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  • 1

    Dabur Research FoundationNew Delhi, INDIA

    www.daburresearch.in

    ONCOLOGY SERVICES

    Provided in Europe by the Exclusive Distributor

    Kinase Logistics ApSStationsvej 805792 AarslevDenmark

  • 2

    Dabur Research Foundation (DRF)

    Parent company was established in 1884, Dabur India Ltd is among the oldest and largest

    healthcare companies in India, has over 5000 employees working in more than 20 countries

    Contract Research Organization focused on Preclinical drug discovery & Development

    Positioned as an Oncology specialist CRO with services in several other therapeutic areas

    Strength of 70 scientists with close to 40 % being PhDs recruited from the top 5

    Universities of India

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

    molecules, Phytochemicals, generics & differentiated formulations in multiple therapeutic

    areas

    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 various

    client requirements

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

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

    teams

  • 3

    Phase I Phase II Phase IIIClinical

    Support

    Drug

    Discovery

    Early

    Preclinical

    Advanced

    Preclinical

    APISynthesis

    & Form. Dev

    Drug

    Manufacture

    CLINICALCLINICAL

    Biochemical & Cell based

    screens

    Target basedscreens

    Signal transduction

    Molecularmodeling

    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

    CRAMCRAMDRUG DISCOVERY & PRECLINICALDRUG DISCOVERY & PRECLINICAL

  • 4

    Cell Biology & Pharmacology

  • 5

    in vitro Screens Cancer

    Cell Lines

    Indias largest tumor bank repository with human cancer cell linesrepresenting 15 20 different cancer types

    Authenticated sources (ATCC / ECACC)

    Screens validated with client defined reference drugs

    Cell lines include -

    Human Cancer lines

    Epidermal cells

    Hepatocytes

    Cardiomyocytes

    Endothelial cells

    Assays

    Validated cell based assays

    Drug resistant cell lines available

    Primary cultures for colon cancers available

    Combination index method for evaluation of synergy

    P-II tissue culture facility capable of screening up to 1000 compounds/month using the 96-well-plate format

  • 6

    Cancer cell lines

    Name of cell line Tumor type Species

    BRAIN

    U-87 MG Glioblastoma, astrocytoma Human

    SK-N-SH Neuroblastoma Human

    SK-N-MC Neuroepithelioma Human

    U373 MG Glioblastoma, astrocytoma Human

    D283 Brain,cerebellum Human

    BREAST

    MCF-7 Breast Adenocarcinoma Human

    T-47D Ductal carcinoma Human

    MDA-MB-453 Mammary gland,Metastatic carcinoma Human

    SK-BR-3 Mammary gland Human

    ZR-75-1 Ductal carcinoma Human

    HBL100 Mammary gland Human

    COLON

    SW620 Colorectal adenocarcinoma Human

    Colo 205 Adenocarcinoma Human

    HT-29 Colon Adenocarcinoma Human

    HCT-15 Colorectal carcinoma Human

    CACO-2 Human Caucasian Colon Adenocarcinomas Human

    MELANOMA

    B16-F10 Melanoma Mouse

    SK-MEL-28 Melanoma, malignant Human

    SK-MEL-2 Malignant melanoma Human

    SK-MEL-5 Melanoma Human

    LEUKEMIA

    HL-60 Leukemia, acute promyelocytic Human

    RAW 264 Leukemic monocyte-macrophage Mouse

    K-562 Leukemia Human

    MOLT-4 Human acute lymphoblastic leukemia Human

    JurgatE6.1 leukemia Human

    KIDNEY

    COS 7 Kidney fibroblast Monkey

    RAG Adenocarcinoma,Kidney Mouse

    293 Kidney Human

    MDCK canine kidney Dog

    LUNG

    DMS 114 Small Cell Lung Cancer Human

    NCI-H460 Lung Carcinoma Human

  • 7

    Cancer cell lines

    A-549 Lung Carcinoma Human

    NCI-H345 Small Cell Lung Carcinoma Human

    NCI-H522 Adenocarcinoma; non-small cell lung cancer Human

    L-132 Embryonic lung tissue (HeLa contaminant) Human

    PROSTATE

    PC-3 Adenocarcinoma, Prostate Human

    DU-145 Prostate, Carcinoma Human

    LNCaP Prostate, Carcinoma Human

    ENDOTHELIAL

    HUVEC Primary human umbilical vein endothelial cells Human

    EAHY 926 Endothelial Human

    ECV304 Umbilical cord, endothelial Carcinoma Human

    OVARY

    PA1 Teratocarcinoma Human

    SK-OV-3 Adenocarcinoma Human

    NIH:OVCAR-3 Adenocarcinoma,Ovary Human

    ORAL

    KB Oral (HeLa contaminant) Human

    SCC-25 Tongue Carcinoma Human

    CAL 27 Tongue Carcinoma Human

    HEART

    H9C2 Heart myocardium Rat

    GASTRO-Intestinal

    INT 407 Intestine Human

    FHs 74 Int Small Intestine Human

    SNU-5 Gastric Carcinoma Human

    HuTu 80 Duodenum,Adenocarcinoma Human

    BONE

    KHOS Osteosarcoma Human

    MG-63 Osteosarcoma Human

    HOS Osteogenic Sarcoma Human

    PANCREAS

    MIA PaCa-2 Pancreas Carcinoma Human

    Capan-2 Pancreatic Carcinoma Human

    HPAC Human Pancreas Human

    LIVER

    HepG2 Carcinoma, hepatocellular Human

    SKIN

    A-431 Carcinoma, epidermoid Human

    HFF-1 Human foreskin, fibroblast Human

    + many more

  • 8

    Test System

    Cancer cell lines

    Method

    Determination of % cytotoxicity & IC50 of individual drugs

    Selection of concentrations around IC50 for

    combination studies

    Testing all possible combinations of drugs for

    combined cytotoxicities

    Calculation of Combination Index (CI)

    CI1 - Antagonism

    End points

    Categorization of drug combinations as

    synergistic/additive/antagonistic for each combination

    tested.

    Evaluation of drug interactions in combination cancer chemotherapy by combination index method

    Drug Combination Studies

    Drug 1

    Drug 2

  • 9

    Xenograft Models1

    Strains

    FoxN1 or NMRI strain athymic nude mice SCID mice C57BL/6 mice

    Xenograft Models

    Ovary Oral Lung Colon Melanoma + other solid cancers incl. Adenocarcinomas & squamous cell carcinomas

    End points Body weight Change Median Tumor Volume % Tumor Growth Inhibition Log Cell Kill %T/C Relative Tumor Volume Tumor Growth Delay Regression Chart

    All studies carried out in a welfare regulations approved rodent animal facility equipped with state-of-the-art barrier facility. We have performed efficacy studies for more than 100 NCEs

    including plant-based, synthetics, biologicals & differentiated products

  • 10

    Xenograft Models..2

    0

    250

    500

    750

    1000

    1250

    1500

    1750

    2000

    18 20 22 24 26 28 30 32 34 36 38

    Days Post inoclulation

    Med

    ian

    Tum

    or V

    olum

    e (m

    m3 ) Vehicle Control

    Tamoxifen Citrate, 10mg/kg, s.c.

    In-vivo anticancer activity of Tamoxifen citrate on nude mice bearing MCF-7 tumor

    Time to Reach 500mm3

    24

    >38

    0 10 20 30 40

    VehicleControl

    TamoxifenCitrate,

    10mg/kg, s.c.

    Days

    Median %T/C

    0

    25

    50

    75

    100

    125

    18 20 22 24 26 28 30 32 34 36 38Days

    Med

    ian

    %T/

    C Tamoxifen Citrate, 10mg/kg, s.c.

    Treatment Partial

    Regression

    Complete

    Regression

    Cure

    XXX, 50mg/kg,p.o.

    Late stage model0 1 0

    XXX, 30mg/kg,p.o.

    Early stage model1 1 2

  • 11

    Xenograft Models3

    Median Tumor Volume

    0

    250

    500

    750

    1000

    1250

    1500

    1 3 5 7 9 11 13 15 17 19 21 23 25 27 29Days

    Med

    ian

    Tum

    or V

    olum

    e (m

    m3)

    Vehicle Control

    Erlotinib, 90mpk,po

    In-vivo anticancer activity of Erlotinib on nude mice bearing A549 tumor

    Time to Reach 500mm3

    30.2

    45.5

    0 10 20 30 40 50

    VehicleControl

    Erlotinib,90mpk,po

    Days

    Median %T/C

    0

    25

    50

    75

    100

    125

    1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

    Days

    Med

    ian

    %T/

    C Erlotinib, 90mpk,po

  • 12

    Xenograft Models..4

    Antitumor activity of DRFXXX on HT-29 (colon) xenograft.

    Antitumor activity of DRFXXX on HuTtu-80 (duodenum) xenograft.

    0

    1000

    2000

    3000

    4000

    5000

    6000

    0 5 10 15 20 25 30

    Days post inoculum

    Tum

    or v

    olum

    e (c

    u.m

    m)

    Untreated

    DRF7295

    T/C% = 19.1%

    0

    1000

    2000

    3000

    4000

    5000

    0 10 20 30

    Days post inoculum

    Tum

    or v

    olum

    e (c

    u.m

    m)

    UntreatedDRF7295 T/C% = 2.8%

  • 13

    in vivo Models Syngeneic..1

    Animals C57BL/6 mice BALB/c

    Syngeneic Models Lung Colon

    Metastasis Model

    Mouse melanoma - lung nodule- I.V Injection of melanoma cells in C57BL/6 mice- Estimation of Number of Lung nodule, Melanin content, etc

    Colon SQ Model ( CT-26 - Colon )- SQ injection of CT-26 in BALB/c mice- Development of Solid tumor & Metastatic tumor at

    colon, pancreas, lung, etc- Tumor growth, Histopathology etc..

    Relative Melanin Content

    0

    25

    50

    75

    100

    G1, SWFI, 10ml/kg,p.o. S1P ReceptorModulator,60mg/kg,p.o.

    in vivo model for assessment of anti-cancer potential of Compounds using syngeneic model

  • 14

    Median Tumor Volume

    0

    1000

    2000

    3000

    4000

    5000

    1 3 5 7 9 11 13 15

    Days

    Med

    ian

    Tum

    or V

    olum

    e (m

    m3 )

    Vehicle Control

    Paclitaxel, 5mg/kg, i.v.

    Median %T/C

    0

    25

    50

    75

    100

    1 3 5 7 9 11 13 15Days

    Med

    ian

    %T/

    C

    Paclitaxel, 5mg/kg, i.v.

    In-vivo anticncer activity of Paclitaxel on BALB/c bearing CT-26 colon tumor

    Time to Reach 500mm3

    14

    0

    0 5 10 15

    VehicleControl

    Paclitaxel,5mg/kg, i.v.

    Days

    Syngeneic Models2

  • 15

    Syngeneic Models3

    Median Tumor Volume Chart

    0

    500

    1000

    1500

    2000

    2500

    3000

    3500

    1 3 5 7 9 11 13 15Days

    Med

    ian

    Tum

    or V

    olum

    e (m

    m^3

    )

    Vehicle Control

    5-FU, 12mg/kg, i.p.

    Median %T/C

    0

    20

    40

    60

    80

    100

    1 3 5 7 9 11 13 15Days

    Med

    ian

    % T

    /C 5-FU, 12mg/kg, i.p.

    In-vivo anticancer activity of 5-FU on BALB/c bearing CT-26 colon tumor

    Median Tumor Volume Chart

    0

    500

    1000

    1500

    2000

    2500

    3000

    3500

    1 3 5 7 9 11 13 15Days

    Med

    ian

    Tum

    or V

    olum

    e (m

    m^3

    )

    Vehicle Control

    Herbal Formulation,500mg/kg

    Herbal Formulation, 500mg/kg

    0

    10

    20

    30

    40

    50

    60

    70

    80

    90

    100

    1 3 5 7 9 11 13 15Days

    Med

    ian

    % T

    /C

    Anticancer activity of Herbal formulation on BALB/c bearing CT-26 colon tumor

  • 16

    in vivo Models leukemia

    in vivo model for assessment of anti-Leukemic potential of Compounds

    Model Assessment of anti-leukemic activity of the compounds in SCID mice

    Test system SCID mice

    Method

    Intravenous inoculation of leukemic cell line Administration of test item and reference item at predetermined

    time Observation of animals for defined time period Observation of animals in satellite group for occurrence of

    chloroma Percentage Change in body weight Survival rate of animals Clinical signs & symptoms Quantification by flow cytometry analysis in bone marrow &

    peripheral blood

    End points Standard surface specific antigen staining - CD45

    Lineage specific antigen staining Histopathology

  • 17

    in vivo Models Angiogenesis

    in vivo & In-vivo model for assessment of anti-angiogenesis potential of Compounds

    Currently available Angiogenesis Model

    In-vivo SQ Model in C57BL/6- SQ injection of Matrigel with growth factors

    (FGF & VEGF)- Histopathology, Hb content etc..

    In-vitro Tube formation assay- Culture of HUVEC on Matrigel plate- Estimation of branch point, Tube area and tube length

    In-vitro cell migration Assay- HUVEC Cell- No. of cells migrated towards wound area

    In-vitro VEGF estimation- Incubation of Test compound with various cells (HUVEC etc..)- Estimation of endogenous VEGF production using ELISA

  • 18

    Test Principle

    The in-vitro studies are not fully predictive of the metastatic behavior of tumor cells due to

    its lack of interaction with the host. B16F10, a murine melanoma selectively colonize the

    lung after intravenous administration.

    Test System

    C57BL/6 Mice; 6-8week

    Method

    B16F10 cells cultured and injected into the lateral tail vein at the conc. of 5-10X104 in 0.2ml.

    The test animals grouped as per experimental design

    Treatment as per recommended dose & regimen

    Humane sacrifice on 3-4weeks and lung will be weighed & fixed in Bouins solution

    End points

    Lung weight

    Lung Nodule

    Relative melanin content

    Lung Metastasis Model

    Evaluation of Anti metastatic potential by lung colonization assay using murine syngenic Melanoma model

  • 19

    Lung Metastasis Model2

    0

    10

    20

    30

    G1, SWFI, 10ml/kg,p.o. S1P ReceptorModulator,60mg/kg,p.o.

    No.

    of l

    ung

    nodu

    les

    Relative Melanin Content

    0

    25

    50

    75

    100

    G1, SWFI, 10ml/kg,p.o. S1P ReceptorModulator,60mg/kg,p.o.

    Weight of Lung

    0

    0.1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7

    0.8

    0.9

    Untreated XXX, 5mg/kg, i.v Normal lungs

    Wei

    ght o

    f lun

    g (g

    ram

    s)

    *

    *Control

    XXX Receptor

    Modulator, 60mg/kg

    Model under Validation

    Spontaneous Meatstasis Model B16F10 cell injection into the foot pad of C57BL6 Mouse

  • 20

    in vivo Models Orthotopic

    Animals Nude and SCID Mice

    Models Colon Liver Breast Spleen Renal

    Currently available Orthotopic Model

    Colon Model- Development of Solid tumor & Metastatic tumor at

    colon, pancreas, lung etc- Tumor growth, Histopathology etc..

    in vivo model for assessment of anti-cancer potential of Compounds using orthotopic model

  • 21

    Mechanistic Studies

  • 22

    Sorting Endosomes

    ACTIVE TARGETING IN CYTOPLASM

    Early Endosomes

    Free Paclitaxel

    6 Tubulin Stabilization

    Nanoparticle micelles containing Paclitaxel

    1

    NIPAM based Micellar polymers with LCST of 52-60 C in plasma

    2Targeting through EPR effect

    3Polymer aided higher cellular

    uptake

    4

    Intracellular trafficking through endosomal lysosomal compartment

    5 Drug Release from micelles at low pH conditions in Endo-

    lysosomal compartment

    Early Endosomes

    7 Apoptosis

    MECHANISTIC PROFILING STRATEGIES

    Nanoxel - A novel Nanopolymer based tumor targeted delivery system for Paclitaxel - ASCO 2008

  • 23

    Complete repertoire of Screens for PCD to enable selection of Proapoptotic molecules

    Apoptotic pathways

    Death receptor and mitochondrial pathway

    Parameters

    Transmembrane Mitochondrial permeability

    Cell Cycle

    Reactive oxygen species (ROS) quantitation

    Levels of pro-apoptotic and anti-apoptotic proteins

    Ras-ERK kinases

    Caspase 3 activation

    PARP cleavage

    DNA fragmentation

    Micronuclei staining

    Apoptosis Screens

  • 24

    Models for Clinical Toxicities

  • 25

    in vivo Model for Cardiotoxicity

    Preclinical Models to compare lead molecules/ formulations to assess cardiac safety

    Model

    Wistar rat/ FVB mice

    Parameters

    Serum markers

    CK-MB

    LDH

    Validated with Dex.

    Myocardial markers

    SOD enzyme

    Catalase enzyme

    Reduced Glutathione

    Glutathione peroxidase

    Lipid Peroxidation

    Histopathology

    Light microscopy

    Electron microscopy

    Heart wt & Body wt. ratio.

    Discrete markers of toxicity in cardiac biopsies

    CK-MB

    Wistar rat

    Biochemical analysis & Histopathology

  • 26

    in vivo Model for Gastrointestinal Safety

    Preclinical in vivo models to compare lead molecules / formulations for their potential to cause Gastrointestinal toxicity during Clinical development

    Validated Screens

    in vivo:

    Adult Wistar rat FOLFOX induced models

    Parameters

    External analysis of scores

    Oral Mucositis Index (OMI)

    Erythema

    Ulceration

    Internal analysis of scores

    Number of ulcers in tongue

    % Ulcerative area in tongue

    Histopathological evaluation

    Loss of keratinized filliform papillae

    Decrease in mucosal thickness

    Necrotic fibrinoid material

    Inflammatory cells

    Active granulation tissue

    External Stomatitis

    Ulcer scoring

    Tongue Histology

  • 27

    Validated Screens in vitro:

    Murine model - Hematopoeitic progenitor cellsfrom Bone marrow of C57/BL6 mice

    Human model - Cord Blood cells

    Parameters

    Scoring of CFU-GM colonies

    Determination of IC 50 values Inhibition of cell proliferation

    Morphology of CFU-GM colonies

    Prediction of Human MTD by determining

    IChuman/ICmurine

    Toxicity to progenitors of megakaryocytic or

    erythrocytic lineages can be predicted using

    Thrombopoietin & Erythropoietin

    in vivo Model for Neutropenia

    Preclinical in vivo models to compare lead molecules / formulations for their potential to cause Neutropenia during Clinical development (ECVAM 2006 approved)

  • 28

    Models in vivo:

    Docetaxel induced neuropathy in Adult Swiss albino mice

    Parameters Pharmacological end points Loss of hind limb extension Paresis of hind limb The Foot Print of Pain (FPP)

    Histology based end points Sciatic nerve histology Fiber density Axonal detachment Regeneration of myelin sheath

    in vivo Model for Peripheral Neuropathy

    Preclinical in vivo models to compare lead molecules / formulations for their potential to cause

    Peripheral Neuropathy during Clinical development

  • 29

    Chemotherapy Induced Alopecia

    in vivo models

    Etoposide and Cyclophosphomide induced models

    Adult Swiss albino

    Adult C57/BL6 mice

    Parameters

    Scale of hair growth by blinded observer

    Comparative hair weight

    Swiss albino mice C57BL6 mice

    Comparative hair weight

    Preclinical in vivo models to compare lead molecules / formulations for their potential to cause

    Alopecia during Clinical development

  • 30

    Toxicology

    ADME-PK

  • 31

    Systemic Toxicity Studies

    Single dose toxicity studies (Guideline: OECD 420,423/425/Revised Schedule Y )

    Species/Sex/No. of animals

    - First mice and then rats to establish body surface area linearity

    - Five animals of either sex

    Route/ Dose/ Duration

    - As intended in humans

    - Four graded doses + control

    - Single dose followed by 14 days observation period

    End Points

    - MLD & MTD; LD50 & LD10, Target organ of toxicity, body weight,

    intoxication, mortality, gross pathological changes.

  • 32

    Repeated dose toxicity studies(Guideline: OECD / Revised Schedule Y)

    Species/Sex/No. of animals

    One rodent species, preferably rat

    15 animals of each sex

    Route/ Dose/ Duration

    As intended in humans

    Highest dose is MTD of single dose study

    (4 graded doses + control + vehicle)

    continuous administration

    End Points

    Intoxication, Target organ of toxicity, body weight, mortality, haematology, biochemistry, gross & microscopic pathological changes,

    Systemic Toxicity Studies

  • 33

    Acute Dermal Toxicity

    Acute dermal irritation / corrosion

    Repeated dose dermal toxicity

    Phototoxicity

    Ocular Toxicity

    Dermal toxicity at site of injection

    Dermal Toxicity

  • 34

    Pharmacokinetic Studies In Rodents

    Several administration routes [oral, i.v.(bolus & infusion), i.p.,]

    Bio-analytical Method development

    Bio-analysis of sample

    Parameters analyzed: Cmax, Tmax, AUC, Clearance, half life

    and volume of distribution

    Tissue distribution studies

    Excretion studies (Urinary and feces)

    We generate information on critical aspects of PK/ADME of a candidate, including half-life, bioavailability, and occurrence of active metabolite

    using pre-formulation early in discovery process

  • 35

    ADME Studies

    We address ADME early in the discovery process, and perform medium-throughput screening using in silico and in vitro methods

    leading to in vivo pharmacokinetics

    Predictive ADMET

    in silico prediction

    in vitro assays

    Aqueous solubility

    Permeability

    Metabolic stability in liver microsomes

    CYP450 inhibition

    CYP450 profiling

    Plasma protein binding

  • 36

    Bio-distribution

    Bio-distribution

    Development of Bioanalytical method in tissue homogenate

    Dosing of animals by appropriate route

    Collection of organs at specified time points.

    Homogenate preparation of organs collected

    Quantitation of drugs in organ homogenate using suitable

    methods

    Calculation and expression in terms of drug/gm of organ

  • 37

    Elimination

    Elimination

    Development of Bioanalytical method in urine, feces and bile

    Dosing of animals

    Collection of urine and feces at specified time intervals using

    metabolic cages

    Determination of drug concentration in urine & feces using suitable

    quantitation method

    For binary excretion study

    Cannulation of bile duct

    Dosing of animals

    Collection of bile at specified time intervals

    Quantitation of drug in bile using suitable quantitative method

  • 38

    Drug-Drug Interaction CYP450 Inhibition

    Determination of Potential for Drug-Drug Interaction by

    cytochrome P450 Inhibition assay

    The assay methodology tested the ability of test compounds at

    different concentrations to inhibit the metabolism of fluorescent

    substrate to its metabolite

    The metabolite is read fluorometrically at their emission &

    excitation wavelength with the help of Multiwell

    spectrophotometer & fluorimeter to determine IC50

  • 39

    CaseStudies

  • 40

  • 41

    ANTICANCER ACTIVITY OF DRF7295: A PEPTIDE COMBINATION TARGETING MULTIPLE NEUROPEPTIDE RECEPTORS IN COLORECTAL

    CANCERManu Jaggi, Anu T. Singh, Sudhanand Prasad, Praveen Rajendran, Sarjana Dutt, Anand C. Burman, Rama MukherjeeDabur Research Foundation, 22, Site 4, Sahibabad, Ghaziabad-201010, Uttar Pradesh, India www.daburpharma.com

    Figure 1: Structures of the component peptides of DRF7295

    Figure 6PTC (colon) cells probed with a polyclonalantibody to Vasoactive Intestinal Peptide(x400).

    Table 1Receptor affinity [KD(M) and number [R(M/L)] ofinidividual peptides on 8 primary tumor cultures(PTC-1 to PTC-8) of human adenocarcinoma.

    Table 2Percent inhibition of the binding of thenative neuropeptides on PTC (colon) byDRF7295

    Fold excess of cold Peptide combination

    Neuropeptide 400 fold(1.2 M)

    1000 fold(3 M)

    30,000 fold(90 M)

    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 asneurotransmitters and neuro-modulators. In a majority of cases, the receptors, which mediate signaling by neuropeptides, aremembers of the superfamily of the G protein coupled seven membrane-spanning receptors [1]. Neuropeptides have beendocumented to play important roles as autocrine /paracrine growth factors for human cancers [2]. The interruption of autocrineand paracrine neuropeptide signaling with specific antagonists or broad spectrum biased antagonists offer new therapeuticapproaches to the treatment of cancer [3]. Neuropeptides and their analogs bind to specific high affinity transmembranereceptors on target cells to initiate a cascade of cytoplasmic signaling events [4]. The role of neuropeptides in cancer and cancerassociated angiogenesis has been extensively reviewed [5-7]. Recently, extensive reviews have also been appeared describingthe neuropeptide receptors as target for cancer treatment as well as diagnosis of cancer [8-10]. Earlier studies have demonstratedthe 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 peptideswould block cell proliferation and lead to cancer cell death. In order to test our hypothesis, we chose to work on colon cancer, asit is the second most common cause of cancer death in the Western world, resulting in 56,730 deaths in the US alone accordingto 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 3Percentage cytotoxicity caused by

    DRF7295 on human tumor cell lines

    BackgroundNeuropeptides play an important role as growth factors for human cancers. The interruption of autocrine and paracrineneuropeptide signaling with specific analogs offer a therapeutic approach for the treatment of cancer.PurposeTo identify neuropeptides that act as growth factors for adenocarcinomas including colorectal cancer and to develop structurallydesigned synthetic peptide analogs with anticancer activity.MethodsWe have developed an ELISA capable of detecting secretion of neuropeptides in the culture supernatants of human colonadenocarcinoma with a sensitivity of 0.5 - 5.0 ng/ml (Jaggi M., Mukherjee R. J Immunoassay 15 (2), 1994). Further, wecharacterized these cells for receptor number and affinity for neuropeptides. A panel of novel analogs of peptides weredesigned, synthesized and characterized. (US 6,316,414, US 6,489,297, US 6,596,692). These peptides were screened forcytotoxicity using the MTT assay and in vivo efficacy determined on tumor xenograft models. Toxicity and Pharmacologystudies were conducted as per regulatory guidelines.ResultsVasoactive Intestinal Peptide, Bombesin, Substance P and Somatostatin were found to be secreted by colon adenocarcinomacells. Moderate to high affinity receptors for the respective peptides were detected on cell surface. The in vitro screening ofpeptides for cytotoxicity led to the identification of four analogs, the combination of which was code-named DRF7295 (US6,828,304). DRF7295 competed with the binding of native peptides to their membrane bound receptors on colonadenocarcinoma cells without interfering with the binding of specific growth factors of the EGF - TGF family. In vitroanticancer activity of DRF7295 in a large panel of human adenocarcinomas showed cytotoxicity ranging from 60 - 95% withcolon cancer cell lines being most sensitive.Efficacy studies for individual peptides and DRF7295 were conducted in vivo on colon xenografts. While individual peptidesshowed a mean T/C% in the range of 2.0-54.8% in primary tumor cells of colon adenocarcinoma (PTC), DRF7295 showedsignificant tumor regressing activity at 320g/kg injected twice daily for 14 days. The mean T/C % was < 1.0 % and 19.1% forPTC and HT29 (colon) xenografts respectively. No tumor recurrence was observed in the normal life span of the treatedanimal. Further, DRF7295 demonstrated better tumor regression when used in combination with standard cytotoxics fortreatment of colon cancer.Acute and long-term toxicity studies as well as safety pharmacology studies indicate the safety of the drug upon systemicadministration with no significant adverse pharmacological effects.ConclusionPreclinical studies demonstrated DRF7295 to have potent in vitro and in vivo anticancer activity with a potential for use asmonotherapy or in combination for treatment of colon cancer. Phase I dose escalation study of DRF 7295 presented at theASCO Meetings (Abstract No: 948, 2003 ASCO Annual Meeting, Abstract No: 3094, 2004 ASCO Annual Meeting) haveshown it to be a well-tolerated anticancer drug devoid of toxicities associated with cytotoxics. DRF7295 is presently in Phase IIclinical trials and is being evaluated in patients with colorectal cancer.

    DRF7295 causes tumor regression in GI cancer xenografts in nude mice

    0200040006000

    8000100001200014000

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    Figure 8Antitumor activity of Peptide 1,2,3 & 4 on

    PTC (colon) xenograft

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    Figure 9 Antitumor activity of DRF7295 on

    PTC (colon) xenograft.

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    HT-29 (colon) xenograft.

    ABSTRACT

    INTRODUCTION

    MATERIALS & METHODS

    RESULTS & DISCUSSION

    0

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    Figure 11Antitumor 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 34.00Time0

    100

    %

    3.55

    5.17

    16.6110.13

    DRF 7295

    Figure 3LC-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-061.841.6 E-

    10 3.062.7 E-08

    Somatostatin 5.914.2 E-10 7.924.1 E-088.704.0 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-proliferativegrowth 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 andthis effect is inhibited by their antagonists/analogs.DRF7295 (combination of four peptide analogs) was developed which could kill greater than 80% of gastrointestinal tumorcells 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 tumorregression of GI cancer xenografts.DRF7295 is devoid of any acute toxicity/mortality and observable untoward effects. The cardiovascular system showed aspecies varied reversible mild to moderate hypotensive response, not associated with any increase in the heart rate orchanges 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 studiedin 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 effectat the same dose.

    The CVS effects in rats show mild reversible hypotension at 15x, while in cats, the CVS, respiratory and autonomicganglionic transmission were not effected at a dose of 1x.

    Effects on isolated tissues were studied wherein DRF7295 produced spasmogenic effect on guinea pig ileum withoutinterfering with cholinergic or histaminergic responses. It also seems to posses oxytocic activity in the estrogen primed ratuterus.

    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 synthesisThe 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 CultureHuman 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 culturedin Dulbeccos modified Eagles medium- DMEM (GibcoBRL, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, USA) and 100U/mL penicillin and100g/mL streptomycin (Hyclone USA) in a humidified atmosphere of 5% CO2 and 95 % air at 37oC.Cytotoxicity assayA modified MTT method [15] was followed. Briefly, 10,000 cells were incubated with growth medium (control) or medium containing DRF7295 (1 M 0.1nM) for 3days 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 supernatantsA sandwich ELISA method was developed by us [16] was used for the detection and identification of each of the four neuropeptides, namely, Vasoactive intestinalpeptide, Somatostatin, bombesin and Substance P. 100 L of Amicon concentrated culture supernatant of PTC (colon cancer) cells was added to round bottomed wellscoated 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 ulH2O2) 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

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    Splenocytes Endothelium L. intestine S. intestine Brain PTC

    Figure 7DRF7295 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 andSubstance P were detected in supernatants of colon cancercell line (PTC) by a highly sensitive and specific sandwichELISA.

    0

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    Indirect ImmunofluorescencePTC (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 with1:200 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 numberPTC cells (0.5 x 106 cells/50 L) 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 1hour. 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 saturationconditions. 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 induplicates to the tubes.Receptor binding assay125I 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 werewashed twice with ice-cold Binding buffer (10mM MgCl2, 1% BSA, 1mM EGTA, 0.25mM phenyl methyl sulfonyl fluoride (PMSF) and 10 M/ml aprotinin in RPMI 1640). Cells were incubated with 3nM of either VIP, Bombesinor 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 weresubsequently 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 gammacounter (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)valuesAnimalsAthymic 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 SpragueDawley 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 Safetypharmacology studies. Due permission was taken by the Institutional Animal Ethics Committee (IAEC) to perform experimentation on the animalsTumor xenograft assayHuman 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 400-800cu.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 tumorswere 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 studiesSingle 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 and50.0 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 andfor 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 threereceived 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 weredone. 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 organsand tissues was studied.

    Figure 4 Detection of peptides in culture supernatant oftumor cells. Four peptides, namely VIP,somatostatin, bombesin and Substance P weredetected in supernatants of colon cancer cellline (PTC) by HPLC

  • 42

    DISCOVERY AND DEVELOPMENT OF BETULINIC ACID DERIVATIVES FOR THE TREATMENT OF CANCER

    Manu Jaggi, MJA Siddiqui, Praveen R, Anand Vardhan, Rama Mukherjee, Anand C.BurmanDabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad. Uttar Pradesh. INDIA

    www.daburpharma.com

    In-vitro Anti-cancer activityS.No Cell line Cytotoxicity of Betulinic acid

    ED50 (g/ml)1 HL 60 (Human myelogenous leukemia) 2.80 0.322 K 562 (Human myelogenous leukemia) 3.25 0.493 MOLT-4 (Human lymphoblastic leukemia) 1.23 0.704 Jurkat E6.1(Human lymphoblastic leukemia) 0.65 0.045 CEM.CM3 (Human lymphoblastic leukemia) 0.98 0.036 U937 (Human histiocytic lymphoma) 0.69 0.017 BRISTOL-8 (Human B-cell lymphoma) 0.84 0.058 MiaPaCa2 (Human pancreas) > 109 HeLa (Human cervical) > 10

    10 PA-1 (Human ovary) > 1011 U87MG (Human glioblastoma) > 1012 U373MG (Glioblastoma) > 1013 MDA.MB.453 (Breast) > 1014 T47D (Breast) > 1015 HT29 (Colon) 1.8 0.016 SW 620 (colon) > 1017 CoLo 205 (colon) > 1018 A549 (lung) > 1019 L132 (lung) 1.30 0.5520 KB (Oral) > 1021 DU145 (Prostate) 1.13 0.3522 Malme 3M (Melanoma) 2.20 0.7023 RPMI 8226 (Myeloma) >10

    S.No. Cell line Cytotoxicity of lead moleculesED50 (g/ml)

    LEAD1 LEAD5 LEAD2 LEAD31 HBL100 2.82 3.94 2.87 6.982 DU145 0.82 3.24 2.37 8.923 KB 16.43 >20 13.53 16.34 SW620 3.4 5.9 9.57 4.775 Hs294T 3.09 >20 11.59 7.876 MiaPaCa-2 3.17 3.11 3.95 7.887 HuTu-80 16.27 10.50 10.99 12.568 U87MG >20 >20 >20 18.919 Hep-2 13.39 7.65 9.59 7.1110 PA-1 3.63 3.4 7.53 6.8111 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 LEAD31 HBL100 4.94 1.82 >6.96 2.152 DU145 17.01 2.22 >8.43 1.663 KB 0.85 0.36 >1.48 0.914 SW620 4.1 1.22 >2.08 3.115 Hs294T 4.5 1.7 1.886 MiaPaCa-2 4.4 2.31 >5.12 1.887 HuTu-80 0.86 0.68 >1.81 1.188 U87MG 0.70 2.1 2.0810 PA-1 13.84 2.1 >2.65 2.17

    5.5

    18.5

    49.221.733.4

    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 analysis 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)

    Metabolicstability

    Plasmaproteinbinding

    CYP450(1A2,2C9,2D6,3A4)

    inhibitionIntravenous Oral

    Poor Poor Good High Does not inhibit keyenzyme isoforms

    2 compartment,1st order elimination

    model

    Poor oralavailability

    Toxicity / Safety

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

    1 LEAD2 150 2002 LEAD6 25 37.53 LEAD1 25 37.54 LEAD5 300 ND5 Betulinic Acid 150 ND6 Vehicle Equivalent to 200 ND

    ND=Not determined (above highest dose tested)

    AcknowledgementsWe would like to acknowledge the contributions made in this work by

    scientists of Molecular Oncology and Analytical Development divisions of Dabur Research Foundation.

    Reference1. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.; Farnsworth, N. R.; Cordell, A. C.; Beecher, C. W. W.; Fong, H. H. S.;

    Kinghorn, A. D.; Brown, D. M.; Wani, M. C.; Wall, M. E.; Hieken, T. J.; DasGupta, T. K.; Pezzuto, J. M. Nat. Med. 1995, 1,1046.

    2. Fulda, S.; Jeremias, I.; Steiner, H. H.; Pietsch, T.; Debatin, K. M. Int. J. Cancer 1999, 82, 435.3. Schmidt, M. L.; Kuzmano., K. L.; Ling-Indeck, L.; Pezzuto, J. M. Eur. J. Cancer 1997, 33, 2007.4. Zuco, V.; Supino, R.; Righetti, S. C.; Cleris, K.; Marchesi, E.; Gambacorti-Passerini, C.; Formelli, F. Cancer Lett. 2002, 175,

    17.5. Thurnher D, Turhani D, Pelzmann M, Wannemacher B, Knerer B, Formanek M, Wacheck V, Selzer E. Head Neck. 2003

    Sep;25(9):732-40.6. Jeremias I, Steiner HH, Benner A, Debatin KM, Herold-Mende C. Acta Neurochir (Wien). 2004 Jul;146(7):721-9. Epub 2004

    May 21.7. Ehrhardt H, Fulda S, Fuhrer M, Debatin KM, Jeremias I. Leukemia. 2004 Aug;18(8):1406-12.8. Fulda, S.; Friesen, C.; Los, M.; Sca.di, C.; Mier, W.; Benedict, M.; Nunez, G.; Krammer, P. H.; Peter, M. E.; Debatin, K. M.

    Cancer Res. 1997, 57, 4956.9. Selzer, E.; Pimentel, E.; Wacheck, V.; Schlegel, W.; Pehamberger, H.; Jansen, B.; Kodym, R. J. Invest. Dermatol. 2000, 114,

    935.10. Fulda S, Scaffidi C, Susin SA, Krammer PH, Kroemer G, Peter ME, Debatin K-M. J Biol Chem 1998;273: 33942 33948.11. Fulda S, Debatin KM. Med Pediatr Oncol 2000;35:616618.12. Melzig, M. F.; Bormann, H. Planta Med. 1998, 64, 655.13. Kwon, H. J.; Shim, J. S.; Kim, J. H.; Cho, H. Y.; Yum, Y. N.; Kim, S. H.; Yu, J. Jpn. J. Cancer Res. 2002, 93, 417.14. Yasukawa K, Takido M, Matsumoto T, Takeuchi M, Nakagawa S. Oncology 1991;48:7276.15. Yasukawa K, Yu SY, Yamanouchi S, Takido M, Akihisa T, Tamura T. Phytomedicine 1995;4:309313.16. Mukherjee R, Jaggi M, Rajendran P, Siddiqui MJ, Srivastava SK, Vardhan A, Burman AC. Bioorg Med Chem Lett. 2004 May

    3;14(9):2181-4.17. Mukherjee, R.; Jaggi, M.; Rajendran, P.; Srivastava, S. K.; Siddiqui, M. J. A.; Vardhan, A.; Burman, A. C. Bioorg. Med.

    Chem. Lett. 2004, 14, 3169.18. Mukherjee R, Jaggi M, Siddiqui MJ, Srivastava SK, Rajendran P, Vardhan A, Burman AC. Bioorg Med Chem Lett. 2004 Aug

    2;14(15):4087-91.

    Abstract

    Betulinic acid is a naturally occurring pentacyclic triterpenoid that has demonstratedselective cytotoxicity against melanoma and glioblastomas. Its application in treatmentof cancer has been limited due to its poor solubility. Betulinic acid is currentlyundergoing advanced preclinical development for treatment of melanoma where it isapplied topically as a cream. We have for the first time established betulinic acid as apotential anti-cancer drug with broad-spectrum anti-cancer activity in leukemia,lymphoma, prostate, ovary, lung, melanoma & colon human tumor cell lines andxenografts.

    In continuation and in line with development of more potent derivatives with improvedphysicochemical properties we have synthesized more than 1000 novel betulinic acid ordihydro-betulinic acid derivatives with modifications in C2, C3, C20, C28 and C29 positionsand identified more than 50 novel molecules with better activity profile as compared tothe parent molecule. We further elucidated the mechanism of action of Lead compoundswherein it was demonstrated that the compounds have anti-apoptotic and anti-angiogenicpotential as well as significant PKC inhibitory activity in cancer cells. These moleculesare being tested for metabolic stability, potential for drug interactions,permeability/absorption, pharmacokinetics and toxicity. Being a natural product derivedmolecule with ready availability of starting material and high yield of synthesis coupledwith 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 cells 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

    Design/Synthesize derivatives

    In vitro cytotoxicity /specificity screening

    In-vitro anti -angiogenic screening

    Efficacy in animalxenograft/angiogenesis

    models

    In vitro ADMEscreening

    DMPK studiesSafety / toxicitystudies

    Mechanism of action

    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 ED50values (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 MatrigelTM (70 L). Compounds were solubilized in DMSO and were added in duplicate wells at non-cytotoxic concentration and incubated at 4 g/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 g/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 g/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 100M (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 20M 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 studiesAdult Balb/c mice, age 6-8 wks, weighing between 20-25gms were selected for the study. 3 animals per groupwere administered a single intravenous dose of the test compound (dissolved in co-solvents) at doses rangingfrom 10 to 300 mg/kg. Mortality, body weight and apparent toxic signs/symptoms were recorded over aperiod of 14 days. The maximum tolerated dose at which no toxic signs were seen was designated as Safedose and the dose at which atleast one mortality was seen was designated as Lethal dose.PharmacokineticsMale Wistar Rats, age 6 -10 weeks, weighing between 100-150 gms were selected for the study. Testcompounds were dissolved in co-solvents at a concentration of 5 mg/ml (intravenous dose) or in 0.5% CMCsuspension 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 timepoints (3 min, 10 min, 30 min, 1 hr, 2hr, 4hr, 6hr, 8hr, and 24 hr). The plasma layer was separated, extractedusing organic solvents, centrifuged and supernatant evaporated to dryness. It was reconstituted with 200 l of10% DMSO in Methanol and analyzed using HPLC. The pharmacokinetic parameters were determined usingWinNonlin 4.0 software

    In vivo anti-tumor activity

    Effect of LEAD1 formulation on colon (PTC) xenograft

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    Effect of LEAD1 formulation on lung (L132) xenografts

    0100200300400500600

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    In vitro Anti-angiogenic acitivityCytotoxicity 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 LEAD31 HBL100 0.8 0.3 0.8 7.12 DU145 0.2 0.3 0.7 9.13 KB 4.7 >1.55 3.7 16.64 SW620 1.0 0.5 2.6 4.95 Hs294T 0.9 >1.55 3.2 8.06 MiaPaCa-2 0.9 0.2 1.1 8.07 HuTu-80 4.7 0.8 3.0 12.88 U87MG >5.73 >1.55 >5.49 19.39 Hep-2 3.8 0.6 2.6 7.3

    10 PA-1 1.0 0.3 2.1 6.911 A549 0.3 >1.55 >5.49 >20.412 HT29 4.1 >1.55 >5.49 >20.413 CHO 4.0 0.6 >5.49 15.114 ECV304 1.0 1.0 1.0 1.0

    ECS less than 10 = Low ECSECS between 10 and 20 = Moderate ECSECS greater than 20 = High ECS

    Mechanism of action

    Effect of Betulinic acid on levels of Bcl-2 in PA-1 (ovarian) cell line

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    Effect of Betulinic acid on Nucleosome release in PA-1(ovarian) cell line

    02468

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    Nucleosome

    Effect of betulinic acid on pro-angiogenic factors in

    K562 (leukemia) cell line at 1ug/ml

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    VEGF bFGF Endostatin% in

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    Effect of Betulinic acid on Protein Kinase(PKC) activity in PA-1 (ovarian)

    cell line

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    Betulinic acid has broad-spectrum anti-cancer activity. The derivatives havebetter 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 havebetter potency and varying degree of specificity to endothelial cells.

    Betulinic acid causes cell death by Apoptosis as demonstrated by inhibition ofbcl-2 and induction of nucleosome release. The apoptotic cell death inducedmay be mediated by inhibition of PKC activity

    One of the derivatives (LEAD1) was shown to inhibit and cause regression ofhuman tumor (colon and lung) xenografts in nude mice.

    The derivatives had poor solubility and permeability with high protein bindingand poor oral bioavailability (as shown in PK studies). However they wereshown to have good metabolic stability and did not inhibit key CYP enzymescapable of causing drug interactions.

    The derivatives show varying levels of toxicity and safety profiles ascompared to Betulinic acid.

    Conclusions

  • 43

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