# Presentation: 950018

## Preclinical Models and their Role

**in Drug Discovery**
**Glenn Merlino**

**Brief History of Preclinical Models in Drug Discovery**
**New Animal Models**
**New Approaches**
**New Technology**
**Preclinical Models in the CCR**

## GOALS OF PRECLINICAL DRUG STUDIES

**Discovery of potential winners (lead structures)**
**Early development: chemistry, pharmacology, efficacy**
**Late development: formulation, dose form, toxicology**
**Bottom line: to get better drugs to patients faster**

## DEFINING EFFECTIVE CANCER DRUGS:

**- PHASE III  CLINICAL TRIAL**
**	****= WINNER**

**- PHASE II**
**	****= POTENTIAL**
**	****   ****WINNER**

**Treatment A**

**- ****PRECLINICAL MODEL**
**(i.e., mouse)**

**Cytotoxic**

- Adapted from Ed Sausville, DTP

## THE DRUG DISCOVERY/DEVELOPMENT LIFE CYCLE

**THE DRUG DISCOVERY/DEVELOPMENT LIFE CYCLE**

**Circumstantially link molecule to disease**

**Show changing target changes disease traits**

**Identify chemicals that modify target**

**Develop lead drug candidate,**
**pharmacology**

**Show drug candidate**
**is efficacious and**
**non-toxic**

**Goals**

- Adapted from Karen Moore, Hypnion Inc

**Text here**

**Text here**

**Text here**

**Biology**

**Chemistry**

**Development**

## POTENTIAL APPLICATIONS OF

**ANIMAL TUMOR MODELS**

**Discovery: Screening for new drugs**
**Tumor Biology: Uncovering novel tumor targets/pathways**
**Validation: Verifying drug interactions with target**
**Development: Predicting drug efficacy and safety**

**Not all models are appropriate for all questions!**

## TYPES OF ANIMAL TUMOR MODELS

**  ****Pharmacology / Efficacy Models:**
**To qualify compounds & define their potential for biologic effect**
**Geared for throughput to evaluate numerous compounds**
**- Rodent / Rodent (Lewis Lung, L1210, P388, B16)**
**Immunocompetent system**
**Allow analysis of metastasis & tumor-host interactions**
** ****Human Xenografts**
**Immunocompromised system (Athymic nude, SCID)**
**Allow assessment of human cells**
**- Site-directed (hollow fiber & subrenal capsule assays)**
** **** ****Biology Models:**
**For analysis of targets/pathways and mechanisms of action**
**Geared for close correspondence to human disease (therefore**
**long latency); not appropriate for evaluation of many compounds**
**- Spontaneous / Carcinogen-induced**
**- Orthotopic: Place tumor cells in site of origin ± remove primary**
**- Genetically engineered animals: Transgenic mice**

- Adapted from Ed Sausville, DTP

## CHANGING APPROACHES TO DRUG DISCOVERY

_**Empirical Drug Discovery**_**:  generally unselected search ****	****for new cytotoxic drugs**
_**Rational  Drug Discovery**_** - development of drugs that act ****	****through defined molecular mechanisms associated ****	****with cancer genesis or progression**

## EMPIRICAL DRUG DISCOVERY

**Screen**

**+ Anti-Tumor**
**Activity**
**(*****in vitro/in vivo*****)**

**Pharmacology**

**Chemistry**

**Optimized Schedule (*****in vivo)***

**Toxicology / Formulation**

**Phase I / II /  III Clinical Trials**

- Adapted from Ed Sausville, DTP

## PROBLEMS WITH EMPIRICAL MODELS

**  ****Lack of predictive power *****in vivo***
**  ****Poor correlation of non-human with human**
**	****pharmacology**
**  ****Divorced from biology**

## EARLY (1975-1985) NCI IN VIVO PANEL

***In Vivo***** Prescreen with P388 Murine Leukemia**

**Breast**
**Colon**
**Lung**
**Melanoma**
**Leukemia**
**Sarcoma**

**Mouse**

**Human**

**CD8F-1**
**colon 38**
**Lewis**
**B16**
**L1210**
**s180**

**MX-1**
**CX-1**
**LX-1**

- Adapted from Ed Sausville, DTP

## IMPROVED NCI STRATEGY (since 1990)

_**PRIMARY SCREEN**_**:**
**In vitro panel of 60 tumor cell lines representing major human cancers**
**Reflects heterogeneity in human clinical situation**
**Screen for agents with (anti-proliferative) activity in different cancers**
**Can screen chemicals and natural products**
**Can indicate compounds with novel mechanisms of action**

_**SECONDARY SCREEN**_**:**
**In vivo panel of 60 solid tumor human xenografts derived from the in vitro panel**
**Follow up positive leads using sensitive cell types suggested by**
**	****the in vitro testing**
**Both the hollow fibre and classical xenograft models are used**

**Promising compounds are further developed for suitable formulation,**
**	****optimal route and schedule, pharmacology and toxicology**

## CORRELATION OF IN VIVO ACTIVITY WITHCLINICAL ACTIVITY BY DISEASE TYPE

- Johnson et al,. 2001 Brit J Cancer 84, 1424-31

## WHY ANIMAL MODELS FAIL TO PREDICTHUMAN CLINICAL OUTCOME

**  ****Differences in etiology, natural history, and/or molecular ****	****pathogenetic events, between tumor model and human disease**
**  ****Differences in the way drugs interact with the host: Pharmaco-**
**kinetic/Pharmacodynamic (PK/PD) differences**
**Species-specific incompatibility between drug and target**
**  ****Most models are not used in the context of advanced disease, therefore do not closely resemble the patient situation**
**   ****Failure to adhere to a more rigorous criteria for declaring a drug to be active, or response (i.e., require at least a net zero increase in tumor burden over treatment/observation period)**

## Comparative OncologyThe study of cancer biology across species boundaries 

**Naturally Occurring Cancer Models**
**Relevant tumor biology**
**Naturally Occurring**
**Large Animal (more similar size, cell kinetics, biologic behavior)**
**Metastatic Biology consistent with human disease**
**Immunocompetant**
**Appropriate model for novel biologically based therapeutics**
**Integrated trials with biological and clinical endpoints are feasible**

**Rodent Models**
**Genetically engineered models**
**Syngeneic murine models**
**Human-Murine xenograft models**
**Feline naturally occurring cancers**
**Canine naturally occurring cancers**
**Human**

**Adapted from Chand Khanna**

## THE ADVENT OF RATIONAL DRUG DISCOVERY

_**Rational Drug Discovery**_** refers to the development of drugs that act through defined molecular mechanisms associated with cancer genesis or progression**

_**Translational Cancer Research**_** refers to the application of basic scientific research toward the improvement of patient care**
**Faster drug development**
**Better risk-value assessment**
**Reduction in drug attrition rate late in development**
**Improved patient care**

## RATIONAL DRUG DISCOVERY

**TARGET-DEPENDENT *****IN VIVO***** MODEL**
**TOXICOLOGY/FORMULATION**
**CLINICAL TRIALS - INCLUDING ASSESSMENT**
**OF AFFECT ON TARGET**

**PHARMACOLOGY**
**(to affect target)**

**CHEMISTRY**

**MOLECULAR TARGET SCREEN**

**Biochemical**
**Engineered cell**
**Animal (yeast/worm/fish)**

## Target Validation or “Credentialing”

**Is the target associated with the disease, and what is****	****the frequency of association?**
**Is the target linked mechanistically to the disease?**
**Does pharmacologic modulation of the target modify ****	****the disease?**

**Genetically engineered mice can play a significant**
**	****role here**

## SOME MOLECULAR TARGETS CHARACTERIZED IN XENOGRAFT MODELS

**Oncogenes (k-ras, c-myc, c-erbB2)**
**Growth factors/Receptors (ER, PR, EGFR, TGF********)**
**Tumor Suppressors (p53)**
**Drug Resistance (Mdr1, Mdr3)**
**Angiogenesis (VEGF, angiogenin)**
**Metastasis (nm23, uPA, TIMP-2, MMPs)**
**Enzymes (telomerase)**

- Adapted from H.H. Fiebig 2002 Tumor Models in Cancer Res

## Common Breakpoints and Molecular Consequences

- From Druker, 2002 Tr Mol Med 8, S14-18

**Common Breakpoints and Molecular Consequences**
**in CML and ALL Leukemia**

## A BCR/ABL Kinase Inhibitor (Gleevec) Eradicates Leukemia Cells In Vivo

**A BCR/ABL Kinase Inhibitor (Gleevec) Eradicates Leukemia Cells In Vivo**

**Tyr phosphorylation *****in vivo***

- le Coutre et al, JNCI 91:163, 1999

**Antitumor activity *****in vivo***

- (hrs)

- (days)

- (days)

**ST1571**

**Prevention**

**Intervention**

## Transgenic MMTV-v-Ha-ras Mice Undergo Regression after Farnesyltransferase Inhibitor (FTI) Treatment

- Kohl et al, Nature Med 1: 792, 1995

**20 mpk/day L-744,832**

**Vehicle**

**10 mpk/day L-744,832**

**40 mpk/day L-744,832**

**2 mpk/day x 9 Doxorubicin**

- Tumor Volume, mm3

**L-744,832**

## FTIs Inhibit Receptor Tyrosine Kinase Driven Transgenic TumorsDrugs Can be Designed to Target Pathways

**FTIs Inhibit Receptor Tyrosine Kinase Driven Transgenic Tumors********Drugs Can be Designed to Target Pathways******

- Nørgaard et al. 1999 Clin Cancer Res 5, 35

- Vehicle

- FTI

- MMTV-TGF
- multiparous

- MMTV-TGF
- virgin + DMBA

- MMTV-TGF/neu
- virgin

- % of initial volume

- Day

## FTIs Allow Pathway Effect Assessment in Transgenic TumorsMolecular Endpoints

- Nørgaard et al. 1999 Clin Cancer Res 5, 35

- Phosphorimager CPM,
- % Pre-FTI

- Tumor

**MAP Kinase**

**Serum TGF-******

- s-hTGF
- pg/ml

##  Conditional erbB2 knock-out helps to explain unique side effect 

*** ******Neu***** transgenic mice develop spontaneous mammary tumors**
** ****HER2-transfected xenografts used to understand tumor biology**
** ****Subrenal capsule assay used to select MAb**
** ****Radiolabeled Herceptin Fab localizes to tumor xenografts**
** ****Herceptin efficacy demonstrated in multiple xenograft models**
** ****Target Herceptin serum concentrations based on PK in mice**
** ****Optimized dose/schedules established for chemo combinations**
** ****Conditional erbB2 knock-out helps to explain unique side effect**** **

**Animal models indispensable for clinical**
**development of Herceptin**

**From Mark Pegram, UCLA**

## Use of Preclinical Models in a Pediatric Testing Program

**Use of Preclinical Models in a Pediatric Testing Program**

- Houghton et al. 2002 Clin. Cancer Res. 8, 3646-57

- MTD, maximum tolerated dose

## Preclinical Model Benchmarks

**      ****histopathological & molecular-genetic features?**
**Is the model reproducible with high penetrance?**
**Does the model exhibit multistage progression (including metastasis)?**
**Does the model provide continuous readout of tumorigenesis (imaging)?**
**Does the model exhibit proper response to known environmental insults?**
** **
**Does the model include an intact immune system?****	**
**Is the model cost feasible: reasonable time frame for studies, simple**
**breeding/treatment strategy?**

**Preclinical Model Benchmarks**

**Preclinical Efficacy: can the model be used to make good therapeutic**
**and preventive predictions for humans?**

## Mouse Melanoma Model vs. Human Histopathology

**Microinvasive melanoma**
**(RGP)**

**Metastatic melanoma**

**Invasive malignant**
**melanoma**
**(VGP)**

**Mouse Melanoma Model vs. Human Histopathology**
******Mouse melanoma stages resemble human melanoma******

**Human                            Mouse**

- Noonan et al. 2001 Nature 413, 271-2
- Recio et al. 2002 Cancer Res. 62, 6724-30

## Tumor Latency as an Issue in Preclinical Modeling

**Tumor Latency as an Issue in Preclinical Modeling**

**Sharp et al. 2002 Nat. Med. 8, 1276-80**

## An Animal Cancer Model Should Resemble its Human

**An Animal Cancer Model Should Resemble its Human**
**Counterpart at the Molecular Pathway Level:**
******A Mouse Model for Human Rhabdomyosarcoma ******

- Sharp et al. 2002 Nat. Med. 8, 1276-80

## New Technologies/New Approaches

## Use of Luciferase Activity to Determine Tumor Size

**Day 4**
***0.5 mm******3***

**Day 6**
***7 mm******3***

**Day 9**
***41 mm******3***

**Day 16**
***324 mm******3***

**Day 12**
***206 mm******3***

- Courtesy of Steve Libutti

## Green Fluorescent Protein Imaging of Liver Metastasis

**Liver Metastatic Model:  Day 9 after Intrasplenic Injection of 10****6**** MC38-GFP Tumor Cell Line in C57BL/6 Mice**

**Left: through intact skin**
**Right: after dissection of body wall**

- Courtesy of Steve Libutti

## GFP Fluorescent Visualization of Spontaneous Mammary Tumors

- WAP-driven
- EGFP expression
- in Py middle T
- mouse tumor

- WAP-driven
- EGFP
- expression
- in Neu
- oncogene
- mouse tumor

- EGFP visualization
- of tumor cell
- locomotion
- toward and into
- blood vessel

- Ahmed et al. 2002 Cancer Res 62, 7166-9

## Glioma in an Ntv-a* mouse identified by bioluminescence imaging for cell proliferation

**Glioma in an Ntv-a* mouse identified by bioluminescence imaging for cell proliferation**

**No glioma**

**Glioma**

- Courtesy of Eric Holland, MSKCC

**Nestin promoter driving expression**
**of an avian retrovirus receptor**

## Signal transduction blockade of glioma with small molecules detected by bioluminescence imaging

**Signal transduction blockade of glioma with small molecules detected by bioluminescence imaging**

**24 hr Tx**

**48 hr Tx**

- Courtesy of Eric Holland, MSKCC

## Effect of a VEGFRII Inhibitor on Mammary Cancer 

**Effect of a VEGFRII Inhibitor on Mammary Cancer **
**Development in C3(1)/Tag Transgenic Mice**

- Percent Tumor
- Incidence

- Survuval

- Tumor Burden

- Tumor Multiplicity

- Courtesy of Jung-im Huh and Jeff Green

## Use of Mouse Tumor Models in Prevention

**p<0.001**

**p<0.001**

- Courtesy of Steve Hursting

**Use of Mouse Tumor Models in Prevention**

## Use of Mouse Tumor Models to Study Metastasis

**Metastasis suppression by transgenic expression of a TGF-******** antagonist**

- R=0.98

- Serum SR2F (ng/ml)

- LIVER

- LUNG

- SPLEEN

- PANCREAS

- # mets/organ

- # mets/liver

- P=0.03

- A.  Site-independent

- B.  Dose-dependent

- SR2F is secreted into the circulation and distributes to all organs except brain

- Extracellular domain of type II TGF- receptor

- Fc domain of human IgG1

- “SR2F”

- Courtesy of Lalage Wakefield

**Use of Mouse Tumor Models to Study Metastasis**

## Knockout mice

**Knockout mice**
**CYP1A1**
**CYP1A2**
**CYP1B1**
**CYP2E1**
**CYP3A11**
**CYP3A23**
**mEH**
**sEH**
**GSTP**
**NQO1**

- Courtesy of Frank Gonzalez

## The Tet System for Inducible Transgene Expression

**The Tet System for Inducible Transgene Expression**

- Bockamp et al. 2002 Phys. Genomics 11, 115-32

## Elimination of Spontaneous Mouse Tumors Using the Tet-On System

**Elimination of Spontaneous Mouse Tumors Using the Tet-On System**
- The Tyrosinase-rtTA / TRE-Ha-ras inducible expression system

- Chin et al. 1999 Nature 400, 468-72

## Use of the Cre

- Bockamp et al. 2002 Phys.
- Genomics 11, 115-32

**Use of the Cre**
**Recombinase in**
**Conditional Gene**
**Inactivation**

## Switchable Gene

- Inactivation -
- Endothelin Receptor B
- Conditional Knockout

- Shin et al. 1999 Nature 402, 496-501

## How to Get Help Getting Started

## http://ccr.nci.nih.gov/tech_initiatives/animalmodels/

**http://ccr.nci.nih.gov/tech_initiatives/animalmodels/**

**The AMI provides expertise and advice to intramural investigators conducting**
**research using animal models. AMI also strives to facilitate interchanges**
**among members of the intramural research community. The AMI steering**
**committee acts in an advisory capacity to the CCR Director, providing in-depth**
**analysis of emerging trends in biomedical research to inform scientific policy**
**decisions. **

_**Office of Mice Advice**_** - Susan Silk (**_[silks@mail.nih.gov](mailto:silks@mail.nih.gov)__)_

## http://ccr.nci.nih.gov/tech_initiatives/animalmodels/db/

**This database is intended to foster collaboration and facilitate exchange of useful animal**
**models between investigators of the NCI Intramural Research Program.  It provides NCI**
**investigators with a list of useful animal models developed within, or available from, the**
**scientific community, and provides access to relevant information about each model, such**
**as the gene of interest, type of mutation, and brief description of their phenotype.**

**http://ccr.nci.nih.gov/tech_initiatives/animalmodels/db/**

## NIH Mouse Imaging Facility

**Micro-PET**

**MRI**

**CT**

**http://ccr.nci.nih.gov/tech_initiatives/animalmodels/MIF/**

- Computed
- Tomography

- Positron
- Emission
- Tomography

- Magnetic
- Resonance
- Imaging

**Steve Libutti (**_[libuttis@mail.nih.gov](mailto:libuttis@mail.nih.gov)_**) & Mark Simpson (**_[simpsoma@mail.nih.gov](mailto:simpsoma@mail.nih.gov)_**)**

## CCR-NCI

**To facilitate the design of clinical investigations in pet animals that will be used in the design of similar trials in human patients**
**Define activity**
**Define surrogates and predictive pharmacodymanic endpoints**

**CCR-NCI**
**Comparative Oncology Program**

**Contact: Chand Khanna (**_[khannac@mail.nih.gov](mailto:khannac@mail.nih.gov)_**)**

## CCR PRECLINICAL MODELING PROGRAM

_**Central Mission:**_
**To facilitate translational science through the development and use**
**of animal models that accurately predict the efficacy of novel**
**therapeutic and preventive strategies in humans**

_**Summary of Current Situation:**_
**Animal models now used to predict the efficacy of anti-cancer**
**agents in the clinic, mostly human xenografts, have yielded highly**
**mixed results.  These models lack the natural history of cancer, as**
**well as appropriate tumor-host interactions.**

_**How can the CCR uniquely contribute?**_

## CCR Preclinical Models Strategy Team 

## Preclinical

**Working**
**Group**

**Biopharm**

**Molecular **
**Pathology Unit**

**PK/PD Core**

**Animal Models**
**Initiative**

**Biologics**
**Working**
**Group**

**Regulatory**
**Affairs**

**Molecular**
**Targets Development**
**Program**

**Imaging**

**Structural**
**Biology**

**Biomarkers**
**(Proteomics)**

**Chemistry**

**Animal**
**Clinical**
**Care**

**Preclinical**
**Models**

**Strategic Planning for Development of Novel Cancer Therapy**

## CCR PRECLINICAL MODELING PRIORITIES?

- <date/time>

**CCR PRECLINICAL MODELING PRIORITIES?**

**Identify novel therapeutic targets**
**Validate candidate targets in transgenic mice with engineered mutations**
**Validate the ability of preclinical models to predict human responses**
**Test therapeutic agents using longer-term, higher-risk preclinical trials**
**Define biomarkers for early detection, early outcome assessment and staging**
**Test combination therapies in collaboration with academia and industry**
**Generate “Humanized” models for assessment of PK/PD, toxicity and efficacy**
**Study gene-environment interactions in appropriate mouse models**
**Prevention: include early stage tumorigenesis in the above initiatives**
**Imaging: broadly incorporate imaging technologies in the above initiatives**

## NCI  SEMINAR

***SPONSORED BY ***
***DEVELOPMENTAL  THERAPEUTICS  PROGRAM******  ***
***APRIL  21,  2003***
***9:30  -  11:30***
***549 Auditorium (NCI-Frederick)***
***with Videoconferencing to EPN 8051***
***PROF.  DR.  HEINZ-HERBERT  FIEBIG***
***Tumor  Biology  Center***
***Universitat  Freiburg***
***Freiburg,  Germany***
***“******IN  VIVO  MODELS  FOR  CANCER  DRUG DEVELOPMENT:***
***THE  FREIBURG  APPROACH”***