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