67/68Ga-labeling agent that liberates 67/68Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels

Article abstract of DOI:10.1021/bc5004058

The renal localization of gallium-67 or gallium-68 (^67/68Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that ⁶⁷Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine (⁶⁷Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental ⁶⁷Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new ^67/68Ga-labeling reagent for LMW probes that liberates ^67/68Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived ⁶⁷Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for ⁶⁷Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. ⁶⁷Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, ⁶⁷Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of ⁶⁷Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of ⁶⁷Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as ⁶⁷Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of ⁶⁷Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of ⁶⁷Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of ⁶⁷Ga-labeled Fabs for low renal radioactivity levels.

Full text of DOI:10.1021/bc5004058

Subscriber access provided by KING MONGKUT'S UNIVERSITY OF TECHNOLOGY THONBURI (UniNet)
Article
A 67/68Ga-labeling agent that liberates 67/68Ga-NOTA-
methionine by lysosomal proteolysis of parental low molecular
weight polypeptides to reduce renal radioactivity levels
T. Uehara, Takemi Rokugawa, Mai Kinoshita, Souki Nemoto,
Francisco Lazaro Guerra-Gomez, Hirofumi Hanaoka, and Y. Arano
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/bc5004058 • Publication Date (Web): 10 Oct 2014
Downloaded from http://pubs.acs.org on October 15, 2014
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A Gaꢀlabeling agent that liberates Gaꢀ
NOTAꢀmethionine by lysosomal proteolysis of
parental low molecular weight polypeptides to
reduce renal radioactivity levels
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Tomoya Uehara *, Takemi Rokugawa , Mai Kinoshita , Souki Nemoto , Guerra Gomez-
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Fransisco Lazaro , Hirofumi Hanaoka , Yasushi Arano
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Graduate School of Pharmaceutical Sciences, Chiba University, 1ꢀ8ꢀ1 Inohana, Chuoꢀku,
Chiba, 263ꢀ8675, Japan.
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Running Head: A
Gaꢀlabeling reagent for low renal radioactivity levels
KEYWORDS: gallium, NOTA, peptide, kidney, reduce
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ABSTRACT
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The renal localization of galliumꢀ67 or galliumꢀ68 (
Ga)ꢀlabeled low molecular weight
(LMW) probes such as peptides and antibody fragments constitutes a problem in targeted
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imaging. Wu et al. previously showed that Gaꢀlabeled Sꢀ2ꢀ(4ꢀisothiocyanatobenzyl)ꢀ1,4,7ꢀ
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triazacyclononaneꢀ1,4,7ꢀtriacetic acid (SCNꢀBzꢀNOTA)ꢀconjugated methionine ( Gaꢀ
NOTAꢀMet) was rapidly excreted from the kidney in urine following lysosomal proteolysis
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of the parental GaꢀNOTAꢀBzꢀSCNꢀdisulfideꢀstabilized Fv fragment (Bioconjugate Chem.,
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(1997) 8, 365ꢀ369). In the present study, a new
Gaꢀlabeling reagent for LMW probes that
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liberates
GaꢀNOTAꢀMet was designed, synthesized, and evaluated using longerꢀlived
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Ga in order to reduce renal radioactivity levels. We employed a methionineꢀisoleucine (MI)
dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCNꢀBzꢀ
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NOTA for Gaꢀlabeling, while the carboxylic acid residue of MI was derivatized to
maleimide for antibody conjugation in order to synthesize NOTAꢀMIꢀMal. A Fab fragment of
the antiꢀHer2 antibody was thiolated with iminothiolane, and NOTAꢀMIꢀMal was conjugated
with the antibody fragment by maleimideꢀthiol chemistry. The Fab fragment was also
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conjugated with SCNꢀBzꢀNOTA (NOTAꢀFab) for comparison. GaꢀNOTAꢀMIꢀFab was
obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the
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biodistribution study using normal mice, GaꢀNOTAꢀMIꢀFab registered significantly lower
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renal radioactivity levels from 1 h to 6 h postꢀinjection that those of GaꢀNOTAꢀFab. An
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analysis of urine samples obtained 6 h after the injection of GaꢀNOTAꢀMIꢀFab showed that
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the majority of radioactivity was excreted as GaꢀNOTAꢀMet. In the biodistribution study
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using tumorꢀbearing mice, the tumor to kidney ratios of GaꢀNOTAꢀMIꢀFab were 4 times
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higher (6 h postꢀinjection) than those of GaꢀNOTAꢀFab. Although further studies including
the structure of radiometabolites and/or cleavable linkages are required, the results of the
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present study indicate that the current chemical design is applicable to the development of
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INTRODUCTION
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Gallium radioisotopes are of great interest for molecular imaging. Galliumꢀ67 ( Ga) is a γꢀ
emitter (halfꢀlife: 3.3 d) that is currently used in the diagnosis of infection and inflammatory
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, 2
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processes as well as tumor imaging by SPECT. Galliumꢀ68 ( Ga) is a PET radioisotope
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that is obtained from longꢀlived Ge/ Ga generator systems, allowing the potentially cost
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effective production of Ga radiotracers far away from a cyclotron facility. Its physical halfꢀ
life of 67.7 min is suitable for labeling low molecular weight (LMW) probes such as small
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peptides and antibody fragments with rapid pharmacokinetics. To prepare
Gaꢀlabeled
LMW probes, a bifunctional chelating agent is conjugated with an LMW probe, followed by
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a complexation reaction between
Ga and the conjugate. Of the various bifunctional
chelating agents so far reported, the macrocyclic chelator, 1,4,7ꢀtriazacyclononaneꢀN,N’,N’’ꢀ
triacetic acid (NOTA) is preferably used due to the formation of a stable gallium complex
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under mild reaction conditions.
Radiolabeled LMW probes allow for rapid targeting and uniform distribution in target
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tissues.
However, the use of LMW probes, particularly those labeled with metallic
radionuclides, has been associated with high and persistent radioactivity levels in the kidneys,
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, 9
which compromises target visualization near the kidneys
Previous studies demonstrated
that radiolabeled antibody fragments were reabsorbed by proximal tubules via luminal
endocytosis after glomerular filtration. Persistent renal radioactivity levels were attributed to
the long residence time of radiometabolites generated following the lysosomal proteolysis of
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radiolabeled antibody fragments in renal cells.
These findings indicated that the in vivo
behaviors of radiometabolites played crucial roles in renal radioactivity levels and also that
the liberation of a radiometabolite by rapid excretion from the kidneys in urine following the
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lysosomal degradation of parental
renal radioactivity levels.
Gaꢀlabeled LMW probes may be useful for reducing
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Approaches using metabolizable linkers cleaved by lysosomal enzymes, ester bonds, or
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diester bonds have been employed as metabolizable linkers.
However, the stability of
ester bonds as metabolizable linkers was shown to be dependent on the molecular size of
probes and the development of plasma stable metabolizable linkers was also needed for
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LMW probes. Moreover, few studies have examined radiolabeling reagents using
macrocyclic chelates that release radiometabolites designed for rapid excretion from the
kidneys in urine following the lysosomal degradation of parental radiolabeled LMW probes.
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Wu et al. previously reported that when a Gaꢀlabeled disulfideꢀlinked Fv fragment of an
antibody (dsFv) using 2ꢀ(pꢀisothiocyanatobenzyl)ꢀ1,4,7ꢀtriazacyclononaneꢀ1,4,7ꢀtriacetic
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acid (SCNꢀBzꢀNOTA) as a bifunctional chelating agent was administered, the GaꢀNOTAꢀ
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BzꢀSCNꢀadduct of methionine ( GaꢀNOTAꢀMet, Figure 1) and GaꢀNOTAꢀBzꢀSCNꢀadduct
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of lysine ( GaꢀNOTAꢀLys, Figure 1) were generated as radiometabolites in the kidneys
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because the Nꢀterminal amino acid of the dsFv was methionine. This study also indicated
that the former had a significantly shorter residence time in the murine kidney than the latter.
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These findings suggested that
Ga labeling reagents for LMW probes that liberate
Gaꢀ
NOTAꢀMet following proteolysis of the parental compound in lysosomes may be useful for
reducing renal radioactivity levels after Gaꢀlabeled LMW probes are injected. Since the
second amino acid from the Nꢀterminal of the light chain of the dsFv used by Wu et al. was
1
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isoleucine (Ile), NOTAꢀMetꢀIleꢀMaleimide (NOTAꢀMIꢀMal, Figure 2) was designed,
synthesized, and conjugated to the Fab fragment of a mAb against Her2 after the treatment of
the antibody fragment with 2ꢀiminothiolane (NOTAꢀMIꢀFab). In this molecular design, Gaꢀ
NOTAꢀMet was released from the conjugate when the peptide bond between methionine and
isoleucine in GaꢀlabeledꢀNOTAꢀMIꢀFab (GaꢀNOTAꢀMIꢀFab) was cleaved. The tissue
distribution of radioactivity and radiometabolites in the kidneys and urine after an injection of
GaꢀNOTAꢀMIꢀFab were compared to those of a GaꢀNOTAꢀSCNꢀconjugated Fab fragment
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(GaꢀNOTAꢀFab). The validity of the new Gaꢀlabeling reagent of LMW probes, NOTAꢀMIꢀ
Mal, in reducing the renal radioactivity levels was estimated.
EXPERIMENTAL SECTION
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Materials. GaCl was supplied by FUJIFILM RI Pharma Co., Ltd. (Tokyo, Japan).
Analytical reversed phase HPLC (RPꢀHPLC) was performed with a Cosmosil 5C18ꢀARꢀ300
column (4.6 mm x 150 mm, Nacalai Tesque Inc., Kyoto, Japan) at a flow rate of 1 mL/min
with a gradient mobile phase starting from 95% A (0.1% aqueous trifluoroacetic acid (TFA)
and 5% B (acetonitrile with 0.1% TFA) to 70% A and 30% B at 20 min. Preparative RPꢀ
HPLC was performed with a Cosmosil 5C18ꢀARꢀ300 column (20 mm x 150 mm, Nacalai
Tesque Inc.) at a flow rate of 5 mL/min with a gradient mobile phase starting from 95% C
(0.05% TFA) and 5% D (acetonitrile with 0.05% TFA) to 55% C and 45% D at 25 min, and
then to 55% C and 45% D at 35 min. Sizeꢀexclusion HPLC (SEꢀHPLC) was performed with
a Cosmosil Diolꢀ300ꢀII column (7.5 x 600 mm, Nacalai Tesque Inc.) eluted with 0.1 M
phosphate buffer (pH 6.8) at a flow rate of 1 mL/min. The eluent was monitored online with
an UV/Visible single beam spectroscopy detector (Lꢀ7405, Hitachi Co. Ltd., Tokyo) coupled
to a NaI(Tl) radioactivity detector (Gabi star, Raytest, Strubenhardt, Germany). Each eluent
was collected with a fraction collector (RediFrac, GE Healthcare BioꢀScience, Tokyo) at 0.5ꢀ
min intervals, and the radioactivity counts in each fraction were determined with an auto well
gamma counter (ARCꢀ380M, Aloka, Tokyo). TLC analyses were performed with silica plates
(
Silica gel 60 F , Merck Ltd., Tokyo) developed with saline. Cellulose acetate
254
electrophoresis (CAE) strips were run in a veronal buffer (pH 8.5, I=0.06) at a constant
current of 1 mA/cm for 30 min. Radioactivity was measured using a MiniGita Star Gamma
TLC Scanner (Raytest) and an auto well gamma counter. Mass spectrometry was carried out
using an Agilent 6130 Series Quadrupole LC/MS electrospray system (Agilent Technologies,
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Tokyo). H-NMR spectra were recorded on a JEOL JNM-ECPꢀ400 (400 MHz) spectrometer
(
JEOL Ltd., Tokyo). High resolution mass spectrometry was carried out using JMSꢀT100LP
JEOL Ltd.). Apoꢀtransferrin (apoꢀTf) Iron Free was purchased from Nacalai Tesque Inc.
Kyoto, Japan). Nꢀ(methoxycarbonyl)maleimide (NMCM) was synthesized according to the
(
(
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procedure of Keller et. al.
Synthesis of the isoleucine methyl ester (1, Ile-OMe). Thionyl chloride (2.0 mL) was
added dropwise to dry methanol (20 mL) at ꢀ15 ˚C. After standing for 10 min at the same
temperature, Lꢀisoleucine (1.0 g, 7.6 mmol) was added to the solution. The temperature of the
solution was gradually increased to room temperature and the solution was mixed for 6 h.
After the solvent was evaporated in vacuo, the residue was suspended in saturated aqueous
NaHCO solution (30 mL). Compound 1 was extracted using chloroform (10 mL x 3) and
3
4
then dried over anhydrous MgSO . After removing the solvent, compound 1 was obtained as
1
a brown oil (0.96 g, 87%). HꢀNMR (CDCl
3
) δ: 0.89 (6H, m, CH
3
2
), 1.30 (2H, dm, CH ), 1.42
+
(
1H, m, CH), 3.36 (1H, d, CH), 3.77 (3H, s, CH
3
). ESIꢀMS m/z [M+H] 146, Found 146.
Synthesis of the tert-butoxycarbonyl-methionyl-isoleucine methyl ester (2, Boc-Met-
Ile-OMe). Compound 1 (742 mg, 5.1 mmol), tertꢀbutoxycarbonylꢀmethionine (BocꢀMetꢀOH,
.27 g, 5.11 mmol), and 1ꢀhydroxybenzotriazole (HOBt, 759 mg, 5.61 mmol) were dissolved
1
in dimethylformamide (DMF, 15 mL) and the mixture was then cooled on ice before the
addition of N,N’ꢀdicyclohexylcarbodiimide (DCC, 1.16 g, 5.61 mmol). The reaction mixture
was stirred for 10 min on ice and for an additional 18 h at room temperature. After removing
the precipitate by filtration, the solvent was evaporated in vacuo. The residue dissolved in
ethylacetate (10 mL) was washed with saturated aqueous NaHCO
3
solution (10 mL x 3) and
5
% citric acid (10 mL x 2). The organic layer was dried over anhydrous MgSO . After
4
1
removing the solvent, compound 2 was obtained as a white solid (1.42 g, 73.8%). HꢀNMR
CDCl ) δ: 0.89 (6H, m, CH ), 1.30 (11H, m, s, CH , Boc), 1.97 (3H, m, CH, CH ), 2.10 (3H,
(
3
3
2
2
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s, CH
3
2 3
), 2.57 (2H, t, CH ), 3.70 (3H, s, OCH ), 4.28 (1H, d, CH), 4.53 (1H, q, CH); ESIꢀMS
+
m/z [M+Na] 399, Found 399.
Synthesis of tert-butoxycarbonyl-methionyl-isoleucine (3, Boc-Met-Ile-OH).
Compound 2 (1.55 g, 3.9 mmol) was dissolved in ethanol (40 mL) and 4N NaOH solution
(1.8 mL) was then added to the solution. After mixing for 8 h at room temperature, ethanol
was evaporated in vacuo. The pH of the solution was adjusted to 3 with 10% citric acid and
the compound was then extracted using ethylacetate (15 mL x 3). The organic layer was dried
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over anhydrous MgSO . After removing the solvent, compound 3 was obtained as a white
1
solid (1.3 g, 92.3%). HꢀNMR (CDCl
3
) δ: 0.85 [6H, m, CH
], 2.58 [2H, t, CH
q, CH]; ESIꢀMS m/z [M+Na] 385, Found 385.
Synthesis of N-tert-butoxycarbonyl-ethylenediamine (4, Boc-ED). Ethylenediamine (1.0
g, 16.6 mmol) was dissolved in chloroform (25 mL) and Boc O (1.82 g, 8.32 mmol)
3
], 1.29 [11H, m, s, CH
2
, Boc],
1.97 [3H, m, CH, CH
2
], 2.10 [3H, s, SCH
3
2
S], 4.28 [1H, d, CH], 4.53 [1H,
+
2
dissolved in chloroform (25 mL) was then added to the solution dropwise on ice. After
mixing for 1 h on ice, the precipitate was removed by filtration. After removing the solvent in
vacuo, the residue was purified with open column chromatography using silica gel and
subsequently eluted with CHCl /MeOH/25% aqueous NH (80/16/3) to obtain compound 4
3
3
1
as a colorless oil (1.21 g, 45.5%). HꢀNMR (CDCl ) δ: 1.41 [9H, s, Boc], 2.76 [2H, t, CH ],
3
2
+
3
.13 [2H, q, CH
Synthesis of N-tert-butoxycarbonyl, N’-maleoyl-ethylenediamine (5, Boc-ED-Mal)
Compound 4 (630 mg, 3.93 mmol) was dissolved in saturated NaHCO solution (10 mL)
2
], 4.88 [1H, s, NH]; ESIꢀMS m/z [M+H] 161, Found 161.
3
and NMCM (610 mg, 3.93 mmol) was then added to the solution on ice. The solution was
mixed for 40 min on ice and for an additional 50 min at room temperature. The pH of the
solution was adjusted to 3 with concentrated H
2 4
SO on ice. The compound was extracted by
ethylacetate (5 mL x 3) and then dried over anhydrous MgSO
4
. After removing the solvent in
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vacuo, compound 5 was obtained as a white solid (870 mg, 92.1%). HꢀNMR (CDCl ) δ:
3
1
2 2
.38 [9H, s, Boc], 3.31 [2H, q, CH ], 3.64 [2H, t, CH ], 4.71 [1H, s, NH], 6.69 [2H, s,
+
maleimide]; ESIꢀMS m/z [M+H] 241, Found 241.
Synthesis of N-Maleoyl-ethylenediamine (6, Mal-ED). Compound 5 (1.0 g, 4.16 mmol)
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was dissolved in 4N HCl/ethylacetate and then mixed for 30 min at room temperature. After
removing the solvent in vacuo, compound 6 was obtained as a white solid (711 mg, 96.7%).
1
HꢀNMR (D
2
2 2
O) δ: 3.09ꢀ3.12 [2H, t, CH ], 3.70ꢀ3.73 [2H, t, CH ], 6.78 [2H, s, maleimide];
+
ESIꢀMS m/z [M+H] 141, Found 141.
Synthesis of Boc-Met-Ile-ED-Mal (7). Compound 3 (234 mg, 1.66 mmol), compound 6
(600 mg, 1.66 mmol), and HOBt (224 mg, 1.66 mmol) were suspended in acetonitrile (5 mL)
and diisopropylethylamine (DIPEA, 289 µL, 1.66 mmol) was then added to the solution on
ice. After 10 min, DCC (343 mg, 1.66 mmol) was added to the solution and mixed for 1 h on
ice and for an additional 18 h at room temperature. After the precipitate was removed by
filtration, the solvent was evaporated in vacuo. The residue was dissolved in chloroform (20
mL) and then washed with 5% NaHCO
3
(20 mL x 2) and 5% citric acid (20 mL x 2). The
. After removing the solvent in vacuo, the
organic layer was dried over anhydrous MgSO
4
residue was purified by open column chromatography using silica gel and subsequently
1
eluted with chloroform to obtain compound 7 as a white solid (402 mg, 49.9%). HꢀNMR
(
CDCl
3
) δ: 0.87 [6H, m, CH
3 2
], 1.21 [11H, m, CH2, Boc], 1.94 [2H, m, CH ], 2.13 [4H, m,
CH , SCH ], 2.57 [2H, t, CH S], 3.44 [2H, t, CH ], 3.67 [2H, t, CH ], 4.22 [2H, m, CH], 6.70
2
3
2
2
2
+
[2H, s, maleimide]; ESIꢀMS m/z [M+Na] 507, Found 507. mp 168ꢀ169 ˚C.
Synthesis of Met-Ile-Mal (8). Compound 7 (85 mg, 0.175 mmol) was dissolved in 4N
HCl/ethylacetate (5 mL) and then mixed for 30 min at room temperature. After removing the
1
solvent, compound 8 was obtained as a white solid (50 mg, 74.4%). HꢀNMR (CD
3
OD) δ:
0.89 [6H, s, CH ], 1.17 [2H, m, CH ], 1.60 [2H, m, CH ], 1.79 [2H, m, CH ], 2.11 [4H, m,
3
2
2
2
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CH, CH
3
], 3.59 [4H, m, CH
2
], 4.04 [1H, t, CH], 6.83 [2H, s, maleimide]; ESIꢀMS m/z
+
[
M+H] 385, Found 385.
Synthesis of NOTA-MI-Mal (9). NOTAꢀBzꢀSCN (5.1 mg, 8.99 µmol) and compound 8
(5.2 mg, 13.5 mmol) were dissolved in dry DMF (300 µL). After triethylamine (TEA, 6.3 µL)
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9
0
was added to the solution, it was mixed for 2 h at room temperature. After the solution was
diluted with H
2
O/acetonitrile (4/1, 3 mL), it was purified by preparative RPꢀHPLC to obtain
1
compound 9 as a white solid (2.2 mg, 30%). HꢀNMR (CDCl
3
) δ: 0.87 [6H, m, CH
3
], 1.94
[
2H, m, CH
2
], 2.13 [4H, m, CH
2
, SCH
3
], 2.57 [2H, t, CH
2
S], 3.44 [2H, t, CH ], 3.67 [2H, t,
2
+
CH
2
], 4.22 [2H, m, CH], 6.70 [2H, s, maleimide]; ESIꢀMS m/z [M+H] 835, Found 835.
+
HRMS m/z [M+H] calculated for C37
H
55
N
8
O
10
S
2
835.34825, observed 835.33769.
Preparation of the NOTA-MI-conjugated Fab fragment (NOTA-MI-Fab). The Fab
18
fragment of herceptin was prepared according to the procedure of Tang et. al. A solution of
the Fab fragment (200 µL, 4 mg/mL) in wellꢀdegassed 0.16 M borate buffer (pH 8.0)
containing 2 mM EDTA was allowed to react with 7.2 µL (1.8 µL x 4) of 2ꢀiminothiolane
solution (2ꢀIT, 2 mg/mL) prepared in the same buffer. After gentle agitation of the reaction
mixture for 30 min at room temperature, excess 2ꢀIT was removed by a centrifuged column
procedure using Sephadex Gꢀ50 fine (GE Healthcare BioꢀScience AB, Tokyo) equilibrated
and eluted with 0.1 M phosphate buffer (pH 6.0) containing 2 mM EDTA. Aliquots of this
mixture were sampled to estimate the number of thiol groups with 2,2’ꢀdipyridyl disulfide
19
(
DPS). NOTAꢀMIꢀMal (1 µL, 100 mg/mL in DMF) was added to the filtrate (100 µL) and
then mixed gently for 2 h at room temperature. Excess NOTAꢀMIꢀMal was removed by the
centrifuged column procedure using Sephadex Gꢀ50 fine equilibrated and eluted with 0.1 M
phosphate buffer (pH 6.0) containing 2 mM EDTA, and aliquots of the filtrate were then
sampled to estimate the number of thiol groups with DPS. After 14.8 µL of iodoacetamide
(10 mg/mL) in phosphate buffer (pH 6.0) was added, the reaction mixture was further
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incubated for 30 min to alkylate the unreacted thiol groups. NOTAꢀMIꢀFab was subsequently
purified by the centrifuged column procedure using Sephadex Gꢀ50 fine equilibrated and
eluted with 0.25 M acetate buffer (pH 5.5).
Preparation of the NOTA-SCN-conjugated Fab fragment (NOTA-Fab). The Fab
fragment (3 mg/mL, 100 µL) was dissolved in borate buffer (0.1 M, pH 9.0) and SCNꢀBzꢀ
NOTA (7.8 µL, 8 mg/mL in DMF) was then added to the solution. After incubating for 18 h
at room temperature, the conjugate was purified by Sephadex Gꢀ50 fine equilibrated and
eluted with 0.25 M acetate buffer (pH 5.5).
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3
Preparation of Ga-NOTA-MI-Fab and Ga-NOTA-SCN-Fab. GaCl (0.05 N HCl,
30 µL) was added to acetate buffer (0.25 M, pH 5.5, 30 µL). After 5 min, NOTAꢀMIꢀFab or
NOTAꢀSCNꢀFab (3 mg/mL, 0.25 M acetate buffer pH 5.5, 30 µL) was added to the solution
and then incubated for 40 min at 37 ˚C. After the addition of EDTA (15 µM, 30 µL), the
6
7
Gaꢀlabeled Fab fragments were purified by Sephadex Gꢀ50 fine equilibrated and eluted
with 20 mM phosphate buffer (pH 7.4). Radiochemical yield and purity were determined by
sizeꢀexclusion HPLC and CAE.
67
67
Plasma stability of Ga-NOTA-MI-Fab. GaꢀNOTAꢀMIꢀFab (20 µL) was added to a
solution of freshly prepared murine plasma (230 µL), and the solution was then incubated at
37 ˚C. Aliquots of samples were collected after being incubated for 1, 3, 6, and 24 h, and
radioactivity was analyzed by TLC and SEꢀHPLC.
Cell line. The squamous cell lung carcinoma cell line NCIꢀH2170 was purchased from the
American Type Culture Collection (ATCC, Manassas, VA). These cells were cultured
according to protocols prescribed by the ATCC.
In vivo study. Animal studies were conducted in accordance with institutional guidelines
20
approved by the Chiba University Animal Care Committee. Sixꢀweekꢀold male ddy mice
67
(
Japan SLC Inc,., Shizuoka, Japan) were injected via the tail vein with either GaꢀNOTAꢀ
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MIꢀFab or GaꢀNOTAꢀFab (100 µL, 11.1 kBq, 20 µg). Animals were then sacrificed and
dissected 10 min, 1 h, 3 h, and 6 h after this administration. Tissues of interest were removed
and weighed, and radioactivity counts were determined by an auto well gamma counter.
Urine and feces were collected for 6 h and radioactivity was also measured. Values were
expressed as the mean (SD) for a group of 3ꢀ5 animals.
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Sixꢀweekꢀold male BALBc nu/nu mice (Japan SLC Inc.) were xenografted by a
6
subcutaneous injection of NCIꢀH2170 squamous cell carcinoma cells (5 x 10 cells/80 µL of
culture medium) into their right hind legs. These mice were then subjected to biodistribution
3
studies when tumor volumes reached 100ꢀ300 mm . Biodistribution studies were conducted
in male BALBc nu/nu mice bearing NCIꢀH2170 xenografts 6 h after the administration of
each radiotracer (n=3).
67
Radiometabolites in urine. Six h after the injection of GaꢀNOTAꢀMIꢀFab (74 kBq, 20
µg) into 6ꢀweekꢀold ddyꢀmice, urine samples were collected and filtered through a
polycarbonate membrane (0.45 µm) before being analyzed by SEꢀHPLC. Urine samples were
also filtered through a 10,000 Da cutꢀoff ultrafiltration membrane before being analyzed by
RPꢀHPLC.
67
Radiometabolites in the kidney. To investigate the metabolites of GaꢀNOTAꢀMIꢀFab or
6
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GaꢀNOTAꢀFab in the kidney, GaꢀlabeledꢀFab (20 µg, 74 kBq) was intravenously injected
into 6ꢀweekꢀold male ddy mice. At 1 h or 6 h postꢀinjection, mice were treated according to
21
the procedure of Arano et. al. with slight modifications. The kidneys were excised, placed in
a plastic tube, and subjected to three cycles of freezing (dry iceꢀmethanol bath) and thawing.
Two milliliters of cold 0.1 M trisꢀcitrate buffer (pH 6.5) containing 0.15 M NaCl, 0.002%
sodium azide, 1 TIU/mL aprotinin, 2 mM benzamideꢀHCl, 2 mM iodoacetamide and 5 mM
diisopropyl fluorophosphate (buffer A), containing an additional 35 mM of betaꢀoctylꢀ
glucoside, was then added to each tube. The kidneys were homogenized by cell disruption
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with a polytron homogenizer (PTꢀ3100 Kinematica GmbH Littau, Switzerland) at full speed
for three 30ꢀsec bursts, followed by centrifugation at 48,000 x g for 20 min (Optima MAX
Ultracentrifuge, TLA110, Beckman Instruments, Inc. CA, USA). Supernatants were
separated from the pellets and radioactivity was counted. Following filtration through a
polycarbonate membrane with a pore diameter of 0.45 µm, the supernatants were analyzed by
SEꢀHPLC. The supernatants were also filtered through a 10,000 Da cutꢀoff ultrafiltration
membrane before being analyzed by RPꢀHPLC.
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RESULTS
Preparation of NOTA-MI-Fab and NOTA-Fab (Scheme 1). NOTAꢀMIꢀMal was
synthesized by reacting SCNꢀBzꢀNOTA with MetꢀIleꢀMal. The conjugation of Fab with
NOTAꢀMIꢀMal was performed by maleimideꢀthiol chemistry using thiol groups generated by
the addition of 2ꢀIT. The number of NOTAꢀMI introduced per molecule of Fab was estimated
to be 0.75ꢀ0.9 by measuring the thiol groups of the Fab fragment before and after the
conjugation reaction. The conjugation of SCNꢀBzꢀNOTA with the Fab fragment was
achieved by reacting isothiocyanate with primary amine residues of the Fab fragment.
6
7
Unpurified NOTAꢀFab conjugates labeled by Ga and subsequent analyses by thin layer
chromatography estimated the number of SCNꢀBzꢀNOTA attached per molecule of Fab to be
1.0.
67
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Preparation of Ga-NOTA-MI-Fab and Ga-NOTA-Fab. Both NOTAꢀMIꢀFab and
6
7
NOTAꢀFab were labeled by Ga in the presence of acetate and the resulting compounds were
6
7
analyzed by TLC, CAE, and SEꢀHPLC. The radiochemical yields of both GaꢀlabeledꢀFabs
6
7
were over 95%. The radiochemical purities of both GaꢀlabeledꢀFabs were over 99% with
centrifugation and column purification after the addition of EDTA.
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Stability estimation of Ga-labeled Fabs. After Gaꢀlabeled Fabs had been incubated for
2
4 h in murine plasma at 37 ˚C, more than 90% of radioactivity remained in intact fractions
in both cases.
Biodistribution study using normal mice. The biodistribution of radioactivity after the
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5
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injection of GaꢀNOTAꢀMIꢀFab and GaꢀNOTAꢀFab to normal mice is summarized in Table
67
67
1
. The radioactivity levels of GaꢀNOTAꢀMIꢀFab in blood were similar to those of Gaꢀ
67
NOTAꢀFab. Although GaꢀNOTAꢀFab showed high radioactivity levels in the kidney from 1
6
7
h to 6 h postꢀinjection, GaꢀNOTAꢀMIꢀFab showed significantly lower renal radioactivity
67
levels during the same time period. More GaꢀNOTAꢀMIꢀFab was excreted in urine (55.63
6
7
%ID) than GaꢀNOTAꢀFab (20.81 %ID).
Radiolabeled species in urine and the kidney. Figure 3 shows radiochromatograms of
67
urine samples obtained 6 h after the injection of GaꢀNOTAꢀITꢀFab. SEꢀHPLC analyses
revealed that 95 % of the radioactivity excreted in urine was observed in the low molecular
weight fraction with a retention time of 24 min. RPꢀHPLC analyses showed that most of the
radioactivity in urine had a retention time that was identical to that of GaꢀNOTAꢀMet (14.5
min).
The supernatant of each kidney homogenate was extracted with efficiencies of over 70%
for all experiments. When the radiolabeled species in the kidney at 1 h and 6 h after the
6
7
injection of Gaꢀlabeled Fabs were analyzed by SEꢀHPLC in each case, over 95% of
radioactivity was eluted in the low molecular weight fractions (data not shown). Figure 4
shows the RPꢀHPLC chromatograms of kidney samples after filtration through a 10,000 cutꢀ
off membrane. The majority of radioactivity in the low molecular weight fractions after the
6
7
administration of GaꢀNOTAꢀMIꢀFab was detected at a retention time that was similar to
that of GaꢀNOTAꢀMet at 1 h postꢀinjection. However, this peak decreased at 6 h postꢀ
injection. On the other hand, the majority of radioactivity in the low molecular weight
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fractions after the administration of GaꢀNOTAꢀFab was detected at a retention time that was
similar to that of GaꢀNOTAꢀLys at both 1 h and 6 h postꢀinjection.
Biodistribution in tumor-bearing mice. The biodistribution of radioactivity after the
67
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administration of GaꢀNOTAꢀMIꢀFab and GaꢀNOTAꢀFab in nude mice bearing NCIꢀ
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H2170 xenografts are summarized in Figure 5. No significant differences were observed in
67
tumor uptake, blood levels, or tumor/blood ratios between the two Gaꢀlabeled Fabs.
67
However, the tumor/kidney ratio of GaꢀNOTAꢀMIꢀFab registered 4 times higher than that
67
of GaꢀNOTAꢀFab at 6 h postꢀinjection.
DISCUSSION
6
7/68
Of the various bifunctional chelating agents reported for
Ga to date, the macrocyclic
chelator, NOTA, is preferably used due to the formation of a stable gallium complex under
3ꢀ6
67
mild reaction conditions.
Wu et al. also demonstrated that GaꢀNOTAꢀMet was rapidly
6
7
excreted from the kidneys in urine following lysosomal proteolysis of the parental Gaꢀ
1
2
NOTAꢀBzꢀSCNꢀdisulfideꢀstabilized Fv fragment. In the present study, the molecular design
of Gaꢀlabeling reagents for LMW probes that liberate GaꢀNOTAꢀMet after proteolysis of the
parental probe in lysosomes was evaluated for their ability to reduce renal radioactivity levels
after an injection of Gaꢀlabeled LMW probes.
A metabolizable linker in Gaꢀlabeling reagents was designed in order to release GaꢀNOTAꢀ
Met after proteolysis of the parental probe. Ester or diester bonds have been used as
metabolizable linkers in the approach using metabolizable linkers cleaved by lysosomal
1
0ꢀ14
enzymes.
However, the stability of ester bonds as metabolizable linkers was shown to
depend on the molecular sizes of probes, and when Fab fragments were used as parental
1
5
probes, ester bonds in the radiolabeling reagent were cleaved in the blood. Thus, peptide
bonds were selected as metabolizable linkers. Since the second amino acid from the Nꢀ
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1
6
terminal of the light chain of the dsFv used by Wu et al. was isoleucine (Ile), the MetꢀIle
sequence (MI) was selected as the metabolizable linker. Moreover, maleimideꢀthiol chemistry
was selected as the conjugation method of NOTAꢀMI to antibody fragments. Because
thiourea bonds between SCNꢀBzꢀNOTA and methionine were previously reported to cause
0
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Edmann degradation under strong acid conditions, acidꢀlabile protecting groups could not
be used for the carboxylic groups of NOTA after the formation of thiourea bonds. Therefore,
difficulties have been associated with synthesizing the NOTAꢀMIꢀadducts of active esters.
Thus, NOTAꢀMIꢀMal was synthesized by reacting SCNꢀBzꢀNOTA with MetꢀIleꢀMal and the
67
67
results obtained with GaꢀNOTAꢀMIꢀFab were then compared with those of GaꢀNOTAꢀ
Fab.
6
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Both GaꢀNOTAꢀMIꢀFab and GaꢀNOTAꢀFab exhibited the stable attachment of
radiolabels to the respective Fab in mice serum. This was reflected in the similar radioactivity
levels of the two radiolabeled Fab fragments in the blood (Table 1). Thus, similar amounts of
the radiolabeled Fab fragments could be filtered through the glomerulus and transported to
the proximal tubules of the kidneys after their administration. Radioactivity levels in the
67
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kidneys 10 min after the injection of GaꢀNOTAꢀMIꢀFab were similar to those of Gaꢀ
6
7
NOTAꢀFab (Table 1). However, although GaꢀNOTAꢀFab exhibited high renal radioactivity
67
levels from 1 h to 6 h postꢀinjection, GaꢀNOTAꢀMIꢀFab demonstrated markedly lower renal
radioactivity levels from 1 h postꢀinjection onward (Table 1). Moreover, the amount of
67
radioactivity excreted in urine after the injection of GaꢀNOTAꢀMIꢀFab (55.63 %ID) was
6
7
higher than that of GaꢀNOTAꢀFab (20.81 %ID). SEꢀHPLC analyses of urine revealed that
6
7
9
5 % of the radioactivity excreted in urine after the administration of GaꢀNOTAꢀITꢀFab
was observed in the low molecular weight fraction with a retention time of 24 min (Figure 3).
RPꢀHPLC analyses of the low molecular weight fraction showed that the majority of
radioactivity was observed at a retention time identical to that of GaꢀNOTAꢀMet (14.5 min,
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Figure 3). These results suggested that the significantly lower renal radioactivity levels
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achieved by GaꢀNOTAꢀMIꢀFab were attributed to the rapid release of GaꢀNOTAꢀMet
from the kidneys.
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To further investigate the low renal radioactivity levels of GaꢀNOTAꢀMIꢀFab, the fates of
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radiometabolites in the kidney were compared with those of GaꢀNOTAꢀFab (Figure 4).
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While both GaꢀNOTAꢀLys and intermediate radiometabolites were observed in the kidneys
67
1
h after the injection of GaꢀNOTAꢀFab, the majority of radioactivity that remained in the
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67
kidneys 6 h after the injection of GaꢀNOTAꢀFab was GaꢀNOTAꢀLys. On the other hand,
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both GaꢀNOTAꢀMet and the intermediate radiometabolites were observed in the kidneys 1 h
6
7
after the injection of GaꢀNOTAꢀMIꢀFab. However, small amounts of radioactivity remained
in the kidneys in the form of GaꢀNOTAꢀMet or other intermediate radiometabolites. This
67
67
result suggested that GaꢀNOTAꢀMet may have had a shorter residence time than Gaꢀ
12
NOTAꢀLys in the kidneys, which was consistent with the findings of Wu et al. On the other
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hand, previous findings indicated that the residence time of Inꢀlabeled diethylenetriamine
111
pentaacetic acid (DTPA)ꢀconjugated methionine ( InꢀDTPAꢀMet) in the kidneys was similar
111
27
to that of the other InꢀDTPAꢀadducts of amino acids so far reported. These findings
67
suggested that the short residence time of GaꢀNOTAꢀMet in the lysosome compartment
may represent one of its specific characteristics.
The ability of NOTAꢀMIꢀMal to reduce renal radioactivity levels without impairing the
target radioactivity levels delivered by the antibody fragment was demonstrated in the
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7
biodistribution study using nude mice. Although GaꢀNOTAꢀMIꢀFab showed similar
6
7
radioactivity levels in the tumor as GaꢀNOTAꢀFab, the tumor/kidney ratios of radioactivity
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after the injection of GaꢀNOTAꢀMIꢀFab were 4 times higher than those of GaꢀNOTAꢀFab
67
6
h postꢀinjection (Figure 5). These results suggested that the metabolizable linker in Gaꢀ
NOTAꢀMIꢀFab at tumor tissues may be stable. Since the Fab fragment of herceptin was
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previous shown to not be internalized in Her2ꢀexpressing cells,
the metabolizable linker,
MI, may not have been cleaved at tumor tissues.
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In conclusion, GaꢀNOTAꢀMIꢀFab registered significantly lower renal radioactivity levels
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from 1 h to 6 h postꢀinjection than those of GaꢀNOTAꢀFab in normal mice. Moreover, the
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tumor/kidney ratios of radioactivity after the injection of GaꢀNOTAꢀMIꢀFab were 4 times
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7
higher than those of GaꢀNOTAꢀFab at 6 h postꢀinjection. Although further studies including
the structure of radiometabolites and/or cleavable linkages are required to reduce renal
67
radioactivity levels shortly after the injection of Gaꢀlabeled Fabs, the results of the present
6
7
study demonstrated that the present chemical design is applicable to the development of Gaꢀ
labeled Fabs for low renal radioactivity levels.
ACKNOWLEDGMENTS
This study was supported in part by a GrantꢀinꢀAid for Young Scientists (A) and Special
Funds for Education and Research (Development of SPECT Probes for Pharmaceutical
Innovation) from the Ministry of Education, Culture, Sports, Science and Technology of
67
Japan. We would also like to thank FUJIFILM RI Pharma Co. Ltd., for providing GaCl .
3
AUTHOR INFORMATION
Corresponding Author
Tomoya Uehara, PhD. Graduate School of Pharmaceutical Sciences, Chiba University, 1ꢀ8ꢀ1
Inohana, Chuoꢀku, Chiba, 260ꢀ8675, Japan. Tel: +81ꢀ43ꢀ226ꢀ2898, FAX: +81ꢀ43ꢀ226ꢀ2897,
Eꢀmail: tuehara@chibaꢀu.jp.
REFERENCES
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Table 1. Biodistribution of radioactivity in mice after an injection of GaꢀNOTAꢀMIꢀFab
and GaꢀNOTAꢀFab .
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a
Time after the injection
10 min
1 h
3 h
6 h
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7
0
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9
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9
0
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9
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Ga-NOTA-MI-Fab
Blood
16.56 (3.90)
3.38 (0.28)*
62.74 (7.29)
0.69 (0.19)
2.94 (0.13)*
1.49 (0.11)
1.02 (0.11)*
0.99 (0.15)
0.59 (0.07)
Liver
0.61 (0.11)*
12.41 (0.39)*
2.41 (0.43)*
55.63 (9.35)*
1.26 (1.41)
Kidney
Intestine
85.34 (11.68)*
1.02 (0.21)
28.34 (5.22)*
1.53 (0.27)*
b
Urine
b
Feces
Kidney/Blood
3.94 (0.89)
28.99 (3.82)
28.18 (6.86)*
20.96 (2.55)*
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Ga-NOTA-Fab
Blood
14.81 (1.17)
4.61 (1.23)
61.59 (7.00)
2.09 (0.59)
3.68 (0.23)
1.30 (0.12)
1.27 (0.16)
2.86 (0.28)
0.65 (0.08)
2.75 (0.49)
Liver
Kidney
Intestine
114.98 (6.25)
3.44 (0.21)
117.41 (10.6)
2.20 (0.20)
101.02 (6.46)
3.28 (0.59)
b
Urine
20.81 (2.72)
0.69 (1.10)
b
Feces
Kidney/Blood
4.19 (0.68)
31.39 (2.74)
92.70 (1.19)
157.59 (22.8)
a
Tissue radioactivity is expressed as % ID/g for each group (n=3ꢀ4); results are expressed
as the mean (SD)
b
Expressed as %ID
Significance was determined by an unpaired Student’s tꢀtest; (*) p<0.05 significantly
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different from GaꢀNOTAꢀFab.
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Scheme 1. Synthetic procedure to prepare NOTAꢀMIꢀMal. (a) SOCl , MeOH; (b) BocꢀMet,
HOBt, DCC; (c) 4N NaOH/EtOH; (d) Boc O; (e) NMCM, NaHCO ; (f) 4N HCl/ethylacetate;
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(g) HOBt, DCC; (h) 4N HCl/ethylacetate; (i) NOTAꢀBzꢀSCN, triethylamine.
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Figure 1. Structures of GaꢀNOTAꢀMet (A) and GaꢀNOTAꢀLys (B).
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Figure 2. Structure of NOTAꢀMIꢀMal.
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SE-HPLC
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membrane
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Figure 3. Radioactivity profiles of urine collected 6 h after the injection of GaꢀNOTAꢀMIꢀ
Fab by SEꢀHPLC and RPꢀHPLC. Urine samples were analyzed after filtration through a
polycarbonate membrane with a pore diameter of 0.45 µm (SEꢀHPLC) and after filtration
through a 10 kDa cutꢀoff membrane (RPꢀHPLC).
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Bioconjugate Chemistry
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67Ga-NOTA-MI-Fab
67Ga-NOTA-Fab
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Figure 4. RPꢀHPLC radioactivity and UV profiles of kidney homogenates 1 h and 6 h after
67
the injection of Gaꢀlabeled Fab fragments. Samples were analyzed after filtration through a
1
0 kDa cutꢀoff membrane.
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