A convenient method for the preparation of radioiodinated meta-iodobenzylguanidine at a no-carrier-added level

Article abstract of DOI:10.1002/jlcr.3348

Radioiodinated meta-iodobenzylguanidine (MIBG) in high effective specific activity was prepared using 3-tributylstannylbenzylguanidine as the precursor. The labeling was carried out in aqueous solution with the insoluble and lyophilized precursor suspende

Full text of DOI:10.1002/jlcr.3348

Research article
Received 11 May 2015,
Revised 18 July 2015,
Accepted 27 August 2015
Published online 22 September 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3348
A convenient method for the preparation of
radioiodinated meta-iodobenzylguanidine at
a no-carrier-added level
*
Gang Wang, Zhiming Chen, Erming Wu, Yang Wang, and Heyun Huang
Radioiodinated meta-iodobenzylguanidine (MIBG) in high effective specific activity was prepared using 3-
tributylstannylbenzylguanidine as the precursor. The labeling was carried out in aqueous solution with the insoluble and
lyophilized precursor suspended in the solvent. Simply by filtration, the starting material and by-products were readily
separated from the labeled solution. Less than 1.15 ppb tin has remained in the filtrate as determined by the atom
fluorescence spectrometry. By this approach, high specific activity (3.4 GBq/μmol) [131I]MIBG was obtained in 72.3 3%
(n = 3) radiochemical yield and 97.3 2% (n = 3) radiochemical purity. The whole preparation could be finished in less than
10 min. According to this method, a kit for the preparation of 123I-MIBG and 131I-MIBG is currently being developed.
Keywords: meta-iodobenzylguanidine; destannylation; no-carrier-added; lyophilization
the polymer precursor is not that simple for most laboratories,
and it is still not commercially available. The Donovan group
has reported a highly fluorinated (fluorous) tin precursor of
Introduction
Meta-iodobenzylguanidine (MIBG) is an analogue of the ad-
renergic neurotransmitter norepinephrine, which shows high
uptake not only in normal sympathetically innervated tissues,
MIBG, and purification was easy by chemoselective filtration
using a fluorous solid-phase extraction cartridge.6 But the elution
such as the heart and salivary glands, but also in neural crest
used to elute the radiolabeled MIBG from the cartridge including
tumors, such as neuroblastoma.1,2 Nowadays, radioiodinated
80% methanol and further purification and re-formulation is
MIBG has been used extensively in the clinic either as an imaging
required before a suitable preparation ready for injection is
agent for diagnosis or a therapeutic agent for tumors, especially
obtained.
123I-MIBG and 131I-MIBG.2–4
We herein present a new method for the no-carrier-added
At present, the radioiodinated MIBG used in the clinic is
MIBG synthesis using 3-tributylstannylbenzylguanidine as the
prepared on the basis of the isotopic exchange reaction
precursor. The labeling reaction was carried out in aqueous
between unlabeled MIBG and radioactive iodine.1,2 However,
solution with the lyophilized and insoluble precursor
the products synthesized this way contains large amount of
suspended in the solvent. The unreacted precursor and
unlabeled MIBG molecules, or ‘cold carriers’. These unlabeled
by-products were readily separated by filtration through a
molecules would competitively inhibit the uptake of
radiolabeled MIBG, greatly lower the drug’s effectiveness, and
might cause a side effect.
Up until now, several methods to synthesize MIBG at a no-
0.22-μm membrane syringe filter. The filtrate contained only
water solvent and thus, after dilution with an isotonic
physiological buffer or saline, had the potential be
administered
intravenously
into
patients.
The
tin
carrier-added level have been developed with reported
specific activities ranging from 9.2 to 59 GBq/μmol.1 The most
preferred precursor of no-carrier-added radioiodinated MIBG
so far is 3-tributylstannylbenzylguanidine, or its derivatives
for the tributylstannyl group could be easily replaced by
iodine.2 Most often, preparative HPLC would be employed
during purification after labeling, which is time consuming
and not readily available at most hospital radiopharmacy
departments.
concentration in the filtration was less than 1.15 ppb as
determined by atom fluorescence spectrometry (AFS). The
whole preparation could be finished in less than 10 min with
72.3 3% (n = 3) radiochemical yield and 97.3 2% (n = 3)
radiochemical purity.
Many efforts have been made to avoid the use of preparative
HPLC. Among these, the Hunter group has developed a method
on the basis of electrophilic radioiodination reaction and solid-
phase technology by using dibutylstannyl benzylguanidine
precursor linked to polymers.5 It is simple and convenient, as
purification is just involving filtration. However, the synthesis of
Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine,
Jiangsu Institute of Nuclear Medicine, Wuxi, 214063 Jiangsu, China
*Correspondence to: Gang Wang, Ministry of Health, Jiangsu Key Laboratory of
Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi,
214063 Jiangsu, China.
E-mail: wanggang@jsinm.org
J. Label Compd. Radiopharm 2015, 58 442–444
Copyright © 2015 John Wiley & Sons, Ltd.

G. Wang et al.
Materials and methods
Reagents
All chemicals were purchased from Sigma-Aldrich (St louis, Missouri,
USA) unless otherwise noted. The sodium [131I]iodide solution with
specific activities of approximate 3.7 GBq/μmol was obtained from
Chengdu Gaotong Isotope Co., Ltd (Chengdu, Chongqing province,
China). Non-radioactive MIBG was synthesized according to
literature procedure.7 The 717 anion exchange resin (exchange
capacity = 1.2 meq/mL) was obtained from Sinopharm Chemical
Reagent Co., Ltd (Beijing, China). Prior to use, the resin was washed
three times with 20 mL of sterile water for injection per milliliter
resin.
Scheme 1. Synthesis of (1) [3-tributylstannylbenzylguanidine].
Non-radioactive (cold) iodination
Into the lyophilized mixture of 3-tributylstannylbenzylguanidine
(0.05 mg, 0.114 μmol) and KH2PO4 (1.36 mg, 10 μmol) was added
100 μL of 0.1 mg/mL KI solution, 50 μL of a solution 0.5 mol/L in
acetic acid, and 0.3 mol/L in H2O2, and 1 mL sterile water for
injection. The mixture was reacted for 5 min at room
temperature and then ended by adding 1 mL of 10 mg/mL
sodium metabisulphite solution. After that, the 717 anion
exchange resin (0.75 mg, 1 mL) was added into the solution for
2 min. Finally, the solution was separated from the resin and
precipitations through a 0.22-μm membrane syringe filter. HPLC:
retention time (RT) = 7.0 min; MS (+): [M + H]+ 276.0, [M-C2N3H4]+
205.0, peak at 439.4 [1 + H]+ absent.
Instrumentation
The preparative HPLC was performed on a Waters (Milford,
Massachusetts, USA) 2545 Binary Gradient Module equipped with
a Waters 2998 Photodiode Array Detector, a Waters Xbridge prep
OBD column (C18, 5 μm, 19 * 150 mm). The elution condition was
as follows: solvent A = H2O, solvent B = CH3OH; 0–10 min 90% B;
10–15min 90% B to 100% B; 15–30 min 100% B. The flow rate
was 10mL/min. The detection wavelength was 230 nm.
Preparation of [131I]MIBG
The analytical HPLC of the precursor was performed on a
Waters 1525 Binary HPLC Pump equipped with a Waters 2487
Dual λ Absorbance Detector and a Waters SunFire column
(C18, 5 μm, 4.6 * 150 mm). The elution condition was as follows:
solvent A = H2O (0.1%CF3COOH), solvent B = CH3OH; 0–10 min
90% B; 10–15 min 90% B to 100% B; 15–30 min 100% B. The flow
rate was 1 mL/min. The detection wavelength was 230 nm.
The analytical HPLC of final [131I]MIBG product was performed
on a Waters 1525 Binary HPLC Pump equipped with a Waters
2487 Dual λ Absorbance Detector, a Perkin Elmer γ-counter, a
Waters SunFire column (C18, 5 μm, 4.6 * 250 mm). The elution
condition was as follows: solvent A = H2O (0.1% H3PO4), solvent
B = CH3CN (0.1% H3PO4); 0–20 min 23% B. The flow rate was
1 mL/min. The detection wavelength was 230 nm.
NMR spectra were recorded on an Avance III 400 MHz Digital
NMR spectrometer (Billerica, Massachusetts, USA). ESI mass
spectra were acquired on a Waters Acquity instrument fitted
with a Waters SQ Detector 2. The lyophilization was performed
on a Labconco Tray Dryer (version 10417A) (Kansas, Missouri,
USA). The tin concentration of the purified product was
measured on an AFS-9700 atomic fluorescence photometer
(Made by Haiguang Instrument corporation, Beijing, China). The
0.22-μm membrane syringe filter (13 mm Nylon 100/pk) was
purchased from Dikma Technologies Inc (Beijing, China).
Into the lyophilized mixture of 3-tributylstannylbenzylguanidine
(0.05 mg, 0.114 μmol) and KH2PO4 (1.36 mg, 10 μmol) was added
1 mL (~0.185 GBq) of Na131I solution and 50 μL of a solution
0.5 mol/L in acetic acid and 0.3 mol/L in H2O2. The mixture was
reacted for 5 min at room temperature and then ended by
adding 1 mL of 10 mg/mL sodium metabisulphite solution. After
that, the 717 anion exchange resin (0.75 mg, 1 mL) was added
into the solution for 2 min. Finally, the solution was separated
from the resin and precipitations through a 0.22-μm membrane
syringe filter. The filtrate was analyzed by analytical HPLC
equipped with both ultraviolet (UV) detector and γ-ray counter.
The UV detector showed a small yet clearly observed peak at
the RT of MIBG (RT = 7.0 min) and two larger peaks near the
solvent front (RT < 3.5 min). The corresponding radioactivity
trace showed one main peak at the RT of MIBG (RT = 7.0 min),
which was coordinated with the UV signal and another small
peak at 3.1 min, which was confirmed by coinjection to be
iodine. The radiochemical purity calculated from the γ-ray signal
spectrum was 97.3 2% (n = 3).
Results and discussion
The precursor 3-tributylstannylbenzylguanidine could be syn-
thesized by reaction of MIBG and bis(tributyltin) catalyzed by bis
(triphenylphosphine)palladium(II) dichloride in one step.8,9 Owing
to the strong polarity of the guanidino group, the product could
not be separated by normal-phase chromatography.10,11 As a
3-Tributylstannylbenzylguanidine (1)
A mixture of MIBG (500 mg, 1.82 mmol), bis(tributyltin) (1.27 g, result, the preparative HPLC was employed. Reversed-phase
2.18 mmol), Pd(PPh3)2Cl2 (127.6 mg, 0.18 mmol) in 30 mL 1,4- preparative HPLC was found to give 3-butylstannylbenzylguanidine
dioxane, and 10 mL dimethylformamide was reflux at 100 °C until in nearly 100% percent chemical purity as determined by analytical
the solution turned black. After cooling down to room temperature, HPLC. The identity of the product was confirmed by 1H NMR and
the solution was filtrated and evaporated. The residues were then comparison with literature spectrum. The peaks at δ = 0.88–1.58,
resolved in 50 mL methanol and washed with 20 mL hexane three 4.39, and 7.24–7.41 coordinated with the hydrogen atoms linked
times. Finally, the preparative HPLC was applied to yield light in the tributyl, benzyl, phenyl groups, respectively. The
1
yellow liquid 576.7 mg (72.4%). H NMR (400 M, CD3OD) δ 0.88– corresponding integrated area ratio of the peaks was 27:2:4, which
0.91(m, 9H), 1.08–1.11(m, 6H), 1.32–1.37 (m, 6H), 1.55–1.58 (m, was coordinated with the numbers of hydrogen atoms linked.
6H), 4.39 (s, 2H), 7.24–7.26 (m, 1H), 7.33–7.37 (m, 1H), 7.41 (m, 2H);
The 3-tributylstannylbenzylguanidine was lyophilized into
white solid powder together with KH2PO4. When the powder
mass spectrometry (MS) (+): [M + H] + 439.4 (Scheme 1).
J. Label Compd. Radiopharm 2015, 58 442–444
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

G. Wang et al.
was resolved into water during the labeling, the insoluble 3- and γ-ray counter to be 3.4 GBq/μmol. It was much higher than
tributylstannylbenzylguanidine was suspended in the solvent. the previously reported ca [*I]MIBG but lower than the nca [*I]
The unreacted starting material and by-product could be easily MIBG. With regard to the fact that the specific activity of the
separated by filtration through a 0.22-μm membrane. Fur- sodium [131I]iodide solution we used as the starting material
thermore, the lyophilized white solid powder had a looser was merely 3.7 GBq/μmol, the relatively low specific activity of
structure, which could increase the interacting surface between our purified product was acceptable. Once the specific activity
3-tributylbenzylguanidine and iodine, and further improve the of the sodium [131I]iodide solution was improved, the specific
chemical yield.
activity of our purified product would increase accordingly.
The H2O2/HOAc was chosen to be oxidant because there was
no need for further purification after labeling. As compared, the Conclusion
chloramine-T could reach the similar radiochemical yield, but its
A convenient method of nca [*I]MIBG synthesis in high specific
by-product could not be easily removed. Another potential
oxidant was iodogen, as it could be coated on the vial bottom
as solid phase and removed easily. Furthermore, it could be
added into the mixture of 3-tributylstannylbenzylguanidine and
KH2PO4 previously and lyophilized into white solid powder
altogether as a kit, and then only the sodium [131I]iodine solution
should be added during radiolabeling. This method would
increase the labeling efficiency and feasibility in the clinic and
was under evaluation.
activity using 3-tributylstannylbenzylguanidine as the precursor
has been developed. The whole synthesis process can be
finished in less than 10 min with good radiochemical yield and
high radiochemical purity. Purification only includes filtration.
MS and AFS data indicate that the precursor and its by-products
can be readily separated from the desired product.
Acknowledgements
The unreacted tin precursor and its by-products that remained in
the purified product are toxic, and the tin concentration must be
determined. The previous document6 has reported a similar [*I]MIBG
injection using 3-tris[2-perfluorohexylethyl]stannylbenzylguanidine
as the precursor in which the tin concentration was determined to
be less than 1 ppm by inductively coupled plasma–MS. In our case,
the tin concentration in the purified product was determined by
AFS to be under the detection limit (1.15 ppb), which is far more less
than 1 ppm.
As verified, the purified product was free of the tin precursor and
its by-products. The oxidant H2O2/HOAc was reduced to water,
which needed no further purification. The purified product already
had KH2PO4 buffer in it, and the PH was measured to be around 5.
The metabisulphite was indexed in the Chinese Pharmacopeia to
be used as pharmaceutical excipient. Thus, the purified product,
after dilution with an isotonic physiological buffer or saline, had
the potential to be administered intravenously into patients and
was fit for the clinic to prepare temporarily. The toxicity, sterility,
and endotoxicity of the purified product were under evaluation.
The high specific activity of no-carrier-added (nca) [*I]MIBG
can reduce the total MIBG molecular amount injected, which
would improve the drug’s effectiveness and lower the possible
side effect. The specific activity of ca [*I]MIBG and nca [*I]MIBG
reported before ranged from 0.037 to 0.185 and 9.25 to
74 GBq/μmol, respectively. The specific activity of our purified
product was measured by HPLC equipped with both UV detector
We would like to thank the Jiangnan University and Jiangsu
Institution of Microbiology Co., Ltd for the H NMR, MS spectra,
and the AFS.
1
References
[1] S. Vallabhajosula, A. Nikolopoulou, Semin Nucl Med 2011, 41, 324–333.
[2] G. Vaidyanathan, Q J Nucl Med Mol Imaging 2008, 52, 351–368.
[3] R. E. Coleman, J. B. Stubbs, J. A. Barrett, M. D. L. Guardia, N. LaFrance,
J. W. Babich, Cancer biotherapy and radiopharmaceuticals 2009, 24
(4), 469–475.
[4] J. A. Barrett, J. L. Joyal, S. M. Hillier, K. P. Maresca, F. J. Femia, J. F.
Kronauge, M. Boyd, R. J. Mairs, J. W. Babich, Cancer biotherapy and
radiopharmaceuticals 2010, 25(3), 299–308.
[5] D. H. Hunter, X. Zhu, J Label Compd Radiopharm 1999, 42, 653–661.
[6] A. C. Donovan, J. F. Valliant, Nucl Med Biol 2008, 35, 741–746.
[7] G. J. Eastland, Drugs Fut 1989, 14(5), 427.
[8] T. J. Manger, J. L. Wu, D. M. Wieland, J Org Chem 1982, 47, 1484–1488.
[9] D. M. Wieland, T. J. Manger, M. N. Inbasekaran, L. E. Brown, J. L. Wu, J
Med Chem 1984, 17, 149–155.
[10] G. Vaidyanathan, D. J. Affleck, K. L. Alston, M. R. Zalutsky, J Label
Compd Radiopharm 2007, 50, 177–182.
[11] G. Vaidyanathan, M. R. Zalutsky, Appl Radiat Isot 1993, 44(3),
621–628.
Supporting information
Additional supporting information may be found in the online
version of this article at the publisher’s web-site.
www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 442–444