copper and zirconium radiopharmaceuticals

1. Copper and Zirconium
Radiopharmaceuticals:
Production, Properties and
Clinical Applications

2. Introduction
For widespread use in medicine of any radioisotope, two factors are
essential
• availability of the isotope
• a stable and effective mode of binding with an appropriate chemical
carrier
With the progress in medical sciences, novel radiopharmaceuticals
derived from radioisotopes such as copper and zirconium have gained a
lot of attention owing to favorable above-mentioned characteristics

3. Copper
• transition metal with atomic number 29, known since ancient times
• In humans, copper plays a role as a cofactor for numerous enzymes, such
as Cu/Zn superoxide dismutase, cytochrome c oxidase, tyrosinase,
ceruloplasmin, and other proteins
• Crucial for respiration, iron transport and metabolism, cell growth, and
hemostasis

4. • Natural copper comprises two stable isotopes
• 63Cu (69.17%) and 65Cu (30.83%)
• 27 known radioisotopes
Five of them are particularly interesting for
• molecular imaging applications (60Cu, 61Cu, 62Cu, and 64Cu)
• In vivo targeted radiation therapy (64Cu and 67Cu)

6. Decay characteristics

7. Cu-60: properties
• 𝛽+ emitter with decay properties making it possible candidate tracer
for positron emission tomography (PET)
• proton-rich nuclide that decays to its stable Ni isotopes through a
combination of positron decay and electron capture processes
• can be produced on a medical cyclotron at relatively low costs using
proton or deuteron induced reactions on enriched Ni-60 targets

8. Cu-61
• can be produced from zinc, nickel or cobalt targets on a medical cyclotron
using proton or deuteron or alpha particles induced reactions
• Necessity of highly enriched Ni and Zn targets or high-energy particle
beams limited accessibility
Half-life longer than that of 60Cu and 62Cu
• makes it better choice for prolonged imaging of processes with slower
kinetics
• Much less popular in today’s biomedical studies than other copper
radioisotopes

9. Cu-62
• almost pure 𝛽+ emitter (98%) with short half-life of 9.7min
• currently the most intensively studied copper radioisotope besides
64Cu
• can be produced on a medical cyclotron using proton or deuteron
induced reactions on enriched 62Ni targets
• can also be produced indirectly through its parent 62Zn in a
62Zn/62Cu generator system: preferred option

10. Zn-62/Cu-62 generator
Parent activity 5-6 GBq:
• 93% of this activity released as 62Cu in the first 3.2mL of eluate
• However relatively short half-life of parent 62Zn
• Hence these generators operable for not more than three days

11. Cu-64
• Highly unusual radioisotope
• Decays by three processes, namely, positron, electron capture, and
beta decays
• Allows either cyclotron or reactor production
• Reactor employed method can lead to
• either low specific activity (n,𝛾) or high specific activity (n,p) products

12. Cu-64 production
• Most commonly employed: cyclotron based
• 64Ni (p,n) 64Cu reaction
• Target: Enriched 64Ni (99.6%).

13. • Typically 10–50mg is prepared and electroplated onto a gold disk
• Typically, 18.5 GBq (500 mCi) of 64Cu are produced with a 40mg
target (Bombardment time of 4 h)
• Enriched 64Ni can be 85–95% recovered and reused for future
bombardments
• Contributes to the cost-efficiency

14. Cu-64 production: reactor
• 64Zn(n,p)64Cu
• fast neutrons are used to bombard the target in a (n,p) reaction
• High specific activity

15. Cu-67
• longest living copper radioisotope
One of the most difficult to produce
• Requires fast neutron flux reactor or high-energy proton beams
• Costly 68Zn target
• 68Zn(p,2p)67Cu: requires dedicated proton beam of 20 – 70 MeV

16. Production of copper isotopes and
radiotracers

18. 60Cu, 61Cu, 62Cu and 64Cu – PET tracers
• 60Cu, 61Cu, 62Cu and 64Cu, for imaging by PET, taking into account:
1. The effect of the radioactive emission characteristics on imaging
performance
2. The impact of the radioactive decay properties on patient radiation
dose
3. Cyclotron production and availability
4. The impact of (a) and (b) on the design of imaging Methodology

19. Eur J Nucl Med Mol Imaging (2005) 32:1473–1480

20. 60Cu and 62Cu – shorter half-lives
• higher positron decay fractions
• suitable for characterising the faster kinetics of smaller tracer
molecules, such as Cu-PTSM or Cu-ATSM
61Cu and 64Cu – longer half-lives
• studying the slower kinetics of labelled peptides, antibodies
and cells
• an extended imaging period to compensate lower sensitivity
• although the loss of image contrast due to poorer counting
statistics is partially compensated by better spatial
resolution
Impact on imaging methodology

21. 64Cu - versatility
the half-life (12.7 h)
flexibility to image both
smaller molecules and larger,
slower clearing proteins and
nanoparticles
easily shipped for PET
imaging studies at sites
remote to the production
facility
decay properties
β+, 0.653 MeV [17.8 %]
→ PET imaging
β−, 0.579 MeV [38.4 %]
of 64Cu → radiotherapy
well established
coordination
chemistry
reaction with a
wide variety of
chelator systems
potentially be
linked to antibodies,
proteins, peptides
Due to the versatility of 64Cu,
abundance of novel research in this area over the past 2 decades,
primarily in the area of PET imaging, but also for targeted radiotherapy of cancer

22. Coordination Chemistry of Copper
• The aqueous coordination chemistry of copper is limited to its three
accessible oxidation states (I-III).
• The lowest oxidation state, Cu(I)
• complexes without any crystal-field stabilization energy.
• Complexes of this type are readily prepared using relatively soft, polarizable
ligands like thioethers, phosphines, nitriles, isonitriles, iodide, cyanide and
thiolates;
• however, Cu(I) complexes are typically not used for in vivo imaging
due to their lability.

23. • Copper (II)
• favours amines, imines, and bidentate ligands like bipyridine to form
various molecular geometries.
• Due to presence of some crystal-field stabilization energy,
• Cu(II) is generally less labile toward ligand exchange as compared to
Cu(I)
• the best candidate for incorporation into radiopharmaceuticals
• A third oxidation state Cu(III) is
• relatively rare and
• difficult to attain

24. Chelating Ligands for 64Cu
• High kinetic inertness to Cu(II) decomplexation are ideal
• Resistance of the 64Cu complex to Cu(II)/Cu(I) reduction
• Reduction of Cu(II) to Cu(I) and subsequent Cu(I) loss
may also be a pathway for loss of 64Cu
• Rapid complexation kinetics
• Must be designed with available functional groups
• covalently linked to targeting peptides, proteins, and
antibodies
High specific activity 64Cu with a chelator
 that forms highly stable complexes in vivo
 critical for high uptake of 64Cu in the target
Properties:

25. Chelators based on cyclam and cyclen backbones
• BFCs for 64Cu complexation and PET are subject to
stringent criteria for in vivo applications.
• Requirements such as
• High thermodynamic and kinetic stability,
• Selectivity,
• Fast complexation and
• Stability against reduction to Cu(I)
Have naturally driven the development of Cu(II) BFCs toward
polyaza macrocyclic ligands such as cyclam and cyclen

26. • Two of the most important chelators studied were
• DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)
• TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid)
• DOTA has been used as a BFC (bifunctional chelator) for
64Cu,
• its ability to bind many different metal ions,
• and its ↓ stability compared to TETA make it less than ideal
• The tetraazamacrocyclic ligand TETA therefore →
extensively used as a chelator for 64Cu,
• successful derivatization of this ligand
• allowed researchers to conjugate it to antibodies, proteins, and
peptides

27. • Although 64Cu-TETA complexes are more stable than 64Cu-DOTA and
64Cu-labeled complexes of acyclic ligands,
• their instability in vivo has been well documented
• Bass et al. demonstrated that when 64Cu-TETA-octreotide (OC) was
injected into normal Sprague- Dawley rats,
• nearly 70% of the 64Cu from 64Cu-TETA-OC was transchelated to a 35 kDa
species believed to be superoxide dismutase (SOD) in the liver
20 h post-injection

28. • it is evident that the in vivo instability of these 64Cu complexes
emphasizes
• the need for more inert 64Cu chelators.
• With this goal, new ligand systems, including those based upon the
• cross-bridged (CB) tetraazamacrocycles have been developed to complex
64Cu more stably

29. The Cross-bridged Tetraamine Ligands
• Weisman, Wong and coworkers in the 1990's,
• originally designed to complex metal cations like Li+, Cu2+, and Zn2+ within
their clamshell-like clefts
• Attachment of two carboxymethyl pendant arms to CB-cyclam to give
• CB-TE2A (4,11-bis (carboxymethyl)-1,4,8,11-etraazabicyclo[6.6.2]hexadecane)
• further ensures complete envelopment of a six-coordinate Cu(II)

31. 64Cu-BFC-peptides: advantages
In comparison to monoclonal antibodies
• good tissue distribution
• fast clearance
• low immunogenicity
• and inexpensive, automated production
Hence appear more a more attractive option for targeted PET imaging

32. • Some of the cell surface proteins that has been investigated include:
1. epidermal growth-factor receptor (EGFR),
2. somatostatin receptors(SSRs), and
3. integrin alpha v beta 3 (αvβ3).

33. Targeting Somatostatin Receptors (SSRs)
• Somatostatin
• 14 amino acid peptide
• regulation and release of a number of hormones,
• SSRs are present in many different normal organ systems such as the
CNS, GIT, and the exocrine and endocrine pancreas.
• presence of SSRs in a tumor is predictive of a good therapeutic response
• Several tumors are SSR positive making it a viable disease target
neuroendocrine system (NET),
CNS,
breast and
lung
• An 8 amino acid analog of somatostatin, octreotide (OC) has
• longer biological half-life
• several times more effective than somatostatin in the suppression of GH
secretion in animals

34. • OC was conjugated with TETA for labeling with 64Cu
• and this agent was compared with 111In-DTPA-D-Phe1-OC (111In-DTPA-OC;
Octreoscan®), a SPECT imaging agent
• Overall, 64Cu-TETA-OC and PET showed greater sensitivity for
imaging neuroendocrine tumors,
• in part due to the greater sensitivity of PET compared to SPECT.

35. • In vitro / in vivo evaluation of a second generation
somatostatin analog, 64Cu-TETA-Y3- TATE (Y3-TATE:
tyrosine-3-octreotate) were conducted.
• Y3-TATE differs from OC in that
• tyrosine (Tyr) replaces phenylalanine (Phe) in the 3-position, and
• C-terminal threonine (Thr) is an acid rather than an alcohol,
shown improved targeting of somatostatin-rich tissues
• In vivo, in CA20948 and AR42J rat pancreatic tumor
models,
• 64Cu-TETA-Y3-TATE had twice as much uptake as 64Cu-TETAOC.
• This new reagent demonstrated superior potential

36. • In vitro / in vivo evaluation of a second generation somatostatin analog, 64Cu-
TETA-Y3- TATE (Y3-TATE: tyrosine-3-octreotate) were conducted.
• Y3-TATE differs from OC in that
• tyrosine (Tyr) replaces phenylalanine (Phe) in the 3-position, and
• C-terminal threonine (Thr) is an acid rather than an alcohol,
• showed improved targeting of somatostatin-rich tissues

37. • The majority of somatostatin analogs are agonists,
• they are internalized into cells via receptor-mediated
endocytosis and mimic the behavior of somatostatin itself.
• The pervasive belief
• greater cellular internalization of a radiolabeled
somatostatin analog in vitro is a predictor of improved
tumor uptake in vivo.
• An interesting characteristic of the SSTr2 antagonist is
that it appears to
• bind to ∼15-fold higher number of receptors than the
agonist (23,000 vs 1551 fmol/mg protein),
• but with ∼17-fold decreased affinity (26 nM vs 1.5 nM)

38. Targeting Epidermal Growth Factor Receptor (EGFR)
• The epidermal growth factor family of membrane
receptors is one of the most relevant targets in the
tyrosine kinase family
• EGFR expression is increased in many human tumors such
as
 breast cancer,
 squamous-cell carcinoma of the head and neck
 prostate cancer
• Activation of EGFR contributes to
• Several tumorigenic mechanisms, and
• EGFR expression may act as a prognostic indicator, predicting
poor survival and/or more advanced disease stage

39. • Immuno-PET offers an exciting imaging option to conform and
quantify selective tumor targeting by mAbs
• This can be used as
• A prelude to Rx
• Selection of right patient pop. and Rx schedules ↑ Rx efficiency
• clues about uptake in critical healthy organ to anticipate toxicity
• Inter-patient variation in PKs and tumor targeting

40. • At present, monoclonal antibodies (mAbs),
• which block the binding of EGF to the extracellular ligand-
binding domain of the receptor and small tyrosine kinase
inhibitors, have shown promise from a therapeutic
standpoint
• Cetuximab (C225, Erbitux®) was the first mAb
targeted against the EGFR
• approved by the Food and Drug administration
• for the Rx of patients with EGFR-expressing metastatic
colorectal carcinoma
• Cetuximab binds to the extracellular domain of EGFR
• with a KD of 1nM, similar to that of the natural ligand, EGF

41. 64Cu-DOTA-cetuximab
for the high-EGFR-expressing
A431 tumor bearing nude
mice.

42. Other 64Cu-labeled mAbs for tumor targeting
• neuroblastoma and melanoma.
• The SarAr chelator was attached to the anti-GD2 mAb, 14.G2a, and its
chimeric analog, ch14.18,
• Target disialogangliosides overexpressed on neuroblastoma and melanoma.
• breast cancer
• scFv-Fc labelled 64Cu
• targeting HER2 in a breast cancer model

43. Copper-64-Labeled Integrin-Targeting Peptides
• Integrins
• transmembrane proteins → cell-cell & cell-matrix interactions
• dimers → two subunits (a & b) that have an extracellular domain
• important roles in angiogenesis and tumor metastasis.
• So far, 24 different integrins have been identified
• constituted by combinations of 18-a and 8-b subunits
• Alpha v beta 3 (avb3)
• Alpha 4 beta 1 (a4b1)
• upregulated in BM derived cells
• may be an imaging biomarker for the pre-metastatic niche,
• micoenvironment for tumor cells → distant metastases

44. • Alpha v beta 3 (avb3)
• the most widely studied integrins,
• a well known cell surface disease biomarker
• up-regulated in
1. tumor angiogenesis,
2. metastasis,
3. inflammation,
4. certain CVS abnormalities, and
5. bone resorption (osteoclasts)
• This discovery has led to the design of many RGD-based agents
• several invest. involving the 64Cu radiolabeled complexes
avb3 integrin binds specific binding 3-AA seq.
to extracell. proteins pocket that recognizes (Arg-Gly-Asp or RGD)
• Tumors
 glioblastomas
 breast
 prostate tumors
 malignant melanomas
 ovarian ca

45. • alternative to imaging angiogenesis
• imaging the up-regulation of αvβ3 in osteoclasts,
• high numbers in osteolytic bone metastases.
• Osteoclasts → ↑αvβ3 integrin compared to other normal cell
• Studies have proposed 64Cu-CB-TE2A-c(RGDyK)
• for detecting osteoclasts in osteolytic bone lesions.
• Selective targeting of 64Cu-CB-TE2A-c(RGDyK)
was demonstrated ex vivo in cell studies using
• αvβ5-positive bone marrow macrophages (BMM) and
• αvβ3-positive osteoclasts → 2.6-fold increase
open the possibility of other applications for imaging avb3 in
osteoarthritis or osteoporosis
osteoclasts

47. Imaging Tumor Hypoxia with 64Cu-ATSM
• There is one class of copper radiopharmaceuticals
• where the stability of the Cu(II) complex is not essential for
successful targeting
• Cu(II) thiosemicarbazones have been evaluated as
blood-flow agents &
imaging tumor hypoxia
• hypoxia can result in
• tumor aggressiveness
• failure of local control
• activation of transcription factors that support cell survival and
migration
• The ability to locate and quantify the extent of hypoxia
• early diagnosis and
• most appropriate treatment for each individual
• determinant of response of the tumor to conventional therapy.

48. Cu64 ATSM: Uptake & Metabolism
Nucleus
Cytochrome P450 reductase
NAD+NADH
HYPOXIA
NORMOXIA
Cu(I)64
Cu(I)64
Cu(II)64
Binding due to –ve charge only under hypoxic
 reduction of Cu(II) to Cu(I) → trapped in hypoxic tissue but
 not in normal mitochondria
 not retained in necrotic tissue

49. Responder Non-responder
Terence Z. Wong, Jeffrey L. Lacy et al, EJNM, 2007

50. Cu64-ATSM/PET in cervical cancer
Cu64-ATSM/PETFDG/PET
Dehdashti .F, Grigsby P.W. Int J Radiat Oncol Biol Phys. 2003; 55(5):1233-8

51. • [64Cu ]Copper(II)-diacetyl-bis(N4-methylthiosemicarbazone (64Cu-
ATSM) was developed as an alternative to [18F]FMISO.
• Overall this agent shows
• rapid delineation of tumor hypoxia and higher tumor to background ratios
(≫2.0)
• thus showing advantages over [18F]FMISO.
.

52. acute vs. chronic tumor hypoxia
• Lewis et al.
• first clinical comparison between 60Cu-ATSM and 64Cu-
ATSM (and [18F]-FDG) in ca cervix
• after Cu-ATSM was approved for study as an investigational
new drug (IND 62,675)
• tumor uptake of Cu-ATSM as measured in images
recorded between 1 and 9 days was reproducible
• showed that Cu- ATSM is a marker for chronic tumor
hypoxia, a opposed to acute hypoxia.
• predictive of the likelihood of disease-free survival post-
treatment in patients with cervical cancer

53. ? universal hypoxia marker
• Yuan et al evaluated 64Cu-ATSM as a hypoxia agent
• This group studied the uptake and immunohistochemistry in
• mammary adenocarcinomas,
• fibrosarcomas and
• gliomas.
• determined that high tumor to muscle ratios
• observed for the adenocarcinomas and gliomas,
• this was not the case for the fibrosarcomas
• This shows that although Cu-ATSM may be a valid hypoxia agent for some tumor types,
• this cannot be extrapolated to all tumors and
• hence this may not be a universal hypoxia marker.

54. Cu-64 targeting vectors
based upon bombesin peptide
• Preparations of new Cu-64 targeting vectors based upon BBN
• resulted in conjugates with biodistribution profiles that are ideal for microPET
imaging.
• MicroPET molecular imaging of xenografted tumor models has
revealed
• focal sites of radioactivity uptake and retention
• with relatively high resolution and low background contrast.

55. Cu-64 Imaging in Wilson disease
• Increased accumulation of copper in liver, brain, kidneys and ocular
tissue
• Produced by mutational defects of a protein (ATP7B) that transports
copper from the liver to the bile for excretion
• Poor incorporation of copper into ceruloplasmin and impaired biliary
copper excretion

57. • Some WD patients
• Copper accumulation in the brain ( gliosis and neuronal loss) and
show neurological disorder, while others do not
• 64CuCl2 PET may change the management of WD
• Could reflect the symptoms variation of WD and thus affect the
treatment strategy

58. ImmunoPET
• (PET) imaging with radiolabeled monoclonal antibodies has been a
dynamic area in molecular imaging
• With decay half-life (3.3 d) well matched to the circulation half-lives of
antibodies (usually on the order of days)
• PET with radiolabeled monoclonal antibodies (mAbs), sometimes termed
as “immunoPET”, is an attractive method for non-invasive tumor detection
• combines the high sensitivity of PET with the high antigen specificity of
mAbs

59. ImmunoPET requires that
• The PET isotope can be attached to the mAb with good in vivo
stability
• The decay half-life of the isotope should match the pharmacokinetics
of the mAb
• some of the commonly used PET isotopes for antibody labeling
include 89Zr (t1/2 = 3.3 d), 124I (t1/2 = 4.2 d), 64Cu (t1/2 = 12.7 h),
86Y (t1/2 = 14.7 h)

60. Zirconium
• transition metal in Group IVB of the periodic table
• The radioisotope of interest Zr-89, decays by positron emission (23%)
and electron capture (77%) to Y-89, with t1/2 = 3.3 days (Emax = 897
keV and Eave = 396 keV)

61. • Ideally, a positron emitter should not have prompt photons with
energy near 511 keV in order to achieve better quantitative accuracy.
• The spontaneous gamma decay of 89Zr gives rise to 908.97 keV
photons (99% abundance)
• Can be easily gated off by setting the energy window of a PET scanner

62. Advantages of Zr-89
• no need to use highly enriched target
• energy required for 89Zr production is lower
• 124I linked directly to mAbs via tyrosine residues can be subjected to
dehalogenation in vivo, which can lead to significant radioactivity
uptake in non-targeted organs unrelated to mAb-antigen binding
mAb -> internalization
• rapid loss of 124I -> lysosomal proteolysis of the mAb
• accumulation of radiometals such as 89Zr

63. Zr-89 Production
Cyclotron based
• Y-89 (p, n) Zr-89
• Y-89 (d, 2n) Zr-89
• Proton beam with 14-14.5 MeV energy
• Inexpensive natural yttrium foil mounted onto an aluminum/copper
disc
• Bombardment duration: 2-3 h

64. PET Imaging with Zr-89
• a wide variety of mAbs have been labeled with 89Zr and several of
these 89Zr-labeled mAbs have entered clinical investigation with
promising results
Currently most extensively studied receptor types of Zr-89 labeling
strategies
• HER2
• EGFR
• VEGF
• PSMA

65. Zr-89 HER2 Imaging
Human epidermal growth factor receptor 2 (HER2)
• a member of the ErbB tyrosine kinase receptor family
• Involved in cell survival, differentiation, proliferation, metastasis, and
angiogenesis
• Overexpression of HER2 has been found in a wide variety of human
cancers

67. • Menard et al
• Annals of Oncology 12 (Suppl. I): S15-S19, 2001

68. • Currently, HER2 overexpression is determined using immunohistochemistry
(IHC) and fluorescence in situ hybridization (FISH) at the time of diagnosis
of the primary tumor
• Trastuzumab (i.e. Herceptin), an anti-HER2 mAb that was approved by the
FDA to treat HER2-positive breast cancer, has been extensively investigated
for imaging applications over the last decade
• Noninvasive imaging of HER2 expression and localization of HER2-
overexpressing tumor lesions using immunoPET could be a practical
method in the clinic to guide HER2-targeted therapy

69. • A) 89Zr-trastuzumab PET scans of a patient already on trastuzumab
• B) 89Zr-trastuzumab PET of a patient with liver and bone metastases at 5 days post-injection

70. EGFR
• critical role in tumor cell proliferation, differentiation, and survival
• overexpression has been associated with a number of cancers such as
breast carcinoma, lung cancer, bladder cancer, and colon carcinoma
• often associated with more aggressive tumors, poor prognosis, and
resistance to treatment with cytotoxic agents

71. • Cetuximab (Erbitux, ImClone System Inc.) is a chimeric IgG1 mAb that can
block EGFR activation by binding to the ligand-binding domain, which
induces internalization of EGFR thereby preventing downstream signaling
• 89Zr-cetuximab PET was used as a scouting procedure before
radioimmunotherapy (RIT) to confirm tumor targeting and allow estimation
of radiation dose delivery to tumors and normal tissues
• could serve as a surrogate for scouting the biodistribution of 90Y- and
177Lu-cetuximab in tumor-bearing mice

72. PSMA Targeting
• PSMA expression levels have been shown to exhibit a positive
correlation with increased tumor progression, development of
castration resistance, and/or resistance to hormone-based therapies
• 89Zr-labeled anti-PSMA mAb, J591, was reported for immunoPET and
quantification of PSMA expression in vivo
• Some studies have taken advantage of the phenomenon known as
the Cerenkov effect (Ruggiero A, et al. J. Nucl. Med. 2010)

73. • Cerenkov luminescence imaging (CLI) of tumors in vivo, using a small
animal optical scanner
• The results obtained from optical scans correlated well with those
obtained from immunoPET studies in a quantitative manner