of

test compounds comprised of

• Tumor Necrosis Factor-

• Interleukin-1-

• Interleukin-6 (IL-6)

• Macrophage-Inflammatory Protein (MIP-1-)

• Interferon-gamma-inducible Protein (IP-10 ).

REFERENCES

CONCLUSIONS

• A library of 100 NCEs belonging to naphthyridine group was

evaluated for anti-inflammatory activity by modulation of pro-

inflammatory cytokines and chemokines.

• The extent of inhibition of elevated levels of pro-inflammatory

cytokines by test molecules was indicative of their anti-inflammatory

properties. The downregulation of chemokine and cytokine levels by >

25% was considered as significant.

TNF- activity:

• Significant down regulation of TNF- -activity was observed for compound no. #59, #62, #66, #68, and #71.

• Screening was carried out over a concentration range of 0.001-10

μg/ml for selected compounds no. #59, #62, #66, #68, and #71.

• IC50 values were found to be <0.001 μg/ml for these compounds.

IL-1- activity:

• Same set of molecules were analyzed for downregulation of

IL-1- activity.

• Several of the molecules demonstrated >50% IL-1 inhibitory

activity.

• This DC based in vitro screening can be used as a mechanism

based assay for early identification of molecules with potential anti-

inflammatory activity.

• Screening molecules using DC assay has resulted in early

identification of potential anti-inflammatory candidates. These

molecules can be further evaluated in an in-vivo model for

inflammatory disorder. Hence less number of animals are required for

early screening studies.

• Action of biological drugs is mediated by targeting molecular

markers on cell surface, for e.g. Abatacept competes with CTLA4 on

T-cells to block T cells activation. This model presents DC as an

excellent cellular target for evaluation of anti-inflammatory activity and

offers scope for exploring and targeting other relevant DC markers

indicative of this ability.

IL-6 activity:

• Molecules exhibiting high TNF- down modulation also

demonstrated IL-6 inhibition, (IC50 < 0.001g/ml).

MIP-1- activity:

• Molecules with potent TNF- and IL-1- activity were investigated

for their effect on MIP-1- activity, a key pro-inflammatory chemokine.

• Compound No. #59, #62, #68, #71 down regulated MIP-1- at

0.1g/ml and 1 g/ml.

1. Xiao-yu R. Song et al.: Coming of Age: Anti–Cytokine Therapies, Molecular Interventions (2002), 2:36-46.

2. D.L. Scott et al.: Tumor Necrosis Factor Inhibitors for Rheumatoid Arthritis, The

New England Journal of Medicine, (2006), Vol. 355:704-712.

3.Kremer JM et al.: Treatment of rheumatoid arthritis by selective inhibition of T-cell

activation with fusion protein CTLA4Ig. N Engl J Med (2003), 349:1907-1915.

4. Banchereau J., Steinmann R.M.: Dendritic Cells and the control of immunity, Nature,

1998); 392:245-52.

5. Abe M, Thomson AW.:Influence of immunosuppressive drugs on dendritic cells.

Transpl Immunol. (2003) Jul-Sep; 11(3-4): 357-65.

6. Holger Hackstein, Angus W. Thomson, Dendritic Cells: Emerging pharmacological

targets of immunosuppressive drug , Nature reviews/ Immunology, (2004) (4) (24-34).

7. C.L.Schlichting. et al.: Dendritic Cells as pharmacological targets for the generation

of regulatory immunosuppressive effectors. New implications for allo-transplantation.

Current Medicinal Chemistry, (2005), 12(16): 1921-30.

8. Norikatsu Mizumoto et al.: Discovery of novel immunostimulants by dendritic-cell–

based functional screening, Blood, 1 November 2005, Vol. 106, No. 9, pp. 3082-3089.

9. Grossi G et al.: 1,8-Naphthyridines v. novel N-substituted 5-amino-N, N-diethyl-9-

isopropyl [1,2,4] triazolo [4,3-a] [1,8] naphthyridine-6-carboxamides, as potent anti-inflammatory and/or analgesic agents completely devoid of acute gastrolesivity. Eur J

Med Chem. (2005) Feb; 40(2): 155-65.

10. Chiara Dianzani et al.: Effects of anti-inflammatory [1, 2, 4] triazolo [4, 3-a] [1,8]

naphthyridine derivatives on human stimulated PMN and endothelial cells: an in vitro

study, Journal of Inflammation (2006), 3:4 1476-9255-3-4.

RESULTS AND DISCUSSION • BMDC based assay was developed to screen new molecules with

potential anti-inflammatory activity.

• Culture conditions were optimized to obtain reproducible DC yields

and minimum inter and intra assay variations.

• Assay system was validated with known anti-inflammatory agents,

such as NS-398 and Indomethacin for downregulation of TNF- ,

and IL-6.

METHODOLOGY

Figure-1: TNF- downregulation

Figure-2: IL-1- downregulation

Figure-4: MIP-1- downregulation

IP-10 activity:

• Compound No. #66 was a potent down regulator of IP-10 activity.

• Modest TNF-inhibitors #43, #47, #50, #51 showed <25% IP-10

downregulation.

Test molecules

Figure-3: IL-6 downregulation

Figure-5: IP-10 downregulation

Naphthyridine class of molecules has previously been reported as

potential anti-inflammatory agents (9,10). We investigated the

potential anti-inflammatory activity of novel derivatives of this class of

molecules using in vitro DC based assay.

Animals: Inbred 6-8 weeks old, male C57BL/6 mice were used.

The animals were bred in house at the specific pathogen free Small

Experimental Animals facility of Dabur Research Foundation. All

animal studies were approved by the Institutional Animal Ethics

Committee of Dabur Research Foundation.

N N

O O

NH

Ph

OH

O

R1

R

X

1

2

345

6

7

8

1'2'

3'

Structure of 1-8 naphthyridines derivatives

wherein X is hydrogen, halo, alkyl, alkoxy, amino or substituted amino; R is hydrogen,

alkyl, aryl or heterocyclic; and R1 is hydroxy, alkoxy, amino or substituted amino

group.

ACKNOWLEDGEMENTS:

The support of Dr. Anand Burman, Chairperson, Dabur research

Foundation is gratefully acknowledged.

DABUR

RESEARCH

FOUNDATION

Molecules tested

-125

-100

-75

-50

-25

0

#39

#40

#41

#43

#44

#45

#46

#47

#48

#49

#50

#51

#52

#53

#59

#60

#61

#62

#63

#66

#67

#68

#69

#70

#71

#72

#73

#74

#75

#76

#81

%c

ha

ng

e T

NF

-alp

ha

1ug/ml

0.1ug/ml

Molecules tested

-125

-100

-75

-50

-25

0

#3

9

#4

0

#4

1

#4

4

#4

5

#4

6

#4

8

#4

9

#5

2

#5

3

#5

9

#6

0

#6

1

#6

2

#6

3

#6

6

#6

7

#6

8

#6

9

#7

0

#7

1

#7

3

#7

5

#7

6

#8

1

%ch

an

ge

IL

-1-b

eta

1ug/ml

0.1ug/ml

Molecules tested

-125

-100

-75

-50

-25

0

#59

#62

#66

#68

#71

%ch

ang

e IL

-6

1ug/ml

0.1ug/ml

Molecules tested

-125.0

-100.0

-75.0

-50.0

-25.0

0.0

#42

#54

#55

#57

#58

#59

#62

#63

#67

#68

#71

#72

#73

#78

%c

ha

ng

e M

IP-1

-alp

ha

1ug/ml

0.1ug/ml

Molecules tested

-125.0

-100.0

-75.0

-50.0

-25.0

0.0

#43

#47

#50

#51

#63

#66

#72

#73

#74

%ch

ange

IP-1

0

1ug/ml

0.1ug/ml

38

Thanks