Copper and Zirconium Radiopharmaceuticals:

Production, Properties and Clinical Applications

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

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

• 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)

Decay characteristics

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

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

Cu-62

• almost pure + emitter (98%) with short half-life of 9.7min𝛽• 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

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

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, ) or high specific activity (n,p) products𝛾

Cu-64 production

• Most commonly employed: cyclotron based

• 64Ni (p,n) 64Cu reaction

• Target: Enriched 64Ni (99.6%).

• 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

Cu-64 production: reactor

• 64Zn(n,p)64Cu

• fast neutrons are used to bombard the target in a (n,p) reaction• High specific activity

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

Production of copper isotopes and radiotracers

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 availability4. The impact of (a) and (b) on the design of imaging Methodology

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

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

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

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.

• 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

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:

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

• 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

• 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

• 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

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)

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

• 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).

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 targetneuroendocrine 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

• 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.

• 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

• 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

• 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)

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

• 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

• 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

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

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

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

• 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

• 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 inosteoarthritis or osteoporosis

osteoclasts

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.

Cu64 ATSM: Uptake & Metabolism

Nucleus

Cytochrome P450 reductase

NAD+NADHHYPOXIA

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

Responder Non-responder

Terence Z. Wong, Jeffrey L. Lacy et al, EJNM, 2007

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

• [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.

.

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

? 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.

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.

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

• 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

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

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)

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)

• 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

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

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

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

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

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

• 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

• 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

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

• 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

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)

• 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