64Cu2+ Ions as PET Probe: An Emerging Paradigm in Molecular

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64Cu2+ Ions as PET Probe: An Emerging Paradigm in Molecular Imaging of Cancer Rubel Chakravarty, Sudipta Chakraborty, and Ashutosh Dash Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00582 • Publication Date (Web): 06 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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Molecular Pharmaceutics

64

Cu2+ Ions as PET Probe: An Emerging Paradigm in Molecular Imaging of Cancer Rubel Chakravarty,* Sudipta Chakraborty and Ashutosh Dash

Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

Short title: 64Cu2+ Ions as PET Probe

*To whom all correspondences must be addressed: Rubel Chakravarty, Ph.D. E-Mail: [email protected], [email protected] Phone: +91-22-25590624 Fax: +91-22-25505151

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ABSTRACT Positron emission tomography (PET) imaging has transformed diagnostic nuclear medicine and become an essential strategy in cancer management. With the expected growth of this molecular imaging modality, there is a recognized need for new PET probes to address the clinical challenges in the early diagnosis and staging of various types of cancers. In this endeavor, the prospect of using 64Cu in the form of simple Cu2+ ions as PET probe is not only a cost-effective proposition, but also seems poised to broaden the palette of molecular imaging probes in the foreseeable future. The usefulness of 64Cu2+ ions as PET probe is based on the fact that Cu is an essential element which plays an important role in cell proliferation and angiogenesis. Over the last few years, there has been continuous flow of evidences based on studies in animal models on the uptake of

64

Cu2+ ions in different types of tumors,

including, hepatoma, colorectal cancer, prostate cancer, lung cancer, breast cancer, head and neck cancer, fibrosarcoma, melanoma, glioblastoma and ovarian cancer. The widespread preclinical success of

64

Cu2+ ions as PET probe has recently resulted in translation of this

radiotracer to clinical settings for non-invasive imaging and staging of prostate cancer in human patients. In this concise review, we have focused on the latest developments in PET imaging of cancer in preclinical and clinical settings using

64

Cu2+ ion as a probe and

discussed the challenges and opportunities for future development.

Keywords: Cancer, 64Cu, Cu2+ ion, Molecular imaging, PET, Radiotracer, Theranostics

2


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INTRODUCTION Molecular imaging has become a vital tool in management of several diseases, including, cancer and is growing rapidly as one of the most exciting areas of scientific research.1-9 Not only it offers the scope for non-invasive and real time in vivo visualization of physiological processes at the cellular and molecular level in living organisms, but also provides an useful means of understanding the integrative biology of the disease process, early detection and staging of the disease, and assessment of therapeutic efficacy. Currently, various imaging modalities, such as, single photon emission computed tomography (SPECT), PET, magnetic resonance imaging (MRI), computed tomography (CT), optical imaging, ultrasound imaging, etc., are being employed to evaluate specific molecular targets in biomedical and clinical settings. 10-14 Among various molecular imaging modalities, the growth of PET imaging technology has been phenomenal over the last decade.

15-21

This is particularly because PET imaging

technology provides the scope to assess living systems by use of probes comprising of positron emitting radioisotopes with high sensitivity of measurement and accurate quantification.

17-21

This technology can aid toward improved and cost-effective decision

making and proceed only the right patients for tailored therapeutic regimens.

15

One of the

most important aspects in molecular imaging with PET is the development of appropriate PET probes,

16, 22-24

which has now engaged a growing cadre of researchers from various

scientific disciplines, including, radiochemistry, medical physics, molecular biology, engineering and medicine. The choice of the radioisotope for development of a PET probe depends on its physical and chemical characteristics, production feasibility, availability, and the timescale of the biological process which is planned to be investigated. Among various radioisotopes studied,

64

Cu (t½ = 12.7 h, E.C. 45%, β- 37.1%, β+ 17.9%) is unique as it decays by three 3



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different routes, namely, electron capture (EC), β- and β+ decays (Figure 1).

25

Due to

simultaneous emission of both β+ and β- particles, this radioisotope holds promise toward development of PET imaging probes for non-invasive visualization of diseases and can also be used in targeted radiotherapy.

25

interest toward development of new efforts from the academia.

26-36

Over the last 2 decades, there has been widespread 64

Cu-based probes for PET imaging mainly due to the

Though the potential of many of these probes could be

effectively demonstrated in preclinical settings, very few of them could actually be translated to the clinics.

37-40

Diacetyl-2,3-bis(N4-methyl-3-thiosemicarbazone) (ATSM) labeled with

64

Cu (64Cu-ATSM) is one such probe that has been extensively utilized in nuclear medicine

clinics for PET imaging of hypoxia.

41-45

PET imaging using this radiotracer provides

clinically significant information which helps in predicting the response of the cancer patients to therapeutic regimens. It has been demonstrated that

64

Cu in its ionic form (as

64

Cu2+ ions) can directly be

used as a probe for PET imaging of various types of cancers. conventional radiopharmaceuticals, utilization of

64

46-48

Unlike majority of

Cu in this form would not require

complexation of the radioisotope with expensive targeting ligands (based on peptides, antibodies, etc.), thereby, rendering the process cost-effective. The absence of the radiolabeling step would provide a unique advantage as normal radiochemical processing in a hot-cell after target irradiation would be adequate to obtain the radiotracer in the desired form for use in PET imaging studies. Also, it would not be difficult to establish the protocol under current good manufacturing (cGMP) compliant settings, which is necessary for getting regulatory approval for clinical use of this radiotracer. The recent surge of interest in the use of 64Cu2+ ions as a cost-effective PET imaging probe has been the source of motivation to present a focussed review on this topic. This article will highlight the different routes for production of

64

Cu with an aim to identify a

4


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viable mode for large-scale production of this radioisotope to meet the global demand. The mechanism of

64

Cu2+ uptake in cancerous lesions will be discussed and the promising data

obtained to date in preclinical settings will be summarized with emphasis on quantitative estimation of tumor-targeted delivery, radioactivity accumulation in normal organs and toxicity issues associated with Cu2+ ions. The preliminary clinical success in utilization of 64

Cu2+ ions as PET probe will be highlighted and limitations of these studies shall be

discussed. Given the multidisciplinary nature of this field, the authors apologize for possible oversights of important contributions. The present review is by no means exhaustive, but intended to serve as a resource not only for the scientists and technologists to offer an impetus for further development but also for clinicians, to become familiar with the expectations, capabilities, constraints, and gratifications involved in the use of 64Cu2+ ions as probe for PET imaging.

PRODUCTION OF 64Cu Copper-64 is one of the few radioisotopes, production of which is possible both in a cyclotron as well as in a nuclear reactor.

25, 49

The different routes for production of 64Cu are

summarized in Table 1 and schematically represented in Figure 2. Production of this radioisotope in a nuclear reactor is feasible by two main routes: (a) thermal neutron capture 63

Cu (n, γ)

64

Cu and (b) fast neutron capture

64

Zn (n, p)

64

Cu reactions.

49-52

Though large-

scale production of 64Cu is possible by thermal neutron capture route, the major limitation of this approach is the low-specific activity of

64

Cu, making it unsuitable for preparation of

receptor specific radiopharmaceuticals. The specific activity of 64Cu can be increased by ~ 45 % by using highly (> 99 %) enriched 63Cu target for neutron activation. It is also relevant to mention here that the specific activity of neutron activated 64Cu can be substantially increased (up to 1000 times) by adopting the Szilard Chalmers process.53, 5


54

However, the specific


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activity of

64

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Cu attained by adopting these approaches would still not be adequate for

preparation of conventional target specific radiopharmaceuticals. Moreover, Szilard Chalmers process suffers from several inherent limitations and cannot be used for large-scale production of 64Cu to meet the routine clinical requirements. On the other hand, high specific activity 64Cu in a ‘no carrier added’ (NCA) form can be produced by irradiation of reactor core.

50

64

Zn target with fast neutrons in the fast flux position of the

Copper-64 produced by this route can easily be utilized for radiolabeling of

receptor specific biomolecules for PET imaging of tumors. However, the fast flux position at the reactor core is generally not accessible for radioisotope production at majority of research reactors in the world.25, 49 Additionally, there are limitations on sample volume that can be irradiated which in turn put restrictions on the activity of

64

Cu that can be produced.

Therefore, this approach is not suitable for industrial-scale production of

64

Cu to meet the

regular demands of the nuclear medicine clinics. Copper-64 can also be produced in a NCA form by 64Ni (p, n) 64Cu nuclear reaction in a cyclotron. 49, 55-57 Nowadays, ‘state-of-the-art’ automated modules are available for fast and highly efficient separation of 64Cu from 64Ni and other extraneous radioisotopes adopting ion exchange chromatography. date have utilized

64

58-60

Majority of the preclinical and clinical studies reported to

Cu produced by this approach in a cyclotron.

25, 61

For developed

countries with excellent cyclotron facilities, this is probably the most practical option for production of

64

Cu for regular use in clinical context. However, this strategy may not be

viable in many developing nations with insufficient or no cyclotron facilities suitable for 64Cu production. Owing to the relatively short half-life of 64Cu, it is not logistically favourable for transportation of

64

Cu from limited number of cyclotron production facilities to distant user

sites, thereby restricting worldwide utility of this radioisotope for preclinical and clinical studies. 6


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For nations which have medium flux nuclear reactors for production of radioisotopes, the issue of unavailability of cyclotron facilities for 64Cu production might not be so severe, especially when imaging.

52

64

Cu is intended to be used in the form of Cu2+ ions as probe for PET

According to the database of the International Atomic Energy Agency (IAEA),

there are ~ 80 medium flux (1 × 1013 - 1 × 1014 n.cm-2.s-1) research reactors in the world already involved in radioisotope production.

62

Owing to their excellent geographic

distribution, many of these research reactors might be utilized for large-scale production and supply of neutron activated 64Cu to meet the regional demand of this radioisotope for use in PET imaging. Utilization of both research reactors as well as cyclotrons for production of 64

Cu would significantly increase the availability of this radioisotope throughout the world

and enhance its popularity in clinical context. After radiochemical processing of the irradiated target (irrespective of the production route),

64

Cu is generally available in the form of

64

CuCl2 (or

64

Cu2+ ions) solution which is

slightly acidic. Before administration in living subjects, it is diluted in saline or phosphate buffered saline medium to achieve pH in the range 6-7.

MECHANISM OF UPTAKE OF 64Cu2+ IONS IN CANCER CELLS Copper ions are essential for multiple biological processes and are indispensable to life.

63-66

They are required as catalytic cofactor of many enzymes or as important structural

component for proteins, and thus play significant roles in crucial biological functions such as biosynthesis of neurotransmitters, mitochondrial respiration, detoxification of free radicals, construction of connective tissues and blood vessels, enzyme activity, transport of oxygen and cell signalling.

65, 66

Even a slight disparity in the bioavailability of copper ions through

genetically inherited mutations or transformed environmental circumstances results in deficiency or toxicity, which might lead to adverse pathological effects. 7


65

Hence, it is


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essential to maintain a tight control over the homeostasis of copper ions in the body to sustain a healthy life. According to an estimate, a healthy adult human being consumes 0.6 - 1.6 mg of copper daily through his diet. 63 The mechanism by which Cu2+ ions are taken up by human cells has not yet been fully understood. The present opinion is that the liver is the main organ which regulates the status of copper ions in the body, managing their distribution to serum and tissues.

63-67

The

clearance of excess copper ions from the biological system primarily takes place through the hepatobiliary route. In the body, Cu2+ ions are bound to plasma proteins (ceruloplasmin albumin, and transcuprein), which carry them to the cell surface where the enzymes (reductases) reduce Cu2+ ions to Cu+ ions, before their uptake into cells (Figure 2). In its reduced form, Cu+ ions are then transported across the cell membrane by the human copper transporter 1 (hCTR1), a 190-amino-acid protein of 28 kDa with 3 transmembrane domains with high affinity for copper (Figure 2). 63, 67 After entry into the cell, the copper ions are tightly bound by copper chaperones [cytochrome c oxidase copper chaperone (COX17), copper chaperone for SOD1 (CCS), and antioxidant protein (ATOX1)], thus keeping them in bound Cu+ state by preventing redox cycling.

63, 67

These copper chaperones deliver Cu+ ions to cytosolic SOD1, cytochrome c

oxidase (COX) in the mitochondria and to copper transporting ATPase A/B (ATP7A/B) at the trans-Golgi network (TGN), respectively (Figure 2).

63, 67

Interestingly, glutathione

(GSH) - a small cysteine containing tripeptide present inside the cell plays a safeguarding role by binding excess Cu+ ions to prevent oxidative damage due to redox cycling and thus protect the cell from copper toxicity.

63, 67

Analogous defensive mechanisms are carried out

by metallothioneins (MTs), a family of cysteine rich small proteins, capable of irreversibly binding Cu+ ions. 63, 67 When intracellular copper is elevated beyond a certain level, hCTR1 is internalized and is then destroyed. During this process, copper transporting ATPase A/B 8


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(ATP7A and ATP7B) transfer from the TGN to the plasma membrane to help in excretion of copper from the cell. Since copper plays an important role in many cellular functions, it is involved not only in cancer development but can also aid toward cancer growth, angiogenesis and metastasis.

63, 67, 68

Several researchers have reported in the past that an elevated level of

copper accumulation is observed in malignant tissues.

63, 67

This is particularly because

hCTR1 is generally overexpressed in several cancer cells, which include, prostate cancer, lung cancer, glioblastomas, liver cancer, breast cancer, and melanoma.

46, 48, 63, 67

Based on

this knowledge, it was proposed that hCTR1 could be used as a promising target for molecular imaging of a wide variety of cancers. 63, 67, 68 Increased copper uptake in the cancer cells could easily be tracked in vivo using radioactive copper (64Cu2+) ion as a radiotracer. This forms the basis of the proposition that 64Cu2+ ion can serve as an effective biomarker for non-invasive assessment of cancer using PET imaging technology. Although increased 64Cu2+ ion uptake in cancerous lesions could be described on the basis of overexpression of hCTR1, it is pertinent to point out here that no attempt has yet been made to understand the exact chemical form of the administered copper ions in blood serum. The in vivo behavior of Cu2+ metal ions can be theoretically understood by speciation modelling of blood serum,

69

and can be experimentally corroborated by various analytical

techniques. 70 Such studies would aid toward exact determination of copper species formed in blood serum after in vivo administration of Cu2+ ions, investigation of the processes leading to their formation or conversion and assessment of these processes in terms of kinetic and equilibrium parameters. The viable modes for large-scale production of 64Cu and its utilization in the form of Cu2+ ion for tumor targeting are schematically illustrated in Figure 2.

9



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PRECLINICAL STUDIES USING 64Cu2+ IONS AS PET PROBE Over the last 10 years, there have been numerous reports on utilization of 64Cu2+ ions as probe for PET imaging in mouse models of cancers, including, hepatoma, colorectal cancer, prostate cancer, fibrosarcoma, melanoma, ovarian cancer, glioblastoma, head and neck cancer, lung cancer and breast cancer. 46-48 The high-affinity mouse copper transporter 1 (mCTR1) plays the same role as hCTR1 and is responsible for increased cellular copper uptake in murine cancers.

46-48

Most of these reported studies were based on the use of

cyclotron produced NCA 64Cu. It was only recently that the utility of neutron activated 64Cu produced in a research reactor could be demonstrated for cancer targeting.

52

These

preclinical studies exploring the utility of (a) cyclotron produced and (b) reactor produced 64

Cu in the form of Cu2+ ions for non-invasive assessment of cancers are summarized in

Table 2 and elaborated in the following text. Cyclotron Produced 64Cu2+ Ions as PET Probe for Cancer Imaging The utility of cyclotron produced NCA 64Cu2+ ion as a probe for cancer imaging was first reported more than 10 years back by Peng et al.

71

In this study, the authors utilized

64

Cu2+ ions as probe for PET imaging of hepatoma xenografts in mouse model. In vivo PET

imaging studies after intravenous administration of the radiotracer showed significantly higher tumor uptake in comparison to the uptake in the muscles at 1 h post-injection (p.i.). However, the PET images also showed high radioactivity uptake in the liver, probably due to significant level of mCTR1 expression in this organ. Though this preliminary study revealed that this radiotracer was not suitable for detection of primary hepatic cancer, it might be a practical choice for revealing extrahepatic metastasis of the primary cancer in the organs or tissues (such as bones, muscles and brain), which otherwise show much lower uptake of 64

Cu2+ ions.

10


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The same group of authors also utilized prostate cancer xenograft model in mice.

47

64

Cu2+ ions as PET probe for imaging of

In vivo PET imaging and biodistribution studies

showed significant accumulation of radioactivity (4.4 ± 1.1 %ID/g) in the tumor at 24 h p.i., with good target-to-background contrast (Figure 3A). As expected, a high uptake (17.5 ± 3.9 %ID/g) of radioactivity in the liver was observed due to elevated level of mCTR1 expression in this organ. Due to high liver uptake, this radiotracer might not be a suitable choice for detection of hepatic metastases of prostate cancer. Also, it was found that this radiotracer cleared from the system mainly through hepatobiliary route, which might lead to issues related to visualization of abdominal metastases. To corroborate the findings of PET imaging studies, the authors carried out immunohistochemistry study with hCTR1-specific antibody and showed significant level of hCTR1 immunoreactivity in the tumor tissue (Figure 3B). This study was further pursued by the same group of authors to investigate whether enhanced uptake of

64

Cu2+ ions in prostate cancer was indeed due to hCTR1 mediated pathway, or

simply because of non-specific binding of

64

Cu2+ ions to tumor tissue.

72

The authors also

evaluated the function of hCTR1 in proliferation and growth of prostate cancer. For this purpose, a lentiviral vector encoding short-hairpin ribonucleic acid (RNA) specific for hCTR1 (lenti-hCTR1-RNA) was made for RNA interference-mediated knockdown of hCTR1 expression in prostate cancer cells. The authors determined the extent of hCTR1 knockdown by Western blot analysis, and examined the outcome of hCTR1 knockdown by in vitro evaluation of 64Cu-uptake in prostate cancer cells and cell proliferation assay. The results of these studies suggested that RNA interference-mediated knockdown of hCTR1 was responsible for decreased uptake of 64Cu in prostate cancer cells and the inhibition of cancer cell proliferation. Further, the consequence of hCTR1 knockdown on the uptake of

64

Cu2+

ions in the tumor was established by in vivo PET imaging and biodistribution studies in mice bearing prostate cancer xenograft (DU-145 and PC-3 tumors). These studies revealed that the 11



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uptake 64Cu2+ ions in the tumors with knockdown of hCTR1 (4.02 ± 0.31 %ID/g in PC-3 and 2.30 ± 0.59 %ID/g in DU-145) was considerably lesser than the radioactivity uptake in the control tumors without knockdown of hCTR1 (7.21 ± 1.48 %ID/g in PC-3 and 5.57 ± 1.20 % ID/g in DU-145) at 24 h p.i. (Figure 3C). Additionally, the volumes of tumors with knockdown of hCTR1 (179 ± 111 mm3 for PC-3 or 39 ± 22 mm3 for DU-145) were much lesser than those without knockdown of hCTR1 (536 ± 191 mm3 for PC-3 or 208 ± 104 mm3 for DU-145). Though the authors could not explain the exact mechanism of inhibition in tumor growth by hCTR1 knockdown, the promising results obtained in this study amply demonstrated that hCTR1 is a promising target for PET imaging of prostate cancer using 64

Cu2+ ions as probe. The utility of

64

Cu2+ ions as a PET radiotracer for non-invasive imaging of hCTR1

overexpression in breast cancer xenograft model was reported by Kim et al. 46 In this study, a lentiviral vector intrinsically expressing the hCTR1 gene was used to infect human breast cancer cells (MDA-MB-231) and positive clones (MDA-MB-231-hCTR1) were chosen for the study. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was confirmed by reverse transcription polymerase chain reaction, Western blot assay, and cellular 64Cu uptake level. The radiotracer was intravenously administered in mice bearing MDA-MB-231 and MDA-MB-231-hCTR1 tumor xenografts. In vivo PET imaging studies showed an increased uptake of 64Cu in MDA-MB-231-hCTR1 tumors (5.373 ± 1.098 %ID/g) than in MDA-MB231 tumors (2.581 ± 0.254 %ID/g) at 48 h p.i. of the radiotracer (Figure 4A). These results were further corroborated by immunohistochemistry study which showed that there was negligible hCTR1 immunoreactivity signal in the MDA-MB-231 tumor tissue, while there was a mixed type of hCTR1 expression in the MDA-MB-231-hCTR1 tumor tissue as indicated by intense spots on some of the cells (Figure 4B). The results of this study further established the potential utility of hCTR1 gene for cancer diagnosis using PET. 12


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In another interesting study, Jorgensen et al determined the degree of

64

Cu2+ ion

accumulation in xenograft models of different types of human cancers, such as, U87MG (glioblastoma), HT29 (colorectal cancer), H727 (neuroendocrine lung cancer), FaDu (head & neck cancer), and A2780 (ovarian cancer).

73

In vivo PET imaging studies showed almost

comparable uptake of the radiotracer in U87MG (3.48 ± 0.35 %ID/g), H727 (3.13 ± 0.12 %ID/g) and FaDu (3.04 ± 0.18 %ID/g) tumors at 1 h p.i., while the uptake in HT29 (2.20 ± 0.18 %ID/g) and A2780 (1.53 ± 0.07 %ID/g) tumors were significantly lower at the same time-point (Figure 5). The authors also compared 64Cu2+ ion accumulation in tumor tissue to hCTR1 expression in the different types of tumors by quantitative real-time polymerase chain reaction measurement. Despite differences in 64Cu2+ ion uptake in the tumor models studied, no definite relationship between the gene expression of hCTR1 and 64Cu2+ ion accumulation could be found. The potential of

64

Cu2+ ions as a radiotracer for both PET imaging and targeted

radiotherapy of malignant melanoma was evaluated for the first time in preclinical settings by Qin et al.

48

In this study, the authors selected two melanoma cell lines (melanotic B16F10

and amelanotic A375M) and confirmed the high level of hCTR1 expression by Western blot analysis. As expected, 64Cu2+ ions showed significantly elevated and specific uptake in both B16F10 as well as A375M cells. The radiotracer was administered in mice bearing melanoma tumors (B16F10 and A375M) and subjected to in vivo PET imaging after different time intervals p.i. (Figure 6A). The PET imaging studies revealed significant radioactivity uptake (~ 4 %ID/g at 4 h p.i.) in both B16F10 and A375M tumors, with good target-to-background ratio. The radioactivity accumulation in the tumor then remained at comparable levels until 72 h p.i. (3.48 ± 0.34 %ID/g), while uptake in the non-targeted organs decreased significantly. On the basis of the promising results obtained from the PET imaging studies, the authors further studied the therapeutic efficacy of 64Cu2+ ions in melanoma (B16F10 and 13



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A375M) tumor bearing mice. For this purpose, mice bearing melanoma tumors were intravenously administered with

64

Cu2+ ions (~ 74 MBq) or phosphate-buffered saline (as

control) and the tumor sizes were examined over the period of therapy. This study showed that the growth of both B16F10 and A375M tumors under

64

Cu2+ ions therapy was much

slower than what was observed in the control group (Figure 6B), thus, establishing the efficacy of this radiotracer for cancer theranostics. In a similar study, Ferrari et al studied the theranostic potential of 64Cu2+ ions in mice bearing glioblastoma tumor (U87MG) xenograft.

74

In vivo PET imaging and biodistribution

studies demonstrated significant tumor uptake [maximum standardized uptake value (SUVmax) = 3.6 ± 0.44] with good target-to-background ratio. In therapy studies, the authors divided the tumor bearing mice into 3 groups as follows: (a) mice not administered with 64

Cu2+ ions (non-treated or control group), (b) mice treated with a single administration of

333 MBq of

64

Cu2+ ions (single-dose group), and (c) mice treated with a multiple-doses of

64

Cu2+ ions (multiple-dose group), each of 55.5 MBq administered daily over 6 days. The

theranostic potential of the radiotracer was examined by performing PET imaging at 1 week and 20 weeks after completion of therapy. From the PET images, the reduction in tumor volume was estimated semi-quantitatively by a direct calculation of the volume of interest (VOI). The authors observed a significant reduction in tumor volume (ranging from 70 - 90 %) over the control group in both single-dose group as well as multiple-dose group. Also, a significant increase in survival rates in mice treated with

64

Cu (both single-dose group and

multiple-dose group) was observed in comparison to the survival rate in the control-group (‫݌‬ < 0.005). However, not much difference in therapeutic efficacy was observed between the results of single- and multiple-dose administration and the relative advantages or disadvantages of one therapeutic protocol over the other could not be established.

14


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Reactor Produced 64Cu2+ Ions as PET Probe for Cancer Imaging Recently, Chakravarty et al. demonstrated the utility of 64Cu (in the form of Cu2+ ions) produced by neutron activation route in a medium flux research reactor for cancer imaging. 52 Since the specific activity of

64

Cu produced by this route is low (~ 3 TBq/g), it was

anticipated that administration of relatively large quantities of Cu2+ ions in the biological system might lead to aggregation of serum proteins incorporating such aggregates after in vivo administration of

64

Cu.75-77 Formation of

64

Cu2+ ions would lead to high radioactivity

uptake in the reticuloendothelial system (RES). Under such situation,

64

Cu-incorporated

protein aggregates would be excreted mainly through the hepatobiliary route and negligible radioactivity accumulation in the tumor would be observed. This issue of aggregation of proteins present in blood serum 75-77 was not a matter of serious concern in earlier preclinical studies which used cyclotron produced NCA

64

Cu. This is because of the extremely low

concentration of Cu2+ ions in NCA 64Cu, which are not expected to play any significant role in protein aggregation. In order to further understand the phenomenon of serum protein aggregation in presence of Cu2+ ions, dynamic light scattering (DLS) studies were performed after addition of different concentrations of Cu2+ ions in mouse serum. 67,78 The DLS studies demonstrated that aggregate formation would occur at a concentration > 20 µg Cu / mL of serum. This observation was further confirmed by examination of the dispersion behavior (adopting magnetic T2 relaxation time measurement and radio-thin layer chromatography assay) of the mouse serum medium containing Cu2+ ions (Figure 7). From these studies, it could be inferred that 64Cu2+ ions at a concentration ≤ 20 µg Cu / mL would not lead to aggregation of serum proteins. Based on these results, the authors argued that the radioactivity corresponding to this threshold concentration of Cu2+ ions was adequate for PET imaging with decent contrast in both preclinical and clinical settings and there should not be any 15



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technical issue toward utilization of low specific activity

Page 16 of 48

64

Cu2+ ions as probe for cancer

imaging. In order to demonstrate the tumor targeting efficacy of low specific activity

64

Cu2+

ions, the radiotracer was intravenously administered in melanoma tumor bearing mice and fibrosarcoma tumor bearing mice.

52

The biodistribution studies showed significant

accumulation of radioactivity in the tumor (7.64 ± 1.61 %ID/g in melanoma, 6.54 ± 1.41 %ID/g in fibrosarcoma) within 4 h p.i., with good target-to-background ratio (Figure 8). It is interesting to note that the biodistribution pattern observed after administration of low specific activity

64

Cu2+ ions was comparable to that observed with NCA

64

Cu2+ ions. This

could probably be attributed to the transporter protein (hCTR1) based uptake mechanism of Cu2+ ions, as discussed earlier. However, it is essential to assess the role of specific activity of 64Cu2+ ions on tumor uptake as such a study might establish whether cold Cu2+ ions present in the radioactive solution have any saturation effect with regards to tumor uptake or exhibit toxicological issues. Such a study might also aid in determining the threshold specific activity limit of reactor produced 64Cu in order to use this radioisotope in the form of its divalent ions as PET probe for cancer imaging. Nevertheless, the encouraging results obtained in this preliminary study might enable extensive use of low specific activity 64Cu for non-invasive imaging of cancer.

CLINICAL STUDIES USING 64Cu2+ IONS AS PET PROBE The utilization of 64Cu2+ ions as an effective probe for PET imaging of human cancer has begun to be explored in the nuclear medicine clinics only recently. Presently, there are very few published reports on clinical deployment of 64Cu2+ ions directly as a radiotracer for cancer imaging and all the clinical studies reported to date have used NCA 64Cu produced in a cyclotron.

79

The first clinical study on utilization of

64

Cu2+ ions as a PET probe was

16


Page 17 of 48

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reported by Capasso et al., imaging using

64

80

wherein the authors have studied the applicability of PET-CT

Cu2+ ions as probe in staging of prostate cancer patients. In this

investigational clinical trial, seven prostate cancer patients (confirmed by histological studies performed earlier) were enrolled. These patients also undertook MRI to confirm the disease. Among these patients, three went through adrenal deprivation therapy (ADT) at time of PET imaging, while the other four patients did not undergo any therapeutic procedure. In all these patients, ~ 339 MBq of 64Cu2+ ions were intravenously administered and PET-CT scans were performed. The PET-CT scans showed that the radiotracer cleared mainly through the hepatobiliary route and excretion through the renal route was bare minimum. This is in fact an undesirable phenomenon because possible cancerous lesions in the pelvic area and abdomen cannot easily be identified by PET imaging. Primary prostate cancer lesions could be detected within 1 h p.i., with appreciably good tumor-to-background ratio. The radiotracer uptake was higher (SUVmax = ~ 7 at 1 h p.i.) in primary tumors of patients without ADT than in patients who had undergone this therapy (SUVmax = ~ 4 at 1 h p.i.). No undesirable or clinically measurable pharmacologic effect was observed in any of these patients. The major limitation of this study was that a small number of patients were enrolled and the PET imaging data were not validated by histological evaluation. However, this preliminary study demonstrated the potential of PET-CT imaging using 64Cu2+ ions as probe for initial staging of prostate cancer patients. Recently, Panichelli et al demonstrated the utility of 64Cu2+ ions for PET imaging of brain tumors in patients affected by glioblastoma.

81

In this study, 19 patients with a known

history and radiological confirmation of cerebral tumors were registered. After initial cerebral MRI, patients were administered with

64

Cu2+ ions (13 MBq/kg). PET-CT imaging of brain

clearly showed the cancerous lesions at 1 h p.i., with steady retention of radioactivity up to 24 h. Good concurrence was found between the results obtained from PET-CT imaging and 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MRI. As expected, the radioactivity cleared quickly from the blood and was mainly excreted through hepatobiliary route. The results of this preliminary clinical study showed the potential of

64

Cu2+ as a diagnostic tracer for glioblastoma multiforme. Interestingly, in a

recent review, PET-CT images from patients with prostate and brain tumors administered with

64

Cu2+ ions were reported.

79

Although, the details of these studies and the clinical

implications of these results were not provided, the PET images reported are certainly outstanding and holds tremendous promise, mainly because they demonstrate the efficacy of simple

64

Cu2+ ions as an effective radiotracer for PET imaging of cancers with excellent

contrast (Figure 9).

TOXICOLOGICAL CONCERNS ON IN VIVO ADMINISTRATION OF 64Cu2+ IONS FOR PET IMAGING An obvious concern while using

64

Cu2+ ions as probe in PET imaging of cancer in

human subjects might be the cytotoxicity of copper.

82-86

Even though copper is a necessary

element that is vitally important for human health, overexposure of copper ions can produce a wide spectrum of side effects.

85, 86

This is particularly because excess copper ions can

contribute toward Fenton-type reaction, resulting in formation of radical species that can cause oxidative stress and consequent damage to lipids, proteins, and nucleic acids in the living system. 85-88 It has been reported that the cytotoxic effects of copper ions are apparent only at concentration ≥ 7.42 mg/L.

85, 86

Considering this as the threshold limit, cytotoxicity effects

are not a matter of serious concern while using 64Cu2+ ions for PET imaging of cancer. 52 For PET imaging in human patients with reasonably good tumor-to-background ratio, only 185 370 MBq (5 - 10 mCi) of 64Cu2+ ions is expected to be administered. 40, 80 This clinical dose required essentially translates into few ng of Cu2+ ions when cyclotron produced NCA 64Cu is 18


Page 18 of 48

Page 19 of 48

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used while it is < 0.5 mg of Cu2+ ions when low specific activity 64Cu produced in a research reactor is used for PET imaging.

52

Hence, after intravenous administration of the dose of

Cu2+ ions required for PET imaging, their overall concentration in human blood would be significantly below the cytotoxic limit since the average volume of blood in a human adult is in the range of 4-5 L.

52

Thus, it could be argued that the level of Cu2+ ions in clinically

relevant doses of the radiotracer when administered in vivo would not manifest cytotoxicity 52, 89

effects.

Safe usage of 64Cu2+ ions for cancer imaging using PET was also proven by the

earlier utilization of this radiotracer for evaluation of copper metabolism in healthy human beings and in patients with copper metabolism problems without observing significant side 90-92

effects.

Systemic toxicity or clinically detectable pharmacologic effects were also not

observed in prostate cancer patients who were administered with 64Cu2+ ions,

80

thus, further

alleviating toxicity concerns regarding use of this radiotracer for clinical PET imaging.

SUMMARY AND FUTURE DIRECTIONS A review of the PET imaging of cancer using 64Cu2+ ions as probe indicates that the field is still in its infancy but is rapidly evolving. This non-invasive molecular imaging approach not only holds significant promise in the detection of primary cancer and its metastases, but can also aid toward accurate staging of cancer and examining the efficacy of the treatment. With further maturity in understanding of the molecular mechanisms of cancer and biochemical phenomenon of copper ion uptake, this approach is supposed to play a progressively important role in cancer management. Recently, the concept of PET imaging using

64

Cu2+ ions as probe has been translated to clinical settings and successfully used for

primary staging of prostate cancer patients. This recent clinical success indicated that more clinically translatable advances can be expected in near future which would lead to a positive impact on care of millions of cancer patients worldwide. 19



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Page 20 of 48

Despite immense potential of 64Cu2+ ions, the cost of a given probe and the logistics associated with a particular radionuclide production route needs to be considered suitably. Though the cyclotron route remains the most viable approach for production of NCA 64Cu for clinical use, simultaneous utilization of several medium flux research reactors located in different parts of the world for production of this radioisotope would enhance the global availability of this radioisotope. The low specific activity of neutron activated 64Cu is not a matter of concern when directly used as a radiotracer in the form of Cu2+ ions for PET imaging. Probably, this is the only approach by which reactor produced low specific activity 64

Cu can be effectively utilized in clinical context. With the availability of neutron activated

64

Cu, clinical use of this PET probe would intensify in future. Direct utilization of

64

Cu2+ ions as a PET probe would offer numerous advantages

over conventional PET-radiopharmaceuticals which involve radiolabeling of complex biomolecules and is poised to lower the cost of molecular imaging of cancer. The unique phenomenon of nuclear decay by emission of both β+ as well as β- particles makes

64

Cu an

ideal candidate for theranostic approaches to cancer management. Though the potential of 64

Cu2+ ions for cancer theranostics have been successfully demonstrated in preclinical

settings, detailed radiation dosimetry and toxicity assessments would be required before administering therapeutically relevant doses of this radiotracer in human subjects. Over the last few years, there is an increasing trend toward implementation of cGMP regulations for PET radiotracer preparations. With a view to achieve such a goal, a systematically planned and appropriately executed quality assurance system is of paramount importance. Also, to be successful in addressing the regulatory issues,

64

Cu production

technologies must be tailored to local legislative, regulatory and institutional conditions. In this premise, utilization of automated radiochemical processing modules will represent an

20


Page 21 of 48

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intuitive vision as automation technology not only guarantees reproducible yield and purity but also complies with regulatory protocols. While most of the studies involving use of

64

Cu2+ ions directly as a radiotracer for

PET imaging remain at a proof-of-concept phase, the potential of this strategy for personalized cancer management in humans is exceedingly attractive. The progress toward the widespread clinical adaptation of this innovative approach is slow as of now but can definitely be expedited by addressing the interdisciplinary challenges and overcoming the biological barriers with concerted efforts of radiochemists, biologists, medical physicists, molecular imaging scientists, clinicians, and the regulatory authorities.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 48

Table 1: Comparative assessment of different routes for production of 64Cu

Production

Target

mode

Nuclear

Cross

Neutron

Specific

Possible

Viability

Refere

reaction

section

flux/

activity

radio-

for large

nces

(b)

beam

(TBq/g

nuclidic

scale

current

of Cu)

impuriti

producti

es

on

(Reactor/ cyclotron)

Reactor

63

Cu

63

13

Cu (n,

Thermal

10 -

γ) 64Cu

4.5 ± 0.1

1014

2–6

65

Yes

52

67

Cu

No

54

Co,

Yes

56, 57

Zn,

65

Ni

Epitherm n.cm-2.s-1 al 6.1 ± 0.3 64

Zn

64

Zn (n,

p) 64Cu

0.031 ±

1013 -

~ 3.7 ×

0.002

1014

103

n.cm-2.s-1 Cyclotron

64

Ni

64

Ni (p,

n) 64Cu

0.035 ±

15 – 60

3.1 × 103

55

0.003

µA

– 1.1 ×

60

105

61

22


Cu, Cu

Page 23 of 48

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Table 2: Overview of 64Cu2+ ions as probe in preclinical PET imaging of cancers

64

Cu

Cancer type

Cancer cell

Maximum

Blood

Tumor-

line used

tumor

uptake

to-

production route

uptake

References

muscle ratio

64

Ni (p, n) 64

Cu

Hepatocellular

Murine

3.7 ± 0.4

carcinoma

hepatoma

SUVmax

reaction in

cells (Hep 1-

cyclotron

6) Prostate cancer

#

~3

71

~6

47

~ 14

72

#

46

~4

73

Human

4.4 ± 1.1

0.8 ±

prostate

%ID/g

0.2

cancer cells

%ID/g

(PC-3) Human

5.6 ± 1.2

1.5 ±

prostate

%ID/g

0.5

cancer cells

%ID/g

(DU-145) Breast cancer

Human breast cancer

5.4 ± 1.1

~ 1.5

%ID/g

%ID/g

#

cells (MDAMB-231) infected with a

lentiviral

vector intrinsically expressing the

hCTR1

gene Colorectal

Human

3.0 ± 0.2

cancer

colorectal

%ID/g

cancer cells 23



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Page 24 of 48

(HT29) Glioblastoma

Human

3.8 ± 0.4

glioblastoma

%ID/g

cells

3.6 ± 0.4

(U87MG)

SUVmax

Head and neck

Human

4.9 ± 0.3

cancer

squamous

%ID/g

#

~4

73,74

#

~6

73

#

~6

73

#

~ 1.5

73

4.1 ± 0.1

48

3.5 ± 1.2

48

52

cell carcinoma cells (FaDu) Lung cancer

Human lung

4.9 ± 0.2

carcinoma

%ID/g

cells (H727) Ovarian cancer

Human

1.3 ± 0.1

ovarian

%ID/g

carcinoma cells (A2780) Melanoma

Murine

4.1 ± 0.2

1.5 ±

melanoma

%ID/g

0.2

cells

%ID/g

(B16F10) Human

3.6 ± 0.4

1.9 ±

malignant

%ID/g

0.6

melanoma

%ID/g

cells (A375M) 63

Cu (n, γ) 64

Cu

reaction in a

Fibrosarcoma

Murine

6.5 ± 1.4

1.0 ±

19.6 ±

fibrosarcom

%ID/g

0.4

3.1

a cells

%ID/g

(MC57G) 24


Page 25 of 48

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nuclear reactor

Melanoma

Murine

7.6 ± 1.6

1.1 ±

22.5 ±

melanoma

%ID/g

0.6

3.8

cells

%ID/g

(B16F10) #

Not reported

25


52


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 48

Figure captions: Figure 1: Nuclear decay scheme of

64

Cu. The radioisotope decays to either

64

Ni by β+

emission or EC or to 64Zn by β- decay.

Figure 2: A schematic representation of production of 64Cu in a nuclear reactor or cyclotron, followed by radiochemical processing of the irradiated target in a hot cell facility. After radiochemical processing, 64Cu exists mainly as Cu2+ ion and is administered in vivo as a PET imaging probe. In the extracellular space, Cu2+ is reduced by reductases on the cell surface to Cu+, which is then transported across the cell membrane by the hCTR1. Adapted from reference 67 with permission.

Figure 3: (A) PET image at 24 h p.i. of 64Cu2+ ions in mouse bearing prostate cancer (PC-3) xenograft.

(B)

Immunohistochemistry

study

shows

significantly

high

hCTR1

immunoreactivity on prostate cancer xenograft tissues after incubation with an antibody specific for hCTR1. (C) PET-CT imaging at 24 h p.i. of 64Cu2+ ions to show the outcome of knockdown of hCTR1 on tumor uptake of 64Cu2+ ions in mice bearing prostate cancer (PC-3 and DU-145) xenografts. (i) Reduced 64Cu uptake in PC-3 tumor with knockdown of hCTR1 (tumor on right marked by *), compared with PC-3 tumors without knockdown of hCTR1 (tumor on left marked by **). (ii) Reduced 64Cu uptake in DU-145 tumor with knockdown of hCTR1 (tumor on right marked by *), compared with DU-145 tumor with no knockdown of hCTR1 (tumor on left marked by **). Adapted from reference 47, 72 with permission.

Figure 4: (A) PET images at different time points p.i. of 64Cu2+ ions in mouse bearing MDAMB-231 (arrow) and MDA-MB-231-hCTR1 (arrowhead) tumors. (B) Immunohistochemistry

26


Page 27 of 48

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of slices from MDA-MB-231 (left) and MDA-MB-231-hCTR1 (right) tumor tissues. Adapted from reference 46 with permission.

Figure 5: Transaxial PET images of different tumor types [U87MG (glioblastoma), HT29 (colorectal cancer), H727 (neuroendocrine lung cancer), A2780 (ovarian cancer), and FaDu (head & neck cancer)] in mice at 1 h and 22 h p.i. of

64

Cu2+ ions. Tumors are indicated by

arrows. Adapted from reference 73 with permission.

Figure 6: (A) PET images at various time points p.i. of

64

Cu2+ ions in mouse bearing

melanoma (B16F10 and A375M) tumors. (B) Representative images of A375M tumor necrosis with and without treatment with

64

Cu2+ ions (left: control mouse; right: treated

mouse). Adapted from reference 48 with permission.

Figure 7: Evaluation of serum protein aggregation in presence of Cu2+ ions. (A) DLS study after addition of increasing quantities of CuCl2 in mouse serum. Particle size information was acquired by investigating the electric field correlation function, [g(τ)], where τ is the correlation time. A significant shift in g(τ) was observed only when the concentration of Cu2+ ions in mouse serum was > 20 µg / mL, representing colloidal aggregate formation. (B) Study of the dispersion behavior of Cu2+ ions in mouse serum by studying the relaxation time over 24 h time period. (C) Percentage of free 64Cu2+ ions in mouse serum over 24 h time period, as determined by radio-TLC study. Inset shows the TLC pattern of 64Cu2+ ions in mouse serum when TLC was developed in sodium citrate solution. Adapted from reference permission.

27


52

with


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 48

Figure 8: Biodistribution of 64Cu2+ ions when intravenously administered in mice bearing (A) melanoma tumors and (B) fibrosarcoma tumors. Adapted from reference 52 with permission.

Figure 9: Representative PET-CT images acquired after intravenous administration of 64Cu2+ ions in patients with (A) prostate cancer, (B) cerebral tumor and (C) glioma. Selective uptake of

64

Cu2+ ions can be seen in all cancerous lesions. Adapted from reference

permission.

28


79

with

Page 29 of 48

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Figures 1

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 48

Figure 2

Reactor Production 64Zn

65

48.63 % σ = 0.76 b

Zn 244 d (n,p)

β(n,γγ ) 64Cu 69.17 % 12.9 h σ = 4.5 b 62

Ni 3.63 % σ = 14.5 b

Zn 27.9 % 65

63 Cu

β+ 63Ni

100 y

Cu 30.83 % σ = 2.17 b

Cyclotron Production

Hot Cell

66

64

Zn 48.63 % σ = 0.76 b

Irradiated target

Irradiated target

65 Zn 244 d

β-

63Cu

69.17 % σ = 4.5 b

64

Ni 0.926 % σ = 1.52 b

62

Ni 3.63 % σ = 14.5 b

Radiochemical processing 64 Cu (as Cu2+ ions)

Reductase

Cu2+

Cu+

hCTR1

hCTR1

hCTR1

30


66 Zn 27.9 %

64

Cu 12.9 h β+ 63

Ni 100 y

65 Cu 30.83 % σ = 2.17 b

64 Ni 0.926 % σ = 1.52 b

Page 31 of 48

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Figure 3

A

B

5 %ID/g

0 %ID/g

C (i))

(ii))

PC-3

DU-145 8 %ID/g

0 %ID/g

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 48

Figure 4

A 2h

4h

12 h

24 h

15 %ID/g

0 %ID/g

B

MDA-MB-231

MDA-MB-231-hCTR1

32


Page 33 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5

U87MG

HT29

H727

A2780

FaDu

%ID/g 15

1h

22 h

1

33



Figure 6 A

4h

24 h

B

B16F10

2h

Control mouse

A375M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 48

34


Treated mouse

Page 35 of 48

Figure 7 A

B 2500

0.9

Relaxation time (T2)

2000

0.6

1500

Blank serum 6.7 µg Cu/mL 10.0 µg Cu/mL 13.3 µg Cu/mL 20.0 µg Cu/mL 26.7 µg Cu/mL 33.3 µg Cu/mL

0.3

1000

500

0.0

0

100

1000

0

20000

40000

C 100

90 80

Radioactivity (%)

2+ Cu (%)

80

60

40

20

70 60 50 40 30 20 10 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Rf

0 0

2

60000

Time (s)

Correlation time (t)

64

10

Unbound

Normalized correlation function [g(τ )]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4

6

8

10 12 14 16 18 20 22 24

Incubation time (h)

35


80000

100000


Figure 8 A

B 60

60

Melanoma

Fibrosarcoma

1h 4h 24 h 48 h

50

1h 4h 24 h 48 h

50

40

%ID/g

40

30

30

20

20

10

10

0

0

Bl oo d Li ve r G Ki IT St dne om y ac H h ea Lu rt ng s Ti M bi us a cl Sp es le Tu en m or

Bl oo d Li ve r G Ki IT St dne om y ac He h a Lu rt ng s T M ibi us a c Sp les le Tu en m or

%ID/g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 48

A

A

36


Page 37 of 48

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Figure 9

A

B

C

37



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Page 38 of 48

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England, C. G.; Hernandez, R.; Eddine, S. B.; Cai, W.

Molecular Imaging of

Pancreatic Cancer with Antibodies. Mol Pharm 2016, 13, 8-24. 7.

Shi, S.; Chen, F.; Cai, W.

Biomedical applications of functionalized hollow

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Miller, J. C.; Thrall, J. H. Clinical molecular imaging. J Am Coll Radiol 2004, 1, 4-

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Mawlawi, O.; Townsend, D. W.

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Cassidy, P. J.; Radda, G. K. Molecular imaging perspectives. J R Soc Interface 2005,

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Cu2+ Ions as PET Probe: An Emerging Paradigm in Molecular Imaging of Cancer Rubel Chakravarty,* Sudipta Chakraborty and Ashutosh Dash

Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

Graphical Abstract

PET Imaging 64Cu2+

64Cu

ions as probe

t½ = 12.9 h

Clinical Preclinical 64

Cu in the form of Cu2+ ions can directly be used as a radiotracer for PET imaging of various

types of cancers in preclinical and clinical settings.

48


64Cu2+ Ions as PET Probe: An Emerging Paradigm in Molecular

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