radiolabelling biomolecules with copper · isotope availability an issue importance of targetry...
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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