Sydney Cancer Institute

University of Sydney 19th Jan 2004

Organisation and Research Direction

– The Sydney Cancer Institute is an independent research institute established in February 2003 by:

• Sydney Cancer Centre

• The Central Sydney Area Heath Service (CSAHS)

• The University of Sydney

• The Sydney National Cancer Foundation

– The primary function of the institute is:

“Conducting research to substantially improve cancer outcomes”

Objectives and Research Directions

• The Sydney Cancer Institute’s research programs are based around a clinically driven set of research priorities

• The Sydney Cancer Institute comprises the following divisions

– Molecular Oncology

– Cancer Biology

– Experimental Therapeutics

– Cancer Care and Control

Research Goals• The Sydney Cancer Institute aims to pursue

research excellence, depth and relevance by:

– Integration with clinical cancer medicine by the development of translational research programs

– Providing core research facilities such as:• A clinical cancer register• Statistician and information technology• Shared instruments and operators• A tumour bank available to all SCI researchers

– Providing more structured national and international connections

Scientific Advisory Board• The SCI scientific advisory board includes researchers with a

diverse array of skills and interests

– Prof. W McCarthy (Director Melanoma Foundation)

– Prof. J Bishop (Director Sydney Cancer Centre)

– Prof. B Armstrong (Head School of Public Health, USyd)

– Prof. G Halliday (Department of Dermatology, USyd)

– Prof. P Lay (Centre for Heavy Metals Research, USyd)

– Prof. B Roufogalis (Pro-Dean Pharmaceutical Chemistry, USyd)

– Prof. J Simes (Director NHMRC Clinical Trials Centre, USyd)

– Prof. J Thompson (Director Sydney Melanoma Unit)

– A/Prof. P Buttow (Executive Director, Medical Psychology Unit, USyd)

– A/Prof. M Millward (Head Clinical Research, SCI)

– Dr S Clarke (Senior Research Fellow, SCI)

Affiliations• The SCI has affiliation agreements with:

– The University of Sydney

– Central Sydney Area Health Service

– The Sydney National Cancer Foundation

– The John Wayne Cancer Institute – Los Angeles

– The National Institutes of Health (USA)

– National Cancer Institute (CTEP, USA)

– Cancer Therapeutics Research Group (Singapore)

– National Cancer Centre (Singapore)

– John Hopkins (Singapore)

Research programs• The SCI has a diverse research program including:

– DNA Methylation (Dr Susan Clarke)

– Cancer Genetics and Drug Resistance (Dr Quihan Dong, Dr John Young)

– Viral Oncology (Professor Y Cossart and Dr Carol Thompson)

– Tumour Pathology and Molecular Biology (A/Prof. Soon Lee)

– Skin Cancer Biology (Prof. Gary Halliday)

– Skin Cancer and Photobiology (Dr Vivienne Reeve)

– Cancer Invasion and Metastasis (Dr Guy Lyons)

– Heavy Metals Research Program (Prof. Peter Lay)

– Clinical Pharmacology (A/Prof. Stephen Clarke)

– Pharmaco Oncology (Prof. Basil Roufogalis)

– Gene Therapy (A/Prof John Rasko)

– Molecular Imaging (A/Prof Michael Fulham)

– Clinical Trials (A/Prof. Michael Millwood)

– Early Detection and Diagnosis (Dr Scott Menzies)

Biometals Section

Centre for Heavy Metals Research

School of Chemistry

University of Sydney

Biometals Section, CHMR• Associate Professor Robert Armstrong• Dr. Rachel Codd• Dr Carolyn Dillon• Dr. Ron Fenton• Professor Hans Freeman• Professor Trevor Hambley• Associate Professor Margaret Harding• Dr. Hugh Harris• Associate Professor Brendan Kennedy• Professor Peter Lay• Professor Len Lindoy• Associate Professor Tony Masters• Dr. Lou Rendina

Research Areas/Interests• Anti-Cancer Drugs

- Boron complexes: boron neutron capture therapy (Rendina)

- Co(III) Hypoxic Agents (Hambley)

- Copper Complexes (Dillon, Hambley, Harris, Kennedy, Lay)

- Metallocenes (Dillon, Harding)

- Metal Complexes of Organic Anti-Cancer Drugs (Hambley,

Harding)

- Metalloporphyrin/Fullerenes (Armstrong, Lay)

- Pt(IV) and Pt(II) (Fenton, Hambley)

- Ruthenium (Armstrong, Dillon, Lay)

• Radiopharmaceuticals- Use of copper-64 in macrocyclic systems for the imaging

and therapy of cancer (Lindoy)

- Improved 99Tc Generators (Masters)

Research Areas/Interests• Metal-Induced Cancers and Toxicity

- Cr- and Ni-induced cancers (Codd, Dillon, Harris, Lay)

- As carcinogenesis and toxicity (Dillon, Harris)

• Anti-Inflammatory Drugs- Cu, Zn, Ni, Zn complexes as anti-inflammatories (Dillon,

Hambley, Kennedy, Lay)

• Anti-Diabetics- Cr(III) (Dillon, Harris, Lay)

- Vanadium (Codd, Lay)

Research Areas/Interests• Metalloproteins

- structure of heme proteins and their roles in the immune system, and heart disease (Armstrong, Lay)

• Chelation Therapy and Metabolic Processes- role of transition metal-sialic acid species in metal

homeostasis/disease (Codd)

- cold-adapted 'super-siderophores' for metal chelation

therapies (Codd).

• Diagnostics

- use of vibrational spectroscopy in the diagnosis of cancer

(Armstrong, Lay)

Facilities/Expertise• Structural Biology and Structural Chemistry X-ray, Neutron & Electron Scattering and Diffraction; Synchrotron Techniques; NMR Spectroscopy;

See separate presentation on CSBSC

• Mass SpectrometryElectrospray (including HPLC front end); GC-MS; Maldi

• Vibrational SpectroscopyIR, Raman and Resonance Raman: including tissue mapping

• EPR SpectroscopyL, Q and X band, ENDOR, He cryogenics, Whole cell

• Cell Biology Cytotoxicity; Genotoxicity; Permeability; Imaging, Spectroscopy

• Animal StudiesPharmacology; Pharmacokinetics; Efficacy; Toxicity

Anti-Cancer Drugs

Boron Neutron Capture - Boron Neutron Capture - RendinaRendina

10B

1n

11B*

4He

7Li

kinetic

5 5

0 2

3

energy+(~2.28 MeV)

tumour cell

selectivedestruction

0n110B

10B

Clinical BNCT AgentsClinical BNCT Agents

Malignant brain tumours Malignant melanoma

B SH

B

NH3+

O

O–

OHHO

2–

BPA

B–H

BSH

Two new classes:

• Platinum(II)-amine complexes

• Metallo-intercalator complexes

Dinuclear Platinum ComplexesDinuclear Platinum Complexes

Woodhouse, S. L.; Rendina, L. M. Chem. Commun., 2001, 2464.

International Patent PCT/AU02/00943

HCl.H2N

HCl.H2N

NH2

NH2

Pt

Cl

Cl

NH3

1. K2CO3

2. K[PtCl3(NH3)] or cis-[PtCl2I(NH3)]–/Ag+/HCl(aq)

Pt

NH3

Cl

Cl

NH2

NH2

Pt

NH3

Cl

NH3

1. K2CO3

2. trans-[PtCl(dmf)(NH3)2]OTf

Pt

NH3

Cl

NH3

2+

CBH

(OTf)2

Metallo-intercalator ComplexesMetallo-intercalator Complexes

Todd, J. A.; Rendina, L. M. Inorg. Chem., 2002, 41, 3331.

International Patent PCT/AU02/00943.

H

NN

N

PtS

(CH2)n

+

(n = 0-3)

H

NN

N

PtCH2S

+

BHC

Control A375 Melanoma Cells

P S Cl K

Ca Cr Fe Ni

Cu Zn ScatteringJ. Aitken, H. Harris, P. A. Lay, USyd, P. Farmer, UC Irvine

CuDSF-treated A375 Melanoma Cells

P S Cl K

Ca Cr Fe Ni

Cu Zn ScatteringJ. Aitken, H. Harris, P. A. Lay, USyd, P. Farmer, UC Irvine

Cu XANES of Melanoma Cells

8.95 8.96 8.97 8.98 8.99 9 9.01 9.02

Control

CuCl

Melanin 1 (Cu II)

Melanin 2 (Cu I)

J. Aitken, H. Harris, P. A. Lay, USyd, P. Farmer, UC Irvine

necrotic

suboxic

normoxic

Pt(IV) Pt(IV) Pt(II)

Selective activation in solid tumours Hambley

MicroXANES of Pt obtained from A2780 ovarian cancer cells treated with Pt(IV)

Identification of Novel Intercalating Platinum Compounds - Fenton, Aldrich-Wright

CISPLATIN & analogs bind to DNA

New Pt compounds intercalate into DNA - IP protection- PCT filing, Priority date February 22nd 2001

0

2

4

6

8

10

12

L1210 DDP 2008 C13 SKOV3 PC3cell line

IC50

uM

Test compound

Cisplatin

L1210 = mouse leukemia; 2008 = human ovarian tumor PC3= prostate tumorDDP = Cisplatin-resistant; C13 = acquired; SKOV3= intrinsic Cisplatin resistance

EFFICACY of lead compound- Comparison with Cisplatin & in Cisplatin-resistant cell lines

Phase I clinical trials

• toxic effects : hypoglycaemia, metallic after taste

• bone marrow largely unaffected

• liver toxicity does-limiting side-effect

Phase II clinical trials

• breast cancer

• renal cell carcinoma

A/Prof Margaret Harding, School of Chemistry

• poor hydrolytic stability in water pH> 4.0

• Ti accumulates in nucleic rich regions tumour cells

• species formed in vivo and how interaction with DNA occurs not understood

• mechanism is distinct from platinum based drugs

Titanocene Dichloride

Antitumour Metallocenes

Ti V

Nb Mo

Re

Cr Mn

TcZr

Hf Ta WY = Cl, Br, I, NCS, N3

• Each complex has independent mechanism of action

• Current studies focus on cellular distribution, interaction with biomolecules of Mo, Nb complexes

• M = Mo targets thiols

A/Prof Margaret Harding, School of Chemistry

Design water soluble, stable derivs

Streptonigrin

A/Prof Margaret Harding, School of Chemistry

Water, pH 6.5

NN

O

O

H2N

CH3O

CO2H

CH3H2N

OCH3

OH

OCH3

Clinical use human cancers until 1977

•broad spectrum antitumor activity

•lymphoma, melanoma•cancers of the breast, cervix, head, neck

•severe and unpredictable side-effects

Streptonigrin Metal Complexes

A/Prof Margaret Harding, School of Chemistry

N

O

O

H3CO

H2N

N

O OH

affectsDNA binding

topo II recognition site

Redox related to DNA cleavage

ACCELERATED BYMETAL IONS

bipyridylzwitterionlabile metal complexes

N

O

O

H3CO

H2N

N

O OH

NH2

CH3

OCH3HO

OCH3

•Ru(II) complex

•Reductively activated to semiquinone

•Strong DNA binding/cleavage predicted

Radiopharmaceuticals

Improved Tc-99 Generators - Masters Understanding the interactions between alumina and molybdates has led to improvements in the process for generating 99Tc for radio-diagnostics

Schematic outline of 99Mo production process at ANSTO.

Metal-Induced Cancers and Toxicity

Cellular Metabolism of Chromium

?

Effect of Oxidation State on Cr Genotoxicity

Dillon, C. T.; et al. Chem. Res. Toxicol. 1998, 11, 119-129; Dillon, C. T.; et al. Chem. Res. Toxicol. 2000, 13, 742-748.

0.0 0.1 0.2 0.3 0.4 5 10 15 20 25 300

20

40

60

80 [CrO

4]2-

[CrO(ehba)2]-

[CrO(mampa)]-

[Cr(en)3]3+

[Cr(glygly)2]-

Inci

denc

e of

MN

/100

0 B

N c

ells

[Cr] (mol/dish)

} Cr(VI)

} Cr(V)

} Cr(III)

Exposure of Whole Cells to Cr(VI) and Cr(III)

Cr(III)-Treated Cell

Cr(VI)-Treated Cell

P

K Cr Zn

P K Cr Zn

Min MaxDillon, C. T.; Lay, P. A.; Kennedy, B. J.; Stampfl, A. P. J.; Cai, Z.; Ilinski, P.; Rodrigues,

W.; Legnini, D. G.; Lai, B.; Maser, J. J. Biol. Inorg. Chem. 2002, 7, 640-645.

Anti-Inflammatory Drugs

• Copper indomethacin is a dimeric copper complex containing 4 indomethacin ligands bound to Cu through the carboxylic acid group.

• Cu-algesic (CuIndo) is an effective anti-inflammatory drug commonly used in dogs and horses.

• Importantly, CuIndo is much less TOXIC in dogs than IndoH.

N

O

Cl

CH2CH3O

CH3

OCuO

O

O

CC

O

O

O OCuR

C

C

R

R

R

L

L

R=

L= solvent ligand

Copper IndomethacinLay, Hambley, Kennedy, Dillon

Assessment of Small Intestinal Ulceration

Copper Indomethacin

Indomethacin

Effect of the Formulation on Gastro-Intestinal Damage

• Equivalent doses of indomethacin (10 mg/kg) were administered to each animal.

• The number of animals tested per treatment ranged from 4-6.

0

25

50 Stomach

CMC micelle powder paste86 paste3 indo (CMC)0

50

100

150 Small Intestine

Ulc

erat

ions

(m

m2 )

Formulation

Anti-Diabetics

Codd, Harris, Lay

Efficacy of Cr Dietary Supplements• Chromium supplements are the second biggest market for dietary

supplements (over $1B industry).• Taken to convert fat into muscle in athletes (used instead of steroids for

humans) and food animals.• Used to help prevent diabetes.• There is no compelling evidence (epidemiology, cell work, or

biochemical assays) that Cr supplements convert fat into muscle or help prevent diabetes.

• The FDA has prevented companies from advertising such health benefits.

• There are anti-diabetic effects exhibited in animal studies for certain Cr

complexes and they are believed to have anti-diabetic effects on humans.

Side-Effects of Cr Dietary Supplements• They react with enzyme systems such as glucose oxidase and xanthine

oxidase to form highly genotoxic mixtures of high oxidation state Cr species, which are very damaging to DNA

• The Cr(VI) generated in these enzymatic processes and strongly inhibits phosphatase enzymes, which is likely to be responsible for the anti-diabetic effects of Cr supplements

• High-oxidation-state Cr and V species appear to act in the same way, through similar intermediates in their anti-diabetic effects

0 50 100 150 200

0

25

50

75

100 Cr(VI) Cr(V) V(V)

Activ

ity, %

Concentration, M

Inhibition of PTP (phosphatase enzyme by Cr(VI), Cr(V) and V(V). Mulyani, Levina, Lay, JACS, submitted

Metalloproteins

Heme Proteins - Armstrong, Harris, Lay

• Characterized NO, CO and O2 adducts of heme proteins, many of which are too unstable to crystallise

• Studied unfolding of cytochrome c• Collaborations with Paul Witting and

Roland Stocker- Studies on indolamine 2,3-dioxygenase (IDO) - important in the immune system

- Studies on the roles of heme proteins in heart disease

Fe-O-O = 146

Np

NpNp

NpFe

O

N

1.832.02

2.05

O

Molecular structure of the active site of horseheart MbO2 at 10 K.

Heme Proteins

Rich, Cheng, Armstrong and Lay

Chelation Therapy and Metabolic Processes

Metal-Sialic Acid Speciation CoddInsight into the nature and role of species formed between sialic acid and transition metal ions.

This polyfunctional carbohydrate is present as theterminal residue in many glycopoteins involved inmetal transport (e.g., transferrin; ceruloplasmin)

Sia Sia2,3 2,3

Gal Gal1,4 1,4

GlcNAc GlcNAc1,2 1,2

Man Man1,3 1,6

Man1,4

GlcNAc1,4

GlcNAc

Asn

Membrane glycoproteins and glyco-lipids: extracellular sugar residues on mammalian plasma membranes

Metal-Sialic Speciation9.76

pH

8.38

7.18

6.21

5.47

4.37

3.14

2.08

1.982 1.980 1.978 1.976

giso

1.982 1.980 1.978 1.976

giso

ba

Cr(V)-sialic acid profile•Development of transition metalEPR Spectroscopy as a diagnostictechnique for glycosylation patterns

•Extension of studies of metal-sialic acid speciation with other biologicallyrelevant transition metal ions(e.g., Cu, Fe, Zn, V).

Cu(II)-sialic acid profile

•Implications for metal transport, homeostasis, and role(s) in sialylglycoprotein-dependent disease

Diagnostics

Vibrational Spectroscopy in Breast Cancer Diagnosis - Tam, Armstrong, Carter, Lay

IDC

IDC

Current Diagnostic TechniquesCurrent Diagnostic TechniquesTriple Assessment & Side EffectsTriple Assessment & Side Effects

• A combination of physical examination, mammogram and fine needle aspiration cytology.

• Standard for breast diagnosis.

• Side effects:– Probable false positive and negative results

from mammogram– Time consuming progress

New Diagnostic For Breast CancerNew Diagnostic For Breast Cancer

• 1H Magnetic Resonance Spectroscopy at IMRR– Simple and quick diagnosis of aspirated breast tissues– The choline-to-creatine peak ratio is being used to compare

various breast disease states

• Vibrational Spectroscopy– Infrared Spectroscopy

• Quick diagnosis by comparing the spectral differences in proteins (amides), lipids and DNA levels of sectioned tissues

– Raman Spectroscopy • By using various laser excitations to compare proteins, lipids

and DNA levels of different breast diseases.• Imaging and mapping provide parallel results often difficult

to determine in pathology.

Cho

Cr

-CH=CH-

-CH2- -CH3

ppm

HODsuppressed

1H Magnetic Resonance Spectroscopy

BenignBenign

MalignantMalignant

Wavenumbers (cm1)

Tra

nsm

issi

on

Am

ide

I

Am

ide

II

Nu

clei

c A

cid

s

Pro

tein

s &

Lip

ids

Collagen Levels

FT-IR Spectra

Benign

Malignant

Benign

t

1455(CH3) + (CH2)proteins

1239 as(PO2

ˉ )Nucleic acids

Wavenumbers (cm1)

Tra

nsm

issi

onFT-IR Spectra

Malignant

Wavenumber (cm1)

Ram

an I

nte

nsi

ty (

a.u

.)3100 2750 1800 80

0

s(CH2)LIPIDS

as(CH2)

as(CH3

)

Am

ide

I

CH

2 & C

H3

def

orm

atio

n

Ph

e

Am

ide

IIIL

IPID

SFT-Raman Spectra

Malignant

Benign

Raman ImagingRaman Imaging• New approach to compare results parallel to the native pathological examinations

• Colour intensity of particular Raman marker bands have potential for identifying the progress of cancer

• Proteins, lipids, DNA and other important components can be detected in tissues that require less preparation than for infrared and magnetic resonance spectroscopy

SummarySummary

• 1H Magnetic Resonance Spectroscopy– Fast and suitable for low-lipid content of breast lesions– Research is in collaboration with The Institute for

Magnetic Resonance Research, University of Sydney

• Vibrational Spectroscopy– Suitable for both high- and low-lipid content of breast

lesions– Inexpensive and non-destructive.– can run in parallel with histopathology

• Multivariate Statistical Analysis– Improve significance for the spectral data– Correlate and classify different breast diseases