99mTc-HYNIC-derivatized ternary ligand complexes for99mTc-labeledpolypeptides with lowin vivo protein binding

Masahiro Onoa, Yasushi Aranob,*, Takahiro Mukaic, Yasushi Fujiokaa, Kazuma Ogawaa,Tomoya Ueharab, Tsuneo Sagac, Junji Konishic, Hideo Sajia

aDepartment of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho,Sakyo-ku, Kyoto 606-8501, Japan

bLaboratory of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1–33 Yayoi-cho, Inage-ku,Chiba 263-8522, Japan

cDepartment of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho, Sakyo-ku,Kyoto 606-8507, Japan

Received 16 September 2000; received in revised form 9 November 2000; accepted 18 November 2000

Abstract

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m (99mTc)-labeled polypeptideswith tricine as a coligand. However,99mTc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only fromthe blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) activeester of [99mTc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare99mTc-labeled polypeptides with higherstability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody andgalactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [99mTc](HYNIC-Fab)(tricine)(BP) showed signif-icant reduction of the radioactivity in high molecular weight fractions compared with [99mTc](HYNIC-Fab)(tricine)2. When injected intomice, [99mTc](HYNIC-NGA)(tricine)(BP) was metabolized to [99mTc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivitydetected in protein-bound fractions in contrast to the observations with [99mTc](HYNIC-NGA)(tricine)2. In addition, [99mTc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [99mTc](HYNIC-NGA)(tricine)2. Similar results were observed with99mTc-labeled Fab fragments where [99mTc](HYNIC-Fab)(tricine)(BP) exhibitedsignificantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated thatconjugation of [99mTc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity fromthe blood and nontarget tissues due to low binding of the [99mTc](HYNIC)(tricine)(BP) complex with proteins in the blood and in thelysosomes. Such characteristics would render the TFP active ester of [99mTc](HYNIC)(tricine)(BP) complex attractive as a radiolabelingreagent for targeted imaging. © 2001 Elsevier Science Inc. All rights reserved.

Keywords:Technetium-99m; HYNIC; Protein; Ternary ligand; Pharmacokinetics

1. Introduction

Radiopharmaceuticals derived from low molecularweight polypeptides and antibody fragments are attractiveas vehicles to deliver radioactivity for diagnostic and ther-apeutic nuclear medicine. Rapid clearance of polypeptidesfrom the circulation allows the use of99mTc as the radio-

nuclide of choice for external imaging. Among the variousbifunctional chelating agents developed to date, 6-hydrazi-nopyridine-3-carboxylic acid (HYNIC) constitutes a repre-sentative agent for99mTc radiolabeling. Since HYNICserves as a monodentate or bidentate ligand, a coligand isnecessary to complete the coordination sphere of the tech-netium core [1,5]. Tricine is often used as the coligandbecause of the production of99mTc-HYNIC-labeled pep-tides and polypeptides with high radiochemical yields andhigh specific activities in a short reaction time [14]. Theradiolabeling procedure has been applied to prepare99mTc-labeled monoclonal IgG [21], human serum albu-

* Corresponding author. Tel.:181-43-290-3024; fax:181-43-290-3025.

E-mail address:arano@p.chiba-u.ac.jp (Y. Arano).

Nuclear Medicine and Biology 28 (2001) 215–224

0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0969-8051(00)00210-9

min [25], Fab’ fragment [23], chemotactic peptide [24]and RGD peptide [18].

However,99mTc-HYNIC-labeled polypeptides and pep-tides using tricine as a coligand showed slow eliminationrates of the radioactivity from the blood, which compro-mised the advantages of selecting low molecular weightpolypeptides or peptides to deliver99mTc to target tissues[7,19]. 99mTc-HYNIC-labeled polypeptides using tricine asa coligand also exhibited long residence times of the radio-activity at sites of polypeptide catabolism such as the liverand kidney [19,21,23]. Recent studies suggested that theslow elimination rates of the radioactivity from the bloodand the liver after administration of99mTc-HYNIC-labeledpolypeptides could be attributed to progression of exchangereactions of tricine coligand with proteins in plasma andlysosomes [19,20].

Recently, several coligands have been developed to im-prove the problems associated with99mTc-HYNIC-derivat-ized small peptides. Liu et al. used a water soluble phos-phine or an imine-N-containing heterocycle as an additionalcoligand to form a ternary ligand system [99mTc](HYNIC-peptides)(tricine)(L) (L 5 water-soluble phosphines orimine-N-containing heterocycles) [16,17]. The radiolabel-ing procedure provided99mTc-HYNIC-labeled small pep-tides with higher stability and fewer isomers in high radio-chemical yields and high specific activities. These studiessuggested that expansion of the ternary ligand system topolypeptide labeling would generate99mTc-HYNIC-labeledpolypeptides that are inert against exchange reactions withproteins in plasma or lysosomes. This may provide99mTc-labeled polypeptides with faster elimination rates of theradioactivity not only from the blood but also from nontar-get tissues. However, application of the procedure topolypeptides was hindered due to the requirement ofharsh radiolabeling conditions such as high temperatureand low pH.

In the present study, an active ester of preformed chelateof 99mTc-HYNIC-derivatized ternary ligand complex wasdesigned and synthesized to conjugate99mTc-HYNIC ter-nary complex to polypeptides under mild reaction condi-tions. Previous studies showed significant protein binding of99mTc-HYNIC-Fab in plasma [19], whereas the radiom-etabolites derived from99mTc-HYNIC-galactosyl-neogly-coalbumin (NGA) exhibited long residence time of theradioactivity in the lysosomes of the liver due to binding toproteins in the organelle [20]. Thus, plasma protein bindingof the 99mTc-HYNIC-labeled polypeptides was estimatedusing the Fab fragment of a monoclonal antibody as amodel. The stability of the99mTc-HYNIC-conjugated radio-metabolites against exchange reactions with proteins in thelysosomes was examined after administration of99mTc-labeled NGA. The abilities of the99mTc-HYNIC-conju-gated polypeptides with a ternary ligand complex to accel-erate elimination rates from the blood and from the liver arediscussed.

2. Materials and methods

2.1. Reagents and chemicals

[99mTc]Pertechnetate (99mTcO42) was eluted in saline so-

lution on a daily basis from Daiichi Radioisotope Labora-tory generators (Chiba, Japan). Reversed-phase HPLC (RP-HPLC) was performed with a Cosmosil 5C18-AR-300column (4.6 x 150 mm, Nacalai Tesque, Kyoto, Japan) at aflow rate of 1.0 mL/min with a gradient mobile phasestarting from 90% A (0.1% aqueous trifluoroacetic acid) and10% B (acetonitrile with 0.1% trifluoroacetic acid) to 50%A and 50% B at 20 min and to 100% B at 30 min (solventsystem 1). RP-HPLC was also performed with a gradientmobile phase starting from 100% A to 45% A and 55% B at30 min and 100% B at 40 min (solvent system 2). Size-exclusion HPLC (SE-HPLC) was performed using a 5 Diol-300 column (7.5 x 600 mm, Nacalai Tesque) connected to a5 Diol-300 guard column (7.5 x 50 mm, Nacalai Tesque),eluted with 0.1 M phosphate buffer (pH 6.8) at a flow rateof 1.0 mL/min. The eluent was collected with a fractioncollector (RediFrac; Pharmacia Biotech, Tokyo) at 30-sec-ond intervals and the radioactivity counts in each fraction(500mL) were determined with an auto well counter (ARC-2000; Aloka, Tokyo). The radioactivity of liver homoge-nates was analyzed by gel permeation chromatography(GPC) using a Sephadex G-50 (Pharmacia Biotech Co. Ltd.,Tokyo, Japan) column (183 400 mm) equilibrated andeluted with 0.1 M phosphate buffer (pH 6.8) at a flow rateof 1.0 mL/min. The eluent was collected with a fractioncollector at 2.5-minute intervals and the radioactivity levelsin each fraction (2.5 mL) were measured with an auto wellcounter. TLC analyses were performed with silica plates(Merck Art 5553) with a mixture of 10% aqueous ammo-nium chloride-methanol (1:1) or saline as the developingsolvent. Cellulose acetate electrophoresis (CAE) strips wererun in veronal buffer (pH 8.6,I 5 0.05) at a constant currentof 0.8 mA/cm for 30 min. Proton nuclear magnetic reso-nance (1H-NMR) spectra were recorded on a Bruker AC-200 spectrometer, and the chemical shifts are reported inppm downfield from an internal tetramethylsilane standard.Fast atom bombardment mass spectra (FAB-MS) were ob-tained with a JMS-HX/HX 110 A model (JEOL Ltd., To-kyo). Tricine and 3-benzoylpyridine (BP) were purchasedfrom Nacalai Tesque, and other reagents were of reagentgrade and used as received. To facilitate collection of urineand feces after administration of radiolabeled polypeptide,mice were housed in metabolic cages (Metabolica, MMtype; Sugiyama-Gen Iriki Co. Ltd., Tokyo).

2.2. Monoclonal antibody

A monoclonal antibody against osteogenic sarcoma(OST7, IgG1), generated by the standard hybridoma tech-nique, was purified by ammonium sulfate precipitation withsubsequent protein A affinity chromatography (Pharmacia

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Biotech Co. Ltd.), as reported previously [10]. The Fabfragment was prepared by the standard procedure usingpapain [11].

2.3. Synthesis of NGA

Cyanomethyl-2,3,4,6-tetra-O-acetyl-1-b-galactopyrano-side, synthesized according to the procedure of Lee et al.[15], was conjugated with human serum albumin (HSA,A-3782; Sigma Co., St. Louis, MO), according to the pro-cedure of Stowell et al. [22]. The phenol-sulfuric acid re-action [9] indicated that 43 galactose units were attached toeach HSA molecule.

2.4. Synthesis of 2,3,5,6-tetrafluorophenyl6-hydrazinopyridine-3-carboxylate (HYNIC-TFP)

6-(tert-Butoxycarbonyl)-hydrazinopyridine-3-carboxylicacid was synthesized according to the procedure of Abramset al. [1]. To a mixed solution of 6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carboxylic acid (253 mg, 1.0 mmol)and 2,3,5,6-tetrafluorophenol (250 mg, 1.5 mmol) in drydimethylformamide (DMF) was added 1,3-dicyclohexylcar-bodiimide (268 mg, 1.3 mmol) with rapid stirring. Thereaction mixture was stirred at room temperature for 18 h.The reaction mixture was then filtered to remove whiteprecipitates, and the filtrate was concentrated to give ayellow oil. The oily residue was chromatographed on silicagel using a mixture of ethyl acetate-hexane (1:2) as theeluent to produce 2,3,5,6-tetrafluorophenyl 6-(tert-butoxy-carbonyl)-hydrazinopyridine-3-carboxylate as white crys-tals (227 mg, 56.6%).1H-NMR (CDCl3):d 1.49 (9H, s,Boc), 6.81 (1H, d, pyridine), 7.05 (1H, m, benzene), 8.27(1H, dd, pyridine), 8.98 (1H, d, pyridine). Anal. calcd forC17H15O4N3F4: C, 50.88; H, 3.77; N, 10.47. Found: C,51.30; H, 3.86; N, 10.27. FAB-MS calcd for C17H16O4N3F4

(MH1): m/z402. Found: 402.2,3,5,6-Tetrafluorophenyl 6-(tert-butoxycarbonyl)-hy-

drazinopyridine-3-carboxylate (100 mg, 0.25 mmol) wasstirred in a mixed solution of trifluoroacetic acid (900mL)and anisole (100mL) for 10 min at room temperature. Afterremoval of trifluoroacetic acidin vacuo, the residue wastreated with dry ether to produce 2,3,5,6-tetrafluorophenyl6-hydrazinopyridine-3-carboxylate as pale yellow crystals(72.8 mg, 96.7%).1H-NMR (DMSO-d6): d 6.95 (1H, dd,pyridine), 7.99 (1H, m, benzene), 8.25 (1H, dd, pyridine),8.92 (1H, d, pyridine). Anal. calcd for C12H7O2N3F4

z CF3COOH: C, 40.50; H, 1.94; N, 10.12. Found: C, 40.57;H, 1.83; N, 9.84. FAB-MS calcd for C12H8O2N3F4 (MH1):m/z302. Found: 302.

2.5. Synthesis of [99mTc](HYNIC-TFP)(tricine)(BP)

A solution of 30mL HYNIC-TFP (3 mg/mL in isopropylalcohol) was mixed successively with 200mL tricine (30mg/mL in 10 mM citrate buffer, pH 5.2), 100mL BP (10

mg/mL in 10 mM citrate buffer, pH 5.2, containing 50%isopropyl alcohol), 100mL 99mTcO4

2 (370 MBq/mL insaline) and 25mL SnCl2 z 2H2O (1.0 mg/mL in 0.1 N HCl).The reaction mixture was heated at 95°C for 15 min, fol-lowed by cooling on ice for 10 min. Analysis of the reactionproducts was performed by RP-HPLC (solvent system 1).

2.6. Preparation of [99mTc](HYNIC-Fab)(tricine)(BP) and[99mTc](HYNIC-NGA)(tricine)(BP)

To 100mL of Fab solution (5.0 mg/mL in 1 M carbonatebuffer, pH 10.0) was added an equal volume of[99mTc](HYNIC-TFP)(tricine)(BP) prepared as describedabove, and the reaction mixture was incubated for 1 h atroom temperature.99mTc-HYNIC-conjugates were then pu-rified by the centrifuged column procedure using a Seph-adex G-50 column equilibrated and eluted with 0.1 M phos-phate buffer (pH 7.4). [99mTc](HYNIC-NGA)(tricine)(BP)was prepared according to the procedure described aboveusing 100 mL of NGA solution (5.0 mg/mL in 0.1 Mcarbonate buffer, pH 9.0) in place of the Fab. Radiochem-ical yields were assessed by SE-HPLC, CAE and TLCdeveloped with saline. The levels of HYNIC moleculesconjugated per molecule of NGA and Fab were determinedby measuring the hydrazino groups withp-nitrobenzalde-hyde according to the method of King et al. [13].[99mTc](HYNIC-NGA)(tricine)2 and [99mTc](HYNIC-Fab)(tricine)2 were prepared by conjugation of 6-hydrazi-nopyridine-3-carboxylate hydrochloride with the respectiveprotein, followed by 99mTc radiolabeling with [99mTc](tricine)2, according to the procedure described previously[19,20].

2.7. Synthesis of [99mTc](HYNIC-lysine)(tricine)2 and[99mTc](HYNIC-lysine)(tricine)(BP)

[99mTc](HYNIC-lysine)(tricine)2 was synthesized ac-cording to the procedure described previously [20].[99mTc](HYNIC-lysine)(tricine)(BP) was synthesized asfollows: To 30 mL HYNIC-lysine (3 mg/mL in 10 mMcitrate buffer, pH 5.2), was added successively 200mLtricine (30 mg/mL in 10 mM citrate buffer, pH 5.2), 100mLBP (10 mg/mL in 10 mM citrate buffer (pH 5.2) containing50% isopropyl alcohol), 100mL 99mTcO4

2 (370 MBq/mL insaline) and 25mL SnCl2 z 2H2O (1.0 mg/mL in 0.1 N HCl).The reaction mixture was heated at 95°C for 15 min. Aftercooling at room temperature for 10 min, the reaction mix-ture was analyzed by RP-HPLC (solvent system 2) and TLCdeveloped with a mixture of 10% aqueous ammonium chlo-ride-methanol (1:1).

2.8. Plasma stability of [99mTc](HYNIC-Fab)(tricine)2 and[99mTc](HYNIC-Fab)(tricine)(BP)

[99mTc](HYNIC-Fab)(tricine)2 and [99mTc](HYNIC-Fab)(tricine)(BP) were diluted 20-fold with freshly prepared

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murine plasma, and the solutions were incubated at 37°C for24 h. After 1, 6 and 24 h incubation, 50mL aliquots of thesamples were drawn, and the radioactivity was analyzed bySE-HPLC.

2.9. In vivo studies

Animal studies were conducted in accordance with ourinstitutional guidelines and were approved by Kyoto Uni-versity Animal Care Committee. Biodistribution studieswere performed by intravenous administration of[99mTc](HYNIC-Fab) conjugates or [99mTc](HYNIC-NGA)conjugates to 6-week-old male ddY mice (27–30 g) [12].The protein concentrations of [99mTc](HYNIC-Fab) conju-gates and [99mTc](HYNIC-NGA) conjugates were adjustedto 200 mg/mL and 90mg/mL, respectively, with saline.Groups of five mice each were administered 20mg (37–74KBq) of [99mTc](HYNIC-Fab) conjugates or 9mg (37–74KBq) of [99mTc](HYNIC-NGA) conjugates prior to sacri-ficing the animals at 10 and 30 min, 1, 3, 6 and 24 hpostinjection by decapitation. Tissues of interest were re-moved, weighed and the radioactivity counts were deter-mined with an auto well gamma counter (ARC 2000). Todetermine the amounts and routes of excretion of radioac-tivity from the body after administration of [99mTc](HYNIC-Fab) conjugates or [99mTc](HYNIC-NGA) conju-gates, mice were housed in metabolic cages for 24 h, andurine and feces samples were collected and the radioactivitywas determined. Radioactivity profiles in the blood afteradministration of both [99mTc](HYNIC-Fab) conjugateswere fitted to a two-compartment model using a nonlinearleast-squares program [26], and the elimination half-lives (bphase) of the two Fab conjugates were calculated. Data inthe biodistribution studies were analyzed using the unpairedt-test. Differences were considered statistically significantwhen thep value was less than 0.05.

The radiolabeled species excreted in the urine for 6 hpostinjection of [99mTc](HYNIC-Fab)(tricine)(BP) were an-alyzed by TLC developed with a mixture of 10% aqueousammonium and methanol (1:1) before and after filtrationthrough a 10 kDa cutoff filtration membrane (Microcon 10;Amicon Grace, Tokyo, Japan). The urine samples were alsoanalyzed by SE-HPLC and RP-HPLC (solvent system 2)after filtration through a polycarbonate membrane with apore diameter of 0.45mm (Cosmonice filter, NacalaiTesque) and a 10 kDa cutoff filtration membrane, respec-tively.

The subcellular distribution of radioactivity in the mu-rine liver was investigated by perfusing the organ in situwith cold 0.25 M sucrose buffered with 10 mM phosphatebuffer (pH 7.4) at 1 and 6 h postinjection of [99mTc](HYNIC-NGA)(tricine)(BP) (133–266 KBq). The liver wastreated according to the procedure described previously[3,4,20]. Briefly, the isolated organ was minced with scis-sors, suspended in 4 volumes of the same buffer prior tohomogenization by hand with a Dounce homogenizer (20

strokes). This was followed by two final strokes in anice-cooled Potter-Elvehjem homogenizer with a Teflon pes-tle rotated at 800 rpm. The resulting homogenate was cen-trifuged twice for 5 min at 340g at 4°C. The isolatedsupernatant was then layered on top of iso-osmotic (0.25 Msucrose) 37.5% Percoll (9 mL: Pharmacia Biotech Co. Ltd.)at a density of 1.08 g/mL. After centrifugation at 20,000g(RP 30 rotor; Hitachi Co. Ltd., Tokyo) for 90 min at 4°C,the gradients were collected in 14 fractions.b-Galactosidasewas used as a marker enzyme for lysosomes [2,6] and itsactivity in each fraction was determined usingp-nitrophenylb-galactopyranoside as the substrate [8]. The density andradioactivity counts of the respective fractions were alsodetermined.

The radiolabeled species remaining in the liver at 1 and6 h postinjection of [99mTc](HYNIC-NGA) conjugates(133–266 KBq) were analyzed according to the proceduredescribed previously [3,4,20]. Briefly, the murine liver wasperfused in situ with cold 0.1 M Tris-citrate buffer (pH 6.5)containing 0.15 M NaCl, 0.02% sodium azide, 1 TIU/mLaprotinin, 2 mM benzamidine-HCl, 2 mM iodoacetamide, 1mM phenylmethylsulfonyl fluoride, and 5 mM disopropylfluorophosphate before isolation of the hepatic samples (1 geach). Each tissue sample was placed in a test tube andsubjected to three cycles of freezing (dry ice-acetone bath)and thawing. After addition of 5 volumes of the same buffercontaining an additional 35 mMb-octyl-glucoside, the sam-ples were homogenized with a Polytron homogenizer (PT10–35, Kinematica GmbH, Littau, Switzerland) at fullspeed with three consecutive 30-sec bursts prior to centrif-ugation at 48,000g for 20 min at 4°C (Himac CS-120Centrifuge; Hitachi Co. Ltd., Tokyo, Japan). The superna-tant was separated from the pellet, and the radioactivity wascounted. The liver homogenate samples were analyzed im-mediately by GPC, CAE and TLC developed with a mixtureof 10% aqueous ammonium chloride-methanol (1:1) afterfiltration through a polycarbonate membrane with a porediameter of 0.45mm (Nacalai Tesque). Each sample wasalso analyzed by RP-HPLC (solvent system 2) after filtra-tion with a 10 kDa cutoff membrane (Microcon-10).

3. Results

3.1. Preparation of ternary ligand99mTc-HYNIC-conjugated polypeptides

The ternary ligand99mTc-HYNIC-conjugated polypep-tides were prepared by conjugation of the TFP active esterof the ternary ligand [99mTc](HYNIC-TFP) complex withthee-amine residues of the polypeptides, as outlined in Fig.1. The synthesis of [99mTc](HYNIC-TFP)(tricine)(BP) wasaccomplished in a one-pot reaction of HYNIC-TFP with[99mTc]pertechnetate, tricine and BP in the presence of Sn21

ions to produce 70–90% radiochemical yields.

218 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 215–224

The RP-HPLC radiochromatogram of [99mTc](HYNIC-TFP)(tricine)(BP) is shown in Fig. 2. The ternary99mTccomplex was eluted at a retention time of 23.9 min, whilethe carboxylic acid of the99mTc-HYNIC ternary complexwas eluted at a retention time of 14.1 min as confirmed bycontrol studies using 6-hydrazinopyridine-3-carboxylicacid. The conjugation reaction of the TFP ester of the99mTc-HYNIC ternary ligand complex with polypeptideswas performed at room temperature for 1 h with radiochem-

ical yields of 30–40%. After purification by the centrifugedcolumn procedure, [99mTc](HYNIC-Fab)(tricine)(BP) and[99mTc](HYNIC-NGA)(tricine)(BP) were obtained with ra-diochemical purities of over 95% as determined by SE-HPLC, TLC and CAE analyses. After preparation of99mTc-labeled NGAs and Fab fragments in the preformed chelatedapproach, the average number of HYNIC molecules conju-gated per molecule of NGA and Fab were 1.7 and 0.6,respectively, as determined by measuring the hydrazinegroups.

3.2. In vitro stability

Fig. 3 shows SE-HPLC radiochromatograms of[99mTc](HYNIC-Fab)(tricine)(BP) and [99mTc](HYNIC-Fab)(tricine)2 before and after incubation in murine plasmaat 37°C. [99mTc](HYNIC-Fab)(tricine)2 displayed a gradualdecrease in radioactivity peaks of the intact Fab and anincrease in the radioactivity peaks at earlier retention timesat 12 min (over 670 kDa) and 17–18 min (approximately70–100 kDa). After 6 and 24 h of incubation, the radioac-tivity in higher molecular weight fractions (retention time of12 min) represented 18.9% and 20.2% of the total radioac-tivity, respectively. On the other hand, [99mTc](HYNIC-Fab)(tricine)(BP) significantly reduced the radioactivitypeak at the earlier retention time up to 24 h of incubation.After 24 h incubation, 6.4% of the total radioactivity wasobserved in a higher molecular weight fraction (retentiontime of 12 min).

3.3. Biodistribution studies

The biodistribution of radioactivity after intravenous ad-ministration of [99mTc](HYNIC-Fab) conjugates is summa-rized in Table 1. [99mTc](HYNIC-Fab)(tricine)(BP) showedradioactivity elimination rates from the blood significantlyfaster than those of [99mTc](HYNIC-Fab)(tricine)2. Theelimination half-lives of b phase from the blood of[99mTc](HYNIC-Fab)(tricine)(BP) and [99mTc](HYNIC-Fab)(tricine)2 were 2.5 and 8.8 h, respectively.[99mTc](HYNIC-Fab)(tricine)(BP) also showed faster elim-ination rates of the radioactivity from the kidney with a peakradioactivity level observed at 1 h postinjection. However,[99mTc](HYNIC-Fab)(tricine)2 displayed long residencetime of the radioactivity in the kidney with 37% ID/g stillobserved at 24 h postinjection. [99mTc](HYNIC-Fab)(tricine)(BP) also demonstrated significantly higher ra-dioactivity levels excreted from the body for 24 h postin-jection when compared with [99mTc](HYNIC-Fab)(tricine)2.

Fig. 4 shows the results of analyses of urine samplesobtained by 24 h postinjection of [99mTc](HYNIC-Fab)(tricine)(BP). SE-HPLC showed two radioactivitypeaks at retention times similar to those of intact[99mTc](HYNIC-Fab)(tricine)(BP) and low molecularweight compounds such as [99mTc](HYNIC-lysine)

Fig. 1. 99mTc labeling of polypeptides using a preformed HYNIC-TFPactive ester.

Fig. 2. Radiochromatogram of [99mTc](HYNIC-TFP)(tricine)(BP) by RP-HPLC using solvent system 1.

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(tricine)(BP). RP-HPLC and TLC analyses showed that theradioactivity in the low molecular weight fraction had aretention time and a Rf value similar to those of[99mTc](HYNIC-lysine)(tricine)(BP).

The biodistribution of radioactivity after intravenous ad-ministration of [99mTc](HYNIC-NGA) conjugates is sum-marized in Table 2. At 10 min postinjection, over 90% ofthe injected radioactivity was accumulated in the liver forboth [99mTc](HYNIC-NGA) conjugates. After this postin-

jection interval, the radioactivity was gradually eliminatedfrom the liver by hepatobiliary excretion as the major ex-cretion route for both [99mTc](HYNIC-NGA) conjugates.However, significant differences were observed in elimina-tion of the radioactivity from the liver. [99mTc](HYNIC-NGA)(tricine)(BP) demonstrated significantly faster elimi-nation rates of the radioactivity from the liver. This wasreflected in the higher radioactivity levels excreted from thebody for 24 h postinjection of [99mTc](HYNIC-NGA)(tricine)(BP) when compared with [99mTc](HYNIC-NGA)(tricine)2. The higher urinary excretion of the radioactivityafter injection of [99mTc](HYNIC-NGA)(tricine)(BP) wasreflected in the higher radioactivity levels in the kidney atearlier postinjection time points (30 min to 1 h). The longerresidence times of the radioactivity in the liver following

Fig. 3. SE-HPLC profiles of radioactivity in murine plasma after incubationat 37°C of [99mTc](HYNIC-Fab)(tricine)(BP) (right panels) and[99mTc](HYNIC-Fab)(tricine)2 (left panels). Under these conditions, thy-roglobulin (670 kDa), glutamate dehydrogenase (290 kDa), IgG (150 kDa),bovine serum albumin (68 kDa), Fab (50 kDa) and cytochrome C (13 kDa)had retention times of 12, 14.5, 16, 18, 19 and 21 min, respectively, onSE-HPLC.

Table 1Biodistributions of radioactivity after intravenous administration of[99mTc](HYNIC-Fab)(tricine)(BP) and [99mTc](HYNIC-Fab)(tricine)2 inmicea

Tissue Time after injection

10 min 30 min 1 h 3 h 6 h 24 h

[99mTc](HYNIC-Fab)(tricine)(BP)Blood 21.06c 11.59c 7.36c 3.26c 1.80c 0.35c

(1.32) (1.75) (0.40) (0.45) (0.11) (0.07)Liver 7.13 6.30 6.25c 6.10 4.85 2.71

(0.23) (0.69) (0.61) (0.97) (0.70) (0.35)Kidney 32.61c 52.98c 55.22 36.88c 22.05c 7.13c

(2.87) (5.60) (5.76) (2.45) (1.62) (1.14)Intestine 1.05 1.22 1.59 1.96 2.92 0.37

(0.09) (0.13) (0.29) (0.23) (0.71) (0.08)Spleen 4.00 3.26 3.26 2.81 2.57 1.71

(0.50) (0.94) (0.80) (0.59) (0.58) (0.07)Stomachb 0.34 0.43 0.61 0.39 0.47 0.20

(0.06) (0.04) (0.12) (0.16) (0.30) (0.14)Urineb 52.90c

(3.26)Fecesb 11.26c

(2.00)[99mTc](HYNIC-Fab)(tricine)2

Blood 25.23 18.38 11.53 5.06 2.82 0.98(1.49) (1.06) (1.10) (0.31) (0.20) (0.07)

Liver 4.61 4.59 4.25 4.46 4.96 4.55(0.34) (0.21) (0.15) (0.38) (0.64) (0.49)

Kidney 22.80 42.05 52.57 65.97 65.13 37.01(2.07) (5.04) (3.36) (6.56) (3.59) (2.02)

Intestine 1.24 1.71 1.69 1.41 1.39 1.36(0.15) (0.21) (0.28) (0.10) (0.06) (0.14)

Spleen 3.80 3.42 2.49 1.86 1.63 1.90(0.79) (0.42) (0.16) (0.21) (0.09) (0.41)

Stomachb 0.56 0.61 1.31 0.55 0.50 0.65(0.13) (0.18) (0.53) (0.16) (0.30) (0.20)

Urineb 37.89(3.91)

Fecesb 5.97(0.63)

a Expressed as % injected dose per gram. Each value represents the mean(s.d.) for five animals at each interval.

b Expressed as % injected dose.c Significance determined by unpairedt-test;p , 0.05.

220 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 215–224

injection of [99mTc](HYNIC-NGA)(tricine)2 were reflectedin the lower radioactivity levels in the intestine.

3.4. Analyses of radioactivity in the liver

The Percoll density gradient centrifugation profiles ofradioactivity in the liver at 1 and 6 h postinjection of[99mTc](HYNIC-NGA)(tricine)(BP) are shown in Fig. 5.Each liver homogenate showed a major radioactivity peak ata density of ca. 1.10 g/mL, which was co-purified with highb-galactosidase activity fractions.

The supernatants of the liver homogenates were obtainedwith radiochemical efficiencies of over 90% at all postin-jection time points following administration of99mTc-la-beled NGAs. Fig. 6 shows GPC and RP-HPLC radiochro-matograms of the liver supernatants at 1 and 6 hpostinjection of [99mTc](HYNIC-NGA)(tricine)2. The liversupernatants showed a time-dependent increase in the ra-dioactivity peak at smaller elution volumes on GPC. OnRP-HPLC, a broad radioactivity peak with a retention timeof around 9 min was observed. At 6 h postinjection, mul-tiple radioactive peaks were observed with retention timesbetween 9 and 16 min.

Fig. 7 shows the radiochromatograms of liver superna-tants after injection of [99mTc](HYNIC-NGA)(tricine)(BP).

On GPC, a major radioactivity peak was observed at anelution volume (82.5 mL) similar to that of [99mTc](HYNIC-lysine)(tricine)(BP). Under these conditions,[99mTc](HYNIC-NGA)(tricine)(BP) and [99mTc](HYNIC-lysine)(tricine)(BP) were eluted at elution volumes of 32.5and 82.5 mL, respectively. On RP-HPLC, a major radioac-tivity peak was observed at a retention time similar to thatof [99mTc](HYNIC-lysine)(tricine)(BP) at both postinjec-tion intervals. More than 90% of injected radioactivity wasrecovered from the column in both analyses. The liverhomogenates at both postinjection intervals also showed asingle radioactivity peak on TLC (Rf5 0.6–0.65) and onCAE (2.0–2.5 cm from the anode) at positions similar tothose of [99mTc](HYNIC-lysine)(tricine)(BP) (data notshown).

Fig. 4. Radiochromatograms of urine samples obtained for 6 h postinjec-tion of [99mTc](HYNIC-Fab)(tricine)(BP) by SE-HPLC (A), TLC (B andD) and RP-HPLC (C). Urine samples were analyzed directly (B), afterfiltration through a polycarbonate membrane with a pore diameter of 0.45mm (A) and after filtration through a 10 kDa cutoff membrane (C and D).

Table 2Biodistributions of radioactivity after intravenous administration of[99mTc](HYNIC-NGA)(tricine)(BP) and [99mTc](HYNIC-NGA)(tricine)2in micea

Tissue Time after injection

10 min 30 min 1 h 3 h 6 h 24 h

[99mTc](HYNIC-NGA)(tricine)(BP)Bloodb 0.46 0.58 0.37 0.20 0.17 0.08

(0.04) (0.06) (0.04) (0.02) (0.03) (0.03)Liver 90.34 59.30c 43.17c 30.27c 18.66c 12.55c

(1.70) (4.10) (1.23) (3.58) (2.59) (1.29)Kidney 0.48 1.71 2.01 0.83 0.38 0.21

(0.16) (0.78) (0.31) (0.23) (0.07) (0.04)Intestine 2.63c 20.29c 35.30c 45.06c 35.62c 1.86c

(0.29) (3.12) (1.35) (4.52) (7.04) (1.00)Spleen 0.82 0.68 0.56 0.50 0.47 0.41

(0.09) (0.21) (0.16) (0.16) (0.15) (0.07)Stomach 0.36 0.28 0.64 0.31 0.58 0.16

(0.13) (0.24) (0.62) (0.11) (0.19) (0.15)Urine 19.88c

(1.21)Feces 51.47c

(1.02)[99mTc](HYNIC-NGA)(tricine)2

Bloodb 0.18 0.18 0.15 0.10 0.06 0.03(0.12) (0.01) (0.02) (0.05) (0.03) (0.01)

Liver 91.50 89.61 85.14 75.11 62.52 37.07(3.19) (1.73) (2.34) (3.46) (5.68) (3.39)

Kidney 0.25 0.34 0.38 0.39 0.47 0.41(0.07) (0.03) (0.06) (0.12) (0.13) (0.09)

Intestine 0.47 4.40 7.56 14.09 19.01 19.28(0.27) (0.67) (0.75) (1.13) (3.66) (6.27)

Spleen 0.05 0.08 0.08 0.06 0.05 0.01(0.04) (0.01) (0.01) (0.05) (0.03) (0.01)

Stomach 0.31 0.51 0.63 0.55 0.88 0.79(0.12) (0.22) (0.25) (0.22) (0.62) (0.26)

Urine 13.42(3.33)

Feces 14.51(3.79)

a Expressed as % injected dose. Each value represents the mean (s.d.) forfive animals at each interval.

b Expressed as % injected dose per gram.c Significance determined by unpairedt-test;p , 0.05.

221M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 215–224

4. Discussion

The procedure for synthesis of ternary complexes of99mTc-HYNIC has been well established by recent studies[7,16,17]. These studies also demonstrated high in vivostability of some of the ternary ligand complexes. Ourprevious study implied that progression of exchange reac-tion between one tricine molecule in [99mTc](HYNIC-polypeptide)(tricine)2 with proteins in plasma would beresponsible for the delayed elimination rates of the radio-activity from the blood [19]. A previous study also sug-gested that the long residence times of the radioactivity inthe liver after administration of [99mTc](HYNIC-NGA)(tricine)2 were caused by exchange reaction of[99mTc](HYNIC-lysine)(tricine)2 with proteins in the lyso-somes [20]. Based on these findings, a TFP active ester ofthe [99mTc](HYNIC)(tricine)(BP) ternary ligand complexwas designed and synthesized to examine whether stabili-zation of99mTc-HYNIC chelate against exchange reactionswould accelerate elimination rates of the radioactivity notonly from the blood but also from nontarget tissues afteradministration of99mTc-HYNIC-conjugated polypeptides.

Since pyridine derivatives may serve as coligands to pro-vide 99mTc-HYNIC ternary ligand complexes with tricine[17], 3-benzoylpyridine was selected as the coligand togenerate lipophilic radiometabolites to facilitate eliminationfrom the lysosomes.

The TFP active ester of99mTc ternary complex wasobtained with radiochemical yields sufficient for the subse-quent conjugation reactions with polypeptides due to thehigh stability of the TFP ester under harsh conditions (Fig.2). 99mTc radiolabeling at low pH (pH 2.5–3) resulted innegligible amounts of intermolecular coupling of an activeester with a hydrazine group due to protonation of thehydrazine group at the reaction pH. Since the level of thehydrolysis product of the active ester was low and it was

Fig. 5. Percoll density gradient centrifugation profiles of liver homogenateat 1 and 6 h postinjection of [99mTc](HYNIC-NGA)(tricine)(BP). In bothpreparations, a single radioactivity peak (v) that coincided withb-galac-tosidase activity (Œ) was detected at a density (f) of ca. 1.10 g/mL.

Fig. 6. Analyses of liver homogenates at 1 and 6 h postinjection of[99mTc](HYNIC-NGA)(tricine)2 by GPC (B and C) and RP-HPLC (E andF) using solvent system 2. Radiochromatograms of [99mTc](HYNIC-lysine)(tricine)2 when analyzed by GPC (A) and RP-HPLC (D). On GPC,blue dextran eluted at 32.5 mL. Liver homogenates were analyzed by GPCafter filtration through a polycarbonate membrane with a pore diameter of0.45 mm and by RP-HPLC after filtration through a 10 kDa cutoff mem-brane, respectively.

222 M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 215–224

easily removed by the centrifuged column procedure, theresulting99mTc-HYNIC-conjugated polypeptides were ob-tained with high radiochemical purity and negligibleamounts of nonspecifically bound99mTc species werepresent in the preparations. This allowed reliable estimationof the 99mTc-labeled polypeptides designed in the presentstudy for subsequent in vitro and in vivo studies.

Plasma incubation of [99mTc](HYNIC-Fab)(tricine)(BP)showed high stability of the ternary ligand99mTc complexagainst exchange reaction with plasma proteins, as demon-strated by the significantly lower amounts of higher molec-ular weight radiolabeled species of [99mTc](HYNIC-Fab)(tricine)(BP) on SE-HPLC as compared with[99mTc](HYNIC-Fab)(tricine)2 controls (Fig. 3).

The progression of exchange reaction of99mTc-HYNIC-

derivatized radiometabolites with proteins in the lysosomeswas estimated after administration of the two99mTc-HYNIC-NGA conjugates into mice as described previously[20]. Analyses of liver homogenates after injection of[99mTc](HYNIC-NGA)(tricine)(BP) showed a single radio-activity peak at a position similar to that of[99mTc](HYNIC-lysine)(tricine)(BP) on both GPC and RP-HPLC (Fig. 7), whereas radiometabolites derived from[99mTc](HYNIC-NGA)(tricine)2 showed a time-dependentincrease in protein-bound (higher molecular weight) frac-tions (Fig. 6). These findings indicated that the[99mTc](HYNIC)(tricine)(BP) ternary ligand complex re-mained stable against exchange reaction with proteins in thelysosomes even after the parental polypeptide (NGA) wascatabolized to monoamino acid (lysine). This provided goodevidence for high stability of the99mTc ternary ligand com-plex, since [99mTc](HYNIC-polypeptide)(tricine)2 exhibitedprotein binding in a manner related to the size of the pa-rental polypeptide [19].

The high stability of the99mTc ternary ligand complexagainst exchange reactions with proteins in plasma or lyso-somes was well reflected in the radioactivity distributionafter administration of99mTc-HYNIC-conjugated NGA andFab fragments in mice. As shown in Table 2,[99mTc](HYNIC-NGA)(tricine)(BP) exhibited significantlyfaster elimination of radioactivity from the liver than[99mTc](HYNIC-NGA)(tricine)2. This was also reflected inhigher amounts of radioactivity excreted from the body by24 h postinjection of [99mTc](HYNIC-NGA)(tricine)(BP).Since the radioactivity was present in the lysosomal com-partment as radiometabolites 1 h after injection of[99mTc](HYNIC-NGA)(tricine)(BP) (Fig. 5) and [99mTc](HYNIC-NGA)(tricine)2 [20], the faster elimination rates ofthe radioactivity indicated faster elimination of [99mTc](HYNIC-lysine)(tricine)(BP) from the lysosomal compart-ment of the liver. This suggested that inert characteristics ofthe 99mTc-HYNIC ternary ligand complex against proteinbinding would have accelerated the elimination rates fromthe organelle, although the lipophilic property of [99mTc](HYNIC-lysine)(tricine)(BP) might also have contributed tothe faster elimination from the liver, as previously observedwith 111In-labeled NGA [3].

The high degree of stability of [99mTc](HYNIC)(tricine)(BP) ternary ligand complex against exchange re-action with proteins in plasma was reflected by the fasterelimination rates of the radioactivity after administration of[99mTc](HYNIC-Fab)(tricine)(BP) from the blood (Table1). This was reflected in higher renal radioactivity levels atearlier postinjection times of [99mTc](HYNIC-Fab)(tricine)(BP). Generation of [99mTc](HYNIC-lysine)(tricine)(BP) asthe major radiometabolite in the kidney after administrationof [99mTc](HYNIC-Fab)(tricine)(BP) (Fig. 4) would be re-sponsible for the faster elimination rates of the radioactivityfrom the kidney and excretion of greater amounts of radio-activity from the body, as discussed above. Thus,99mTc-labeled Fab fragments with inert [99mTc](HYNIC)(tricine)

Fig. 7. Analyses of liver homogenates at 1 and 6 h postinjection of[99mTc](HYNIC-NGA)(tricine)(BP) by GPC (B and C) and RP-HPLC (Eand F) using solvent system 2. Radiochromatograms of [99mTc](HYNIC-lysine)(tricine)(BP) when analyzed by GPC (A) and RP-HPLC (D). Liverhomogenates were analyzed by GPC after filtration through a polycarbon-ate membrane with a pore diameter of 0.45mm and by RP-HPLC afterfiltration through a 10 kDa cutoff membrane, respectively.

223M. Ono et al. / Nuclear Medicine and Biology 28 (2001) 215–224

(BP) ternary complex significantly accelerated the elimina-tion rates of the radioactivity not only from the blood butalso from the kidney. Such characteristics would render theTFP active ester of [99mTc](HYNIC)(tricine)(BP) complexuseful for radiolabeling polypeptides for targeted imaging.

In conclusion, the findings in this study reinforced ourprevious hypothesis that prolonged elimination rates of99mTc-HYNIC-conjugated polypeptides from the blood andnontarget tissues were attributable to the progression ofexchange reaction between [99mTc](HYNIC)(tricine)2 withproteins in the blood and lysosomes. The present study alsoindicated that conjugation of inert [99mTc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides significantlyaccelerated the elimination rates of the radioactivity notonly from the blood but also from nontarget tissues such asthe liver or kidney. Although further studies are required,the TFP active ester of inert [99mTc](HYNIC)(tricine)(BP)may constitute an alternative to prepare99mTc-labeled poly-peptides for targeted imaging.

Acknowledgments

This study was supported in part by a Grant-in-Aid forScientific Research from the Ministry of Education, Sci-ence, Sports and Culture of Japan.

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