Juyoun Jin, D.V.M., Ph.D. Institute for Refractory Cancer Research, Samsung Medical Center
Overview of Anticancer Drug Development Discovery Non-clinical development Clinical Trial Target Identification & Validation Lead Optimization Animal Models for Efficacy IND Synthesis and Formulation Development PHASE I PHASE II PHASE III NDA Assay Development Animal PK and PD Dose Escalation and Initial PK Proof of Concept and Dose Finding Large Efficacy Trials with PK Screen PK/PD Studies in Special Populations Non-Clinical Development Clinical Development PHASE IV
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 There is no perfect tumor model Validity Selectivity/Specificity Predictability Reproducibility Similarity
Animal Model in Cancer 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)
Syngeneic vs xenograft model Human cancer cell Immunedeficient animals Tumor growth
Human Tumor Xenografts 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 SC
Immune-deficient animals Athymic nude mice NOD-SCID (NOD.CB17- Prkdc scid /NCrCrl) mouse 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 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-2Rγ null ) 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
Subcutaneous Xenograft Study Endpoints 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
Experimental Design: Xenograft model (S.C.) Athymic nude mice S.C. injection of Human cancer cells Several days (After tumor formation) Control Test Treatment of test agents Extract Protein/RNA - Target Validation Make Tissue Slides - H&E, IHC - Target Validation 1. Measure Tumor size 2. Measure Tumor weight 3. Measure Body weight Tumors 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 b 2 )/2 a = tumor length b = tumor width T/C < 40-50% is considered significant
Formula representing Tumor Size Size Diameter Area Formula (L+W)/2 L*W L*W 2 /2 π/6*[(l+w)/2] 3 Volume (weight) π /2*L*W*H L: long diameter; W: short diameter; H: thickness; d: mean diameter π /6*(mean d) 3 ½*L*W*H
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
Unmet Need for Translational Research in Cancer Therapeutics 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 교모세포종 / 뇌전이암 유방암 대장암 폐암방광암전립선암
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 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 Appropriately mimics human tumor microenvironment Can predict the drug response of a tumor in a human patient Provides realistic heterogeneity of tumor cells 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 DISADVANTAGES Mice are immuno-compromised, providing a less realistic tumor microenvironment Expensive Technically complicated 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)
Brain Tumor Orthotopic Model- Intracranial injection 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
Experimental Design For In Vivo Study ex. Brain tumor Orthotopic Model 2 X 10 5 U-87MG cells I.C. implantation Tumor volume measurement (B) 0 W 1 W 2 W 3 W 4 W 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 Tumor mass Cell injection into 2 nd MFP Mammosphere
Lung Cancer Orthotopic Model Left lung parenchyma Cell injection into Lung Single cell suspension Cell implantation 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 Procedure Incision : edge of the rib cage near the chest Draw out the stomach and injection or implantation into the stomach wall
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 Spleen Head 50 mm 100 mm T Tail T T: Tumor region
Bone metastatic model by Intracardiac injection CANCER RESEARCH 52. 2304-2309, April 15, 1992 After 8 weeks Hip joint Pelvis Knee joint Hind leg paralysis
Spontaneous Breast Cancer Lung Meta Model 1 X 10 6 MDA-MB-435 LvBr1 cells M.F.P. implantation 0W 1W 2W 3W 4W 5W 6W 7W 8W 9W 10W 11W 12W Pulmonary metastases mesurement After primary tumor formation (1.3~1.5 cm), tumor resection perform
Disseminated Lymphoma model Intravenous injection Mouse: NOD/Shi-scid/IL-2Rγnull (NOG) Single cell suspension I.V. injection of B cell lymphoma cells
Subcutanous Xenograft Model 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..
In vivo optical imaging and PET imaging In vivo optical imaging PET imaging
In vivo optical imaging and PET imaging