Non-Standard PET Radionuclides in Molecular Imaging
Suzanne Lapi, PhD, and Mai Lin, PhD, Department of Radiology, Washington University School
of Medicine, St. Louis, MO
Molecular imaging has been described in the literature as a multidisciplinary field that combines
imaging research, molecular biology, chemistry and medical physics. Although various imaging
modalities have been used in molecular imaging, to date the majority of clinical applications
have been in the field of nuclear medicine [1]. In fact, this concept actually began with the first
use of I-131 to image recurrent thyroid carcinoma [2].
Over the past decade, positron emission tomography (PET) has played an important role in the
field of molecular imaging. The capability of imaging quantification and the high sensitivity (10-1110-12 mole/L) without a limitation in tissue penetration help to facilitate the development of PET
imaging probes. In addition to the chemical form and biological parameters of the desired PET
imaging probes, the choice of a suitable radionuclide is vital in developing
radiopharmaceuticals. Because PET works by detecting 511 keV gamma rays that are
produced by the annihilation of positrons (β+) emitted by a radionuclide and nearby electrons, an
ideal PET radionuclide should have a low β+ energy and high β+ branching ratio. Moreover, the
labeling procedures should minimally alter the properties of the molecule of interest, and the
physical half-life of a radionuclide should match the biological half-life of the molecule to be
labeled.
While C-11, N-13, O-15, and F-18 are typically categorized as “standard PET radionuclides,” the
requirement of an onsite cyclotron or shipment from a closely located commercial source
decreases the availability and flexibility of the radiopharmaceuticals labeled with these
radionuclides. As such, the use of “non-standard PET radionuclides” has increased in recent
years for the development of PET imaging probes. This article is intended to provide an
overview on current research using the non-standard PET radionuclides, Cu-64, Ga-68, and Zr89, for preclinical and clinical applications. Interested readers are referred to recent excellent
review articles for more in-depth discussions of this topic [3-5].
Copper-64
Copper has several radioisotopes including Cu-60, Cu-61, Cu-62, Cu-64, and Cu-67.
Commercially available medical cyclotrons have the capability to produce Cu-60 (t1/2: 23.4 min;
β+%: 93%), Cu-61 (t1/2: 3.3 h; β+%: 62%) and Cu-64 (t1/2: 12.7 h; β+%: 17%) for PET imaging.
Copper is a transition metal that generally exists in two oxidation states, +1 and +2 [6]. Copper
ions in aqueous solution have an oxidation state of +2 with coordination number as 4, 5, or 6 [6].
It has been reported that Cu(II) is rapidly released to bind to proteins in human serum when
complexed with non-macrocyclic chelators such as EDTA (ethylenediaminetetraacetic acid) and
DTPA (diethylene triamine pentaacetic acid) [7]. Meares et al. first introduced cyclam and cyclen
(Figure 1) for Cu-67 labeling of monocolonal antibodies [8]. Since then, DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TETA (1,4,8, 11-tetraazacyclododenane1,4,7,10-tetraacetic acid) (Figure 1) derivatives have been widely used for Cu-64/67 labeling of
biomolecules [9]. However, in vivo dissociation of Cu-64 from the DOTA and TETA complexes
leading to high liver uptake has been observed [6]. In order to enhance the stability of Cu-64/67
conjugates, derivatives based on cross-bridged cyclam (Figure 1) have been developed. It has
been reported that Cu-64 labeled CB-TE2A-bombesin conjugates have better in vivo stability
compared to their DOTA counterparts [10]. Nonetheless, this cross-bridged chelator requires
high temperature (95oC) to achieve sufficient complexation of Cu(II) [9], which precludes its use
for antibodies and certain peptides. Other chelates such as NOTA and other derivatives show
high stability while undergoing radiolabeling under mild conditions [11].
Cu-64-labeled diacetylbis(N4-methylthiosemicarabaone) (Cu-64-ATSM) has been shown to be
selective for hypoxia in tumor imaging (Figure 2) [12]. Tumor oxygenation is an important factor
for cancer treatment, as hypoxia is related to poor prognosis [13]. Although the mechanisms
underlying Cu-64-ATSM retention are not completely understood, Fujibayashi et al. first
suggested that Cu(II)-ATSM reduction occurred only in hypoxic cells and was then irreversibly
trapped [14]. Obata et al. observed that reduction of Cu-64-ATSM in normal brain cells occurred
in mitochondria, whereas it occurred mainly in the microsome or cytosol fraction in tumors. The
retention involves the enzymatic reduction which is enhanced by hypoxia [15]. According to
Dearling et al. [16], the reduction of Cu(II)-ATSM takes place in both normoxic and hypoxic cells.
The resulting Cu(I) slowly dissociates from ATSM. Once the dissociation occurs, it is irreversible
and Cu(I) is trapped. In normoxic conditions, Cu(I)-ATSM could be reoxidized by electron
transport chain on the mitochondria and diffuse back out of the cell. However, reoxidation is
much less likely in hypoxic cells and therefore the retention of Cu-64-ATSM is higher.
Gallium-68
Interest in using Ga-68 (t1/2: 68 min; β+%: 89%) for clinical PET comes from its ready availability
from a generator. The Ge-68/Ga-68 generator was first described in 1960 [17]. While the overall
physical characteristics of Ga-68 are not necessarily superior to those of F-18, the availability of
Ga-68 from a Ge-68/Ga-68 generator eliminates the need of an onsite cyclotron and makes Ga68 an attractive alternative to F-18. As the generator is commercially available and Ge-68 has a
physical half-life of 270.8 days, the generator is ideal for clinical settings with the lifespan of over
a year.
Gallium-68 is usually eluted from the generator with a hydrochloric acid solution in the chemical
form of GaCl3. While macrocyclic chelators for Ga(III) have not been investigated to the same
extent as for Cu(II), DOTA is usually adequate to conjugate a biomolecule for subsequent
labeling with Ga-68 [6]. However, it has been noticed that compared to DOTA-Ga(III) complex,
NOTA-Ga(III) complex (NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid) (Figure1) is much
more thermodynamically stable (log KNOTA-Ga(III) = 31.0 vs. log KDOTA-Ga(III) = 21.3) [6]. As such,
Eisenwiener et al. have designed and synthesized a NOTA-based scaffold to conjugate
targeting molecules for Ga-68-labeling [18].
Recently, the use of Ga-68-DOTATOC (DOTA-D-Phe1-Tyr3-octreotide) and other derivatives for
imaging human neuroendocrine tumors provides an excellent example of the application of Ga68 in the design of a targeted radiotracer (Figure 3) [19]. In a study involving imaging of 51
patients with known or suspected neuroendocrine tumors, Ruf et al. demonstrated the
diagnostic value of Ga-68-DOTATOC PET and observed the overall high sensitivity (72.8%) and
specificity (97.4%) [20].
Zirconium-89
Due to the relatively long half-life and low positron emission energy, Zr-89 (t1/2: 78.41 h; β+%:
23%) is an ideal radionuclide for the labeling of compounds with long blood circulation times
such as antibodies and nanoparticles. However, because the requirement of strong acidic
solutions to maintain the oxidation state of Zr-89 (IV) and the coordination chemistry of Zr-89
requiring octadentate chelators to form stable complexes, the role of Zr-89 in PET imaging was
not well established until recently [4].
In order to achieve high radiochemical purity of Zr-89, Verel et al. have overcome the problem
by using a thin-foil Y-89 plate and employed a hydroxamate resin to expedite the separation
procedure [21]. The introduction of Df-Bz-NCS (p-isothiocyanatobenzyl-desferrioxamine B) by
Perk et al. further facilitated the use of PET studies for Zr-89-labeled antibodies [22], as this
bifunctional chelator allows efficient and easy preparation of the antibody conjugates. Various
Zr-89-labeled antibodies have been investigated for their potential clinical applications, in which
Dijkers et al. and us have observed that relative HER2 expression levels in orthotopic and
metastatic breast cancer models can be assessed by PET imaging using the Zr-89-trastuzumab
(Figure 4) [23, 24]. Moreover, Zr-89 could also be an alternative choice for the dosimetry
analysis of Y-90-labeled antibodies when the conjugates display slow in vivo kinetics.
Conclusion
The availability of radionuclides with potential clinical applications continues to expand with
advances in cyclotron targetry and radiochemistry. Due to the full spectrum of half-lives
(minutes – days) and increased accessibility of Cu-64, Ga-68, and Zr-89, many of the new
radiopharmaceuticals based on these radionuclides are under development. As “accelerated
approval” by the U.S. Food and Drug Administration (FDA) allows alternative endpoints to be
assessed on the basis of biomarkers that are measurable as indicators of clinical and
therapeutic efficiency [25], PET imaging plays an important role by providing the information on
the presence, efficacy, tissue distribution profile, and pharmacokinetics of drug candidates. This
will in turn further enhance the use of “non-standard PET radionuclides” for both preclinical and
clinical studies.
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US Code of Federal Regulations 21 CFR, Food and Drug Administration.
Figure legend
Figure 1. Chemical structures of EDTA, DTPA, cyclen, cyclam, cross-bridged cyclam, TETA,
CB-TE2A, DOTA, and NOTA.
Figure 2. Comparisons between 64Cu-ATSM uptake and hypoxia measured by immunostaining
in R3230Ac and FSA. Close correlation between 64Cu-ATSM uptake and EF5-stained hypoxic
area was observed in R3230Ac tumor (left), whereas no correlation was found in FSA tumor
(right). Images include 64Cu-ATSM microPET image, autoradiography (AR) section from same
tumor, EF5 and Hoechst immunostaining from adjacent section, fused image from
autoradiography and EF5 images, H&E staining, and correlation plot between autoradiography
and EF5 staining images. EF5-stained hypoxic area is indicated by orange, perfused vessels
are marked by blue fluorescent Hoechst 33342 dye, and 64Cu-ATSM distribution in AR is
indicated by green in fused image. In FSA, a large amount of 64Cu-ATSM accumulated in wellperfused areas, which are indicated in Hoechst perfusion image. The spatial correlation
between autoradiography and EF5 staining images in this specific FSA tumor is 0.05, whereas
the spatial correlation is 0.78 in the shown R3230Ac tumor. Reprinted with permission from
reference [12].
Figure 3. A 56-y-old woman with multiple liver and lymph node metastases was referred for
restaging after surgery and chemotherapy. CT presented these tumor lesions; however, it was
negative for bone lesions. Beside the visceral metastases, some additional osteoblastic and
osteolytic bone metastases were clearly depicted with 68Ga-DOTA-TOC (A). Only some of these
bone metastases were delineated by conventional scintigraphy (B, anterior view; C, posterior
view). Osteoblastic bone lesions were confirmed by 18F-Na-fluoride PET (D). Retrospective CT
analysis after image fusion revealed some of these bone metastases. Reprinted with permission
from reference [19].
Figure 4. Examples of noninvasive small-animal PET images (dorsal presentation). 89Zrtrastuzumab (5 MBq per mouse) uptake in human SKOV-3 xenografts in 3 mice at 6 h (A), day
1 (B), and day 6 (C, metastasized tumor) after injection is shown. Primary tumors are indicated
by arrows. Reprinted with permission from reference [23].
Figure 1
Figure 2
Figure 3
Figure 4