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 Ian F Tannock MD, PhD F T k 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: Causes of clinical drug resistance are multifactorial Causes of clinical drug resistance are multifactorial 1. 1. There are marked variations in drug concentration 2. within solid tumours Failure to reach all of the cancer cells is an F il t h ll f th ll i 3. 3 important and neglected cause of drug resistance Drug concentration in normal tissues is more g 4. uniform, putting tumours at a disadvantage Repopulation between cycles of chemotherapy may 5. counter effects of cell killing and lead to drug counter effects of cell killing and lead to drug resistance There are strategies to modify drug resistance in 6. tumours to improve therapeutic effects tumours to improve therapeutic effects 11/11/2008 PMH Anniversary Meeting
Drug development Drug resistance Slides Slides courtesy of Krupa Patel 11/11/2008
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 g (e.g. drug distribution) 11/11/2008
TUMOUR MICROENVIRONMENT Growth factors High IFP High IFP Chemokines ECM ECM 11/11/2008 Modified from Minchinton and Tannock, Nat Rev Cancer. 2006 Aug;6(8):583-92
TUMOUR MICROENVIRONMENT Decreasing Decreasing nutritients and O 2 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 O 2 Decreasing pH pH Decreasing cell Decreasing cell proliferation Modified from Minchinton and Tannock, Nat Rev Cancer. 2006 Aug;6(8):583-92 11/11/2008
DRUG DISTRIBUTION Drug diffusion: molecular size, shape and Drug consumption: solubility u y - drugs that bind avidly to DNA - basic drugs basic drugs Drug delivery: D d li sequestered in concentration acidic organelles and time in - antibodies that b d h blood bind to target antigens high cell high cell ECM proliferation ↑ IFP DRUG low cell proliferation p hypoxic cells Slide courtesy of Olivier Trédan 11/11/2008 PMH Anniversary Meeting
Liver metastasis (arrow) from a xenograft in a nude mouse following injection of (fluorescent) mitoxantrone g j ( ) 11/11/2008 PMH Anniversary Meeting
Tissue Penetration in vitro gas inlet sampling port Multicellular layer (MCL) d drug (in green) (i ) MCL - penetrating permeable through the MCL membrane membrane receiving compartment p M Measurement of drug penetration: su m nt f d u p n t ti n: Drug (green) added to compartment above MCL Sampled from lower receiving reservoir Sampled from lower receiving reservoir 11/11/2008 PMH Anniversary Meeting
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) 11/11/2008 PMH Anniversary Meeting
DRUG DISTRIBUTION in MCL no drug 15 min 1 hour 2 hours 5 min 30 min Concentration of mitoxantrone in lower i i l 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 11/11/2008 PMH Anniversary Meeting
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 immunohistochemistry 11/11/2008 PMH Anniversary Meeting
Fluorescent imaging: Drug Anti-CD-31 Anti CD 31 (blood vessels) Anti-EF5 Anti EF5 (hypoxia) Bar = 100 μ m Composite Composite 11/11/2008 PMH Anniversary Meeting
Doxorubicin gradients in solid tumours (Primeau et al, Clin Cancer Res 2005;11:6702-8) Blue: Doxorubicin Red: Blood vessels G Green: Hypoxia : H p xi EMT6 Mouse Sarcoma Sarcoma Prostate cancer PC-3 xenograft xenograft Mouse mammary 16/C 11/11/2008
11/11/2008 PMH Anniversary Meeting
Image Quantification Image Quantification (Program created by Augusto Rendon) distance distance 11/11/2008 PMH Anniversary Meeting
Quantification of doxorubicin gradients Doxorubicin concentration falls exponentially with p y distance from a blood vessel Distance to half I 0 = 40-50 μ (+/- 0.1-1.6 μ ) Distance to hypoxia = 90-140 μ 11/11/2008 PMH Anniversary Meeting
This contrasts with uniform distribution of doxorubicin in normal liver … Data of Patel et al … and other d th normal tissues such as skin such as skin and muscle … but there is poor is poor distribution in brain 11/11/2008 PMH Anniversary Meeting
Gradients of drug concentration in tumor tissue tumor tissue Method can be applied to: Method can be applied to: � other 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
Therapeutic monoclonal p antibodies (e.g. cetuximab, trastuzumab, rituximab) Th i l Their large size might inhibit i i ht i hibit distribution in tumours… … but they have long half lives 11/11/2008 PMH Anniversary Meeting
Cetuximab (Data of Lee et al) A431 MDA-MB-231 Intermediate EGFR Intermediate EGFR Hi h EGFR High EGFR Cetuximab applied to tumour sections Cetuximab applied to tumour sections 11/11/2008 PMH Anniversary Meeting
In vivo distribution of Cetuximab In vivo distribution of Cetuximab 4 hours after injection 30 min after injection Blue: cetuximab Blue: cetuximab Red: blood vessels Green: hypoxia Green: hypoxia A431 xenograft 11/11/2008 PMH Anniversary Meeting
Gradients of drug concentration in tumour tissue tumour tissue Method can be applied to: Method can be applied to: � other 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
Modifiers of drug distribution might be used might be used… …to increase therapeutic index e.g. proton pump inhibitors such as pantoprazole 11/11/2008 PMH Anniversary Meeting
Modifiers of Endosomal sequestration of basic drugs i f b i d 11/11/2008 PMH Anniversary Meeting
Pantoprazole increases endosomal pH Pantoprazole increases endosomal pH 6.5 6.0 dosomal pH 5.5 End 5.0 5 0 4.5 4.0 1 10 100 1000 10000 Concentration ( μ M) PMH Anniversary Meeting Results provided by Carol Lee 11/11/2008
Pantoprazole prevents sequestration of drug in acidic compartments in acidic compartments nucleus drug drug nucleus DOX PTP + DOX PMH Anniversary Meeting (Images taken by Carol Lee) 11/11/2008
…and increases toxicity of doxorubicin for cultured tumour cells f lt d t ll Pantoprazole 1 vival nogenic Surv 0.1 Doxorubicin 0.01 Clon Pantoprazole + Doxorubicin 0.001 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 Tim e (hours) 11/11/2008 PMH Anniversary Meeting
Multilayered Cell Cultures y Multilayered Cell Cultures Experimental Setup 11/11/2008 PMH Anniversary Meeting
Drug penetration studies – MCCs 0.5 Doxorubicin 0.45 0.4 0.35 netration of D 0.3 Cell Free 0.25 DOX PTP + DOX 0.2 Relative Pen 0.15 0.1 0.05 0 0 2 4 6 8 10 12 Time (hours) ( ) 11/11/2008 PMH Anniversary Meeting
Effect of PTP on distribution of doxorubicin in breast cancer xenografts in breast cancer xenografts DOX PTP + DOX 11/11/2008 PMH Anniversary Meeting
Pantoprazole alters drug distribution in solid tumours distribution in solid tumours 14 PTP + DOX 12 ensity DOX icin 10 ence inte Doxorubi 8 6 Mean D fluoresc 4 2 0 0 20 40 60 80 100 120 Distance to nearest blood vessel ( um) Distance to nearest blood vessel ( um) 11/11/2008 PMH Anniversary Meeting
Effect of pantoprazole to modify growth of breast cancer xenografts treated f g f with doxorubicin 1200 1000 3 ) m r Volume (mm 800 Control 600 DOX Mean Tumou PTP DOX+PTP 400 200 0 0 0 5 10 15 20 25 Time (days) 11/11/2008 PMH Anniversary Meeting
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