Poor drug distribution and Poor drug distribution and repopulation - - PowerPoint PPT Presentation

Poor drug distribution and Poor drug distribution and repopulation as neglected and modifiable causes of drug modifiable causes of drug resistance in solid tumours I F T k MD PhD Ian F Tannock MD, PhD

Princess Margaret Hospital and University of Toronto University of Toronto

11/11/2008 PMH Anniversary Meeting

Goals of Presentation Goals of Presentation

To recognize that:

1.

Causes of clinical drug resistance are multifactorial

1.

Causes of clinical drug resistance are multifactorial

2.

There are marked variations in drug concentration within solid tumours

3

F il t h ll f th ll i

3.

Failure to reach all of the cancer cells is an important and neglected cause of drug resistance

4.

Drug concentration in normal tissues is more g uniform, putting tumours at a disadvantage

5.

Repopulation between cycles of chemotherapy may counter effects of cell killing and lead to drug counter effects of cell killing and lead to drug resistance

6.

There are strategies to modify drug resistance in tumours to improve therapeutic effects

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tumours to improve therapeutic effects

Drug development

Drug resistance

Slides

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Slides courtesy of Krupa Patel

Drug resistance Drug resistance

• Cellular and molecular causes of drug

resistance resistance

• Proliferative causes of drug resistance

(e.g. repopulation)

• Microenvironmental causes of drug resistance

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g (e.g. drug distribution)

TUMOUR MICROENVIRONMENT

ECM

Growth factors Chemokines

High IFP

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ECM

Modified from Minchinton and Tannock, Nat Rev Cancer. 2006 Aug;6(8):583-92

High IFP

TUMOUR MICROENVIRONMENT

Decreasing Decreasing nutritients and O2 Decreasing pH pH

11/11/2008 Modified from Minchinton and Tannock, Nat Rev Cancer. 2006 Aug;6(8):583-92

TUMOUR MICROENVIRONMENT

Decreasing Decreasing nutritients and O2 Decreasing pH Decreasing cell pH Decreasing cell proliferation

11/11/2008 Modified from Minchinton and Tannock, Nat Rev Cancer. 2006 Aug;6(8):583-92

DRUG DISTRIBUTION

Drug diffusion: Drug consumption: molecular size, shape and solubility

• drugs that bind

avidly to DNA basic drugs D d li u y

• basic drugs

sequestered in acidic organelles b d h Drug delivery: concentration and time in high cell

• antibodies that

bind to target antigens blood

ECM ↑IFP

high cell proliferation low cell proliferation DRUG

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p hypoxic cells

Slide courtesy of Olivier Trédan

Liver metastasis (arrow) from a xenograft in a nude mouse following injection of (fluorescent) mitoxantrone g j ( )

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Tissue Penetration in vitro

gas inlet sampling port d (i )

Multicellular layer (MCL)

drug (in green)

• penetrating

through the MCL MCL permeable membrane receiving compartment membrane

M su m nt f d u p n t ti n:

p

Measurement of drug penetration: Drug (green) added to compartment above MCL Sampled from lower receiving reservoir

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Sampled from lower receiving reservoir

MCL form an extracellular matrix (ECM)

(Tannock et al Clin Cancer Res 2002;8:878 84) (Tannock et al, Clin Cancer Res 2002;8:878-84)

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DRUG DISTRIBUTION in MCL

no drug 15 min 30 min 1 hour 2 hours 5 min

Concentration of i i l mitoxantrone in lower compartment : through the membrane g alone through the MCL

11/11/2008 PMH Anniversary Meeting Modified from Tunggal and Tannock, Clin Cancer Res. 1999 Jun;5:1583-6

Penetration of drugs depends on packing density (and IFP) packing density (and IFP)

(Grantab et al: Cancer Res 2006;66:103-9)

HCT-8-E11 HCT-8-1R1

Doxorubicin

Tightly-packed Loose packed 15min 6 hrs

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There is evidence for limited drug g penetration in solid tumours

(Lankelma et al, Clin Cancer Res 1999;5:1703-7; Primeau et al, Clin Cancer Res 2005;11:6702-8)

Tumor-bearing mice g injected with doxorubicin & EF5 Tumors removed Tumors removed, frozen, & sectioned for immunohistochemistry

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immunohistochemistry

Fluorescent imaging:

Drug Anti-CD-31 Anti CD 31 (blood vessels) Anti-EF5 Anti EF5 (hypoxia)

Bar = 100μm

Composite

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Composite

Doxorubicin gradients in solid tumours

(Primeau et al, Clin Cancer Res 2005;11:6702-8)

Blue: Doxorubicin Red: Blood vessels G : H p xi Green: Hypoxia EMT6 Mouse Sarcoma Sarcoma Prostate cancer PC-3 xenograft

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xenograft Mouse mammary 16/C

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Image Quantification Image Quantification

(Program created by Augusto Rendon)

distance distance

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Quantification of doxorubicin gradients

Doxorubicin concentration falls exponentially with p y distance from a blood vessel Distance to half I0 = 40-50μ (+/- 0.1-1.6μ)

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Distance to hypoxia = 90-140 μ

This contrasts with uniform distribution of doxorubicin in normal liver …

Data of Patel et al

d th … and other normal tissues such as skin such as skin and muscle … but there is poor is poor distribution in brain

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Gradients of drug concentration in tumor tissue tumor tissue

Method can be applied to: Method can be applied to:

• ther fluorescent agents

(e.g.mitoxantrone,topotecan) fluorescent antibodies (including targeted agents) modifiers of drug distribution modifiers of drug distribution drug combinations distribution of drug effects such as DNA distribution of drug effects such as DNA adducts and markers of cell proliferation and apoptosis

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Therapeutic monoclonal p antibodies

(e.g. cetuximab, trastuzumab, rituximab)

Th i l i i ht i hibit Their large size might inhibit distribution in tumours… … but they have long half lives

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Cetuximab

(Data of Lee et al)

A431 MDA-MB-231 Hi h EGFR Intermediate EGFR High EGFR Intermediate EGFR

Cetuximab applied to tumour sections

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Cetuximab applied to tumour sections

In vivo distribution of Cetuximab 30 min after injection In vivo distribution of Cetuximab 4 hours after injection Blue: cetuximab Blue: cetuximab Red: blood vessels Green: hypoxia Green: hypoxia A431 xenograft

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Gradients of drug concentration in tumour tissue tumour tissue

Method can be applied to: Method can be applied to:

• ther fluorescent agents

(e.g.mitoxantrone,topotecan) fluorescent antibodies (including targeted agents) modifiers of drug distribution modifiers of drug distribution drug combinations distribution of drug effects such as DNA distribution of drug effects such as DNA adducts and markers of cell proliferation and apoptosis

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Modifiers of drug distribution might be used might be used…

…to increase therapeutic index

e.g. proton pump inhibitors such as pantoprazole

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Modifiers of Endosomal i f b i d sequestration of basic drugs

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Pantoprazole increases endosomal pH Pantoprazole increases endosomal pH

6.0 6.5

dosomal pH

5 0 5.5

End

4.5 5.0

Concentration (μM)

1 10 100 1000 10000 4.0

11/11/2008 PMH Anniversary MeetingResults provided by Carol Lee

Pantoprazole prevents sequestration of drug in acidic compartments in acidic compartments

nucleus drug nucleus drug

DOX PTP + DOX

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…and increases toxicity of doxorubicin f lt d t ll for cultured tumour cells

vival

1

Pantoprazole

nogenic Surv

0.01 0.1

Doxorubicin

1 2 3 4 5 6 7

Clon

0.001

Pantoprazole + Doxorubicin

Tim e (hours)

1 2 3 4 5 6 7

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Multilayered Cell Cultures y

Multilayered Cell Cultures Experimental Setup

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Drug penetration studies – MCCs

0.5 0.35 0.4 0.45 Doxorubicin 0.2 0.25 0.3 netration of D Cell Free DOX PTP + DOX 0.05 0.1 0.15 Relative Pen

2 4 6 8 10 12

Time (hours)

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( )

Effect of PTP on distribution of doxorubicin in breast cancer xenografts in breast cancer xenografts

PTP + DOX DOX

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Pantoprazole alters drug distribution in solid tumours distribution in solid tumours

14 10 12

icin ensity

PTP + DOX DOX

6 8

Doxorubi ence inte

2 4

Mean D fluoresc

20 40 60 80 100 120

Distance to nearest blood vessel ( um)

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Distance to nearest blood vessel ( um)

Effect of pantoprazole to modify growth

• f breast cancer xenografts treated

f g f with doxorubicin

1200 1000

m

3)

600 800

r Volume (mm

Control DOX 400

Mean Tumou

PTP DOX+PTP 200

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5 10 15 20 25

Time (days)

Pantoprazole and doxorubicin

• Pantoprazole modifes the distribution of doxorubicin

p in tumour cells and increases its toxicity

• Pantoprazole improves the distribution of doxorubicin

i lid t in solid tumours

• Pantoprazole increases growth delay of breast

cancer xenografts treated with doxorubicin without cancer xenografts treated with doxorubicin, without apparent increase in toxicity

• We plan a phase I/II trial in women with metastatic

p p breast cancer with minimal prior (adjuvant) exposure to anthracyclines

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Gradients of drug concentration in tumour tissue tumour tissue

Method can be applied to: Method can be applied to:

• ther fluorescent agents

(e.g.mitoxantrone,topotecan) fluorescent antibodies (including targeted agents) modifiers of drug distribution modifiers of drug distribution drug combinations distribution of drug effects such as DNA distribution of drug effects such as DNA adducts and markers of cell proliferation and apoptosis

11/11/2008 PMH Anniversary Meeting

Mitoxantrone

• First chemotherapy drug approved for

First chemotherapy drug approved for treatment of HRPC

• Docetaxel superior as first line
• Docetaxel superior as first-line

treatment PSA t t it t

• PSA response rate to mitoxantrone

after docetaxel ~ 15%

• Are there strategies which could

augment the activity of mitoxantrone?

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AQ4N is an experimental pro-drug reduced under hypoxia to AQ4 – similar to mitoxantrone

N H N O OH H OH N H N

+

O OH O-

AQ4N under hypoxia to AQ4 similar to mitoxantrone

Mitoxantrone

N H N O OH H OH N H N

+

O- O OH

AQ4N

(prodrug)

(C P450 )

Tumor Hypoxia

DNA Binding

AQ4

(Cy P450 enzymes)

N H N O OH

+

Topoisomerase II Inhibition

(activated metabolite)

N H N O OH

+

Inhibition Cytotoxicity

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Cytotoxicity

MITOXANTRONE &AQ4N

AQ4N

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100 μm

Mitoxantrone 10min 48h AQ4N/AQ4 10min 10min 48hr

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Combined treatment with mitoxantrone and AQ4N

10min 48h Concentration of drugs at 10 Concentration of drugs at 10 minutes after injection

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Therapeutic effects of AQ4N + Mitoxantrone i t MDA MB 231 X ft against MDA-MB-231 Xenografts

Green = AQ4N Black = Control (Diluent) Blue = Mitoxantrone Red = Combination

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Blue = Mitoxantrone Red = Combination

Therapeutic effects of AQ4N + Mitoxantrone i t MDA MB 231 X ft against MDA-MB-231 Xenografts

Green = AQ4N Black = Control (Diluent) Blue = Mitoxantrone Red = Combination

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AQ4N/Mitoxantrone

• Targeting mitoxantrone to oxygenated regions and

AQ4 to hypoxic regions results in effective drug xp t th nti t m exposure to the entire tumour

• Studies of tumour growth delay in mice following

combined treatment suggest improved Therapeutic combined treatment suggest improved Therapeutic Index

• We have proposed a phase I trial of the combination

We have proposed a phase I trial of the combination to be followed by a phase II trial as second line treatment for men with hormone-refractory prostate cancer cancer

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Repopulation

The process that allows recovery of bone marrow and other rapidly proliferating p y p g normal tissues between courses of chemotherapy… … but also allows increase in number of tumour cells between courses of chemotherapy

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Potential influence of accelerating Potential influence of accelerating repopulation on tumour volume repopulation on tumour volume repopulation on tumour volume repopulation on tumour volume

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Cycles of chemotherapy at 3 week intervals

Modelling the f process of repopulation

(Kim & Tannock, Nature Rev Cancer 2005;5:516 25) 2005;5:516-25)

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Administering cytostatic targeted agents between courses of chemotherapy is a potential m rapy a p n a method of inhibiting tumour cell repopulation repopulation

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Conclusions

Important and neglected causes of drug Important and neglected causes of drug resistance in solid tumours include:

• Poor Drug Penetration through tissue

g g

• Repopulation of surviving cells between

courses of chemotherapy These processes might be modified to improve therapeutic index We plan to evaluate this hypothesis in clinical trials

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Representation in the scientific and li i l lit t clinical literature

• Cellular causes of drug resistance

95% M l f d

• Microenvironmental causes of drug

resistance 4% (e g drug penetration) (e.g. drug penetration)

• Proliferative causes of drug

Proliferative causes of drug resistance 1% (e.g. repopulation)

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A biased view of clinical importance A biased view of clinical importance

• Cellular causes of drug resistance

33% M l f d

• Microenvironmental causes of drug

resistance 33% (e g drug penetration) (e.g. drug penetration)

• Proliferative causes of drug

Proliferative causes of drug resistance 33% (e.g. repopulation)

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Thanks to

• Dr. David Hedley
• Dr. Lothar Lilge

D . L L g Augusto Cesar Rendon Susann Piechatzek Advanced Optical Microscope Facility: James Jonkman and James Jonkman and colleagues

Tannock Lab (past & present)

Pathology Research Carol Lee Licun Wu Olivier Tredan Rama Grantab Andrea Fung Andy Primeau Pathology Research Program Funding from: Alaina Garbens Hira Mian Jonathan Tunggal g f CIHR, NCIC, Novacea