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a stream of nitrogen gas and in the presence of concentrated HNO3. Hot nitric acid
V
standard22
V
standard22
21
EV
standard
41
..
standard422
1
27
..
decomposes oxalic acid/oxalate, and subsequent reconstitution with 0.1 M HCl gave a
89
‘
Zr-chloride’ formulation. The exact chemical nature of the ‘89Zr-chloride’ species in solution remains unknown, but it is possible that the tetrameric zirconyl chloride structure,
[Zr
(OH)8(H2O)16]Cl8(H2O)12, is present. Although successful, this method has not been
4
widely adopted. In addition, if the mixture is not heated to complete dryness, any residual
oxalic acid will likely mean that the formulation is better described as ‘
89
Zr-oxalate’ in a
HCl mixture. The two dierent speciations can be observed by the dierent distribution
89
of ‘
Zr-oxalate’ and ‘89Zr-chloride’ in mice[39, 51].
In 2018, Graves etal. reported a streamlined approach toward chloride-based
89
Zr radio-
chemistry using a tributyl phosphate (TBP)-functionalized extraction resin[52]. This
method eliminated the need for a QMA extraction and thermolysis, and an average Y/Zr
separation factor of 1.5 × 10
impurities, including radiochemically poisonous Fe
5
(n=3) was obtained. One downside was that trace metal
3+
ions, were higher than in the original
two-step QMA/thermolysis approach. Nevertheless, as work continues on new methods for
accessing dierent initial formulations of
89Zr4+
ions, it is likely that these studies will facili-
tate the investigation of a wider range of radiolabelling chemistry using dierent chelates.
Although zirconium complexes are known in which the metal ion exists in oxidation states
of 1+, 2+, 3+, and 4+, for the vast majority of zirconium complexes, the metal ion is found
in the group oxidation state (Zr
the ion has a very high charge-to-size ratio. In compounds containing Zr
centre exhibits a small covalent radius of 0.145 nm and ionic radius of 0.072 nm, and it
has a value of 1.45 on the Pauling scale of electronegativity, making it an extremely electropositive element. Such high electropositivity is illustrated in the reduction potentials
of various Zr
4+
species (Eqs.(11.5)–(11.8)) and has important implications in the aqueous
phase chemistry of zirconium.
OHeZrHOE
ss
4+
). The chemistry of Zr4+ ions is dominated by the fact that
)HOe Zr(s)HOE
44
4
aq ZrOs HO kJmol
44 236.
aq eZr(s)
553() () .
45()
(11.7)
G
58() ()
4+
ions, the metal
(11. 5)
(11.6)
(11. 8 )
Chapter 11: The Radiochemistry ofZirconium 355
The strongly negative reduction potentials of Zr4+ species mean that in aqueous envi-
ronments, Zr ions with 1+, 2+, or 3+ charge reduce water and/or spontaneously disproportionate to give Zr
89
Zr resides exclusively with the 4+ ion.
4+
ions and Zr(s)[1, 2]. Hence, existing aqueous phase radiochemistry of
Beyond radiochemistry, very few examples exist of Zr complexes that are stable in
aqueous conditions. However, in non-aqueous environments, coordination complexes
of Zr are more extensive, though still dominated by the 4+ ion. The organometallic
compound zirconocene hydrochloride (Schwartz’s reagent, (C
sic example of a covalent coordination complex containing Zr
5H5)4Zr2H2
4+
ions. This compound is
a common reagent used in organic reactions for chemical transformations of alkenes/
alkynes and is also capable of selective reduction of amide bonds in the presence of other
groups such as reducible esters. However, as with many organometallic compounds,
Schwartz’s complex is unstable in water and illustrates the major problem of zirconium
chemistry: namely, how can the Zr
4+
ion be stabilised against hydrolysis?
An additional complexity in zirconium coordination chemistry is that, in spite of the
relatively small size, Zr
4+
ions can form complexes with between six and eight donor atoms
in the rst coordination sphere. In 2010, co-workers and I reported the rst computational
study on the structure of the zirconium desferrioxamine B complex ([Zr(HDFO)(H
(CH3)2) is a clas-
]+
2O)n
Figure 11.11
Calculated relative
energies of the
[Zr(HDFO)(H
2O)n
]2+
(n=0, 1, or 2)
complexes.
1
00
–1
–20
–40
–60
Relative Electronic Energy/kJ mol
–80
–100
–202-eq
–612-ax
–953-cis
Additional Water Molecules
356 Handbook of Radiopharmaceuticals
where n=0, 1, or 2) in which the possibility of introducing coordinated water molecules was
explored using density functional theory (DFT)[53]. Optimised structures and relative free
energies (in kJ mol
−1
) for four possible [Zr(HDFO)(H2O)n]2+ species are shown in Figure11.11.
Note that in all structures, the DFO ligand is in the Λ-N-cis-cis geometric isomer. In total,
eight geometric isomers can be made when DFO coordinates around a central metal cation,
and each is optically active. Upon taking the thermodynamically most stable geometric
isomer of the hexacoordinate [Zr(HDFO)]
2+
complex (1), the addition of the rst water molecule can occur in either a pseudo-axial or pseudo-equatorial site. Interestingly, the pseudoaxial site in which the water binds in the centre of a trigonal face produced by the carbonyl
(C=O) oxygen atoms leads to a stable structure, but the opposite trigonal face (formed by
the three N─ O donors) is inaccessible due to increased electron density that repels the
water ligand. Addition of the water molecule was predicted to be thermodynamically feasible and spontaneous. The [Zr(HDFO)(H
O)]2+ structure with a pseudo-axial water (2-ax)
2
molecule was found to be more stable than the addition of the water molecule in a pseudoequatorial site (2-eq) and was calculated to be ~61 kJ mol
−1
more stable than the hexadentate complex. The combination of both a pseudo-axial molecule and a pseudo-equatorial
water molecule in [Zr(DFO)(H
]2+ stabilised the complex even further. However, it should
2O)2
be noted that the Zr─O bond length of the water ligand in the pseudo-equatorial position
of the 8-coordinate complex was very long (0.248 nm compared to 0.234 nm for the pseudoaxial Zr─O bond length), indicating a ‘loose’ coordination and likely facile exchange with
bulk solvent. The conclusions from this work were that (i) unlike most Zr complexes and
many other radiometal complexes, coordination of a water molecule actually stabilises the
O1
C1
O3
N1
Zr
O2
C2
N2
O4
Figure 11.12 Structure of the 8-coordinate Zr(MeAHA)4 complex. Sources:
Guérard, F., Lee, Y.S., Tripier, R. et al. [54]; and Holland, J.P. and Vasdev, N. [55].
Chapter 11: The Radiochemistry ofZirconium 357
O
YM103
DFO-Star (DFO*) oxoDFO-Star 2,3-HOPO-p-Bn-NCS
NCS
n
O
H
S
O
N
H
OH
N
OH
N
SCN
NH
NN
O
H
2
CO
p-SCN-DTPA
C
2
HO
N
R =
O
O
OH
O
O
O
F
Orn3hx-NCS (n = 1)
F
F
O
O
OH
N
NCS
OO
OH HO
NN
NN
OO
N
N
HO
O O
OH
HN
N
Orn4hx-NCS (n = 2)
O
s
O
NH
F
N
OOO
L5
H
N
OH
N
NCS
O
3
+
N
H
S
N
H
OH
OH
OOO
O
5
O
OH
O
H
N
5
N
O
Desferrioxamine B (DFO) Mesylate
N
H
5
N
OH
N
H
5
NN
OH
O
OMe
O
Zr-based
89
O
N
H
S
O
HN
NN
p-SCN-Bn-HOPO
N
O
HN
HO
NH
HO
OH
OO
HN
OH
NH
O
O
N
HN
NH
O
O
N
O
O
HN
NN
N
O
NH
O
O
OH
OH
OO
OH
O
N
O
O
N
H
O
N
OH
O
O
H
N
O
OH
O
N
O
N
H
Desferrioxamine—squaramide (HDFOSqOEt)
N
OH
O
O
H
N
OO
O
O
N
H
EtO
O
H
N
5
OH
N
Desferrioxamine-p-SCN
OO
N
H
5
NN
MeO
2
NH
2
NH
O
O
N
OH
O
OH
N
N
O
H
O
OH
O
O
HN
O
N
OH
OH
N
N
H
O
Figure 11.13 Chemical structure of selected bifunctional chelates that are potentially useful in the design of
radiotracers.
R
R
R
N
H
O
N
H
O
O
N
O
OH
O
OH
H
N
N
N
O
H
N
H
N
O
NN
OO
OH OH
O
NN
H
2
CO
N
H
2
CO
N
N
C
2
HO
SCN
Handbook of Radiopharmaceuticals
Zr(DFO) complex; (ii) one water molecule ts comfortably in a pseudo-axial site, forming a
potentially thermodynamically robust but still kinetically labile 7-coordinate complex; and
(iii) Zr(DFO) can potentially expand the coordination sphere to accommodate up to eight
donor atoms in the rst shell.
Subsequent to these calculations, it was suggested that the comparatively high bone
uptake observed in mice when using many dierent [
89
Zr][Zr(DFO)]+ labelled antibodies
is potentially the result of incomplete saturation of the rst coordination sphere of
89Zr4+
the
89
Zr radioactivity in bone is a phenomenon that has been observed in mice but is not
ion, which facilitates hydrolysis. It should be noted that high accumulation of
evident in human trials. Nevertheless, there is a denite need and scope for advancing
89
Zr radiochemistry by synthesising new ligands with dierent donor atom sets and with
higher denticity (from six to eight donors).
In 2013, Guérard etal. reported the synthesis and single-crystal X-ray structure of
the eight-coordinate Zr(MeAHA)
complex (Figure11.12)[54]. This work conrmed that
4
when steric constraints are not an issue (MeAHA ligands are very small), hydroxamate
ligands can transchelate oxalate ligands from Zr
4+
ions in eight-coordinate complexes.
Further computational work by myself mapped out the thermodynamics of the MeAHA/
oxalate exchange reaction using simulated ‘acidic’ and ‘basic’ conditions[55]. Indeed, the
DFT studies found that the substitution reaction was thermodynamically feasible above
pH values of approximately 4.19 (corresponding to the second pKa value of oxalic acid).
The calculations also yielded a set of design criteria for the design and synthesis of new
ligands for
89Zr4+
complexation.
In the last few years, many groups around the world have been working actively on the
design and synthesis of new bifunctional ligands for
89Zr4+
radiochemistry[11–20]. Structures of a number of promising bifunctional chelates are shown in Figure11.13[56–68].
The reader should note that the list is by no means comprehensive but is rather a selection of dierent bifunctional chelates that illustrate the current chemical scope of
ligands for Zr coordination. Almost without exception[64], the reports claim that the new
ligands, and in particular those that represent octadentate scaolds, exhibit “superior”
stability, imaging quality, and distribution in mice, which conrms that there is still room
for improvement in Zr coordination chemistry. In spite of the very interesting chemistry
that is emerging from exploring the expanded coordination space of Zr, the success of
DFO in the clinical setting, and the fact that desferrioxamine is readily available as an
approved GMP source, mean the jury is still out on whether any of these new chelates will
supersede the existing technology.
Methods for bioconjugation of proteins with chelates like DFO have been reported elsewhere and are not discussed further here[69]. In addition,
radionuclide for developing alternative protein conjugation strategies[70–73]. The advantages of using [
89
Zr][Zr(C2O4)4]4− (aq.) solutions in radiosynthesis include (i) stabilisation
89
Zr has been employed as a
Chapter 11: The Radiochemistry ofZirconium
of the 89Zr4+ ion against hydrolysis and/or colloidal formation over a wide pH range; (ii)
2+
–
facile exchange of the oxalate ligands by multidentate chelates bearing powerful carboxylate, hydroxamate, or hydroxypyridinonate (HOPO) donors; and (iii) rapid radiolabelling kinetics (Scheme11.2). In most common radiolabelling methods, stock solutions of
89
[
Zr][Zr(C2O4)4]4− containing 1 M oxalic acid are rst neutralised carefully by using a base
(for example, ~1 M solutions of Na
, NaHCO3, or NaOH, etc.). Neutralised solutions can
2CO3
be buered by using carbonate solutions for pH values in the range ~7–9, or alternatively
by using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for tighter control of
pH values ~6.8–8.2. Other buer systems may also work, but it is advisable to avoid those
that contain phosphate groups, which may act as competitive ligands for Zr
4+
ions.
Scheme 11.2
Radiosynthesis
89
of [
Zr][Zr(HDFO)
(H
]2+.
2O)n
O
89
[
Zr]Zr-oxalate
or
89
Zr]Zr-chloride
[
1 h, RTP, pH6 - 9
+
NH
3
N
OH
O
N
5
H
O
OH
H
N
N
5
O
O
⊝
O
S
O
N
O
OH
HN
5
O
N
O
OO
89
Zr
H
OOH
2
O
N
N
5
H
O
N
O
2
O
Desferrioxamine B mesylate
In the manual synthesis of
89
Zr-radiolabelled antibodies conjugated with the DFO chelate,
NH
+
3
2 X
reactions are typically incubated at room temperature or heated up to 37 °C for between
30 and 60 minutes. Mixtures may be agitated gently by hand or mixed by using a rocker but
are not normally stirred or vortexed to avoid protein occulation. Ecient mixing does
increase radiolabelling rates, as illustrated by the work of Wright etal., who used microuidic apparatus to prepare single patient doses of
89
Zr-radiolabelled trastuzumab[74]. After
radiolabelling reactions are complete, mixtures are typically quenched with excess DTPA or
EDTA solutions to strip any non-specically bound
89
Zr-radiolabelled antibody component is puried and formulated for injection using manual
89
Zr from the protein fraction. Then, the
or automated size-exclusion chromatography methods. For manual purication, columns
containing PD-10 (Sephadex gel) are often used, or centrifugation spin-ltration is possible.
Alternatively, preparative size-exclusion HPLC can be used. The advantage of HPLC methods
is that chromatographic resolution can facilitate quantication (and separation) of any
dimeric or multimeric protein aggregates that may form during the bioconjugation or radiolabelling steps. However, HPLC methods are more expensive and time-consuming, require
specialist equipment, and are more challenging to maintain in a GMP environment.
If stock solutions of ‘
89
Zr-chloride’ are employed for radiolabelling, care must be taken
to ensure that the pH of the mixture remains acidic. In the absence of oxalate anions
(or other supporting ligands like citrate), hydrated Zr
4+
ions are likely to aggregate and
form metal clusters that may impact the success of radiolabelling reactions[39, 52].
360 Handbook of Radiopharmaceuticals
If one excludes the early human studies reported by Mealey[23], the rst pilot experiments using
Since then, many preclinical studies and clinical trials using dierent
89
Zr-radiolabelled antibodies in patients were reported in 2006[75, 76].
89
Zr-radiolabelled
antibodies have been performed across the world. A non-comprehensive list of relevant reports is given in Table11.3. Two recent articles by Jauw etal.[129] and Bensch
etal.[130] have provided comparative (meta) studies on the experience gained from
clinical trials with multiple dierent
89
Zr-radiolabelled antibodies. These comparative
assessments provide a retrospective view of the in vivo biodistribution and tumour
targeting of
89
Zr-immuno-PET radiotracers. As 89Zr-immuno-PET radiopharmaceuticals
advance beyond early-stage clinical trials, one important aspect of multicentre trials
is harmonising the methods used to administer the radiotracers and acquire/interpret
the images. Makris etal. pointed out that harmonisation of image quality is achievable,
but more studies will be required before a standard
89
Zr-immuno-PET protocol can be
adapted in a wider setting[131].
TECHNOLOGIES
With the evident success of 89Zr-immuno-PET in the clinic, it is safe to say that 89Zr-based
radiopharmaceuticals are not only breaking new ground in nuclear medicine but also
spurring the development of new chemistry and radiochemistry. The introduction of
advanced technologies such as alternative chelates with improved thermodynamic,
kinetic, and metabolic stability for Zr
4+
ion coordination (vide supra); facile conjugation
methods[132, 133]; automated production[134]; and an increasing number of highly
specic radiotracers against emerging biomarkers means that zirconium-based radiopharmaceutical research (both fundamental and clinical) is accelerating. Two recent
advances that have the potential to impact future production of
89
Zr-radiolabelled anti-
bodies are given next.
In 2018, the team in Amsterdam reported a major advance in the automated radiosynthesis and purication of
cetuximab and [
89
Zr]Zr-DFO-N-suc-rituximab were produced with isolated radiochemical
yields of 74.6% ± 2.0% and 62.6% ± 3.0%, respectively. In both cases, radiochemical purity
was >95%, and the protein remained biochemically active with immunoreactive fractions
>87%. The importance of having an automated production method cannot be overstated.
89
Zr-radiolabelled antibodies. In this work, [89Zr]Zr-DFO-N-suc-
Chapter 11: The Radiochemistry ofZirconium 361
Table 11.3 Overview of some inuential studies using 89Zr-radiolabelled antibodies/proteins in animals
and in human trials that have been completed since 2006.
Tar g e t /
Radiotracer
biomarker Animal model/patient cohort Year/reference
Animal models
89
[
Zr]mcAb-U36 chi-
meric monoclonal
CD44v6 Head and neck squamous cell
carcinoma (HNSCC): HNX-OE
2003, Verel etal.[38]
antibody (cmAb)
89
[
Zr]DN30 c-MET (CD) Head and neck cancer: GLT-16 (high)/
2008, Perk etal.[77]
FaDu (low)
89
Zr]trastuzumab HER2/neu SK-OV-3
[
BT-474/negative
NCI-N87/negative MKN74
MDA-435
MDA
-MB-468
2009, Dijkers etal.[78]
2010, Holland etal.[79]
2012, Chang etal.[80]
2013, Janjigian etal.[81]
2010, Oude Minnink etal.[82]
89
[
Zr]trastuzumab-
HER2/neu SKBR3/FaDu 2012, Oude Minnink etal.[83]
F(ab’)2
89
Zr]cetuximab EGFR U-373 MG/HT29/T-47D 2008, Aerts etal.[84]
[
2005, Perk etal.[85]
89
[
Zr]transferrin CD71 (TRFC) Prostate cancer: HiMYC transgenic
mi
ce and MycCaP tumours
Hormone-sensitive prostate cancer
ce
ll lines CWR22Pc and MDA PCa 2b
Gliomas: TS543, U87-MG, LN-18, SF268
2012, Holland etal.[86]
2013, Evans etal.[87]
2016, Doran etal.[88]
2017, Aggarwal etal.[89]
2018, Henry etal.[90]
Diuse large B-cell lymphoma: TDM8
Burkitt’s lymphoma: Raji
Breast cancers: MDA-MB-157/MDAMB-231/Hs578T
89
[
Zr]7E11 PSMA internal
epit
89
Zr]AC-10 CD30 Lymphoma: Karpas 299/negative
[
89
Zr]J591 PSMA LNCaP/negative PC-3 2011, Holland etal.[53]
[
89
[
Zr]Mb (dimeric
Fv-C
sc
3)
H
89
[
Zr]Cys-Db (dimeric
Fv)
sc
89
Zr]RO5323441 PlGF Hepatocellular cancer: Huh7
[
ope
PSMA LNCaP/negative PC-3 2014, Viola-Villegas etal.[93]
Prostate cancer: LNCaP 2011, Ruggiero etal.[91]
2016, Rylova etal.[92]
431
A-
2013, Oude Minnink etal.[94]
Human renal cell carcinoma: ACHN
89
Zr]bevacizumab VEGF Ovarian cancers: SK-OV-3, A2780/
[
resistant
CP
70
Colorectal cancer: Colo205
89
[
Zr]rituximab CD20 Human B-cell lymphoma: huCD20TM 2012, Natarajan etal.[98]
89
[
Zr]cG250 -F(ab9)2 CAIX (hypoxia) Head and neck: SCCNij3 2010, Hoeben etal.[99]
89
[
Zr]cG250 CAIX (hypoxia) Clear cell renal cell carcinoma: SK-RC-
2007, Nagengast etal.[95]
2010, Nagengast etal.[96]
2011, Nagengast etal.[97]
2013, Stillebroer etal.[100]
52 or NU-12
362 Handbook of Radiopharmaceuticals
Table 11.3 (Continued)
Tar g e t /
Radiotracer
biomarker Animal model/patient cohort Year/reference
[89Zr]TRC105 CD105 Breast cancer lung metastasis: 4T1 2012, Hong etal.[101]
89
[
Zr]R1507 IGF-1R Triple negative breast cancer 2010, Heskamp etal.[102]
89
[
Zr]onartuzumab c-MET MKN-45 (high), SNU-16 (moderate),
7-MG (low)
89
Zr]059–053 CD147 Pancreatic cancer: MIA Paca-2, PANC-
[
U8
2012, Jagoda etal.[103]
2013, Sugyo etal.[104]
1, BxPC-3, AsPC-1 (negative mouse A4)
89
Zr]panitumumab EGFR Breast cancer: MDA-MB-468 2013, Bhattacharyya etal.[105]
[
Wei e
89
2014,
[
Zr]RG7356 CD44 MDA-MB-231/negative HepG2 2014, Vugts etal.[107]
89
[
Zr]αGPC3 Glypican-3 (GPC3) Hepatocellular carcinoma: HepG2/
2014, Sham etal.[108]
tal.[106]
negative HLF and RH7777
89
Zr]-AMA Anti-mesothelin Pancreatic cancer: HPAC and CAPAN-2 2015, ter Weele etal.[109]
[
Human trials
89
[
Zr]
Zevalin(ibritumomab
CD20 CD20+ B-cell non-Hodgkin’s lym-
phoma (NHL)
2006, Perk etal.[75]
2012, Rizvi etal.[110]
tiuxetan)
89
[
Zr]mcAb-U36 chi-
meric monoclonal
CD44v6 Head and neck squamous cell
carcinoma (HNSCC)
2006, Börjesson etal.[76]
2009, Börjesson etal.[111]
antibody (cmAb)
89
[
Zr]trastuzumab HER2/neu Metastatic breast cancer
Esophagogastric cancer
2007, Dijkers etal.[112]
2010, Dijkers etal.[113]
2016, Laforest etal.[114]
2017, O’Donoghue etal.[115]
89
[
Zr]J591 PSMA Prostate cancer 2013, Morris etal.[116]
2014, Pandit-Tasker etal.[117]
2015, Pandit-Tasker etal.[118]
2016, Pandit-Tasker etal.[119]
89
[
Zr]Df-IAB2M mini-
PSMA Prostate cancer 2016, Pandit-Tasker etal.[120]
body
89
Zr]panitumumab EGFR Metastatic colon cancer 2017, Lindenberg etal.[121]
[
89
[
Zr]bevacizumab VEGF Breast cancer 2012, van der Bilt etal.[122]
2013, Gaykema etal.[123]
2014, van Asselt etal.[124]
2015, Oosting etal.[125]
89
[
Zr]fresolimumab Transforming
owth factor β
gr
Glioma 2015, den Hollander etal.[126]
(TGF-β)
89
[
Zr]rituximab CD20 B-cell lymphoma 2015, Muylle etal.[127]
89
[
Zr]cetuximab EGFR Colorectal cancer 2015, van Oordt etal.[128]
Note: the list is not intended to be comprehensive but rather demonstrates a range of compounds/targets that have been
investigated.
Chapter 11: The Radiochemistry ofZirconium 363
Scheme 11.3 Three
conceptual
approaches toward
radiolabelled
antibodies.
Although radiometal labelling reactions are generally simple to perform, it remains true
that manual synthesis of radiopharmaceuticals is not possible in most nuclear medicine facilities. Automation is highly desirable. If a disposable, cartridge-based system
can be made available, it is conceivable that many more hospitals will be able to access
89
Zr-radiolabelled antibodies using in-house facilities.
Conceptually, radiolabelled antibodies can be produced via three dierent routes
(Scheme11.3). The vast majority of radiolabelled antibodies are made via a two-step
pre-conjugation approach. In the rst step (bioconjugation), the protein is normally
puried from source and conjugated to a suitable chelate, and then the intermediate
Chelate
Bioconjugation
Linker
Pre-puried
proteins (mAbs)
Pre-puried or formulated
protein (mAbs)
Simultaneous
(one-step)
<10 min.
Radiolabelling
radiolabelled protein (mAbs)
Bioconjugation
Pre-radiolabelling
Functionalised
protein (mAbs)
Formulated
Chelate
Linker
Radionuclide
364 Handbook of Radiopharmaceuticals