A novel bifunctional chelating agent based on bis(hydroxamamide) for 99mTc labeling of polypeptides

Article abstract of DOI:10.1002/jlcr.1954

This paper describes the synthesis and biological evaluation of a novel bifunctional chelating agent (BCA) based on bis(hydroxamamide) for ^99mTc labeling of polypeptides. We successfully designed and synthesized C₃(BHam)₂·COOH as a new BCA. C ₃(BHam)₂·COOH formed a stable ^99mTc complex and enabled us to prepare ^99mTc-labeled polypeptides by using a 2,3,5,6-tetrafluorophenol (TFP) active ester of C₃(BHam) ₂·COOH. ^99mTc-C₃(BHam) ₂·HSA prepared with C₃(BHam)₂·TFP was stable in both murine plasma and an excess of l-cysteine without any dissociation of ^99mTc from polypeptides. Furthermore, the blood clearance of ^99mTc-C₃(BHam)₂·HSA in mice was similar to that of ¹²⁵I-HSA, suggesting that C ₃(BHam)₂·COOH retained stable binding between ^99mTc and the polypeptides in vivo. When ^99mTc-C ₃(BHam)₂·NGA was injected into mice, the radioactivity showed high hepatic uptake early on and a rapid clearance from the liver, indicating that C₃(BHam)₂·COOH did not affect the pharmacokinetics of polypeptides in vivo and gave radiometabolites, which displayed a rapid elimination from the liver. Such characteristics would render C₃(BHam)₂·COOH attractive as a new BCA for ^99mTc labeling of polypeptides. Copyright

Full text of DOI:10.1002/jlcr.1954

Research Article
Received 5 September 2011,
Revised 7 November 2011,
Accepted 13 November 2011
Published online 12 December 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1954
A novel bifunctional chelating agent based on
99m
bis(hydroxamamide) for Tc labeling of
polypeptides
a
a
b
Masahiro Ono,a,b Masatsugu Ohgami, Mamoru Haratake, Hideo Saji,
*
a**
and Morio Nakayama
This paper describes the synthesis and biological evaluation of a novel bifunctional chelating agent (BCA) based on
bis(hydroxamamide) for 99mTc labeling of polypeptides. We successfully designed and synthesized C3(BHam)2-COOH as a
new BCA. C3(BHam)2-COOH formed a stable 99mTc complex and enabled us to prepare 99mTc-labeled polypeptides by using
a 2,3,5,6-tetrafluorophenol (TFP) active ester of C3(BHam)2-COOH. 99mTc-C3(BHam)2-HSA prepared with C3(BHam)2-TFP was
stable in both murine plasma and an excess of L-cysteine without any dissociation of 99mTc from polypeptides. Furthermore,
the blood clearance of 99mTc-C3(BHam)2-HSA in mice was similar to that of 125I-HSA, suggesting that C3(BHam)2-COOH
retained stable binding between 99mTc and the polypeptides in vivo. When 99mTc-C3(BHam)2-NGA was injected into mice,
the radioactivity showed high hepatic uptake early on and a rapid clearance from the liver, indicating that C3(BHam)2-COOH
did not affect the pharmacokinetics of polypeptides in vivo and gave radiometabolites, which displayed a rapid elimination
from the liver. Such characteristics would render C3(BHam)2-COOH attractive as a new BCA for 99mTc labeling of polypeptides.
Keywords: 99mTc; bifunctional chelating agent; polypeptide; hydroxamamide
(BHam) possessed square base pyramid coordination geometry,
Introduction
and the equatorial plane was formed by two-amine nitrogen
Many polypeptides such as antibodies, single-chain Fv frag-
and two-oxime oxygen atoms in a trans-orientation, whereas
the oxo core of the Tc(V) occupied the apical position.25 Indeed,
ments, diabodies, affibodies, minibodies, and bioactive peptides
have been used as scaffolds of radiolabeled probes for targeted
C3(BHam)2-NH2 provided stable 99mTc-labeled antibodies in
imaging of sites of tumors, infection, and thrombosis.1–10 Among
radionuclides for radiolabeling of these polypeptides, 99mTc is
ideal for scintigraphic imaging because of its excellent physical
properties, low cost, and ready availability.11,12 Generally, poly-
peptides do not possess binding sites to form 99mTc chelates
of high stability in vivo. To prepare 99mTc-labeled peptides for
application in vivo, therefore, one must incorporate appropriate
chelating agents into polypeptide molecules.
mild conditions to image tumor sites in tumor-bearing mice.23
However, as C3(BHam)2-NH2 cannot directly conjugate to poly-
peptides, N-(6-maleimidocaproyloxy)succinimide (EMCS) and
2-iminothiolane (2-IT) were essential for the preparation of
99mTc-labeled polypeptides by using C3(BHam)2-NH2 as a BCA.
When C3(BHam)2-NH2 is applied to 99mTc labeling for lower
molecular weight polypeptides, the incorporation of spacers
(EMCS and 2-IT) between 99mTc-C3(BHam)2 and polypeptides
may affect the pharmacokinetics and bioactivity of the
polypeptides.26,27
99mTc labeling using bifunctional chelating agents (BCAs),
which possess both a binding site for polypeptides and a site
for complexation with 99mTc, is required. It has been reported
that tetradentate ligands with N3S or N2S2 (containing one or
two thiol groups)13–15 and hydrazino nicotinamide (HYNIC)
(thiol-free chelating agent)16–19 serve as a BCA for 99mTc labeling.
However, some N3S or N2S2 ligands require harsh 99mTc complexa-
tion (elevated temperatures or high pH) to prepare 99mTc chelates
with high radiochemical yields.13–15 When 99mTc-HYNIC-labeled
polypeptides were administered in vivo, they showed not only
localized radioactivity in target tissues but also strong, persistent
radioactivity in non-target tissues.17
aGraduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan
bGraduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida
Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
*Correspondence to: Masahiro Ono, Graduate School of Pharmaceutical
Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto
606-8501, Japan.
To improve BCAs, we have developed 4′-aminomethyl-N,N′-
trimethylene bisbenzohydroxamamide [C3(BHam)2-NH2], which
has C3(BHam)2 for chelating with 99mTc and a primary amino
group for binding with polypeptides (Figure 1).20–24 A recent
E-mail: ono@pharm.kyoto-u.ac.jp
**Correspondence to: Morio Nakayama, Graduate School of Biomedical Sciences,
Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
report showed that the 99mTc complexes of benzohydroxamamide E-mail: morio@nagasaki-u.ac.jp
J. Label Compd. Radiopharm 2012, 55 71–79
Copyright © 2011 John Wiley & Sons, Ltd.

M. Ono et al.
NH2
NH HN
OH HO
NH HN
OH HO
N
N
N
N
C3(BHam)2-NH2
C3(BHam)2
EMCS
2-IT
Lys- -amino residue
C3(BHam)2-NH2
O
O
O
H
N
N
N
H
S
NH HN
NH
99mTc
OH HO
N
N
99mTc-C3(BHam)2-EMCS-IT-polypeptide
Figure 1. Chemical structure of C3(BHam)2, C3(BHam)2-NH2, and 99mTc-C3(BHam)2-EMCS-IT-polypeptide.
The objective of this study was to develop a novel BCA based on with ethyl acetate. After drying of the organic layer on Na2SO4, eva-
C3(BHam)2, which can be introduced into polypeptides without poration gave 16.6 g of BHam (83.1%). 1H NMR (DMSO-d6)
a long linker such as EMCS and 2-IT. We designed and synthe- d: 5.81 (s, 2H, -NH2), 7.36–7.38 (m, 3H, aromatic), 7.66–7.69 (m, 2H,
sized 4′-carboxyl-N,N′-trimethylene bisbenzohydroxamamide aromatic), 9.64 (s, 1H, -NOH).
[C3(BHam)2-COOH], which has carboxylic acid for binding with
the lys-e-amino group of polypeptides. Next, using C3(BHam)2-COOH,
Synthesis of O-carbethoxybenzohydroxamamide (1)
we investigated the labeling of 99mTc with human serum albumin
To a solution of BHam (19.0 g, 140.0 mmol) in dry acetone
(90 mL) was gradually added a solution of ethyl chlorocarbonate
(16.8 g, 156.0 mmol) in acetone (30 mL) in an ice bath for 1 h.
After a 5% NaOH solution (126 mL) was added, the mixture was
stirred at room temperature for 1 h. The precipitate formed was
filtered, and the residue was recrystallized from H2O–acetone
(4:3) to give 15.9 g of 1 (54.9%). 1H NMR (CDCl3) d: 1.37 (t, 3H, –CH3),
4.33 (q, 2H, J = 7.2 Hz, –CH2), 5.11 (s, broad, 2H, -NH2), 7.41–7.48
(m, 3H, aromatic), 7.68–7.71 (m, 2H, aromatic).
(HSA) as a model polypeptide and evaluated the stability of
99mTc-C3(BHam)2-HSA in vitro and in vivo. Furthermore, we used
galactosyl-neoglycoalbumin (NGA) as a model polypeptide and
evaluated the pharmacokinetics of radiometabolites formed in
the liver after the injection of 99mTc-C3(BHam)2-NGA into mice.
Materials and methods
Reagents and chemicals
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Varian Gemini 300 (300 MHz) (Varian Medical Sys-
tems, Inc., Calfornia, USA). Electron impact mass spectra (EI-MS)
and fast atom bombardment mass spectra (FAB-MS) were
obtained with a JEOL IMS-DX300 mass spectrometer (JEOL Ltd.,
Tokyo, Japan). Na[125I]I (3.7GBq/mL, 0.01 N NaOH solution) was
obtained from MP Biomedicals. [99mTc]Pertechnetate (99mTcO4À)
(111 MBq/mL) was purchased from Nihon-Medi-Physics (Tokyo,
Japan). Size-exclusion HPLC (SE-HPLC) was performed using a
TSK G3000SW (7.8Â 300 mm) column (Tosoh Corporation, Tokyo,
Japan), eluted with 0.1M phosphate buffer (PB) (pH 7.0) containing
0.3 M sodium chloride at a flow rate of 0.5mL/min. Reversed-phase
HPLC (RP-HPLC) was performed with a Cosmosil 5 C18-MS column
(4.6 Â 150mm, Nacalai Tesque, Kyoto, Japan) at a flow rate of
1 mL/min with a gradient mobile phase from 85% A (PB, 0.01 M,
pH7.4) and 15% B (acetonitrile) to 20% A and 80% B in 10 min.
Cellulose acetate electrophoresis (CAE) was run on Separax SP
(Joko, Tokyo, Japan) at a constant current of 0.8mA/cm for
30min in 0.072 M veronal buffer (pH 8.6). The distance migrated
by HSA was determined by Ponceau 3R staining.
Synthesis of 3-phenyl-Δ2-1,2,4-oxadiazolin-5-one (2)
To a solution of 1 (15.8 g, 75.7 mmol) in 5% NaOH solution
(80 mL) was added excess acetic acid (26.3 mL, 460 mmol). The
precipitate formed was filtered to give 10.7 g of 2 (87.5%). H
NMR (DMSO-d6) d: 7.56-7.67 (m, 3H, aromatic), 7.81–7.85 (q, 2H,
J = 6.3 Hz, aromatic).
1
Synthesis of potassium 3-phenyl-Δ2-1,2,4-oxadiazolin-5-one (3)
To a solution of 2 (7.50 g, 46.3 mmol) in MeOH (20 mL) was
added a solution of KOH (2.60 g, 46.3 mmol) in MeOH (30 mL).
After the mixture was stirred at room temperature for 1 h,
evaporation gave 8.97 g of 3 (96.8%). 1H NMR (DMSO-d6) d:
7.37–7.42 (m, 3H, aromatic), 7.76–7.80 (m, 2H, aromatic).
Synthesis of 3-phenyl-4-(3-bromopropyl)-Δ2-1,2,4-oxadiazolin-5-one (4)
To a solution of 3 (9.28 g, 32.8 mmol) in DMF (31 mL) was added
1,3-dibromopropane (19.9 g, 98.4 mmol) in DMF (25 mL). The
reaction mixture was stirred at room temperature for 3 days.
After the filtrating precipitate of potassium bromide was filtered
and the solvent was removed, the residue was purified by silica
gel chromatography (ethyl acetate/hexane = 1:5) to give 4.56 g
of 4 (49.1%). 1H NMR (CDCl3) d: 2.22–2.26 (m, 2H, -NCH2CH2CH2Br),
3.35 (t, 2H, -NCH2CH2CH2Br), 3.86 (t, 2H, -NCH2CH2CH2Br), 7.58–7.63
(m, 5H, aromatic).
Synthesis of benzohydroxamamide
To a solution of hydroxylammonium chloride (10.5 g, 145.2 mmol)
and NaHCO3 (12.3 g, 146.0 mmol) in H2O (40 mL) was gradually
added a solution of benzonitrile (15.3g, 145.2 mmol) in EtOH
(100 mL), and the reaction mixture was stirred at 80ꢀC for 4 h.
EtOH was removed in vacuo, and the mixture was extracted
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M. Ono et al.
Synthesis of 4-carboxylbenzohydroxamamide methyl ester
(BHam-COOMe, 5)
stannous tartrate (375 mL, 3 Â 10À4 M) in H2O and Na99mTcO4
(74 MBq/mL, 125 mL) was then added. After incubation for
15 min, radiochemical yields of 99mTc-C3(BHam)2-COOH were
determined by RP-HPLC, CAEP, and TLC.
The reaction mixture of 4-cyanobenzoic acid methyl ester (10.0g,
61.8 mmol), NaHCO3 (5.35 g, 66.7 mmol), and hydroxylammonium
chloride (4.30 g, 61.8 mmol) in MeOH (120 mL) was stirred at room
temperature for 30min. The mixture was then stirred at 80–90 ꢀC
for 3 h. After it had cooled to room temperature, 200 mL of water
was added to produce a precipitate. 5 was obtained after washing
the precipitate with ether in a yield of 68.6% (8.24 g). 1H NMR
(CDCl3) d: 3.94 (s, 3H, –CH3), 4.89 (s, broad, 2H, -NH2), 7.72 (t, 2H,
aromatic), 8.89 (t, 2H, aromatic).
Preparation of 99mTc-C3(BHam)2-TFP
2,3,5,6-Tetrafluorophenol (TFP) (2 mg) was added to a solution
of 99mTc-C3(BHam)2-COOH in saline (200 mL). Next, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (4 mg)
was added, the reaction mixture was stirred at room temperature
for 30min, DMSO (60 mL) was added, and the mixture was purified
using Sep-Pak. The Sep-Pak was replaced with water. The reaction
mixture (260 mL) was applied, the column was washed with water
(6mL) and diethylether (1mL), and 99mTc-C3(BHam)2-TFP was
eluted with acetonitrile (1 mL). After evaporation of the acetonitrile,
the radioactivity in the residue was analyzed by SE-HPLC and CAEP.
Synthesis of 3-(4-carboxylphenyl)-Δ2-1,2,4-oxadiazolin-5-one
methyl ester (6)
To a suspension of 5 (4.96 g, 25.5 mmol) in 1,4-dioxane (35 mL)
was added 1′-carbonyldiimidazole (5.30 g, 30.6 mmol). The mix-
ture was stirred at 110 ꢀC for 30 min. The solvent was removed,
the residue was dissolved in water, and 3 N HCl was added to
produce a precipitate. The precipitate was washed with water,
ether, and ethyl acetate to give 4.91 g of 6 (87.5%). 1H NMR
(DMSO-d6) d: 3.90 (s, 3H, –CH3), 7.96 (d, 2H, J = 8.7 Hz, aromatic),
8.15 (t, 2H, aromatic).
Preparation of 99mTc-C3(BHam)2-HSA
To 200 mL of HSA solution (10 mg/mL in 0.1 M carbonate buffer,
pH 9.5) was added an equal volume of 99mTc-C3(BHam)2-TFP
prepared above, and the reaction mixture was incubated for
1 h at room temperature. 99mTc-C3(BHam)2-HSA was then puri-
fied by the centrifuged column procedure using a Sephadex
G-50 column equilibrated and eluted with PB (0.1 M, pH 7.4).
The radiochemical yield was assessed by SE-HPLC and CAEP.
Synthesis of potassium 3-(4-carboxylphenyl)-Δ2-1,2,4-oxadiazolin-5-
one methyl ester (7)
To a solution of 6 (8.62 g, 39.2 mmol) in MeOH (23 mL) was
added a solution of KOH (2.25 g, 40.0 mmol) in MeOH (28 mL).
After the reaction mixture was stirred at room temperature for
1 h, evaporation of the solvent gave 9.78 g of 7 (96.6%). 1H
NMR (DMSO-d6) d: 3.87 (s, 3H, –CH3), 7.93 (t, 2H, aromatic), 8.00
(t, 2H, aromatic).
Preparation of 99mTc-C3(BHam)2-NGA
Galactosyl-neoglycoalbumin was synthesized by conjugation of
cyanomethyl-2,3,4,6-tetra-O-acetyl-1-thio-b-D-galactopyranoside,
synthesized according to the procedure of Lee et al.,28 with HSA.
The phenol–sulfuric acid reaction indicated that 25 galactose units
were attached to each HSA molecule.29 99mTc-C3(BHam)2-NGA was
prepared similar to 99mTc-C3(BHam)2-HSA, and radiochemical yield
was assessed by SE-HPLC and CAEP.
Synthesis of methyl 4-(4,5-dihydro-5-oxo-4-(3-(5-oxo-3-phenyl-1,2,4-
oxadiazol-4-yl)propyl)-1,2,4-oxadiazol-3-yl)benzoate (8)
To a solution of 4 (1.15 g, 4.06 mmol) in DMF (20 mL) was added
a solution of 7 (1.11 g, 4.30 mmol) in DMF (20 mL) at room tem-
perature. The reaction mixture was then stirred at 40 ꢀC for
7 days. The solvent was evaporated, water was added, and the
mixture was extracted with ethyl acetate. The organic layer was
dried over Na2SO4 and filtered. The residue was purified by silica
gel chromatography (ethyl acetate/hexane = 1:2) to give 850 mg
Preparation of 125I-HSA
To 200 mL of HSA solution (4mg/mL in 0.1 M PB, pH 7.4) was added
1 mL of Na125I (3.7GBq/mL) solution and 10 mL of chloramine-T
solution (2mg/mL in 0.1M PB, pH7.4). The reaction mixture was
incubated for 10 min. A solution of NaHSO3 (1mg/mL, 6 mL) was
added, and the mixture was purified by the centrifuged column
procedure using a Sephadex G-50 column equilibrated and eluted
with PB (0.1M, pH7.4). The radiochemical yield of 125I-HSA was
assessed by CAEP and TLC.
1
of 8 (49.6%). H NMR (DMSO-d6) d: 1.77 (m, 2H, –CH2CH2CH2-),
3.55–3.62 (q, 4H, J = 7.2 Hz, –CH2CH2CH2-), 3.93 (s, 3H, –CH3),
7.56–7.60 (m, 4H, aromatic), 7.64–7.69 (m, 1H, aromatic), 7.75
(d, 2H, J = 7.8 Hz, aromatic), 8.12 (d, 2H, J = 8.1 Hz, aromatic).
EI-MS calc C21H18N4O6 (M+): m/z 422, found: 422.
Synthesis of C3(BHam)2-COOH
Stability of 99mTc-C3(BHam)2-HSA in vitro
8 (850 mg, 2.01 mmol) was added to 5 mL of 1 N NaOH, and the
mixture was heated with stirring at 90–100 ꢀC for 1.5 h, then
allowed to cool to room temperature. The pH was adjusted to
3 with 1 N HCl. The mixture was neutralized with 1 N NaOH,
and the solvent was removed in vacuo. The residue was purified
by silica gel chromatography (MeOH/CHCl3/acetic acid = 10:50:1)
99mTc-C3(BHam)2-HSA (50 mL, 8.3 kBq) was diluted 20-fold with
0.1 M PB (pH 7.4, 200 mL) or freshly prepared murine plasma,
and the solution was incubated at 37 ꢀC. After 1, 3, 6, and 24 h
of incubation, the radioactivity of 99mTc-C3(BHam)2-HSA was ana-
lyzed by CAEP. To a solution of 99mTc-C3(BHam)2-HSA (12.2 kBq)
in 0.1 M PB (pH 7.4, 135 mL) was added L-cysteine (15 mL,
5 Â 10À6 to 5 Â 10À2 M). After incubation for 1 h at 37 ꢀC, the
radioactivity of the reaction mixture was analyzed by SE-HPLC.
99mTc-cysteine was prepared by incubating for 1 h at room tem-
perature after mixing a solution of 0.1 M L-cysteine (100 mL) with
1
to give 620 mg of C3(BHam)2-COOH (61.5%). H NMR (DMSO-d6)
d: 1.57 (m, 2H, –CH2CH2CH2-), 3.00–3.08 (m, 4H, –CH2CH2CH2-),
7.53–7.62 (m, 7H, aromatic), 8.05 (d, 2H, J = 9.0Hz, aromatic), 8.92
(s, 1H, -COOH). FAB-MS calc C18H21N4O4 (MH+): m/z 357, found: 357.
Preparation of 99mTc-C3(BHam)2-COOH
A solution of C3(BHam)2-COOH in DMSO (0.1 M) was prepared. an aqueous solution of stannous tartrate (75 mL, 3 Â 10À4 M) and
This solution (5 mL) was added to H2O (495 mL). A solution of Na99mTcO4 (25 mL, 3.7 MBq).
J. Label Compd. Radiopharm 2012, 55 71–79
Copyright © 2011 John Wiley & Sons, Ltd.
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M. Ono et al.
Experiments in vivo
the biodistribution experiments were analyzed using the unpaired
t-test. Differences were considered statistically significant when
Animal experiments were conducted in accordance with our insti- the p value was less than 0.05.
tutional guidelines and approved by the Nagasaki University Ani-
The concentration of 99mTc-C3(BHam)2-NGA was adjusted to
mal Care Committee. Biodistribution experiments were performed 90 mg/mL with PB (66 mM, pH 7.4). Biodistribution experiments
by intravenously administering 99mTc-C3(BHam)2-HSA (37 MBq/mL were performed by intravenously administering 99mTc-C3
in 66 mM PB, pH7.4) or 125I-HSA (37 kBq/mL in 66 mM PB, pH 7.4) (BHam)2-NGA to 6-week-old male ddY mice (25–30 g). Groups
to 5-week-old male ddY mice (20–25 g). Groups of four to five mice of five to eight mice each were administered 9 mg of 99mTc-C3
each were administered 100mL (3.7kBq) of 99mTc-C3(BHam)2-HSA (BHam)2-NGA prior to sacrifice at 5, 10, 30, 60, 180, and
prior to sacrifice at 5, 10, 30, and 60 min postinjection by decapita- 360 min postinjection by decapitation. Tissues of interest were
tion. Tissues of interest were removed and weighed, and radioac- removed and weighed, and radioactivity was measured with an
tivity was measured with an auto well gamma counter. Data in auto well gamma counter.
O
F
F
F
F
O
OH
O
O
HN
NH
O
HN
NH
F
F
F
F
Tc
Tc
N
N
N
N
EDC
O
-
O
HO
O
O
99mTcO4
polypeptide
( TFP )
O
O
OH
N
H
NH HN
OH HO
O
HN
NH
N
N
Tc
N
N
O
O
C3(BHam)2-COOH
EDC
99mTc-C3(BHam)2-polypeptide
F
F
F
F
HO
-
99mTcO4
( TFP )
F
F
F
O
O
O
N
H
NH HN
NH HN
OH HO
F
N
N
N
polypeptide
OH HO
Figure 2. Two schemes for 99mTc labeling of polypeptides using C3(BHam)2-COOH.
HONH3Cl
NaHCO3
ClCO2Et
NaOH
NaOH
AcOH
H
N
NH2
OH
NH2
O
CN
OEt
O
O
N
N
O
N
Benzonitrile
BHam
(1)
(2)
O
Br(CH2)3Br
/ DMF
KOH
/ MeOH
OH
K
N
DMF
N
Br
N
N
O
O
N
O
N
O
N
N
O
O
O
O
(3)
(4)
(8)
O
O
KOH
/ MeOH
OMe
OMe
H
N
K
N
NaOH
O
O
N
O
N
O
(6)
(7)
O
CDI
OH
/ 1,4-dioxane
O
O
NH HN
OH HO
HONH3Cl
NaHCO3
OMe
OMe
N
N
H2N
NC
N
HO
(5)
4-Cyanobenzoic acid methyl ester
C3(BHam)2-COOH
Scheme 1. Synthesis of C3(BHam)2-COOH.
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J. Label Compd. Radiopharm 2012, 55 71–79

M. Ono et al.
(C)
(A)
(B)
*
*
*
0
10
20
30
0
1
2
3
4
5
6
7
8
0
0.2 0.4 0.6 0.8 1.0
Rf Value
Retention Time (min)
Distance from Origin (cm)
Figure 3. Radiochromatograms of 99mTc-C3(BHam)2-COOH obtained by RP-HPLC (A), CAEP (B), and TLC (C). Arrows with single asterisks show 99mTcOÀ4 .
indicated that C3(BHam)2-COOH can function as the coordinat-
Results and discussion
ing site of 99mTc in mild labeling conditions.
In the present study, in order to introduce 99mTc-C3(BHam)2 into
polypeptides, we designed and synthesized C3(BHam)2 which has
a carboxylic acid for binding to the lys-e-amino group of polypep-
tides. As shown in Figure 1, we tried two methods of preparing
99mTc-labeled polypeptides by using C3(BHam)2-COOH as a BCA.
Considering the half-life of 99mTc, and convenience of labeling
experiments or radiation exposure, a method of labeling poly-
peptides with 99mTc after conjugation with a BCA is preferred.
Therefore, first of all, we tried to synthesize a TFP active ester of
C3(BHam)2-COOH in order to conjugate polypeptides with a BCA
before 99mTc labeling (Figure 2, the scheme shown with a dotted
line). We performed the reaction of (BHam)2-COOH with TFP under
several different conditions. However, all the reactions gave multi-
ple products, and we could not obtain C3(BHam)2-TFP. This may be
one of the reasons why C3(BHam)2-TFP reacts with an active
secondary amino group or hydroxy group in the hydroxamamide
scaffold. As we had difficulty producing C3(BHam)2-TFP, we used
After the active esterification of 99mTc-C3(BHam)2-COOH with
TFP using EDC, we analyzed the radioactivity eluted from the
Sep-Pak column by RP-HPLC. The peak for 99mTc-C3(BHam)2-TFP
was observed at a retention time of 14 min, which was later than that
(A)
(B)
**
**
0
1 2 3 4 5 6 7 8
0
10
20
30
Retention Time (min)
Distance from Origin (cm)
Figure 4. Radiochromatograms of 99mTc-C3(BHam)2-TFP obtained by RP-HPLC (A)
an alternative method to prepare 99mTc-labeled polypeptides, and CAEP (B). Arrows with double asterisks show 99mTc-C3(BHam)2-COOH.
through the conjugation of polypeptides with 99mTc-C3(BHam)2-TFP
after active esterification of 99mTc-C3(BHam)2-COOH prepared by
labeling C3(BHam)2-COOH with 99mTc (Figure 2). Previous papers
(A)
(C)
** *
have reported that 99mTc-labeled polypeptides were successfully
prepared using N2S2 and N3S type ligands as BCAs by such a pre-
chelating method without a loss of bioactivity.13,14,30 Furthermore,
as the chemical form of 99mTc-labeled polypeptides is the same
even using different methods, in the present study, we synthesized
99mTc-labeled polypeptides through the latter approach, and eval-
uated the utility of C3(BHam)2-COOH as a BCA for 99mTc labeling.
C3(BHam)2-COOH was synthesized by using benzonitrile and
4-cyanobenzoic acid methyl ester as the starting materials
according to the route shown in Scheme 1. Compounds 4 and
6 were synthesized as reported previously. 6 was converted to
its potassium salt (7). 7 was reacted with 4 to give 8. 8 was
hydrolyzed in a 5% NaOH solution to give C3(BHam)2-COOH in
a total yield of 5.7%.
***
***
-
0
1 2 3 4 5 6 7 8
0
10
20
30
Retention Time (min)
Distance from Origin (cm)
(B)
(D)
(-)
(+)
8
The radioactivity of the reaction mixture of C3(BHam)2-COOH
and 99mTc was analyzed by RP-HPLC. The radioactivity of
99mTcOÀ4 at 3 min disappeared, and a new peak derived from
99mTc-C3(BHam)2-COOH was detected at a retention time of
7 min (Figure 3(A)). A new peak was detected 4 cm (anode) from
the origin in the analysis by CAEP (Figure 3(B)) and at Rf = 0.4 in
the analysis by TLC (Figure 3(C)). The results suggested that
99mTc-C3(BHam)2-COOH was produced, because radioactivity of
99mTcOÀ4 was detected at 6 cm (anode) by CAEP and at Rf = 1.0
by TLC. The radiochemical yield of 99mTc-C3(BHam)2-COOH was
over 93% in the RP-HPLC, TLC, and CAEP analyses. These results
3
2 1 0 1 2 3 4 5 6 7
Distance from Origin (cm)
0
10
20
30
Retention Time (min)
Figure 5. SE-HPLC profiles of 99mTc-C3(BHam)2-HSA (radioactivity, A) and unmodi-
fied HSA (UV absorbance 254 nm, B). A typical radioactivity profile of 99mTc-C3
(BHam)2-HSA (C) and Ponceau 3R staining of unmodified HSA analyzed by CAEP
(D). Arrows with single, double, and triple asterisks show 99mTcO4À, 99mTc-C3
(BHam)2-COOH, and 99mTc-C3(BHam)2-TFP, respectively.
J. Label Compd. Radiopharm 2012, 55 71–79
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

M. Ono et al.
100
80
60
40
20
0
0 M
5 x 10-6 M
5 x 10-5 M
5 x 10-4 M
0
2
4
6
Incubation Time (h)
5 x 10-3 M
5 x 10-2 M
Figure 6. Percent radioactivity in HSA fractions following incubation of 99mTc-C3
(BHam)2-HSA in phosphate buffer (●) and murine plasma (○) at 37 ꢀC. Each value
was determined by CAEP.
of 99mTc-C3(BHam)2-COOH (Rt = 7 min) (Figure 4(A)). In the analysis
by CAEP, almost all radioactivity existed at the origin different from
the site where the radioactivity of 99mTcO4À and C3(BHam)2-COOH
was detected, suggesting the production of 99mTc-C3(BHam)2-TFP
(Figure 4(B)). The recovery rate of radioactivity was ca. 50% after
purification by Sep-Pak, and the rest remained in the Sep-Pak
column.
We performed by gel filtration chromatography after the
conjugation of 99mTc-C3(BHam)2-TFP with HSA and analyzed
the radioactivity eluted from the gel filtration column by SE-HPLC
and CAEP (Figure 5). In the SE-HPLC analysis, the peaks of radio-
activity at retention times of 7 (dimer) and 8 min (monomer) corre-
sponded with the peaks of UV (254 nm) for unmodified HSA
(Figure 7(A, B)). In the CAEP analysis, over 95% of radioactivity
was detected at the same position as HSA stained with Ponceau
3R (Figure 5(C, D)). The radiochemical yield and purity were
Cys-complex
0
5
10
15
20
25
30
Retention Time (min)
Figure 7. SE-HPLC profiles of 99mTc-C3(BHam)2-HSA after incubation with L-
cysteine for 1 h at 37 ꢀC.
ca. 75% and >95%, respectively. The results suggested that C3
(BHam)2-COOH may serve as a BCA for the labeling of polypeptides
with 99mTc.
Next, we tested the stability of 99mTc-C3(BHam)2-HSA in PB or
murine plasma at 37 ꢀC by CAEP (Figure 6). The amount of
radioactivity bound to HSA was over 95% until 6 h. We also
Table 1. Biodistribution of radioactivity after injection of 99mTc-C3(BHam)2-HSA in mice
Percentage of injected dose per tissue
Tissue
Blooda
5 min
10 min
30 min
1 h
3 h
35.60
(2.69)
7.39
32.92
(2.01)
8.72
31.42
(1.47)
8.07
27.94
(3.17)
7.28
19.54
(2.62)
5.68
Liver
(1.60)
1.68
(0.41)
2.04
(0.45)
0.33
(0.01)
0.51
(1.09)
1.47
(0.29)
2.62
(0.35)
0.35
(0.05)
0.63
(0.70)
1.88
(0.21)
4.81
(0.39)
0.32
(0.06)
1.00
(1.07)
1.76
(0.29)
7.27
(1.18)
0.30
(0.04)
1.02
(0.62)
1.31
(0.08)
11.56
(0.78)
0.21
(0.03)
0.95
(0.11)
1.80
Kidney
Intestine
Spleen
Stomach
Lung
(0.10)
3.50
(0.02)
3.65
(0.24)
2.62
(0.10)
3.13
(0.47)
(0.72)
(0.38)
(0.44)
(0.52)
Each value represents the mean (SD) for four to five animals.
aExpressed as% injected dose per gram.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 71–79

M. Ono et al.
40
serves as a BCA that produces stable 99mTc-labeled polypeptides
in vivo.
When radiolabeled polypeptides were administered in vivo,
persistently high levels of radioactivity were observed in the liver
and kidney where catabolism of the parent proteins and pep-
tides occurs, which compromises the diagnostic accuracy of
these radiopharmaceuticals. Previous investigations regarding
the radiometabolites produced in the liver and kidney indicated
that slow elimination rates are responsible for the persistent
localization of radioactivity in the lysosomal compartment of
hepatic and renal cells.31–34 Several recent studies suggested
that an experimental system using NGA, which is incorporated
by hepatic parenchymal cells via receptor-mediated endocytosis
immediately after its administration, is suitable for investigating
radiometabolites formed in the liver.31–34 To investigate the
pharmacokinetics of the radiometabolites produced in the meta-
bolic tissues after the injection of 99mTc-labeled polypeptides by
using C3(BHam)2-COOH as a BCA, NGA was selected as a model
polypeptide, 99mTc-C3(BHam)2-NGA was prepared, and the
biodistribution in mice was evaluated. The biodistribution of
radioactivity after the injection of 99mTc-C3(BHam)2-NGA is sum-
marized in Table 2. At 5-min postinjection, more than 89% of
the radioactivity was accumulated in the liver. The radioactivity
was rapidly eliminated from the liver by hepatobiliary excretion.
At 6-h postinjection, the radioactivity retained in the liver was
just 7% of the injected dose. No marked accumulation in the
blood or the other tissues was observed.
30
20
10
0
0
10
20
30
40
50
60
Time after Injection (min)
Figure 8. Blood clearance of radioactivity after injection of 99mTc-C3(BHam)2-HSA (●)
and 125I-HSA (○) in mice.
analyzed the radioactivity of 99mTc-C3(BHam)2-HSA by SE-HPLC
after incubation with excess L-cysteine (Figure 7). No marked
change in the peak of radioactivity was observed at
5 Â 10À5 M, 5 Â 10À4 M, 5 Â 10À3 M, or 5 Â 10À2 M of L-cysteine.
These results suggested that 99mTc stably binds to HSA via C3
(BHam)2-COOH.
We compared the clearance of 99mTc-C3(BHam)2-NGA from the
liver with that of 99mTc-(HYNIC-NGA)(tricine)2 (Figure 9).17 Radioac-
tivity in the liver cleared faster after the injection of 99mTc-C3
(BHam)2-NGA than that of 99mTc-(HYNIC-NGA)(tricine)2, indicating
that 99mTc-labeled polypeptides prepared with C3(BHam)2-COOH
produce radiometabolites, which show rapid elimination from
the liver. We have not determined the radiometabolites produced
in the liver after the injection of 99mTc-labeled polypeptides
by using C3(BHam)2-COOH as a BCA. Previous studies using
111In-labeled NGAs with cDTPA or SCN-Bz-EDTA or 99mTc-HYNIC-NGA
showed that radiolabeled NGAs generated lysine-adducts ([111In]
Furthermore, we determined the biodistribution of radioactivity
after the injection of 99mTc-C3(BHam)2-HSA into mice (Table 1). At
5-min postinjection, 36% ID/g was observed in the blood, and at
180 min, 20%ID/g. The profile of radioactivity was similar to that
for 125I-HSA (Figure 8). The results suggested that 99mTc-C3(BHam)
2-HSA may reflect the pharmacokinetics of HSA without the disso-
ciation of 99mTc from HSA, and in addition, C3(BHam)2-COOH serves
as a BCA, which can give stable 99mTc-labeled polypeptides. As no
marked increase in radioactivity was detected in the stomach and
lungs, the reoxidation to 99mTcO4À or production of 99mTc-colloid
did not seem to occur, supporting the notion that C3(BHam)2-COOH
Table 2. Biodistribution of radioactivity after injection of 99mTc-C3(BHam)2-NGA in mice
Percentage of injected dose per tissue
Tissue
Blooda
5 min
10 min
30 min
1 h
3 h
6 h
1.35
(0.28)
89.43
(4.15)
0.36
(0.11)
0.93
(0.03)
0.19
(0.07)
0.41
(0.13)
0.24
0.89
(0.39)
80.52
(7.71)
0.33
(0.04)
2.28
(0.34)
0.16
(0.03)
0.51
(0.11)
0.17
1.84
(0.39)
59.96
(2.79)
0.45
(0.09)
22.33
(1.87)
0.18
(0.03)
0.58
(0.16)
0.19
1.49
(0.48)
32.66
(2.87)
0.53
(0.13)
43.66
(6.16)
0.14
(0.01)
0.74
(0.28)
0.21
0.98
(0.42)
14.64
(3.05)
0.50
(0.13)
62.44
(4.05)
0.13
(0.06)
0.51
(0.22)
0.21
0.75
(0.31)
7.12
(0.62)
0.36
(0.01)
27.74
(11.02)
0.11
(0.03)
0.27
Liver
Kidney
Intestine
Spleen
Stomach
Lung
(0.09)
0.12
(0.13)
(0.03)
(0.05)
(0.03)
(0.01)
(0.03)
Each value represents the mean (SD) for five to eight animals.
aExpressed as% injected dose per gram.
J. Label Compd. Radiopharm 2012, 55 71–79
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

M. Ono et al.
100
90
80
70
60
50
40
30
20
10
0
Acknowledgements
The study was supported by the Funding Program for Next Gen-
eration World-Leading Researchers (NEXT Program), and a Grant-
in-aid for Young Scientists and Exploratory Research from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan.
Conflict of Interest
The authors did not report any conflict of interest.
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