drug studies using mouse models melinda hollingshead, d.v.m., ph.d. biological testing branch...
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Drug Studies Using Mouse Drug Studies Using Mouse ModelsModels
Melinda Hollingshead, D.V.M., Ph.D.Melinda Hollingshead, D.V.M., Ph.D.
Biological Testing BranchBiological Testing Branch
Developmental Therapeutics ProgramDevelopmental Therapeutics Program
Division of Cancer Treatment and DiagnosisDivision of Cancer Treatment and Diagnosis
NCINCI
Why should the taxpayers fund Why should the taxpayers fund this type of work?this type of work?
Cancer is the second leading cause of death in America. It accounts for one of every four deaths. More than 565,000 - that’s more than 1,500 people a day - die annually from cancer. Close to 1.4 million new cases are diagnosed each year. This estimate does not include
pre-invasive cancer or the more than 1 million cases of non-melanoma skin
cancer expected to be diagnosed annually.
Why do we use mouse Why do we use mouse models?models?
geneticsgenetics progressionprogression preventionprevention diagnosticsdiagnostics therapeuticstherapeutics physiologically complex vs in vitro studiesphysiologically complex vs in vitro studies cost effectivecost effective develop human clinical protocolsdevelop human clinical protocols
To prevent, diagnose and treat human disease
What models are available?What models are available?
Spontaneous Spontaneous Virus-inducedVirus-inducedTransgenicTransgenicKnock-out/inKnock-out/inInduced/carcinogensInduced/carcinogensTransplantedTransplanted
Spontaneous ModelsSpontaneous Models
Natural history is most “normal”Natural history is most “normal”Random tumorsRandom tumorsDifficult to predict occurrenceDifficult to predict occurrenceMonitor through lifespan ~ 2 yr in mice Monitor through lifespan ~ 2 yr in mice Large number of animals may be Large number of animals may be
required for a small experimentrequired for a small experimentTumors may be heterogeneousTumors may be heterogeneousSignificant animal holding space neededSignificant animal holding space needed
These allow the study of the biological history of natural disease. They can be applied to many types of studies.
Virus-Induced ModelsVirus-Induced Models
Classic models of virus-induced leukemia:Classic models of virus-induced leukemia: RauscherRauscher MoloneyMoloney LP-BM5LP-BM5 FriendFriend
Non-leukemic tumors including:Non-leukemic tumors including: Mammary tumors due to MMTV Mammary tumors due to MMTV Thymomas of AKR miceThymomas of AKR mice
High tumor occurrence rateHigh tumor occurrence rate Predictable time to tumor developmentPredictable time to tumor development Well-characterized disease states/natural historyWell-characterized disease states/natural history Do not accurately reflect the natural history of most Do not accurately reflect the natural history of most
human tumorshuman tumors
Early studies of cancer, both as a disease state and for therapeutic intervention, used virus-induced models. These models continue
to be applied in a variety of studies.
Transgenic ModelsTransgenic Models
Many available through commercial and collaborative Many available through commercial and collaborative arrangementsarrangements
http://emice.nci.nih.gov/emice/mouse_models http://mouse.ncifcrf.gov/ http://jaxmice.jax.org/query/f?p=205:1:1510299228434659165
Not all patents have expired - check with OTT regarding legalitiesNot all patents have expired - check with OTT regarding legalities Remember MTAs are needed to receive or ship material that may Remember MTAs are needed to receive or ship material that may
have patent or licensing rights. This also applies to mice. have patent or licensing rights. This also applies to mice.
Rate of tumor occurrence may be lowRate of tumor occurrence may be low Time to tumor occurrence may be difficult to predictTime to tumor occurrence may be difficult to predict Breeding schemes may be complex e.g., 3, 4 or more Breeding schemes may be complex e.g., 3, 4 or more
intermediate genetic crossesintermediate genetic crosses Genetic error(s) are generally well characterizedGenetic error(s) are generally well characterized Disease may follow a more natural course e.g., time to Disease may follow a more natural course e.g., time to
significant tumor burden may more accurately mimic the significant tumor burden may more accurately mimic the human; lesions likely relevant to their site of occurrencehuman; lesions likely relevant to their site of occurrence
Transgenic models aid our understanding of the genetics of cancer and are being pursued as models for intervention.
Induced/CarcinogensInduced/Carcinogens
EpithelialEpithelial tumorigenesistumorigenesis 7,12-dimethylbenz[a]anthracene [initiator mutagen] followed 7,12-dimethylbenz[a]anthracene [initiator mutagen] followed
by multiple applications of 12-by multiple applications of 12-OO-tetradecanoylphorbol-13--tetradecanoylphorbol-13-acetate [pro-inflammatory]acetate [pro-inflammatory]
GI tumorigenesisGI tumorigenesis 1,2 dimethlhydrazine-2-HCl1,2 dimethlhydrazine-2-HCl azoxymethaneazoxymethane
Sarcoma inductionSarcoma induction MethylcholanthreneMethylcholanthrene
Lung tumorigenesisLung tumorigenesis nitrosamine 4- (methyl-nitrosamino)-1-(3-pyridyl)-1-butanonenitrosamine 4- (methyl-nitrosamino)-1-(3-pyridyl)-1-butanone
These models aid studies of the local, systemic, and environmental factors that influence tumor susceptibility,
growth, and progression.
Induced/CarcinogensInduced/Carcinogens
Time to tumor development may be Time to tumor development may be variablevariable
Mimics some human diseases very Mimics some human diseases very wellwell
Requires technical work with known Requires technical work with known carcinogenic agents thus special carcinogenic agents thus special handling/equipment/facilities/training handling/equipment/facilities/training requiredrequired
Provides the entire natural history of Provides the entire natural history of tumor development to studytumor development to study
Transplanted ModelsTransplanted Models
Tissue SourceTissue Source Syngeneic - same inbred strain – e.g., B16 - same inbred strain – e.g., B16
tumors in C57Bl/6 micetumors in C57Bl/6 mice Allogeneic - same species, different strain - same species, different strain
(genetically diverse) – e.g., M5076 sarcomas in (genetically diverse) – e.g., M5076 sarcomas in athymic mice produces an allograathymic mice produces an allograftft
Xenogeneic - different species e.g., human, rat - different species e.g., human, rat or dog tumors grown in immunocompromised or dog tumors grown in immunocompromised mice produces a xenogramice produces a xenograftft
Implant SiteImplant Site OrthotopicOrthotopic HeterotopicHeterotopic
EndpointsEndpoints
These models are commonly applied in studies of genetics, diagnostics, and medical interventions.
Transplanted ModelsTransplanted Models
Easy to control time of tumor occurrenceEasy to control time of tumor occurrenceMany tumor types/lines availableMany tumor types/lines availableWell accepted modelsWell accepted modelsDo not accurately recapitulate human diseaseDo not accurately recapitulate human diseaseMetastatic lesions are difficult to findMetastatic lesions are difficult to findTumor growth rates may preclude multiple Tumor growth rates may preclude multiple
treatment cyclestreatment cycles
production In vitro studies
efficacy trials
Discovery & DevelopmentDiscovery &
Development
New therapy
Pharmacology & toxicology
• Human Tumors– Hollow fiber– Subcutaneous– Intravenous– Intraperitoneal– Orthotopic
• Mammary fat pad• Intracranial• Intrarenal• Intrahepatic• Intracecal
• Rodent Tumors– Subcutaneous– Intravenous– Intraperitoneal– Orthotopic– Metastatic– Transgenic– Knock-in/out– Induced– Spontaneous
In Vivo Efficacy ModelsIn Vivo Efficacy Models
• Tumor sources – Cells cultured in vitro– Serially passaged tumor – Cryopreserved tumor
Issues faced during Issues faced during Efficacy EvaluationsEfficacy Evaluations
ModelVehicle, formulation, stability
Dose, route and scheduleExperimental protocolEndpoints
What are you assessing? What are you assessing? Which type of model is most appropriate?Which type of model is most appropriate? Is the treatment designed to:Is the treatment designed to:
impact the tumor biochemically, e.g., impact the tumor biochemically, e.g., cytotoxiccytotoxic
impact the tumor genetically, e.g., impact the tumor genetically, e.g., modulatormodulator
impact the stroma e.g., vasculatureimpact the stroma e.g., vasculatureimpact the immune systemimpact the immune systemact as an adjuvantact as an adjuvantsynergize with known drugssynergize with known drugsinteract with specific proteinsinteract with specific proteins
ModelModel Selection Selection
Model Model SelectionSelection
A
0
200
400
600
800
1000
1200
1400
1600
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22 25 28 31 35 39 43 46 49 54 61
Day Post-Implantation
Med
ian T
umor
Weig
ht
B
-200
0
200
400
600
800
1000
1200
1400
1600
1800
13 19 22 26 29 33 36 39 46 50 53 56 60 63
Day Post-implantation
Med
ian
Tum
or W
eigh
t
B
-5 00
0
5 00
1 000
1 5 00
2 000
1 3 1 9 2 2 2 6 2 9 3 3 3 6 3 9 4 6 5 0 5 3 5 6 6 0 6 3
D a y P o st-im p la n t a tio n
100% sesame oil 45 mg/kg tamoxifen
22.5 mg/kg tamoxifen 11.25 mg/kg tamoxifen
estrogen independent
estrogen dependent
Experimental ProtocolExperimental Protocol
When will treatment start?When will treatment start?When will treatment end?When will treatment end?Will samples be collected for ex vivo Will samples be collected for ex vivo
evaluation?evaluation?Will tumors be monitored visually, by Will tumors be monitored visually, by
imaging techniques?imaging techniques?What will terminate the experiment, What will terminate the experiment,
i.e., what are the endpoints?i.e., what are the endpoints?How will the animals be randomizedHow will the animals be randomized
What is published/prior knowledge? What is published/prior knowledge? What is proposed/expected What is proposed/expected
mechanism?mechanism? How much exposure is required for How much exposure is required for
effect?effect? Is the material soluble/stable in Is the material soluble/stable in
aqueous solution?aqueous solution? What routes of administration are What routes of administration are
technically feasible?technically feasible? Options Options
What is the maximum tolerated dose (MTD)?What is the maximum tolerated dose (MTD)? IP, IV, SC, PO?IP, IV, SC, PO? QDx?; BIDx?; TIDx?QDx?; BIDx?; TIDx?
Dose, Route, ScheduleDose, Route, Schedule
Kaplan-Meier Plot of mice bearing intraperitoneal OVCAR-5 human ovarian cancer xenografts. Mice were treated with vehicle (PBS or lipid vehicle) with a therapeutic solubilized in each of the vehicles. Note that the lipid vehicle alone was as effective in improving survival as was the therapeutic prepared in the lipid vehicle and it was more effective than the therapeutic prepared in PBS.
0 20 40 60 800
20
40
60
80
100
PBS lipid vehicle
treatment in PBS treatment in lipid vehicle
day
Pe
rce
nt
su
rviv
al
EndpointsEndpoints
Tumor sizeTumor sizeWeight lossWeight lossTime to sacrificeTime to sacrificeImagingImagingPre-defined time of terminationPre-defined time of terminationTime post-treatmentTime post-treatment
PC-3 Tumor
12
0
10
0
80
60
40
20
0PS-341 (mg/m2)
0.9 1.8Vehicle
20
S A
cti
vit
y (
% V
eh
icle
)
120
100
80
60
40
20
0
0.3 0.9 3.0 9.0VCTLPS-341 (mg/m2)2
0S
Acti
vit
y (
% V
eh
icle
)
Mouse WBC
GENOMICSTumor RNA
BIOMARKER
SURROGATEBIOMARKER
DRUG ANALYSIS
Plasma
In Vivo StudiesIn Vivo Studies
IHC IHC Original tumor and each passageOriginal tumor and each passage Antibodies for specific gene productsAntibodies for specific gene products
Molecular analysis Molecular analysis genetics : NGS, RNA-Seq, microarray (LCM), RT-genetics : NGS, RNA-Seq, microarray (LCM), RT-
PCRPCR proteins/phosphoproteins: reverse arrays, IHCproteins/phosphoproteins: reverse arrays, IHC biomarkers: serum proteomicsbiomarkers: serum proteomics
Analysis of host responseAnalysis of host response immune system responseimmune system response
(syngeneic v.s. immunosuppressed)(syngeneic v.s. immunosuppressed) Angiogenesis (MFP v.s. sc)Angiogenesis (MFP v.s. sc)
Stem cell searchStem cell search OCT
IHC
Snap-Frozen (RNA)
Harvest and freeze cell suspension
Tools for AnalysisTools for Analysis
Colo-829 SC Tumors Collected Post Dose 4
Drug Vehicle Q12hx5D PO
Topotecan 5mg/kg QDx5 IP
ABT-888 25mg/kg Q12hx5D PO
Poly-ADP-ribose (Trevigen)
Min 20.0Max 400.04 min exp
Mou
se 5
Mou
se 9
Mou
se 1
1
Mou
se 1
3
Mou
se 1
5
Mou
se 1
8
AA
XR
2-7
#1
Mou
se 3
Mou
se 7
Mou
se 1
6
Mou
se 1
7
Mou
se 1
9
Mou
se 2
1
AA
XR
2-7
#1
Mou
se 1
Mou
se 2
Mou
se 4
Mou
se 6
Mou
se 8
Mou
se 1
0
AA
XR
2-7
#1
200
140
100
80
513 517 521
b-Actin
Topotecan 5mg/kg QDx5 IP
ABT-888 25mg/kg Q12hx5D PO
Poly-ADP-Ribose(Trevigen)Min 20.0
Max 400.04 min exp
Mou
se 2
6
Mou
se 2
7
Mou
se 3
0
Mou
se 3
4
Mou
se 3
6
AA
XR
2-7
#1
Mou
se 1
2
Mou
se 1
4
Mou
se 2
5
Mou
se 2
8
Mou
se 3
2
Mou
se 3
3
AA
XR
2-7
#1
519 523
Drug Vehicle Q12hx5D PO
Mou
se 2
0
Mou
se 2
3
Mou
se 2
4
Mou
se 2
9
Mou
se 3
1
Mou
se 3
5
AA
XR
2-7
#1
200
140
100
80
515
b-Actin
Colo-829 SC Tumors Collected Post Dose 10
Many Models – Many OptionsMany Models – Many Options
With few exceptions, every rodent model, even if conducted with hundreds of experimental
mice, represents a single patient. Interpreting the preclinical results and applying these
outcomes to human clinical trials continues to prove challenging for those charged with
translating the preclinical experience into viable drug candidates.
Statistically valid model assessing relevant endpoints on an optimal
schedule with clinically appropriate doses.
PROBLEMSPROBLEMS
The appropriateness of animal models to The appropriateness of animal models to identify, qualify and promote new identify, qualify and promote new therapies for cancer has been under therapies for cancer has been under review, and in some ways under attack, for review, and in some ways under attack, for many years. Continuing concerns about many years. Continuing concerns about the failure rate of agents being sent to the the failure rate of agents being sent to the clinic has led to a flurry of publications on clinic has led to a flurry of publications on the irreproducibility of published preclinical the irreproducibility of published preclinical data and their over-prediction of activity. data and their over-prediction of activity.
What can you do?What can you do? 1. use well powered animal studies1. use well powered animal studies 2. reproduce your own data2. reproduce your own data 3. have 2 separate operators generate the data3. have 2 separate operators generate the data 4. provide adequate details in publications4. provide adequate details in publications 5. don’t over-interpret your data5. don’t over-interpret your data 6. stage tumor studies correctly6. stage tumor studies correctly 7. don’t selectively use/present your data7. don’t selectively use/present your data 8. remember the clinical situation and what can be 8. remember the clinical situation and what can be
assessed in manassessed in man
Clinical Endpoints In ManClinical Endpoints In Man
Toxicity -Toxicity - W What harmful effects are induced?hat harmful effects are induced? Tumor response - Tumor response - Does the cancer respond to the treatment?Does the cancer respond to the treatment?
Biomarker modulation as a measure of the effect of a Biomarker modulation as a measure of the effect of a treatment that may correlate with a traditional clinical treatment that may correlate with a traditional clinical endpoint (PFS; TR) endpoint (PFS; TR)
Progression-free survival (stable disease)Progression-free survival (stable disease) Tumor regressionTumor regression Statistically significant improvement in survivalStatistically significant improvement in survival
Survival - Survival - how long does the person live?how long does the person live? Quality of life - Quality of life - how does the treatment affect a person's how does the treatment affect a person's
overall enjoyment of life and sense of well being?overall enjoyment of life and sense of well being?
Progression, Stable, RegressionProgression, Stable, Regression