dabur research foundation new delhi, india...
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Dabur Research FoundationNew Delhi, INDIA
www.daburresearch.in
ONCOLOGY SERVICES
Provided in Europe by the Exclusive Distributor
Kinase Logistics ApSStationsvej 805792 AarslevDenmark
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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
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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
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Cell Biology & Pharmacology
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in vitro Screens … Cancer
Cell Lines
India’s 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
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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
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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
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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)
CI<1 - Synergy
CI=1 - Additive
CI>1 - 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
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Xenograft Models…1
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
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Xenograft Models..2
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Days Post inoclulation
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Tamoxifen Citrate, 10mg/kg, s.c.
In-vivo anticancer activity of Tamoxifen citrate on nude mice bearing MCF-7 tumor
Time to Reach 500mm3
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>38
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VehicleControl
TamoxifenCitrate,
10mg/kg, s.c.
Days
Median %T/C
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50
75
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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
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Xenograft Models…3
Median Tumor Volume
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750
1000
1250
1500
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29Days
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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
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VehicleControl
Erlotinib,90mpk,po
Days
Median %T/C
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25
50
75
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125
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Days
Med
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%T/
C Erlotinib, 90mpk,po
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Xenograft Models..4
Antitumor activity of DRFXXX on HT-29 (colon) xenograft.
Antitumor activity of DRFXXX on HuTtu-80 (duodenum) xenograft.
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DRF7295
T/C% = 19.1%
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Tum
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UntreatedDRF7295 T/C% = 2.8%
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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
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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
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Median Tumor Volume
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1000
2000
3000
4000
5000
1 3 5 7 9 11 13 15
Days
Med
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Vehicle Control
Paclitaxel, 5mg/kg, i.v.
Median %T/C
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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 Models…2
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Syngeneic Models…3
Median Tumor Volume Chart
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1500
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3000
3500
1 3 5 7 9 11 13 15Days
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5-FU, 12mg/kg, i.p.
Median %T/C
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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
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1000
1500
2000
2500
3000
3500
1 3 5 7 9 11 13 15Days
Med
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Vehicle Control
Herbal Formulation,500mg/kg
Herbal Formulation, 500mg/kg
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10
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30
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90
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1 3 5 7 9 11 13 15Days
Med
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% T
/C
Anticancer activity of Herbal formulation on BALB/c bearing CT-26 colon tumor
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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
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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
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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 Bouin’s 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
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Lung Metastasis Model…2
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10
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30
G1, SWFI, 10ml/kg,p.o. S1P ReceptorModulator,60mg/kg,p.o.
No.
of l
ung
nodu
les
Relative Melanin Content
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50
75
100
G1, SWFI, 10ml/kg,p.o. S1P ReceptorModulator,60mg/kg,p.o.
Weight of Lung
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0.2
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Untreated XXX, 5mg/kg, i.v Normal lungs
Wei
ght o
f lun
g (g
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s)
*
*Control
XXX Receptor
Modulator, 60mg/kg
Model under Validation
Spontaneous Meatstasis Model – B16F10 cell injection into the foot pad of C57BL6 Mouse
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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
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Mechanistic Studies
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Sorting Endosomes
ACTIVE TARGETING IN CYTOPLASM
Early Endosomes
Free Paclitaxel
6 Tubulin Stabilization
Nanoparticle micelles containing Paclitaxel
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NIPAM based Micellar polymers with LCST of 52-60 C in plasma
2Targeting through EPR effect
3Polymer aided higher cellular
uptake
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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
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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
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Models for Clinical Toxicities
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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
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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
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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)
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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
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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
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Toxicology
ADME-PK
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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.
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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
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Acute Dermal Toxicity
Acute dermal irritation / corrosion
Repeated dose dermal toxicity
Phototoxicity
Ocular Toxicity
Dermal toxicity at site of injection
Dermal Toxicity
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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
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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
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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
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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
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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
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Case Studies
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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 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 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
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Pept ide 2
Pept ide 3
Pept ide 4
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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Figure 10Antitumor activity of DRF7295 on
HT-29 (colon) xenograft.
ABSTRACT
INTRODUCTION
MATERIALS & METHODS
RESULTS & DISCUSSION
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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
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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 Dulbecco’s modified Eagles medium- DMEM (GibcoBRL, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, USA) and 100U/mL penicillin and100µg/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 µM – 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 µL 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%
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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.
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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 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 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 µM/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 (µg/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 (µg/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 <0.36 >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 <0.36 1.0 0.789 Hep-2 1.04 0.93 >2.1 2.0810 PA-1 13.84 2.1 >2.65 2.17
5.5
18.5
49.221.733.4
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Betulinic acid
LEAD3
LEAD4
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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;35:616–618.12. 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;48:72–76.15. Yasukawa K, Yu SY, Yamanouchi S, Takido M, Akihisa T, Tamura T. Phytomedicine 1995;4:309–313.16. 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. It’s 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 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
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 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 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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u.m
m)
ControlLEAD1
Effect of LEAD1 formulation on lung (L132) xenografts
0100200300400500600
0 10 20 30
Days post inoculum
Tum
or v
olum
e (c
u.m
m)
ControlLEAD1
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
0
50
100
150
5 20 100ug/ml
% o
f con
trol
Bcl-2
Effect of Betulinic acid on Nucleosome release in PA-1(ovarian) cell line
02468
5 20 100ug/ml
Fold
incr
ease
vs
con
trol
Nucleosome
Effect of betulinic acid on pro-angiogenic factors in
K562 (leukemia) cell line at 1ug/ml
0
10
20
VEGF bFGF Endostatin% in
hibi
tion
Effect of Betulinic acid on Protein Kinase(PKC) activity in PA-1 (ovarian)
cell line
0
50
100
150
5 20 100ug/ml
% o
f con
trol
• 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
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