Nuclear Medicine and Biology 28 (2001) 155–164

Plasma protein binding of 99mTc-labeled hydrazino nicotinamide

derivatized polypeptides and peptides
Masahiro Onoa, Yasushi Aranob,*, Takahiro Mukaic, Tomoya Ueharab, Yasushi Fujiokaa,
Kazuma Ogawaa, Shinji Nambaa, Morio Nakayamad, Tsuneo Sagac, Junji Konishic,
Kazuko Horiuchia, Akira Yokoyamaa, Hideo Sajia
a
Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-shimoadachi-cho, Sakyo-ku,
Kyoto 606-8501, Japan
b
Laboratory of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1–33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan
c
Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho, Sakyo-ku,
Kyoto 606-8507, Japan
d
School of Pharmaceutical Sciences, Nagasaki University, 1–14 Bunkyo-machi, Nagasaki 852-8521, Japan

Received 3 July 2000; received in revised form 10 August 2000; accepted 9 October 2000

Abstract
6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare 99mTc-labeled polypeptides
and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, 99mTc-HYNIC-conjugated IgG
showed biodistribution of radioactivity similar to that of 111In-DTPA-conjugated IgG. However, recent studies indicated significant plasma
protein binding when the 99mTc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of
99m
Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this
99m
Tc-labeling method. In mice, [99mTc](HYNIC-IgG)(tricine)2 and [99mTc](HYNIC-Fab)(tricine)2 showed persistent localization of
radioactivity in tissues when compared with their 125I-labeled counterparts. [99mTc](HYNIC-IgG)(tricine)2 eliminated from the blood at a
rate similar to that of 125I-labeled IgG, while [99mTc](HYNIC-Fab)(tricine)2 showed significantly slower clearance of the radioactivity than
125
I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of
[99mTc](HYNIC-IgG)(tricine)2 in murine plasma. However, [99mTc](HYNIC-Fab)(tricine)2 and [99mTc](HYNIC-RC160)(tricine)2 demon-
strated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight
species was reduced when [99mTc](HYNIC-RC160)(tricine)2 was stabilized with nicotinic acid (NIC) to generate [99mTc](HYNIC-
RC160)(tricine)(NIC). [99mTc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the
blood than [99mTc](HYNIC-RC160)(tricine)2. These findings suggested that one of the tricine coligands in 99mTc-HYNIC-labeled
(poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that exhibit slow blood clearance. In
addition, the molecular sizes of parental peptides played an important role in the progression of the exchange reaction of one of the tricine
coligands with plasma proteins. © 2001 Elsevier Science Inc. All rights reserved.

Keywords: 99mTc; HYNIC; (poly)peptide; Protein binding; Tricine; Coligand

1. Introduction polypeptides and peptides for visualization of the sites of

tumors, infections and thromboses at early postinjection
6-Hydrazinopyridine-3-carboxylic acid (HYNIC) consti- times. Previous studies indicated that HYNIC acts as a
tutes one of the most attractive bifunctional coupling agents monodentate or bidentate ligand to form a mixed ligand
(BCAs) to prepare technetium-99m (99mTc)-labeled 99m
Tc complex in the presence of appropriate coligands [1].
Tricine was usually selected as a coligand due to generation
* Corresponding author. Tel.: ⫹81-43-290-3024; fax: ⫹81-43-290- of 99mTc-HYNIC-labeled (poly)peptides with high radio-
3025. chemical yields and high specific activities in a short reac-
E-mail address: arano@p.chiba-u.ac.jp (Y. Arano). tion time. Indeed, tricine has been used to prepare 99mTc-

0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 9 6 9 - 8 0 5 1 ( 0 0 ) 0 0 2 0 0 - 6
156 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164

HYNIC-labeled (poly)peptides such as monoclonal IgG an isocratic mobile phase of 80% A (0.01 M PB, pH 7.0)
[20], human serum albumin [24], Fab⬘ fragment [22], an- and 20% B (acetonitrile) for 5 min, and with a gradient
nexin V [6], chemotactic peptide [23] and RGD peptides [17]. mobile phase starting from 80% A and 20% B to 20% A and
99m
Tc-HYNIC-conjugated IgG showed radioactivity dis- 80% B at 30 min (solvent system 1). Analytical RP-HPLC
tribution and inflammation imaging properties similar to was also performed with a gradient mobile phase starting
those of 111In-labeled IgG using cyclic diethylenetriamine- from 100% water to 100% acetonitrile in 30 min (solvent
pentaacetic acid (DTPA) as a bifunctional chelating agent in system 2). The eluent was collected with a fraction collector
rats [1], rabbits [5], monkeys [9] and normal human subjects (RediFrac; Pharmacia Biotech, Tokyo, Japan) at 30-second
[7]. However, 99mTc-HYNIC-labeled single-stranded DNA intervals and the radioactivity levels in each fraction (500
oligonucleotides showed slower elimination rates of the ␮L) were determined with an auto well counter (ARC-2000;
radioactivity from the blood than those of 111In-DTPA- Aloka, Tokyo, Japan). Cellulose acetate electrophoresis
conjugated DNA, due to an increase in serum protein bind- (CAE, Separax; Joko Co. Ltd., Tokyo) was performed at an
ing of the former [11]. Similar findings were observed when electrostatic field strength of 0.8 mA/cm for 45 min in
HYNIC was applied to 99mTc radiolabeling of small molec- veronal buffer (I ⫽ 0.06, pH 8.6; Nacalai Tesque). TLC
ular weight peptides [8]. analyses were performed with silica plates (Merck Art
Low molecular weight polypeptides and peptides are 5553) with 80% aqueous methanol or saline as developing
useful as vehicles to enable target visualization using 99mTc solvents. Fast atom bombardment mass spectra (FAB-MS)
as the radionuclide of choice due to their rapid elimination were obtained with a JMS-HX/HX 110 A model (JEOL
rates from the circulation. However, plasma protein binding Ltd., Tokyo). [Lys5-Boc]-RC160 was a gift from interna-
of 99mTc-HYNIC-conjugated polypeptides and peptides tional atomic energy agency (IAEA). Glutamate dehydro-
prolongs elimination rates of the radioactivity from the genase and N-(⑀-maleimidocaproyloxy)succinimide (EMCS)
blood, which compromises their clinical applicability. Since were purchased from Oriental Yeast Co. Ltd. (Tokyo, Ja-
HYNIC constitutes an attractive BCA for preparing 99mTc- pan) and Dojindo Labs (Kumamoto, Japan), respectively.
labeled polypeptides and peptides, further understanding of IgG-mercaptalbumin conjugate was prepared by maleimide-
the mechanisms responsible for the protein binding charac- thiol chemistry. Briefly, IgG treated with EMCS was re-
teristics of 99mTc-HYNIC-conjugated polypeptides and pep- acted with mercaptalbumin prepared by reducing a cysteine
tides would provide a good basis for further design of residue in human serum albumin (A-3782, Sigma Chemical
appropriate BCAs or coligands for 99mTc-labeled peptides Co. St. Louis, MO) [19]. Other reagents were of reagent
and polypeptides. grade and used as received. To facilitate collection of urine
In the present study, protein binding of 99mTc-HYNIC- and feces after administration of radiolabeled (poly)pep-
conjugated polypeptides and peptides was investigated us- tides, mice were housed in metabolic cages (Metabolica,
ing polypeptides or a peptide of different molecular weights MM type; Sugiyama-Gen Iriki Co. Ltd., Tokyo). Succin-
(intact IgG antibody, Fab fragment and the synthetic soma- imidyl 6-hydrazinopyridine-3-carboxylate hydrochloride
tostatin analog RC160). The pharmacokinetics of the radio- (SHNH) and N-succinimidyl 3-[125I]iodobenzoate ([125I]SIB)
activity derived from each 99mTc-labeled polypeptide and were synthesized as reported previously [1,4].
the peptide were compared with their radioiodinated coun-
terparts in mice. Mechanisms of protein binding of the 2.2. Monoclonal antibody. The monoclonal antibody
99m
Tc-HYNIC-conjugated polypeptides and peptide were against osteogenic sarcoma (OST7, IgG1), generated by the
investigated. standard hybridoma technique, was purified by ammonium
sulfate precipitation with subsequent protein A affinity
chromatography (Pharmacia Biotech Co. Ltd.), as reported
2. Materials and methods previously [12]. The Fab fragment was prepared by the
standard procedure using papain [10].
2.1. Reagents and chemicals. [99mTc]Pertechnetate
(99mTcO⫺ 4 ) was eluted in saline solution on a daily basis 2.3. Preparation of 99mTc-HYNIC-labeled IgG and Fab.
from the Daiichi Radioisotope Laboratory generators The conjugation reaction of SHNH with IgG or Fab and
(Chiba, Japan). Na[125I] was obtained from Daiichi Kagaku subsequent 99mTc-labeling were performed according to the
(Tokyo, Japan), and was diluted with 0.1 M phosphate procedure reported previously [18] with slight modifica-
buffer (PB) (pH 7.4) to 3.7 MBq/␮L. Size-exclusion (SE) tions. Briefly, 9 or 11 ␮L of SHNH (10 mg/mL) in dimeth-
HPLC was performed using a Cosmosil 5 Diol-300 column ylsulfoxide (DMSO) was added dropwise to 1 mL of IgG
(7.5 ⫻ 600 mm, Nacalai Tesque, Kyoto, Japan) connected solution (5 mg/mL) or Fab solution (2 mg/mL) in 0.15 M
to a Cosmosil 5 Diol-300 guard column (7.5 ⫻ 50 mm, borate buffer (pH 8.5). The solutions were stirred gently for
Nacalai Tesque), eluted with 0.1 M PB (pH 6.8) at a flow 2 h at room temperature protected from the light. The
rate of 1 mL/min. Analytical reversed phase (RP) HPLC HYNIC-IgG and HYNIC-Fab conjugates were purified by
was performed with a Cosmosil 5 C18-AR column (4.6 ⫻ the centrifuged column procedure using Sephadex G-50
150 mm, Nacalai Tesque) at a flow rate of 1 mL/min with (Pharmacia Biotech Co. Ltd.) columns equilibrated and
 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164 157

eluted with 10 mM citrate buffer (pH 5.2). The number of with [99mTc]tricine2 and the radioactivity was analyzed as
HYNIC groups attached per molecule of IgG or Fab was described above.
determined by measuring the hydrazino groups with p-
nitrobenzaldehyde, according to the method of King et al. 2.7. Preparation of [99mTc](HYNIC-RC160)(tricine)(NIC) ter-
[14]. nary mixed ligand complex. A solution of [99mTc](HYNIC-
To a solution of HYNIC-IgG (50 ␮L; 5 mg/mL) and RC160)(tricine)2 (100 ␮L) was mixed with 50 ␮L of nico-
HYNIC-Fab (50 ␮L; 2 mg/mL) in 10 mM citrate buffer (pH tinic acid (NIC) solution (20 mg/mL) in 10 mM citrate
5.2) was added an equal volume of [99mTc]tricine2 prepared buffer (pH 5.2), and the solution was reacted at 95°C for 15
by the method of Larsen et al. [15], and the mixture was min. The reaction mixture was analyzed by analytical RP-
incubated for 1 h at room temperature. [99mTc](HYNIC- HPLC (solvent system 1 and 2), CAE and TLC developed
IgG)(tricine)2 and [99mTc](HYNIC-Fab)(tricine)2 were pu- with saline.
rified by the centrifuged column procedure using Sephadex
G-50 columns equilibrated and eluted with 0.1 M phosphate 2.8. Radioiodination of RC160. Radioiodination of RC160
buffer (pH 7.4). Radiochemical purities of [99mTc](HYNIC- was performed according to the procedure of Uehara et al.
IgG)(tricine)2 and [99mTc](HYNIC-Fab)(tricine)2 were as- [21]. Radiochemical purity was analyzed by analytical RP-
sessed by SE-HPLC, CAE, and TLC developed with saline. HPLC (solvent system 2) and TLC developed with 80%
In a control study, unmodified IgG and Fab were labeled methanol.
with [99mTc]tricine2, according to the procedures described
above. 2.9. In vivo studies. The animal studies were conducted in
accordance with our institutional guidelines and approved
2.4. Preparation of radioiodinated IgG and Fab. [125I]SIB by Kyoto University Animal Care Committee. Biodistribu-
was conjugated with IgG and Fab according to the proce- tion studies were performed by intravenous administration
dure reported previously [3]. [125I]SIB-IgG and [125I]SIB- of a mixed solution of [99mTc](HYNIC-IgG)(tricine)2 and
Fab were then purified by the centrifuged column procedure [125I]SIB-IgG, or [99mTc](HYNIC-Fab)(tricine)2 and
using Sephadex G-50 columns equilibrated and eluted with [125I]SIB-Fab to 6-week-old male ddY mice (27–30 g) [13].
0.1 M PB (pH 7.4). Radiochemical purities of [125I]SIB-IgG The concentrations of IgG or Fab were adjusted to 200
and [125I]SIB-Fab were assessed by SE-HPLC, CAE and ␮g/mL with saline. Groups of five mice each were admin-
TLC developed with 80% aqueous methanol. istered 20 ␮g of radiolabeled IgG or Fab prior to sacrificing
the animals at 10 and 30 min, 1, 3, 6 and 24 h postinjection
2.5. Synthesis of HYNIC-RC160. To a solution of [Lys5- by decapitation. Tissues of interest were removed, weighed
Boc]-RC160 (30 mg, 26 mmol) in 1 mL dimethylform- and the radioactivity counts were determined with an auto
amide (DMF) was added a solution of Boc-SHNH (9.3 mg, well gamma counter (ARC 2000). To determine the
26.4 mmol) in 1 mL of DMF, and the reaction mixture was amounts and routes of excretion of radioactivity from the
stirred for 18 h at room temperature. The resulting reaction body, mice were housed in metabolic cages for 24 h after
mixture was purified by a preparative RP-HPLC (50 ⫻ 250 administration, and urine and feces were collected and the
mm, 5C18-MS, Nacalai Tesque) using a mixed solution of radioactivity was determined. Biodistributions of radioac-
acetonitrile and water, and the fractions containing the de- tivity after injection of [99mTc](HYNIC-RC160)(tricine)2 or
sired products were collected and lyophilized to afford Boc- [99mTc](HYNIC-RC160)(tricine)(NIC) (0.1 ␮g each) were
HYNIC-[Lys5-Boc]-RC160 in 30.7% yields. FAB-MS also determined according to the procedures described
calcd for C73H91N15O14S2, [MH⫹], m/z 1466, found 1466. above.
A mixture of 5% anisole and 95% trifluoroacetic acid To analyze the radiolabeled species in plasma, blood was
(TFA) was added to Boc-HYNIC-[Lys5-Boc]-RC160, and withdrawn from the heart with a heparinized syringe at 10
the reaction mixture was stirred for 30 min at room tem- min, 1 and 6 h postinjection for [99mTc](HYNIC-
perature. After removing TFA in vacuo, ether was added to IgG)(tricine)2 and [125I]SIB-IgG, and at 10, 30 min and 3 h
the residue to precipitate TFA salt of HYNIC-RC160 as a postinjection for [99mTc](HYNIC-Fab)(tricine)2 and
white solid in yields of over 90%. FAB-MS calcd for [125I]SIB-Fab. After centrifugation at 800 g for 15 min at
C63H75N15O10S2, [MH⫹] m/z 1266, found 1266. 4°C, plasma samples were filtered through a polycarbonate
membrane with a pore diameter of 0.45 ␮m (Nacalai
2.6. 99mTc-labeling of HYNIC-RC160. To a solution of Tesque). The radiolabeled species in plasma were then an-
HYNIC-RC160 (100 ␮L; 100 ␮g/mL) in 10 mM citrate alyzed by SE-HPLC.
buffer (pH 5.2) was added an equal volume of [99mTc]
tricine2. The mixture was incubated for 1 h at room tem- 2.10. In vitro studies. [99mTc](HYNIC-IgG)(tricine)2 (0.5
perature. Radiochemical purity of [99mTc](HYNIC- mg/mL), [99mTc](HYNIC-Fab)(tricine)2 (0.5 mg/mL),
RC160)(tricine)2 was determined by analytical RP-HPLC [99mTc](HYNIC-RC160)(tricine)2 (10 ␮g/mL) or
(solvent system 1 and 2), CAE and TLC developed with [99mTc](HYNIC-RC160)(tricine)(NIC) (10 ␮g/mL) was
saline. In a control study, unmodified RC160 was reacted mixed with their radioiodinated counterparts. The mixtures
158 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164

were diluted 20-fold with 20 mM phosphate buffered saline Table 1

Biodistribution of radioactivity after intravenous administration of
(pH 7.4) or freshly prepared murine plasma. After incubat-
[99mTc](HYNIC-IgG)(tricine)2 and [125I]SIB-IgG in micea
ing the solutions at 37°C, 50 ␮L aliquots were drawn at 1,
6 and 24 h and the radioactivity was analyzed by SE-HPLC. Tissue Time after administration
The plasma after incubation of [99mTc](HYNIC- 10 min 30 min 1h 3h 6h 24 h
RC160)(tricine)2 was reacted with NIC, and the reaction [99mTc](HYNIC-IgG)(tricine)2
products were analyzed to further characterize the 99mTc Blood 31.48 29.32 27.39 22.38 20.83 12.37
species bound with the plasma protein. The plasma samples (2.18) (1.68) (1.31) (0.89) (1.05) (0.87)
(200 ␮L) obtained at 6 h postincubation of [99mTc](HYNIC- Liver 14.68 14.24 13.31 13.25 12.22 9.80
(1.00) (0.78) (0.33) (0.74) (0.41) (0.58)
RC160)(tricine)2 were mixed with 100 ␮L of a solution of Kidney 5.57 5.59 5.86 5.29 5.24 4.48
NIC (10 mg/mL) in 10 mM citrate buffer (pH 5.2). The (0.52) (0.34) (0.30) (1.18) (0.38) (0.61)
reaction mixture was incubated at 37°C for 1 h. The reaction Intestine 0.61 0.97 1.26 1.90 2.35 1.54
mixture was analyzed by SE-HPLC after filtration through a (0.07) (0.07) (0.08) (0.20) (0.37) (0.31)
Spleen 5.60 5.36 5.36 5.06 5.92 4.78
polycarbonate membrane with a pore diameter of 0.45 ␮m, (0.67) (0.26) (0.49) (0.41) (0.35) (0.55)
and was also analyzed by RP-HPLC (solvent system 1) after Stomachb 0.33 0.37 0.37 0.75 0.68 0.65
filtration through a 10 kDa cutoff ultrafiltration membrane (0.06) (0.09) (0.07) (0.22) (0.10) (0.05)
(Microcon-10, Amicon). Urineb 4.57
(0.66)
Fecesb 1.71
(0.49)
[125I]SIB-IgG
3. Results
Blood 31.98 29.59e 27.80e 23.02c 21.50d 13.68e
(2.24) (1.59) (1.39) (0.92) (1.00) (0.37)
3.1. Radiolabeling of IgG, Fab and RC160 Liver 14.18c 12.34 11.12d 10.02 8.80d 4.73c
(1.04) (0.77) (0.37) (1.56) (0.77) (0.73)
Kidney 5.72 6.36d 6.68e 4.80e 4.12d 3.483
The average numbers of HYNIC groups attached per (0.55) (0.50) (0.76) (1.04) (0.21) (0.45)
molecule of IgG and Fab were 1.4 and 1.8, respectively, as Intestine 0.64e 1.08d 1.41c 2.12 2.28e 1.10e
determined from the hydrazine groups. 99mTc labeling of (0.08) (0.10) (0.05) (0.37) (0.64) (0.19)
Spleen 5.43 4.90e 5.06 4.08e 3.91c 2.80c
HYNIC-IgG and HYNIC-Fab were performed by reaction (0.36) (0.45) (0.35) (0.41) (0.40) (0.54)
with [99mTc]tricine2. Both [99mTc](HYNIC-IgG)(tricine)2 Stomachb 0.39 0.49c 0.54c 0.88 0.80e 0.57e
and [99mTc](HYNIC-Fab)(tricine)2 were obtained with ra- (0.05) (0.09) (0.09) (0.16) (0.17) (0.05)
diochemical purities over 95% as determined by SE-HPLC, Urineb 15.58c
(2.63)
CAE and TLC. [99mTc](HYNIC-RC160)(tricine)2 was pre- Fecesb 1.20
pared by reaction with [99mTc]tricine2 for 30 min at room (0.55)
temperature. [99mTc](HYNIC-RC160)(tricine)(NIC) was ob- a
Expressed as % injected dose per gram. Each value represents the mean
tained by reaction of [99mTc](HYNIC-RC160)(tricine)2 with (s.d.) for five animals at each time point.
NIC. [99mTc](HYNIC-RC160)(tricine)2 and [99mTc](HYNIC- b
Expressed as % injected dose.
RC160)(tricine)(NIC) were obtained with radiochemical puri- c
Significance determined by paired t-test; p ⬍ 0.001
ties over 95% as determined by analytical RP-HPLC. On the
d
Significance determined by paired t-test; p ⬍ 0.01.
e
Significance determined by paired t-test; p ⬍ 0.05.
other hand, reaction of [99mTc]tricine2 with unmodified IgG,
Fab or RC160 resulted in less than 4% of the radioactivity
associated with the polypeptides/peptide. The radiochemical IgG are shown in Table 1. [99mTc](HYNIC-IgG)(tricine)2 was
purities of [99mTc](HYNIC-IgG)(tricine)2, [99mTc](HYNIC- eliminated from the blood at a rate similar to that of [125I]SIB-
Fab)(tricine)2, [99mTc](HYNICRC160)(tricine)2 and [99mTc] IgG. On the other hand, [99mTc](HYNIC-IgG)(tricine)2
(HYNIC-RC160)(tricine)(NIC) remained unchanged when in- showed significantly higher radioactivity levels in all tissues
cubated in a neutral buffered solution at 37°C for 24 h. at 24 h postinjection. While both [125I]SIB-IgG and [99mTc]
Both [125I]SIB-IgG and [125I]SIB-Fab were obtained (HYNIC-IgG)(tricine)2 excreted small amounts of radioac-
tivity in the feces at 24 h postinjection, [125I]SIB-IgG
with radiochemical purities over 97% after purification by
showed higher levels of radioactivity in the urine as com-
the centrifuged column procedure. 125I-RC160 was obtained
pared with [99mTc](HYNIC-IgG)(tricine)2.
with radiochemical purity over 95%.
The biodistributions of radioactivity after concomitant ad-
ministration of [99mTc](HYNIC-Fab)(tricine)2 and [125I]SIB-
3.2. Biodistribution study in mice Fab are summarized in Table 2. [99mTc](HYNIC-Fab)(tricine)2
demonstrated significantly higher radioactivity levels in
The biodistributions of radioactivity after concomitant ad- the blood than [125I]SIB-Fab. [99mTc](HYNIC-Fab)
ministration of [99mTc](HYNIC-IgG)(tricine)2 and [125I]SIB- (tricine)2 also showed longer residence times of radioac-
 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164 159

Table 2 Table 3
Biodistribution of radioactivity after intravenous administration of Biodistribution of radioactivity after intravenous administration
[99mTc](HYNIC-Fab)(tricine)2 and [125I]SIB-Fab in micea of [99mTc](HYNIC-RC160)(tricine)2 and [99mTc](HYNIC-
RC160)(tricine))NIC) in micea
Tissue Time after administration
Tissue Time after administration
10 min 30 min 1h 3h 6h 24 h
10 min 30 min 1h 3h 6h 24 h
[99mTc](HYNIC-Fab)(tricine)2 99m
[ Tc](HYNIC-RC160)(tricine)2
Blood 25.47 16.81 12.61 6.90 3.17 0.55
Blood 4.20 2.68 2.30 1.43 1.08 0.36
(1.82) (0.93) (0.95) (1.04) (0.33) (0.09)
(0.55) (0.45) (0.20) (0.42) (0.21) (0.03)
Liver 5.63 6.08 6.41 6.61 6.70 2.26
Liver 36.34 31.95 31.56 30.48 17.03 13.66
(0.43) (0.39) (0.67) (0.78) (1.02) (0.39)
(3.43) (3.39) (3.42) (2.69) (1.93) (2.31)
Kidney 17.50 29.85 35.87 40.68 41.97 40.89
Kidney 13.24 11.13 8.30 6.37 4.98 2.53
(1.51) (3.35) (5.12) (4.61) (6.66) (2.09)
(1.02) (1.71) (0.33) (0.53) (0.59) (0.22)
Intestine 1.11 1.34 1.56 1.61 1.43 1.03
Intestine 1.52 1.75 3.12 6.60 12.04 4.41
(0.19) (0.11) (0.19) (0.17) (0.32) (0.61)
(0.13) (0.15) (0.80) (0.39) (1.18) (3.30)
Spleen 4.21 3.35 3.48 2.59 2.45 0.91
Spleen 14.02 8.93 9.66 7.60 9.54 3.65
(0.32) (0.19) (0.73) (0.48) (0.37) (0.15)
b (3.44) (2.10) (1.60) (1.75) (1.99) (0.62)
Stomach 0.86 1.11 1.39 1.15 0.68 0.50
Pancreas 2.23 1.64 1.87 1.32 1.31 0.53
(0.27) (0.42) (0.54) (0.45) (0.34) (0.10)
b (0.10) (0.81) (0.11) (0.24) (0.12) (0.14)
Urine 35.24
Stomachb 0.28 0.70 0.96 1.18 1.78 0.63
(1.86)
(0.07) (0.16) (0.25) (0.09) (0.39) (0.34)
Fecesb 4.51
Urineb 27.84
(2.15)
(0.13)
[125I]SIB-Fab
Fecesb 6.86
Blood 21.86d 12.52c 8.36c 3.56c 2.24d 0.16c
(1.76)
(1.05) (0.41) (0.65) (0.30) (0.45) (0.01)
Liver 5.17e 4.37c 3.18c 1.66c 1.15c 0.19c [99mTc](HYNIC-RC160)(tricine)(NIC)
(0.37) (0.46) (0.35) (0.18) (0.33) (0.03) Blood 2.20c 1,33d 1.00c 0.74d 0.47d 0.10c
Kidney 36.68c 45.10c 28.31e 10.52c 6.20c 0.11c (0.21) (0.15) (0.15) (0.16) (0.12) (0.04)
(1.19) (2.32) (2.58) (1.98) (1.08) (0.02) Liver 35.97 34.08 33.64 35.36 33.42e 24.23c
Intestine 0.96d 1.30d 1.51 1.48 0.97c 0.07d (3.58) (4.73) (4.82) (2.30) (4.87) (3.40)
(0.16) (0.17) (0.18) (0.39) (0.31) (0.04) Kidney 12.71 9.96 7.83 4.08d 2.32c 0.85c
Spleen 4.87e 4.73e 3.35 1.48d 0.85c 0.19c (0.88) (1.82) (1.39) (0.73) (0.62) (0.25)
(0.62) (0.55) (0.59) (0.39) (0.31) (0.05) Intestine 1.52 1.96 2.86 5.95 5.96c 1.89
Stomachb 0.50e 1.43d 2.21e 2.40e 1.29 0.11c (0.10) (0.27) (0.52) (1.27) (0.93) (0.76)
b
(0.09) (0.05) (0.04) (0.36) (0.55) (0.06) Spleen 13.05 13.77e 15.10d 14.10c 13.22 9.43c
Urine 71.45c (2.09) (3.31) (2.51) (1.72) (3.73) (0.26)
c
(2.92) Pancreas 1.45 1.00 0.80c 0.50c 0.23c 0.12c
2.26 (0.15) (0.09) (0.15) (0.07) (0.06) (0.11)
b
Fecesb (0.39) Stomach 0.76c 0.82 0.78 1.15 0.84d 0.46
(0.10) (0.16) (0.12) (0.11) (0.20) (0.26)
a Expressed as % injected dose per gram. Each value represents the
Urineb 27.66
mean (s.d.) for five animals at each time point.
b (8.03)
Expressed as % injected dose.
Fecesb 23.98c
c
Significance determined by paired t-test; p ⬍ 0.001.
(8.49)
d
Significance determined by paired t-test; p ⬍ 0.01
e
Significance determined by paired t-test; p ⬍ 0.05. a
Expressed as % injected dose per gram. Each value represents the mean
(s.d.) for five animals at each time point.
b
Expressed as % injected dose.
tivity in all tissues as compared to [125I]SIB-Fab, which
c
Significance determined by unpaired t-test; p ⬍ 0.001.
d
Significance determined by unpaired t-test; p ⬍ 0.01.
was typically observed in the kidney. This was reflected e
Significance determined by unpaired t-test; p ⬍ 0.05.
in the amounts of radioactivity excreted from the body
where [125I]SIB-Fab indicated significantly higher radio- 3.3. In vivo analysis of the radioactivity in the blood
activity levels in the urine than [99mTc](HYNIC-
Fab)(tricine)2 at 24 h postinjection. Fig. 1 shows SE-HPLC radiochromatograms of plasma
The biodistributions of radioactivity after administration radioactivity at 10 min, 1 and 6 h after concomitant admin-
of [99mTc](HYNIC-RC160)(tricine)2 and [99mTc](HYNIC- istration of [99mTc](HYNIC-IgG)(tricine)2 and [125I]SIB-
RC160)(tricine)(NIC) are shown in Table 3. [99mTc] IgG into mice. No significant differences were observed
(HYNIC-RC160)(tricine)(NIC) showed faster rates of between [99mTc](HYNIC-IgG)(tricine)2 and [125I]SIB-IgG
elimination of radioactivity from the blood than [99mTc] at any postinjection time point.
(HYNIC-RC160)(tricine)2. [99mTc](HYNIC-RC160)(tricine) Fig. 2 shows SE-HPLC radiochromatograms of plasma
(NIC) also showed higher levels of radioactivity in the feces as radioactivity after concomitant administration of [99mTc]
compared with [99mTc](HYNIC-RC160)(tricine)2. (HYNIC-Fab)(tricine)2 and [125I]SIB-Fab. [99mTc](HYNIC-
160 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164

Fig. 2. SE-HPLC profiles of blood radioactivity after injection of

Fig. 1. SE-HPLC profiles of blood radioactivity after injection of
[99mTc](HYNIC-Fab)(tricine)2 (left panels) and [125I]SIB-Fab (right
[99mTc](HYNIC-IgG)(tricine)2 (left panels) and [125I]SIB-IgG (right pan-
panels).
els). Under these conditions, thyroglobulin (670 kDa), glutamate dehydro-
genase (290 kDa), IgG-mercaptalbumin conjugate (230 kDa), immuno-
globulin (150 kDa), human serum albumin (68 kDa), Fab fragment (50
kDa) and cytochrome C (13 kD) had retention times of 12, 14.5, 15, 16, 18, min) after 6 and 24 h incubation. On the other hand,
19 and 21 min, respectively, on SE-HPLC.
[99mTc](HYNIC-Fab)(tricine)2 displayed a gradual decrease
in radioactivity peaks of intact [99mTc](HYNIC-
Fab)(tricine)2 and a time-dependent increase in the radioac-
Fab)(tricine)2 showed a time-dependent increase in the ra-
dioactivity peaks at retention times of 12 (over 600 kDa) tivity peaks at earlier retention times (12 and 17 min). The
and 17 min (70 – 80 kDa), whereas [125I]SIB-Fab showed an radioactivity peak at 12 min represented 10.7% of the total
increase in the radioactivity peaks only at a retention time of radioactivity at 1 h postincubation and increased to 20.2% at
17–18 min. 24 h postincubation.
Fig. 5 shows SE-HPLC radiochromatograms of [99mTc]
(HYNIC-RC160)(tricine)2, 125I-RC160 and [99mTc]
3.4. In vitro analysis of radioactivity in the blood
(HYNIC-RC160)(tricine)(NIC) after incubation in plasma.
All the radiolabeled RC160 showed migration of the radio-
Fig. 3 shows SE-HPLC radiochromatograms of
activity from ca. 25–30 min to earlier radioactivity peaks
[99mTc](HYNIC-IgG)(tricine)2 and [125I]SIB-IgG at 1, 6
with incubation time. However, while both 125I-RC160 and
and 24 h after incubation in freshly prepared murine plasma.
Little changes were observed in the radioactivity peaks of [99mTc](HYNIC-RC160)(tricine)(NIC) showed a major ra-
both [99mTc](HYNIC-IgG)(tricine)2 and [125I]SIB-IgG be- dioactivity peak at a retention time of 17–19 min,
fore and after 24 h incubation in plasma. [99mTc](HYNIC-RC160)(tricine)2 showed a major radioac-
SE-HPLC chromatograms of radioactivity after concom- tivity peak at a retention time of 12 min. Although
itant incubation of [99mTc](HYNIC-Fab)(tricine)2 and [99mTc](HYNIC-RC160)(tricine)(NIC) showed a small
[125I]SIB-Fab in murine plasma are shown in Fig. 4. amount of radioactivity at a retention time of 12 min, the
[125I]SIB-Fab showed new small radioactivity peaks at re- proportion of this radioactivity fraction was much lower
tention times earlier and longer than the parental Fab (19 than those of [99mTc](HYNIC-RC160)(tricine)2 and 125I-
 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164 161

Fig. 4. SE-HPLC analysis of radioactivity of [99mTc](HYNIC-

Fig. 3. SE-HPLC analysis of radioactivity of [99mTc](HYNIC- Fab)(tricine)2 (left panels) and [125I]SIB-Fab (right panels) in murine
IgG)(tricine)2 (left panels) and [125I]SIB-IgG (right panels) in murine plasma after incubation at 37°C.
plasma after incubation at 37°C.

elimination rates of radioactivity from the blood after ad-

RC160. [99mTc](HYNIC-RC160)(tricine)2 also generated a ministration of 99mTc-HYNIC-conjugated IgG and Fab
new radioactivity peak at a retention time of ca. 15 min. fragment. While [99mTc](HYNIC-IgG)(tricine)2 showed
Radiochromatograms of the NIC-treated plasma are elimination of the radioactivity from the blood at a rate
shown in Fig. 6. The amount of radioactivity recovered in similar to that of [125I]SIB-IgG (Table 1), a significant delay
the filtrates was 87.3% after filtration of the NIC-treated was observed in clearance of the radioactivity of
plasma through a polycarbonate membrane. On SE-HPLC, [99mTc](HYNIC-Fab)(tricine)2 from the blood as compared
significant decreases were observed in the radioactivity with [125I]SIB-Fab (Table 2). [99mTc](HYNIC-Fab)
peaks at retention time of 12 and 17–18 min in comparison (tricine)2 also showed delayed accumulation of radioactivity
with the radiochromatogram after incubation of in the excretory tissues such as the kidney. As a result,
[99mTc](HYNIC-RC160)(tricine)2 for 6 h (Fig. 6-A). On [99mTc](HYNIC-Fab)(tricine)2 showed significantly altered
RP-HPLC using solvent system 1 shown in Fig. 6-B, almost biodistribution of the radioactivity when compared with
all the radioactivity of the NIC-treated plasma showed a [125I]SIB-Fab. The persistent localization of the renal radio-
peak at a retention time of 14 min, which was identical to activity of [99mTc](HYNIC-Fab)(tricine)2 could be attrib-
that of [99mTc](HYNIC-RC160)(tricine)(NIC) (Fig. 6-C). uted to slow elimination rate of the radioactivity from the
This was confirmed by co-chromatographic analyses. lysosomal compartment of renal cells following reabsorp-
tion of glomerularly filtered Fab fragment to renal cells, as
described previously [2,18].
4. Discussion The number of HYNIC groups attached per molecule of
the polypeptides were more than one, and the majority of
This study demonstrated that molecular sizes of the pa- HYNIC groups remained in unchelated form after 99mTc
rental (poly)peptides played an important role in plasma radiolabeling reaction. Thus, reactions of 99mTc-unchelated
protein binding of 99mTc-HYNIC-conjugated (poly)peptides hydrazine moieties in [99mTc](HYNIC-Fab)(tricine)2 with
using tricine as the coligand. This was clearly reflected in sugar moieties of plasma proteins may constitute the pri-
162 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164

Fig. 6. Radiochromatogram of the plasma at 6 h postincubation with

[99mTc](HYNIC-RC160)(tricine)2 after NIC treatment by SE-HPLC (A).
Radiochromatogram of the plasma at 6 h postincubation with
[99mTc](HYNIC-RC160)(tricine)2 after NIC treatment by RP-HPLC using
solvent system 1 (B). Radiochromatograms of [99mTc](HYNIC-
RC160)(tricine)2 (E) and [99mTc](HYNIC-RC160)(tricine)(NIC) (F) ob-
tained under similar conditions are also shown (C).
Fig. 5. SE-HPLC analysis of radioactivity of [99mTc](HYNIC-
RC160)(tricine)2 (left panels), 125I-RC160 (center panels) and
[99mTc](HYNIC-RC160)(tricine)(NIC) (right panels) in murine plasma af-
ter incubation at 37°C. (HYNIC-Fab)(tricine)2 with plasma proteins would be re-
sponsible for the generation of the high molecular weight
fractions (retention time of 12 min).
mary reason for the plasma protein binding of The plasma protein binding of 99mTc-HYNIC-conju-
[99mTc](HYNIC-Fab)(tricine)2. However, this was not gated polypeptides and peptides with tricine as coligand was
likely since [99mTc](HYNIC-RC160)(tricine)2 lacking the further investigated using RC160 as a model because of its
99m
Tc-unchelated hydrazine moiety also demonstrated sig- simple structure and high protein binding characteristics
nificant plasma protein binding. Thus, factors other than the (Fig. 5). To estimate hydrophobicity-mediated protein bind-
binding of free hydrazine with plasma proteins would con- ing originated from the peptide backbone, 125I-RC160 was
stitute major reasons for the plasma protein binding of incubated in murine plasma. SE-HPLC radioactivity pro-
99m
Tc-HYNIC-conjugated (poly)peptides using tricine as files of 125I-RC160 remained unchanged from 1 h to 6 h
the coligand. postincubation with the majority of the radioactivity ob-
Both [99mTc](HYNIC-Fab)(tricine)2 and [125I]SIB-Fab served at a retention time of 18 min. [99mTc](HYNIC-
showed a radioactivity peak at a retention time of 19 min in RC160)(tricine)2 registered a radiochromatogram on SE-
vivo. However, an additional radioactivity peak at a reten- HPLC similar to that of 125I-RC160 after 1 h incubation,
tion time of 12 min was observed only with [99mTc] suggesting that the 99mTc-labeled RC160 preserved hydro-
(HYNIC-Fab)(tricine)2 in both in vivo and in vitro plasma phobic characteristics of the parental peptide. However, the
99m
analyses (Fig. 2 and 4). Thus, the generation of radioactive Tc-labeled peptide showed migration of the major peak
fractions at the retention time of 17 min would be attribut- of radioactivity to higher molecular weight after 6 h incu-
able to plasma protein binding originating from the parental bation, which indicated that the 99mTc-labeled peptide
Fab fragment. Since the radioactive fractions were signifi- bound to higher molecular weight plasma proteins with
cantly reduced following in vitro plasma incubation studies times following binding to smaller molecular weight plasma
of both [99mTc](HYNIC-Fab)(tricine)2 and [125I]SIB-Fab, proteins.
in vivo metabolism of Fab fragments might be responsible To further investigate the mechanisms responsible for
for the plasma protein binding. On the other hand, binding the time-dependent transfer of radioactivity from lower to
of [99mTc](HYNIC)(tricine)2 chelate moiety in [99mTc] higher molecular weight plasma proteins of [99mTc]
 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 155–164 163

(HYNIC-RC160)(tricine)2, plasma protein binding of Acknowledgments

[99mTc](HYNIC-RC160)(tricine)(NIC) was examined (Fig.
5). A recent study showed that [99mTc](HYNIC-peptide- This study was performed as part of the coordinated
)(tricine)2 reacts with NIC to provide a new ternary mixed research program on “99mTc labeled peptides for peripheral
ligand complex, where one tricine coligand is replaced with receptors” by the International Atomic Energy Agency
one NIC molecule [16]. [99mTc](HYNIC-peptide)(trici- (JPN-8969). This study was supported in part by Grants-in-
ne)(NIC) was reported to possess much higher stability than Aid for Developing Scientific Research from the Ministry of
its precursor, [99mTc](HYNIC-peptide)(tricine)2. [99mTc] Education, Science, Sports and Culture of Japan and from
(HYNIC-RC160)(tricine)(NIC) also showed migration of Terumo Life Science Foundation.
radioactivity to a retention time of 18 min after 1 h incu-
bation, as observed with the other two radiolabeled RC160s.
However, the [99mTc](HYNIC-RC160)(tricine)(NIC) showed References
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