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Juyoun Jin, D.V.M., Ph.D.
Institute for Refractory Cancer Research, Samsung Medical Center
Overview of Anticancer Drug Development
IND NDA
Synthesis and Formulation Development
Animal Models for Efficacy
Assay Development
Animal PK and PD
Dose Escalation
and Initial PK
Proof of Concept and Dose Finding
Large Efficacy Trials with PK Screen
PHASE I
Non-Clinical Development Clinical Development
PK/PD Studies in Special Populations
PHASE IV
Discovery Non-clinical development Clinical Trial
Target Identification & Validation
Lead Optimization
PHASE II PHASE III
The Goal of In Vivo Study using Animal Model
• Efficacy: Proof of therapeutic principle
• Toxicology: Toxicity profile
• Practical Issues:
– Animal pharmacokinetics and pharmacodynamics
– Starting dose and schedule for clinical trials
Ideal Animal Model for Cancer Therapy
Validity
Selectivity/Specificity
Predictability
Reproducibility
Similarity
“There is no perfect tumor model”
Spontaneous tumors Idiopathic
Carcinogen-induced
Transgenic/gene knockout animals: p53, RB, etc
Transplanted tumors Animal tumors syngenic: Lewis lung, S180 sarcoma, etc
Human tumor xenografts: Human tumor lines implanted in immunodeficient mice
(current NCI standard in vivo efficacy testing system)
Animal Model in Cancer
Syngeneic vs xenograft model
Human cancer cell
Immunedeficient animals
Tumor growth
Athymic “nude”mice developed in 1960’s
Human cancers grown in immune-deficient animals.
First human tumor xenograft of colon adenocarcinoma by
Rygaard & Poulson, 1969
Subcutaneous Xenograft model
Human Tumor Xenografts
SC
Immune-deficient animals
Athymic “nude” mice
Developed in 1960’s
Mutation in nu gene on chromosome 11
Lack thymus gland, T-cell immunity, Macrophage and NK cells are active
NOD-SCID (NOD.CB17-
Prkdcscid/NCrCrl) mouse
NOD scid Spontaneous mutant model was developed by the Fox Chase Cancer Center by transferring the scid mutation from a C.B-17 congenic background to a diabetes-susceptible non-obese diabetic background
T cell, B cell deficiency and depressed NK cell activity
NOG (NOD/Shi-scid/ IL-2Rγnull) mouse
New generation of severely immunodeficient mouse, Developed in 2000
No activity of T cell, B cell and NK cell, Dysfunction of macrophage, DC
Lack of NK cells, dendritic cell dysfunctions, and other unknown deficiencies due to inactivation of the IL-2Rγ gene
Efficacy Endpoints Clonogenic assay
Tumor growth assay (corrected for tumor doubling time)
Treated/control survival ratio
Tumor weight change
Toxicity Endpoints Drug related death
Net animal weight loss
Subcutaneous Xenograft Study Endpoints
Experimental Design: Xenograft model (S.C.)
Athymic nude mice
S.C. injection of Human cancer cells
Control Test
Several days (After tumor formation)
Treatment of test agents
1. Measure Tumor size 2. Measure Tumor weight 3. Measure Body weight
Tumors
Extract Protein/RNA - Target Validation
Make Tissue Slides - H&E, IHC - Target Validation
Distribution study - Optical imaging
Subcutaneous Xenograft Tumor Weight Change
Tumor weight change ratio
(used by the NCI in xenograft evaluation)
Defined as: treated/control x 100%
Tumor mass volume= (a x b2)/2
a = tumor length
b = tumor width
T/C < 40-50% is considered significant
Formula representing Tumor Size
Size Formula Diameter (L+W)/2
Area L*W
Volume (weight)
L*W2 /2
π/6*[(L+W)/2]3
π /2*L*W*H
π /6*(mean d)3
½*L*W*H
L: long diameter; W: short diameter; H: thickness; d: mean diameter
Subcutaneous Xenograft Advantages
Many different human tumor cell lines transplantable
Wide representation of most human solid tumors
Allows for evaluation of therapeutic index
Good correlation with drug regimens active in human lung,
colon, breast, and melanoma cancers
Subcutaneous Xenograft Dissdvantages
Different biological behavior, metastases rare Survival not an ideal endpoint: death from bulk of tumor, not
invasion
Shorter doubling times than original growth in human
Difficult to maintain animals due to infection risks
Host directed therapies (angiogenesis, immune modulation)
may not be applicable
Human vs. murine effects
Increasing unmet medical need of developing cancer therapeutics
Increasing new anticancer drugs under R&D projects in pharmaceutical companies
Current subcutaneous xenograft models do not translate the clinical outcome
Need to develop clinically relevant organ-specific orthotopic tumor models to
develop effective targeted therapies
Unmet Need for Translational Research in Cancer Therapeutics
교모세포종/뇌전이암
대장암
유방암
폐암 방광암 전립선암
Advantages and Disadvantages of Orthotopic Model
Advantages Resembles the original tumors morphologically, biologically and
biochemically
Important for the research of cancer metastasis
Short-term screening of variable cancer therapy strategy
Disadvantages
Necessary to have skillful technique
Wide variation
Types of murine model for studying human cancers.
ADVANTAGES DISADVANTAGES Allows for rapid analysis of human
tumor response to a therapeutic regime
Can predict the drug response of a tumor in a human patient
Provides realistic heterogeneity of tumor cells
Mice are immuno-compromised, providing a less realistic tumor microenvironment
Appropriately mimics human tumor microenvironment
Can predict the drug response of a tumor in a human patient
Provides realistic heterogeneity of tumor cells
Expensive Technically complicated
Tumor exists in the presence of competent immune system (realistic microenvironment)
Defined mutations can mimic those identified in human tumors
Can follow tumor development from early time points
Targets a limited number of genes which is usually not reflective of the complex heterogeneity of human tumor cells
Development is costly and time consuming, often requiring years of work before validation
Tumor development in animals is slow and variable
Disease Models & Mechanisms 1, 78-82 (2008)
Novel device for the translational research
7 mice injection/30min 1 mice injection/30 min
VS.
• Device invented for the translational research for the brain tumor orthotopic model • Cells with same condition were injected into seven mice simultaneously
Brain Tumor Orthotopic Model- Intracranial injection
Experimental Design For In Vivo Study ex. Brain tumor Orthotopic Model
2 X 105 U-87MG cells I.C. implantation
1 W 2 W 3 W 4 W 0 W
Tumor volume measurement (B)
21~25d Survival length (C)
I.C.injection of Human GBM cells
Treatment of test agents
1. Measurement of tumor volume 2. IHC study (PCNA, TUNEL) 3. IHC (Target validation) 4. Survival length 5. Distribution study 6. Measurement of body weight
Tumor volume IHC (PCNA/TUNEL) IHC (Target vali.)
Survival length Distribution
Brain Metastases Model- Internal Carotid Artery injection
Breast Cancer Orthotopic Model – Mammary Fat Pad
Tumor mass inoculate to 4th MFP Cell injection into 2nd MFP
Tumor mass Mammosphere
Lung Cancer Orthotopic Model – Left lung parenchyma
Single cell suspension
Cell implantation
Cell injection into Lung
Left lung : One single lobe Right lung: Cranial, middle, caudal and accessory lobes.
Colon Cancer Orthotopic Model – Cecal wall
Cell injection Mass implantation
Gastric Cancer Orthotopic Model
Incision : edge of the rib cage near the chest Draw out the stomach and injection or implantation into the stomach wall
Procedure
Prostate Cancer Orthotopic Model
Procedure
Histopathology (H&E)
Ovarian Cancer Orthotopic Model
Intrabursal inj. Gonadal Fat Pad(GFP) Subrenal Capsule
Pancreatic Cancer Orthotopic Model
Colorectal Cancer Liver Metastasis Model
Liver metastasis
50 mm
Spleen
100 mm
Head
Tail
T
T
T: Tumor region
Bone metastatic model by Intracardiac injection
CANCER RESEARCH 52. 2304-2309, April 15, 1992
After 8 weeks…
Hind leg paralysis
Knee joint
Hip joint Pelvis
0W 1W 2W 3W 4W 5W 6W 7W 8W 9W 10W 11W 12W
1 X 106 MDA-MB-435 LvBr1 cells M.F.P. implantation
After primary tumor formation (1.3~1.5 cm), tumor resection perform
Pulmonary metastases mesurement
Spontaneous Breast Cancer Lung Meta Model
Single cell suspension I.V. injection of B cell lymphoma cells
Mouse: NOD/Shi-scid/IL-2Rγnull (NOG)
Disseminated Lymphoma model – Intravenous injection
50-60 human derived cancer cell line Brain tumor (U87-MG, U373-MG, U251- MG….) Breast cancer cell line (MDA-MB-435, MDA-MB-231, MCF-7….) Colon Cancer (Lovo, SW480, Colo205, HT29, HCT116…..) Lung Cancer (PC14-PE6, A549, H23, H460…….) Lymphoma (Raji, Ramos, Daudi, BJAB, Toledo, SKW 6.3…….) Other Cancer Cell lines..
Subcutanous Xenograft Model
In vivo optical imaging and PET imaging
In vivo optical imaging PET imaging
In vivo optical imaging and PET imaging