DEPARTMENT OF CHEMISTRY

Radiolabelling Biomoleculeswith Copper

Jon Dilworth

Cu radioisotopesCu radioisotopes

Isotope Production Half life

60Cu Cyclotron 24min

61Cu Cyclotron 3.3hrs

62 Cu Cyclotron 9.74min

64Cu Cyclotron 12.7h

67Cu Cyclotron/reactor 62hrs

All positron emitters except 67-Cu (beta), 62-Cu available via 62-Zn/62-Cu generator

PETPET RadionuclidesRadionuclides

Non-metals

18F , 11C , 13N, 15O 68Ga, 124 I,

Metals

89 Zr, 94mTc, 64Cu, 86Y, 44Sc, 45Ti, 55Co,

Copper is unusual among common metallic PETradionuclides:

1. Has facile conversion between two oxidation states2. Is an essential biological metal

CyclotronCyclotron 6464Cu productionCu production

• All chemistry in aqueousmedia [Cu] ca 10-8M

64Ni

cyclotronirradiation 64Cu

ion exchange to separateNi and Cu in HCl

64CuCl2 ca 10-8M

64Ni electroplatedgold target disc

Production via solution targets?Isotope availability an issue Importance of targetry chemistry

BifunctionalBifunctional LigandLigand Design CriteriaDesign Criteria

• Cu species completely stable with respect to ligandexchange/loss despite possible redox

• Reasonable ligand synthesis with easy incorporationof conjugation sites (Carboxyl/amino/acetyleneetc)

• For sensitive biomolecules and (62-Cu) radiolabellingneeds to be high radiochemical yield, rapid, at roomtemperature, post conjugation. Facile separation ofany excess ligand.

Coping with the Cu(II)/Cu(I)Coping with the Cu(II)/Cu(I) redoxredox/copper/copperbiochemistrybiochemistry

1. Ligands which give stable Cu(II) and even ifredox not reversible Cu(I) retained

2. Stable Cu(II) complexes which cannot bebiologically reduced to Cu(I)

3. Both Cu(II) and Cu(I) forms stable with fullyreversible redox

4. Stable Cu(I) complexes which cannot bebiologically oxidised to Cu(II)

Is it necessarily a disadvantage to lose the Cu?

Stabilising Cu(II)Stabilising Cu(II)

C. Anderson et al, Q. J.Nucl. Med. Mol. Imaging, 52, 185,2008.R.E. Mewis and S.A. Archibald, Coord. Chem. Rev., 254,1686, 2010

Stabilities andStabilities and redoxredox propertiesproperties

C. Anderson et al, Q. J.Nucl. Med. Mol. Imaging,52, 185, 2008

How many if any of these reduced in vivo? Potentials measured inacetate buffer –pH dependence? Why only quasi-reversible?

In vivo stabilityIn vivo stability

M. Shokeen and C Anderson, Accts. Chem. Res., 42,832-841, 2009

Conjugate with peptides has one carboxylate coordinated (is theamide involved?) but still shows high in vivo stability. Strong stericshielding. Increasing chain length for carboxylate has little impacton stability. Radiolabelling 40-60o for 1h (MAb’s??)

CuCB-TE2A1

N11-Cu-N4 - 177.5o

CuCB-DO2A2

N3-Cu-N1 – 159.0o

CrossCross--bridgedbridged cyclamscyclams

1. Wong et al J. Amer. Chem. Soc, 122, 10561, 20002. Boswell et al, J.Med Chem, 47, 1465, 2004

In vivo studies with CBIn vivo studies with CB--TE2ATE2A

sst2ANTY3-TATE

Biodistribution studies in rats with AR42J tumours

Cu-CB-TE2A –c(RGDyk) also studied in vivo for targeting integrinin osteoclasts

M. Shokeen and C Anderson, Accts. Chem. Res., 42,832-841, 2009

Tumour to blood ratios 4h pi:Cu-TETA-Y3-TATE - 8.2 (1.6)Cu-CB-TE2A –Y3-TATE – 155 (55)

Tumour to blood ratios 24hpi:Cu-CB-TE2A-Y3-TATE - 20Cu-CB-TE2A-sst2ANT - 72

Radiolabelling at ca40-75 degrees inEtOH for 15-45mins high rcy

The DIAMSARThe DIAMSAR bifunctionalbifunctional chelatorchelator

Complex has 2+charge –protonationminimised. Irrev.Reduction -1.1V.Steric shieldingof Cu

S.V. Smith, J. Inorg. Biochem.,98, 1874, 2004

P.S. Conti et al, Bioconjug. Chem., 22, 256, 2011

PET Images of PC-3 tumours in micewith 64-Cu labelled conjugates–left diamsar derivative right DOTA

DOTA-DGEA conjugates much less stablethen diamsar – competition issues withcarboxylates on peptide fragment.

In vivo imaging withIn vivo imaging with DiamsarDiamsar DGEA conjugatesDGEA conjugates

DGEA peptide targets α2β1 integrinstrongly expressed in PC-3

P. Conti et al, Bioconjug. Chem., 21, 1423, 2010

DiamsarDiamsar conjugates with RGD peptidesconjugates with RGD peptides

Images in micewith humanU87MG gliomaxenograftsDiamsar upperDOTA lower

Biodistribution determinedfrom PET images after 20h.Similar tumour [Cu] uptake butblood higher for DOTA

In vivoIn vivo stability studiesstability studies diamsardiamsar and DOTA derivativesand DOTA derivatives

P. Conti et al, Bioconjug. Chem., 21, 1423, 2010

Stabilising Cu(I) and Cu(II)Stabilising Cu(I) and Cu(II)

Structure ofmonocationicCu(I) complexCu(II) complexalso fourcoordinate.Facile reductionof CuII) to Cu(I)

K. Karlin, J Zubieta et al , Chem. Comm., 465, 1979

Blue copper proteins –facile reduction Cu(II)to Cu(I) – entacticstate, compromisedonors for Cu(II) andCu(I) and distortedgeometriesSteric constraint ofaccess to Cu site

Stabilising Cu(I)Stabilising Cu(I)

R2P

Cu

R2P PR2

PR2

Formed in rapid reaction with Cu(II)Stable for >24h in serumCell uptake modified by R groupsNo oxidation to Cu(II), stabilisation by PNo protonation of ligands

Potential forbioconjugation via use ofmaleic anhydride backbone

P.J. Blower et al, Chem. Comm., 1093, 1996P.J. Blower et al, Nucl. Med. Biol., 23, 957, 1996

RedoxRedox based stabilisation of Cu(II)based stabilisation of Cu(II)

Very stable in serum and towardsglutathione and reductive challenges

Forms stable Ga, Tc complexes

Barnard, Dilworth et al Inorg Chem, 48, 7177, 2009

H Maecke et al, Inorg Chem., 46, 3144, 2007

LigandSpecificActivity(mCi/10μg)

pHa Conditionsb IncubationTime (min)

RadiolabelingYield (%)

PB-TATE 2.0 5.5 rt 60 95.2

PB-TATE 2.0 5.5 95 °C 30 96.4

PB-TATE 2.0 5.5 MWI* 10 99.8

DOTA-TATE 1.0 5.5 MWI* 30 55.9

DOTA-TATE 1.0 7.0 95 °C 60 60.2

CB-TE2A-TATE 2 8.0 MWI* 30 24.8

CB-TE2A-TATE 1 8.0 MWI* 60 43.8

CB-TE2A-TATE 0.5 8.0 MWI* 60 62.3

CB-TE2A-TATE 0.5 8.0 95 °C 120 56.2

RadiolabellingRadiolabelling of PBof PB--TATETATE

Structure of PB-TATE

Radiolabellingresults

P. Barnard, J.R. Dilworth, (Oxford) R. Schibli (Zurich), HMaecke (Basle), 2009-

In vivoIn vivo imagesimages forfor 6464CuCu--PBPB--TATETATE

8h p.i._Blocked8h p.i.

Radioligand: 150 pmol/0.7 MBq/100

μL/mouse

Blocking: 20 nmol TATE/mouse

H. Maecke, P Barnard et al , unpublished2009

Copper(II)Copper(II) bisbis((thiosemicarbazonatethiosemicarbazonate) complexes) complexes

Square planar copper(II), highly delocalised. Associated insolid state via Cu-S interactions, weak axial ligationFirst reported ca 1950’s and shown to have a range ofbiological activity including high cytotoxicity.Can be radiolabelled with Cu in >1min at room temperature,stable in serum. 99m-Tc complex also known and stable.

CuATSM

N N

NH2

HN

HN

NH2S

+

HN

SMeHN

N OHN

SMeHN

NH

S NH

NH2

ATSMA

N NHN

S

NH

SNH

NH2

N NHN

S

NH

SNH

ATSMen

ATSMac

MeHN

MeHN

NewNew bifunctionalbifunctional thiosemicarbazonesthiosemicarbazones

M Christlieb and J R. DilworthChem. Eur. J., 12, 6194–6206, 2006,

DFT studiesshowed thatelectronicstructure leastperturbed bysubstitution at theexocyclic site

UV/vis of transmetallation.Complete in <1min at RT

Inorg Chem 2007, 465

TransmetallationTransmetallation forfor radiolabellingradiolabelling

Holland, Dilworth et al Inorg Chem, 46, 465, 2007

Also effective for transmetallation of macrocycleswith Cu and for Tc and Re based radiolabelling

P. Barnard, S. Bayly, H. Betts, J Dilworth J. Holland, AngewChem. 2008

Solid Phase SeparationSolid Phase Separation

R.Hueting et al, Dalton Trans, 2010, 39, 3620

J. Holland et al, Inorg Chem, 2007, 46, 465

Synthesis of linker groupSynthesis of linker group

Peptide LabelingPeptide Labeling –– Conjugation to GRPR binding bombesin peptideConjugation to GRPR binding bombesin peptide

Fmoc 3hAsp- Ala- Ala-BBS

3hAsp- Ala- Ala-BBSN NHN NH

NH

SSNH

HN

NH

O

O

O

c

3hAsp- Ala- Ala-BBS-NHN NHN NH

NH

SSNH

HN

NH

O

O

O

1 n=0

2 n=1

n

n

n=0 H2ATSM-BBS-1

n=1 H2ATSM-BBS-2

BBS = Gln-Trp-Ala-Val-Gly-His-Cha-Nle

b) 1: BOP, DIPEA, DMF

2: DCC, HOBt, DMF

Reagents and Conditions: a) piperidine/DMF 2:8, 2x10min b) 1: BOP, DIPEA,

DMF, 8h rt 2: DCC, HOBt, DMF, 3h, rt, c) TFA with thioanisole/ethanedithiol 7:3,

3h, rt

a, b

chelator introducedas final amino acid insequence suitable for solidphase peptidesynthesis (piperidine,TFA)

Solid phase peptide synthesis

+

BBS suitable benchmark (18F, 64Cu, 68Ga, 99mTc, 177Lu, 111In and 188Re labelled BBS in

literature)

Active binding sequence = BBS fragment (7-14) Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-

Met14-NH2

Peptide sequence: Dr V. Maes (VUB Belgium), L. Brans et al, Chem Biol Drug Des2008, 72, 496-506 C.Schweinsberger et al Bioconj Chem, 2008, 19, 2432, R.Huetinget al, Dalton Trans, 2010, 19, 2432

6464--Cu radiolabeling of conjugate with BBSCu radiolabeling of conjugate with BBS

64CuCl2 in 0.25M NH4OAc, 1mM ligand in DMSO, Sep-pak-C18

J.R Dilworth, R.Hueting et al, Dalton Trans, 2010, 39, 3620

time

• rapid labeling at RT (5mins)• RCY 90%• easy purification

Receptor Binding AssayReceptor Binding Assay InIn--vivovivo biodistributionbiodistribution

-3 -2 -1 0 1 2 3 4 5 60

20

40

60

80

100 CuATSM-BBS-1CuATSM-BBS-2

Log [nM]

Bin

din

g(%

)

competition assay, 124I-BBS in r-positive PC-3 cells (37C,1h)

Dr Elisa Garcia-Garayoa, Mr Alain Blanc, Prof Roger Schibli ETH Zurich 2009L. Brans et al, Chem Biol Drug Des, 2008, 72, 496-506

Blo

od

Hea

rt

Lung

Spleen

Kid

neys

Pancr

eas

Stom

ach

Smal

l inte

stin

e

Colo

n

Liver

Musc

le

Bone

Tu. PC-3

0

2

4

6

8

10

12

1 h

24 h

13

23

33

43

53

Up

take

(%ID

/gti

ssu

e)

64CuATSM-BBS-2 in nude mice with PC-3 tumour xenografts.

Tumour uptake (~7%) greater than Cu-labelledDOTA (4%) and NOTA (4%) analogues at 1h p.i.blocking study confirms receptor specificityuptake in receptor positive organs and liverunusually high lung uptake (~40%)

Binding affinities for the GRP/BN2 receptor:comparable to natural bombesin (1.9nM)IC50= 2.9nM (ATSM-BBS-1)IC50= 3.8nM (ATSM-BBS-2)

R.Hueting et al, Dalton Trans, 2010, 39, 3620

Synthesis of BTSC with pendantSynthesis of BTSC with pendant maleimidemaleimide

Rebekka Huting, unpublished 2010

ConclusionsConclusions

Cross-bridged cyclams and Diamsar show good in vitroand in vivo behaviour. Possible issue of labellingconditions?

Cu diamidediamino macrocycle shows promising labellingand biodistribution in preliminary results

Cu bis(thiosemicarbazonates) can be used for the rapidRT Cu radiolabelling and show retention of specificityin vitro and in vivo. Metabolites?

V Keersemans, M Christlieb, R Muschel, S.Smart, K Hussein, GIROB, OxfordSt Thomas Hospital, WBIC CambridgeProf P Blower, Dr G. Mullen, St Thomas’ HospitalProf R Schibli, Prof H Maecke

TSB, EPSRC, Siemens, CRUK

AcknowledgementsAcknowledgements