Evaluating PotentialDrug Therapies

Mike Shuler

Biomedical Engineering

Can we use tissue engineeringand related approaches

to evaluate potentialeffectiveness of drugs?

Can they be adapted forpersonalized medicine?

Claudia Fischbach-TeschlAbe StroockLarry BonassarJonathan Butcher

Drug responsiveness in 3-D tumor cell culture

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2-D 2-D,drug

3-D 3-D,drug

Increase in cell death(propidium iodide)

Tumor cells cultured in biomimetic tumor microenvironments

are less responsive to cytotoxic therapy

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In vitro In vivo

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In vitro In vivo

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In vitro In vivo

Tissue engineered tumors recreate conditions

in vivo

Observations

• Individual cancers vary/many possible combination of mechanisms

• Metabolizing tissue – significant variation throughout human population – metabolites can vary in amount and kind

• Dose-limiting normal tissues; tolerance varies

Premise

• Single drugs are unlikely to be broadly effective

• Combination therapy should be more effective

• How can we predict accurately the best therapy for an individual?

In vitro Replacements for Animals and Humans

• Animal studies are expensive, long, and not particularly predictive of human response

• Currently only 1 in 10 drugs entering human clinical trials emerge as FDA approved products

“Microscale Cell Culture Analog”

We can model our bodyas combinations of

tissue culture reactors(physiologically based pharmacokinetic model)

CCA: a physical replica of the PBPK

model

1”

1”

fat

lung

Other

Tissues

liver

Combination Therapies forCancer Treatment

• Could exposure to multiple agents more effectively treat cancer?

• With multiple agents the potential number of combinations and scenarios to be tested is impracticable for animal studies

• Could CCA with PBPK explore a broad experimental range to predict a testable subset for detailed study?

Multidrug Resistant (MDR) Cancer

• Tumor responds initially but reoccurs and in non-responsive or MDR

• Multiple causes of MDR; may need multiple agents to control

• Best studied case is P-glycoprotein (P-gp) overexpression: Pump protein intercepts chemotherapeutic agent before it enters cell

MDR Suppressing Agents Fail

• No MDR suppressing agent has passed clinical trials

• Toxicity to normal tissue/altered pharmacokinetics for chemotherapeutic

• Animal studies not good predictor- Rat has 3 P-gp isoforms; humans have 2

Human Cell Culture Analog

Organ Rationale Cell Lines Characteristics

Liver p450 activity Hep-G2/C3A hepatomaMarrow sensitive to chemo MEG-01 megakaryoblast line(Hematopoietic) dose limiting toxicity attachment/suspension

inducible attachmentTumor initial tumor primary MES-SA uteran sarcoma(Sensitive) type sensitive to doxorubicinTumor (MDR) resistant tumors can MES-SA/DX-5 variant(Resistant) selected for resistance

to doxorubicin

Model Drugs Used

• Doxorubicin as chemotherapeutic agent (naturally fluorescent)

• Cyclosporin, Nicardipine as MDR suppressors

Sensitive Tumor Cells (MES-SA)

Resistant Tumor Cells (MES-SA/DX-5)

Bone Marrow Blood Cells (MEG-01)

Liver Cells (Hep-G2/C3-A)

Micro Cell Culture Analog

Device on peristaltic pump in incubator

All cells labeled with celltracker green before experiment

Other Tissues/ Debubbler

Application to Study Multidrug Resistance Suppressors

Proliferative Toxicity StudyWe challenged the MDR CCA device to 3 day exposure to mixtures of Doxorubicin and 2 modulators: nicardipine and cyclosporine A in McCoys 5A medium with 10% FBS.

The ratio of final cell density to initial cell density for each condition is displayed below.

Result: Modulators have strong response on resistant cell line, moderate in others, and a synergistic effect is observed between the two modulators in the resistant cell type.

Relative Proliferation of Cells in CCA device during 3 Day

Experiment

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C3A DX5 MESSA MEG01

Relative Growth

Flow Control 0.5 Dox 1 Dox

1 Dox/10 NCB 1 Dox/10 CSP 1 DOX/5 CSP/5 NCB

Can we use biopsy tissue from the cancer target, the liver, and other relevant tissue to test patient specific response using a microCCA?

Relevant Features

• Can be made disposable/polystyrene

• Requires few cells – multiple tests possible from modest tissue sample

• Screen large set of drug combinations

• Could also be used to study mechanisms?

Challenges

• Maintenance of tissue specific characteristics in vitro

• Automated processing and “simple” to use

• Validation?