In Vitro System To Estimate Renal Brush Border Enzyme-Mediated

Kyoto 606-8501, Graduate School of Medicine, Gunma University, 3-39-22 Showa-machi, Maebashi 371-8511, and Graduate School of Pharmaceutical ...
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Bioconjugate Chem. 2005, 16, 1610−1616

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In Vitro System To Estimate Renal Brush Border Enzyme-Mediated Cleavage of Peptide Linkages for Designing Radiolabeled Antibody Fragments of Low Renal Radioactivity Levels Yasushi Fujioka,† Satoshi Satake,‡ Tomoya Uehara,‡ Takahiro Mukai,† Hiromichi Akizawa,‡ Kazuma Ogawa,† Hideo Saji,† Keigo Endo,§ and Yasushi Arano‡,* Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Graduate School of Medicine, Gunma University, 3-39-22 Showa-machi, Maebashi 371-8511, and Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Received July 20, 2005; Revised Manuscript Received September 5, 2005

Renal localization of radiolabeled antibody fragments presents a problem in targeted imaging and radiotherapy. We recently reported that Fab fragments labeled with 3′-[131I]iodohippuryl N-maleoylL-lysine (HML) demonstrated markedly low renal radioactivity levels from early postinjection in mice. Previous studies suggested that low renal radioactivity levels were attributable to cleavage of the glycyl-lysine sequence in HML by the action of renal brush border enzymes, followed by urinary excretion of the resulting m-iodohippuric acid. In this study, an in vitro system using brush border membrane vesicles (BBMVs) isolated from the rat kidney cortex was developed to estimate renal brush border enzyme(s)-mediated cleavage of the peptide linkage. Low molecular weight HML derivatives, 3′-[125I]iodohippuryl L-lysine (HL), 3′-[125I]iodohippuryl N-tert-butoxycarbonyl-L-lysine (HBL), and their D-amino acid counterparts, were synthesized and incubated in BBMVs. Both [125I]HL and [125I]HBL generated m-[125I]iodohippuric acid after incubation in BBMVs at 37 °C while the latter liberated significantly higher amounts of the metabolite. [125I]D-HL and [125I]D-HBL failed to release the metabolite under similar conditions. The liberation of m-[125I]iodohippric acid from [125I]HL was significantly facilitated or completely inhibited by the addition of an activator or an inhibitor for carboxypeptidase M. The release of m-[125I]iodohippuric acid from [125I]HBL increased by the addition of the activator, whereas the inhibitor partially inhibited the release of the metabolite from [125I]HBL. The BBMV-mediated release of m-[125I]iodohippuric acid from [125I]HBL was not impaired by the addition of inhibitors for neutral endopeptidase or renal dipeptidase. These findings showed that the glycyl-L-lysine sequence in HML would be recognized and cleaved by metalloenzymes and nonmetalloenzymes on the renal brush border even when iodine was incorporated into a benzene ring and the N-amine residue of lysine was chemically modified, which supported the hypothesis that low renal radioactivity levels of HML-conjugated Fab fragments would be attributed to the release of m-iodohippuric acid by renal brush border enzymes. This study suggested that this in vitro system using BBMVs would be useful to estimate radiolabeling reagents of antibody fragments or peptides designed to reduce renal radioactivity with a variety of radionuclides.

INTRODUCTION

Recent clinical studies of 90Y- and 131I-labeled intact antibodies against CD20 demonstrated significant response rates in patients with chemotherapy-refractory non-Hodgkin’s lymphoma (1). Although the 90Y-labeled antibodies caused myelosuppresion as a result of long circulation times, this could be reduced by using antibody fragments in place of intact antibodies, due to the faster clearance of antibody fragments from the systemic circulation. The uniform intratumor distribution of antibody fragments would also be advantageous to increase the therapeutic efficacy (2). However, radiolabeled antibody fragments showed high and persistent localization of radioactivity in the kidneys from early postinjection onward, which causes therapeutic limitations (3-6). * Corresponding author. Tel.: 81-43-226-2896; fax: 81-43226-2897; e-mail: [email protected]. † Kyoto University. ‡ Chiba University. § Gunma University.

Figure 1. Chemical structures of [125/131I]HML and [125/131I]HML-conjugated Fab fragments.

To circumvent this problem, we recently developed a radioiodination reagent, 3′-iodohippuryl N-maleoyl-Llysine (HML, Figure 1) based on the hypothesis that glycyl-lysine linkage in HML would be cleaved by brush border enzyme(s) present on the lumen of the renal tubule while the antibody fragments are taken up by renal cells (7). In tumor-bearing nude mice, [131I]HMLconjugated Fab fragments (Figure 1) significantly reduced renal radioactivity levels from early postinjection onward without impairing radioactivity levels in the

10.1021/bc050211z CCC: $30.25 © 2005 American Chemical Society Published on Web 11/01/2005

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tumor (7, 8). A follow-up in vivo study showed that cleavage of the glycyl-lysine sequence in [125/131I]HMLconjugated Fab fragments occurred at the membrane fractions of renal cells from an early postinjection time (9). These in vivo studies supported the validity of this chemical design to prepare radiolabeled antibody fragments of low renal radioactivity levels. However, it remains uncertain whether a renal enzyme(s) on the brush border membrane was responsible for cleavage of the glycyl-lysine sequence in HML-conjugated antibody fragments. In addition, since the ability of HML to release m-iodohippuric acid was only estimated by biodistribution studies after conjugation with antibody fragments, an in vitro experimental system is highly warranted to estimate the release of m-iodohippuric acid prior to conjugation with antibody fragments. Such an experimental system would provide an insight into better understanding the mechanism of releasing m-iodohippuric acid from HML-conjugated antibody fragments. Such an in vitro system would also be useful to develop new radiolabeling reagents for antibody fragments or peptides with a variety of radionuclides. Renal brush border membrane vesicles (BBMVs) have been used to investigate the transport mechanisms of drugs in the renal brush border membrane (10, 11). BBMVs have a predominantly luminal side-out orientation with a variety of enzymes on their surface (10, 12). Furthermore, enzymes in renal lysosomes can be separated from those on BBMVs during preparation (12). Such characteristics rendered BBMVs attractive as an in vitro system to estimate the radiolabeling reagents for antibody fragments or peptides of low renal radioactivity levels utilizing enzymes on renal brush border membrane. In this study, 3′-[125I]iodohippuryl L-lysine ([125I]HL), 3′-[125I]iodohippuryl N-tert-butoxycarbonyl-L-lysine ([125I]HBL), and their D-lysine counterparts, 3′-[125I]iodohippuryl D-lysine ([125I]D-HL) and 3′-[125I]iodohippuryl Ntert-butoxycarbonyl-D-lysine ([125I]D-HBL) were synthesized as HML derivatives, and the brush border enzymemediatedreleaseofm-[125I]iodohippuricacidwasinvestigated using BBMVs prepared from the rat kidney cortex. The BBMV-mediated release of m-[125I]iodohippuric acid was also determined by the presence of an activator for carboxypeptidase M (CPM) or inhibitors of some enzymes on BBMVs. The findings in this study showed that the in vitro system using BBMVs would be useful to evaluate newly developed radiolabeling reagents designed to liberate radiometabolites by the action of renal brush border enzymes prior to conjugation to antibody fragments. MATERIALS AND METHODS

Reagents and Chemicals. Na[125I]I was purchased from Biomedicals, Inc. (California). Reversed phase highperformance liquid chromatography (RP-HPLC) was performed with a Cosmosil 5C18-AR-300 column (4.6 × 150 mm, Nacalai Tesque, Kyoto, Japan) eluted at a flow rate of 1 mL/min with a gradient mobile phase starting from 100% water to 100% acetonitrile in 30 min (solvent system 1) or from 80% A (0.1% aqueous trifluoroacetic acid; TFA) and 20% B (acetonitrile with 0.1% TFA) to 20% A and 80% B in 30 min (solvent system 2). Sizeexclusion high-performance liquid chromatography (SEHPLC) was performed with a Cosmosil Diol-120 column (7.5 × 600 mm, Nacalai Tesque, Kyoto) eluted with 0.1 M phosphate buffer (pH 6.8) at a flow rate of 1 mL/min. Each eluent was collected with a fraction collector (RediFrac; Pharmacia Biotech, Tokyo) at 30-s intervals, and

Scheme 1a

a Reagents: (a) 1-hydroxybenzotriazole, triethylamine, Ntert-butoxycarbonyl-L-lysine methyl ester hydrochloride, 1-ethyl3-(3-dimethylaminopropyl)-carbodiimide hydrochloride; (b) NaOH; (c) HCl; (d) N-chlorosuccinimide, Na[125I]I; (e) TFA.

the radioactivity levels in each fraction (500 µL) were determined with an auto well counter (ARC-2000; Aloka, Tokyo). TLC analyses were performed with silica plates (Merck Art 5553) developed with a mixture of chloroform, methanol, and water (15:8:1) (solvent system 3) or a mixture of chloroform, methanol, and acetic acid (50:10: 1) (solvent system 4). 1H NMR spectra were recorded on a Bruker AC-200 spectrometer, and chemical shifts are reported in ppm downfield from an internal tetramethylsilane standard. Mass spectra were obtained with the JMS-HX/HX110 A model (JEOL Ltd., Tokyo), API III model (Perkin-Elmer Sciex Instruments, Canada), or LCMS-QP8000R model (Shimadzu, Kyoto). Sn-HML was synthesized according to the procedure of Wakisaka et al. (13). DL-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), phosphoramidon, and tienam were purchased from Calbiochem (La Jolla, CA), Peptide Institute (Osaka, Japan) and Banyu Pharmaceutical Co., Ltd. (Tokyo), respectively. L-γ-Glutamyl-p-nitroanilde, L-leucine-p-nitroanilide, p-nitrophenyl-β-D-galactopyranoside, and glycyl-D-phenylalanine were obtained from Wako Pure Chemical Industries Ltd. (Osaka), ICN Pharmaceutical Inc. (Costa Mesa, CA), Nacalai Tesque, and Bachem AG (Bubendorf, Switzerland), respectively. Other reagents were of reagent grade. Synthesis of 3′-(Tri-n-butylstannyl)hippuryl NEtert-Butoxycarbonyl-L-lysine (Sn-HBL). 3-(Tri-n-butylstannyl)hippuric acid was synthesized as described previously (13). 3-(Tri-n-butylstannyl)hippuric acid (300 mg, 0.64 mmol), 1-hydroxybenzotriazole (118 mg, 0.77 mmol), triethylamine (143 mg, 1.41 mmol), and N-tertbutoxycarbonyl-L-lysine methyl ester hydrochloride (190 mg, 0.64 mmol) were dissolved in dry chloroform (10 mL) (Scheme 1). After the mixture was cooled to 0 °C, a solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (147 mg, 0.77 mmol) in 3 mL of dry chloroform was added to the solution. After being stirred for 12 h at 0 °C, the reaction mixture was washed with

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5% aqueous citrate (10 mL × 3), saturated NaHCO3 (10 mL × 3), and saturated NaCl (10 mL × 3) successively before drying over anhydrous calcium sulfate. The organic layer was removed in vacuo to produce 3′-(tri-nbutylstannyl)hippuryl N-tert-butoxycarbonyl-L-lysine methyl ester (250 mg, 55%) as a colorless oil. 1H NMR (CDCl3): δ 0.85-1.94 (33H, m, SnBu3, (CH2)3), 1.42 (9H, s, Boc), 3.10 (2H, q, NHCH2), 3.74 (3H, s, COOCH3), 4.19 (2H, d, NHCH2), 4.62 (1H, q, NHCH), 7.39 (1H, t, aromatic), 7.60 (1H, d, aromatic), 7.70 (1H, d, aromatic), 7.90 (1H, t, aromatic). Electro spray ionization mass spectra (ESI-MS) calcd for C33H58N3O6Sn (MH+): m/z 712. Found: 712. 3′-(Tri-n-butylstannyl)hippuryl N-tert-butoxycarbonylL-lysine methyl ester (250 mg, 0.35 mmol) in methanol (10 mL) was added to a solution of 1 N NaOH (2 mL), and the mixed solution was refluxed with stirring for 1 h. After removal of methanol in vacuo, the solution was neutralized with 1 N HCl at 0 °C before extraction with ethyl acetate (10 mL). The organic layer was dried over anhydrous calcium sulfate before removing the solvent in vacuo to produce 3′-(tri-n-butylstannyl)hippuryl Ntert-butoxycarbonyl-L-lysine (138 mg, 56%) as a colorless oil. 1H NMR (CDCl3): δ 0.85-1.82 (33H, m, SnBu3, (CH2)3), 1.42 (9H, s, Boc), 3.08 (2H, q, NHCH2), 4.25 (2H, d, NHCH2), 4.55 (1H, q, NHCH), 7.31 (1H, t, aromatic), 7.58 (1H, d, aromatic), 7.72 (1H, d, aromatic), 7.91 (1H, t, aromatic). Fast atom bombardment mass spectra (FABMS) calcd for C32H55N3O6Sn (MH+): m/z 698. Found: 698. Synthesis of 3′-(Tri-n-butylstannyl)hippuryl NEtert-Butoxycarbonyl-D-lysine (D-Sn-HBL). This compound was synthesized by the reaction of N-tertbutoxycarbonyl-D-lysine methyl ester hydrochloride with 3-(tri-n-butylstannyl)hippuric acid as described above. 1H NMR (CDCl3): δ 0.85-1.88 (33H, m, SnBu3, (CH2)3), 1.42 (9H, s, Boc), 3.01 (2H, q, NHCH2), 4.26 (2H, d, NHCH2), 4.50 (1H, q, NHCH), 7.32 (1H, t, aromatic), 7.57 (1H, d, aromatic), 7.66 (1H, d, aromatic), 7.85 (1H, t, aromatic). ESI-MS calcd for C32H55N3O6Sn (MH+): m/z 698. Found: 698. Synthesis of 3′-Iodohippuryl NE-tert-Butoxycarbonyl-L-lysine (HBL). 3-Iodohippuric acid was synthesized as described previously (13). A mixture of 3-iodohippuric acid (305 mg, 1.00 mmol), 1-hydroxybenzotriazole (184 mg, 1.20 mmol), triethylamine (223 mg, 2.20 mmol), and N-tert-butoxycarbonyl-L-lysine methyl ester hydrochloride (297 mg, 1.00 mmol) dissolved in dry acetonitrile (10 mL) was cooled to 0 °C. A solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (230 mg, 1.20 mmol) in 10 mL of acetonitrile was added, and the reaction mixture was stirred for 7 h at 0 °C. After removal of the solvent in vacuo, the residue was dissolved in ethyl acetate (10 mL) and the organic phase was washed with 5% aqueous citrate (10 mL × 3), saturated NaHCO3 (10 mL × 3), and saturated NaCl (10 mL × 3) successively. After the solvent was dried over anhydrous calcium sulfate, the organic layer was removed in vacuo to produce 3′-iodohippuryl N-tertbutoxycarbonyl-L-lysine methyl ester (443 mg, 81%) as a colorless oil. 1H NMR (CDCl3): δ 1.12-1.95 (6H, m, (CH2)3), 1.42 (9H, s, Boc), 3.10 (2H, q, NHCH2), 3.75 (3H, s, COOCH3), 4.19 (2H, d, NHCH2), 4.60 (1H, q, NHCH), 7.18 (1H, t, aromatic), 7.78 (1H, d, aromatic), 7.85 (1H, d, aromatic), 8.17 (1H, t, aromatic). FAB-MS calcd for C21H30N3O6I(MH+): m/z548.Found: 548.Anal.(C21H30N3O6I) C, H, N. 3′-Iodohippuryl N-tert-butoxycarbonyl-L-lysine methyl ester (425 mg, 0.78 mmol) was added to a mixture of 1 N NaOH (1.55 mL) and methanol (10 mL), and the solution

Fujioka et al.

was refluxed with stirring for 1 h. After removal of methanol in vacuo, the solution was neutralized with 1 N HCl at 0 °C before extraction with ethyl acetate (10 mL). The organic layer was dried over anhydrous calcium sulfate before removing the solvent in vacuo to produce 3′-iodohippuryl N-tert-butoxycarbonyl-L-lysine (290 mg, 70%) as a colorless oil. 1H NMR (CD3OD): δ 1.12-1.99 (6H, m, (CH2)3), 1.42 (9H, s, Boc), 3.02 (2H, q, NHCH2), 4.05 (2H, d, NHCH2), 4.41 (1H, q, NHCH), 7.25 (1H, t, aromatic), 7.84 (1H, d, aromatic), 7.89 (1H, d, aromatic), 8.22 (1H, t, aromatic). ESI-MS calcd for C20H28N3O6I (MH+): m/z 534. Found: 534.Anal.(C20H28N3O6I‚1/2H2O‚1/ 4CH3COOC2H5) C, H, N. Synthesis of 3′-Iodohippuryl NE-tert-Butoxycarbonyl-D-lysine (D-HBL). D-HBL was synthesized by the reaction of N-tert-butoxycarbonyl-D-lysine methyl ester hydrochloride with 3-iodohippuric acid as described above. 1H NMR (CD3OD): δ 0.88-1.98 (6H, m, (CH2)3), 1.42 (9H, s, Boc), 3.03 (2H, q, NHCH2), 4.07 (2H, d, NHCH2), 4.42 (1H, q, NHCH), 7.22 (1H, t, aromatic), 7.85 (1H, d, aromatic), 7.90 (1H, d, aromatic), 8.25 (1H, t, aromatic). ESI-MS calcd for C20H28N3O6I (MH+): m/z 534. Found: 534. Synthesis of 3′-Iodohippuryl L-Lysine (HL). 3Iodohippuric acid (450 mg, 1.48 mmol), 1-hydroxybenzotriazole (239 mg, 1.77 mmol), triethylamine (328 mg, 3.25 mmol), and N-tert-butoxycarbonyl-L-lysine tert-butyl ester hydrochloride (500 mg, 1.48 mmol) were dissolved in dry acetonitrile (10 mL). After the mixture was cooled to 0 °C, a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (339 mg, 1.77 mmol) in 15 mL of acetonitrile was added to the solution. After being stirred for 7 h at 0 °C, the solvent was removed in vacuo and the residue was dissolved in ethyl acetate (10 mL). The organic phase was washed with 5% aqueous citrate (10 mL × 3), saturated NaHCO3 (10 mL × 3), and saturated NaCl (10 mL × 3) successively. After the solvent was dried over anhydrous calcium sulfate, the organic layer was removed in vacuo to produce 3′iodohippuryl N-tert-butoxycarbonyl-L-lysine tert-butyl ester (679 mg, 78%) as a colorless oil. 1H NMR (CDCl3): δ 1.26-2.04 (6H, m, (CH2)3), 1.44 (9H, s, t-Bu), 1.47 (9H, s, Boc), 3.13 (2H, q, NHCH2), 4.17 (2H, d, NHCH2CO), 4.50 (1H, q, NHCH), 7.18 (1H, t, aromatic), 7.79 (1H, d, aromatic), 7.85 (1H, d, aromatic), 8.18 (1H, t, aromatic). Electron Impact mass spectra (EI-MS) calcd for C21H30N3O6I: m/z 589. Found: 589. Anal. (C24H36N3O6I) C, H, N. 3′-Iodohippuryl N-tert-butoxycarbonyl-L-lysine tertbutyl ester (180 mg, 0.31 mmol) was dissolved in TFA (15 mL) containing anisole (66 µL) and stirred for 1 h at room temperature. After removal of TFA in vacuo, the residue was treated with ether to precipitate 3′-iodohippuryl L-lysine (102 mg, 59%) as a white crystal. 1H NMR (DMSO-d6): δ 1.05-2.04 (6H, m, (CH2)3), 3.44 (2H, q, NHCH2), 4.04 (2H, d, NHCH2CO), 4.22 (1H, q, NHCH), 7.30 (1H, t, aromatic), 7.91 (1H, d, aromatic), 8.08 (1H, d, aromatic), 8.29 (1H, t, aromatic). EI-MS calcd for C15H20N3O4I: m/z 433. Found: 433. Anal. (C15H20N3O4I‚7/ 4CF3COOH‚1/2H2O‚1/2C2H5OC2H5) C, H, N. Synthesis of 3′-Iodohippuryl D-Lysine (D-HL). 3′Iodohippuryl D-lysine was synthesized by the reaction of N-tert-butoxycarbonyl-D-lysine tert-butyl ester hydrochloride with 3-iodohippuric acid as described above. 1H NMR (CD3OD): δ 1.05-2.02 (6H, m, (CH2)3), 3.28 (2H, q, NHCH2), 4.09 (2H, d, NHCH2CO), 4.49 (1H, q, NHCH), 7.22 (1H, t, aromatic), 7.85 (1H, d, aromatic), 7.91 (1H, d, aromatic), 8.23 (1H, t, aromatic). ESI-MS calcd for C15H21N3O4I (MH+): m/z 434. Found: 434.

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Synthesis of [125I]HL and [125I]HBL. Sn-HBL was radioiodinated in the presence of N-chlorosuccinimide (NCS) as an oxidant. Sn-HBL was dissolved in dry methanol containing 1% acetic acid (0.5 mg/mL), and 44.6 µL of this solution was mixed with 9.9 µL of NCS in methanol (0.5 mg/mL) in a sealed vial, followed by the addition of Na[125I]I (2 µL). After incubation for 25 min at room temperature, [125I]HBL was purified by RPHPLC (solvent system 1). The radiochemical yield and purity of [125I]HBL were determined by RP-HPLC (solvent system 2) and TLC (solvent system 4), respectively. [125I]HL was prepared by treating RP-HPLC-purified [125I]HBL with TFA (100 µL). After standing for 10 min at room temperature, [125I]HL was purified by RP-HPLC (solvent system 1). The radiochemical yield and purity of [125I]HL were determined by RP-HPLC (solvent system 2) and TLC (solvent system 3), respectively. Synthesis of D-[125I]HL and D-[125I]HBL. D-[125I]HL and D-[125I]HBL were prepared from D-Sn-HBL as described above. Preparation of BBMVs. BBMVs were isolated from the renal cortex of male Wistar rats according to the Mg/ EGTA precipitation method reported previously (11). All steps were performed on ice or at 4 °C. The cortex was homogenized with a Polytron homogenizer (PT10-35, Kinematica GmbH Littau, Switzerland) at full speed for 2 min in an appropriate volume of 300 mM mannitol, 12 mM Tris-HCl (pH 7.1), and 5 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), followed by the addition of the same buffer to make a 10% homogenate. After the mixture was diluted with distilled water (1:1), MgCl2 was added to a final concentration of 10 mM, and the homogenate was allowed to stand for 15 min. The homogenate was centrifuged at 1900g for 15 min in a Hitachi High-Speed Refrigerated Centrifuge SCR 20B (rotor RPR20-2). The supernatant was centrifuged at 24 000g for 30 min. The pellet was resuspended in an appropriate volume of buffer composed of 150 mM mannitol, 6 mM Tris-HCl (pH 7.1), and 2.5 mM EGTA and homogenized in a Potter-Elvehjem homogenizer with a Teflon pestle at 1000 rpm (10 strokes). MgCl2 was added to a final concentration of 10 mM, and the suspension was centrifuged at 1900g for 15 min. The supernatant was centrifuged at 24000g for 30 min. The final pellet (purified brush border membrane) was resuspended in a 0.1 M phosphate buffer (pH 7.0) by 10 passages through a fine needle (0.4 × 19 mm) attached to a plastic syringe. For incubation studies, the vesicles were diluted with 0.1 M phosphate buffer (pH 7.0) to give a final protein concentration of 10 mg/mL. The γ-glutamyltransferase, aminopeptidase, and renal dipeptidase activities on BBMVs, determined according to the procedures of Glossmann et al. (14), Kramers et al. (15), and Hooper et al. (16), using L-γ-glutamyl-p-nitroanilide, L-leucine-p-nitroanilide, and glycyl-D-phenylanaline as substrates, were found to be 5.22 µmol/min/mg protein, 893 and 6.22 nmol/min/mg protein, respectively. The β-galactosidase activity on BBMVs, determined accoroding to the procedure of Wallner et al. using p-nitrophenyl β-D-galactopyranoside (17), was not detectable, which indicated that BBMVs were free from cross-contamination by lysosomal enzymes. Preparation of Cilastatin. One gram of tienam (a mixture of imipenem 0.5 g and sodium cilastatin 0.5 g) was stirred in 20 mL of ethanol for 3 min at room temperature. After filtration, the filtrate was removed in vacuo to produce 120 mg of cilastatin as a sodium salt. 1 H NMR (D2O); δ 0.71-0.87 (2H, m, (CH3)2C-CH2), 0.95-1.04 (6H, m, C(CH3)2) 1.38-1.55 (5H, m, (CH2)2,

Figure 2. Radioactivity profiles of [125I]HL after incubation in BBMVs (center panels) in the presence of 1 mM Co2+ (left panels) or 1 mM MGTA (right panels) at 37 °C as determined by (A) TLC (solvent system 3) or (B) RP-HPLC (solvent system 2). Under these conditions, m-iodohippuric acid and m-iodobenzoic acid had an Rf value of 0.35-0.40 and 0.60, and a retention time of 11 and 16 min, respectively.

(CH3)C-CH), 1.96 (2H, q, CH2CH2S), 2.47 (2H, t, SCH2CH), 2.85-2.96 (2H, m, CHC2CH2), 3.75 (1H, q, CHCOOH), 6.31 (1H, t, CCHCH2). FAB-MS calcd for C16H25N2NaO5S (MH)+: m/z 381. Found: 381. In Vitro Studies. Synthetic substrates were incubated with BBMVs as follows: a solution of BBMVs (20 µL) was preincubated for 2 h at 4 °C, followed by the addition of each substrate solution (20 µL) in 0.1 M phosphate buffer (pH 7.0). After incubation for 3 and 6 h at 37 °C, samples were taken from the solution and analyzed immediately by TLC (solvent system 3 or 4). Each sample was also analyzed by RP-HPLC (solvent system 2) after ultrafiltration with a 10-kDa cutoff membrane (Microcon10, Millipore). A solution of Co2+, MGTA, and phosphoramidon or cilastatin in 0.1 M phosphate buffer (pH 7.0) was added to preincubating solution with a final concentration of 1 mM, and the release of [125I]iodohippuric acid from [125I]HL or [125I]HBL was estimated by TLC and RPHPLC as described above. RESULTS

Figure 2 shows TLC radiochromatograms of [125I]HL after incubation in BBMV solution at 37 °C using solvent system 3. Under this condition, HL had an Rf value of 0.1, whereas m-iodohippuric acid and m-iodobenzoic acid had Rf values of 0.35-0.4 and 0.6, respectively. After 3 h incubation, a radioactivity peak at an Rf value of 0.350.4 was observed. On the other hand, this was not detected when [125I]HL was incubated in BBMVs at 4 °C or in the absence of BBMVs at 37 °C (data not shown). The radioactivity peak at an Rf value of 0.35-0.4 significantly increased when [125I]HL was incubated with BBMVs at 37 °C in the presence of Co2+. A radioactivity peak at an Rf value of 0.6 was also observed; however, no radioactivity peak other than [125I]HL was observed when [125I]HL was incubated in BBMV solution at 37 °C in the presence of MGTA. Similar results were obtained when the reactions were analyzed by RP-HPLC as shown in Figure 2. Under these conditions (solvent system 2), HL and m-iodohippuric acid had a retention time of 7.5 and 11 min, respectively. Figure 3 summarizes the

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Figure 3. Liberation of m-[125I]iodohippuric acid from [125I]HL after incubation in BBMVs at 37 °C for 3 h in the presence of an activator of CPM, or an inhibitor of CPM (MGTA), neutral endopeptidase (phosphoramidon), or dipeptidase (cilastatin), respectively. Columns and bars show the means and SD of three experiments. Significance was determined by unpaired Student’s t-test [(*) p < 0.01 compared to [125I]HL].

Figure 4. Radioactivity profiles of [125I]HBL after incubation in BBMVs (center panels) or in the presence of 1 mM Co2+ (left panels) or 1 mM MGTA (right panels) at 37 °C, as determined by (A) TLC (solvent system 4) or (B) RP-HPLC (solvent system 2). Under these conditions, m-iodohippuric acid and m-iodobenzoic acid had an Rf value of 0.20 and 0.60 on TLC, and a retention time of 11 and 16 min, respectively.

amount of radioactivity in m-iodohippuric acid fractions after [125I]HL incubation in the presence of BBMVs at 37 °C for 3 h, as determined by TLC. The addition of an inhibitor of neutral endopeptidase (phosphoramidon) had no effect on the generation of a radioactivity peak at an Rf value of 0.35-0.4. D-[125I]HL remained unchanged when incubated in BBMV solution in the presence or absence of Co2+. TLC radiochromatograms of [125I]HBL after incubation in BBMV solution at 37 °C are depicted in Figure 4. Under these conditions (solvent system 4), HBL, miodohippuric acid, and m-iodobenzoic acid had an Rf value of 0.35, 0.2, and 0.6, respectively. No radiometabolites were detected when [125I]HBL was incubated in BBMVs at 4 °C or in the absence of BBMVs at 37 °C (data not shown). The radioactivity peak at an Rf value of 0.2 increased when [125I]HBL was incubated in the presence of Co2+ (Figure 4). An additional radioactivity peak was observed at an Rf value of 0.6 in the presence of Co2+. Contrary to [125I]HL, [125I]HBL generated a radioactivity peak at an Rf value of 0.2 when incubated in the presence

Fujioka et al.

Figure 5. Liberation of m-[125I]iodohippuric acid from [125I]HBL after incubation in BBMVs at 37 °C for 3 h in the presence or absence of an activator of CPM (Co2+), or an inhibitor of CPM (MGTA), neutral endopeptidase (phosphoramidon) or dipeptidase (cilastatin), respectively. Columns and bars show the means and SD of three experiments. Significance was determined by unpaired Student’s t-test [(*) p < 0.01 compared to [125I]HBL].

of MGTA although the amount of the radioactivity at this Rf value was significantly reduced (Figure 5, right panel). Similar results were observed by RP-HPLC analyses where [125I]HBL and m-[125I]iodohippuric acid had a retention time of 18 and 11 min (solvent system 2), respectively (Figure 4). Figure 5 summarizes the amount of radioactivity in the m-[125I]iodohippuric acid fraction after incubation of [125I]HBL in BBMV solution at 37 °C for 3 h, when determined by TLC. [125I]HBL generated a significantly higher amount of radioactivity in m-iodohippuric acid fractions than did [125I]HL. The amount of radioactivity in m-iodohippuric acid fractions increased with the presence of Co2+, as also observed with [125I]HL. However, radioactivity fractions were still observed when [125I]HBL was incubated in the presence of MGTA. Both phosphoramidon and cilastatin had no effect on the generation of m-[125I]iodohippuric acid from [125I]HBL. D-[125I]HBL hardly liberated any radiometabolites when incubated in BBMV solution at 37 °C in the presence or absence of Co2+. DISCUSSION

HML was designed based on the hypothesis that the glycyl-lysine sequence in HML would be recognized and cleaved by CPM on the renal brush border membrane during the internalization process of the antibody fragment, since it is well-known that glycyl-lysine is one of the substrates for CPM on the renal brush border membrane (18). Although hippuryl lysine is also recognized and cleaved by CPM (18), HML differs from hippuryl-lysine in that the former has an iodine atom at the meta-position of hippuric acid and the N-amine residue of HML is converted to the maleoyl group, which is further converted to the succinimidyl group after conjugation to thiolated Fab fragments. The latter modification would significantly affect CPM recognition since the presence of a positive charge in the side-chain of the C-terminal lysine residue of glycyl-lysine prefers CPM recognition (19). To estimate the effect of iodination at the meta-position of hippuric acid on CPM recognition, [125I]HL was incubated in BBMVs in the presence or absence of Co2+ or MGTA, an activator and inhibitor of the enzyme, respectively (18, 20). As summarized in Figure 3, the BBMV-

Enzymatic Cleavage of Protected Glycyl-lysine

mediated release of m-[125I]iodohippuric acid from [125I]HL showed temperature dependency. D-[125I]HL was not recognized as a substrate for enzymes on BBMVs. Both Co2+ and MGTA were effective to facilitate and inhibit the hydrolysis reaction. These results suggested that CPM recognized the glycyl-lysine sequence of hippurylL-lysine when iodine was introduced at the meta-position of hippuric acid. To investigate the effect of the modification of N-amine residue of L-lysine on CPM recognition, [125I]HBL was incubated in the presence of BBMVs. [125I]HBL liberated much higher amounts of m-[125I]hippuric acid than [125I]HL (Figures 3 and 5). The hydrolysis reaction of [125I]HBL also showed temperature dependency. D-[125I]HBL was hardly recognized as a substrate by enzymes on BBMVs. Although the addition of Co2+ facilitated the hydrolysis reaction, MGTA acted as a partial inhibitor of the reaction (Figure 5). These studies implied that chemical modification of the N-amine residue of HL made the resulting compound, HBL, a substrate for more than two enzymes on BBMVs at the expense of reduced specificity for CPM. Enzymes on the renal brush border membrane are classified into two types: transmembrane enzymes and glycophosphatidylinositol (GPI)-anchored enzymes. Since the N-amine residue in [125I]HBL was modified with the lipophilic Boc group, [125I]HBL would gain access to not only GPI-anchored enzymes but also transmembrane enzymes on BBMVs, which may account for the significantly higher release of m-[125I]iodohippuric acid from [125I]HBL compared with [125I]HL. Antibody fragments are internalized into renal cells through the brush border of renal tubules. Thus, the iodohippuryllysine moiety in HML-conjugated Fab fragments would also gain access to both types of enzymes during the internalization process. This suggested that a variety of enzymes on BBMVs were involved in the in vivo cleavage of the glycyl-lysine sequence in HML-conjugated Fab fragments. To further investigate the enzymes responsible for the release of m-iodohippuric acid from HML-conjugated antibody fragments, [125I]HBL was incubated in BBMVs in the presence of representative inhibitors for enzymes that cleave the C-terminal peptide sequence. The lack of inhibition of the BBMV-mediated release of m-[125I]iodohippuric acid from [125I]HBL by phosphoramidon and cilastatin indicated that neither neutral endopeptidase nor renal dipeptidase catalyzed the hydrolysis reaction. The acceleration or partial inhibition of the BBMVmediated hydrolysis of [125I]HBL by the presence of Co2+ or MGTA showed that both metalloenzymes and nonmetalloenzymes were involved in the release of m-[125I]iodohippric acid from [125I]HBL and HML-conjugated Fab fragments. These results suggested the involvement of CPM in the hydrolysis of the glycyl-lysine sequence in HBL- and HML-conjugated antibody fragments although the N-amine residues of the two compounds were chemically modified. However, since both activation by Co2+ and inhibition by MGTA are not specific to CPM but rather typical to a variety of metalloenzymes (21, 22), further studies are required for confirmation. In conclusion, the findings in this study suggested that N-modified iodohippuryl-lysine would be recognized as a substrate by enzymes on the renal brush border membrane, which supported our hypothesis that low renal radioactivity levels of [125I]HML-conjugated Fab would be attributable to enzyme-mediated hydrolysis during the internalization process of the conjugate into renal cells. Thus, this study showed that this in vitro system using renal BBMVs would constitute a useful

Bioconjugate Chem., Vol. 16, No. 6, 2005 1615

approach to the development of radiolabeling reagents for antibody fragments and peptides based on the notion of the renal brush border enzyme-mediated release of radiometabolites for low renal radioactivity levels. ACKNOWLEDGMENT

This study was supported in part by a Grant-in-Aid for Scientific Research (B) by the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant-in-Aid from the Fugaku Trust for Medicinal Research. The authors are also grateful to Mr. Takio Kobayashi for financial support. LITERATURE CITED (1) Marcus, R. (2005) Use of 90Y-ibritumomab tiuxetan in nonHodgkin’s lymphoma. Semin. Oncol. 32, S36-S43. (2) Yokota, T., Milenic, D. E., Whitlow, M., Wood, J. F., Hubert, S. L., and Schlom, J. (1993) Microautoradiographic analysis of the normal organ distribution of radioiodinated single-chain Fv and other Immunoglobulin forms. Cancer Res. 53, 37763783. (3) Akizawa, H., and Arano, Y. (2002) Altering pharmacokinetics of radiolabeled antibodies by the interposition of metabolizable linkages. Metabolizable linkers and pharmacokinetics of monoclonal antibodies. Q. J. Nucl. Med. 46, 206-223. (4) Behr, T. M., Behe, M., Stabin, M. G., Wehrmann, E., Apostolidis, C., Molinet, R., Strutz, F., Fayyazi, A., Wieland, E., Gratz, S., Koch, L., Goldenberg, D. M., and Becker, W. (1999) High-linear energy transfer (LET) alpha versus lowLET beta emitters in radioimmunotherapy of solid tumors: therapeutic efficacy and dose-limiting toxicity of 213Bi- versus 90Y-labeled CO17-1A Fab’ fragments in a human colonic cancer model. Cancer Res. 59, 2635-2643. (5) Lambert, B., Cybulla, M., Weiner, S. M., Van De Wiele, C., Ham, H., Dierckx, R. A., and Otte, A. (2004) Renal toxicity after radionuclide therapy. Radiat. Res. 161, 607-611. (6) Olafsen, T., Cheung, C. W., Yazaki, P. J., Li, L., Sundaresan, G., Gambhir, S. S., Sherman, M. A., Williams, L. E., Shively, J. E., Raubitschek, A. A., and Wu, A. M. (2004) Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications. Protein Eng. Des. Sel. 17, 21-27. (7) Arano, Y., Fujioka, Y., Akizawa, H., Ono, M., Uehara, T., Wakisaka, K., Nakayama, M., Sakahara, H., Konishi, J., and Saji, H. (1999) Chemical design of radiolabeled antibody fragments for low renal radioactivity levels. Cancer Res. 59, 128-134. (8) Nakamoto, Y., Sakahara, H., Saga, T., Sato, N., Zhao, S., Arano, Y., Fujioka, Y., Saji, H., and Konishi, J. (1999) A novel immunoscintigraphy technique using metabolizable linker with angiotensin II treatment. Br. J. Cancer 79, 1794-1799. (9) Fujioka, Y., Arano, Y., Ono, M., Uehara, T., Ogawa, K., Namba, S., Saga, T., Nakamoto, Y., Mukai, T., Konishi, J., and Saji, H. (2001) Renal metabolism of 3′-iodohippuryl Nmaleoyl-L-lysine (HML)-conjugated Fab fragments. Bioconjugate Chem. 12, 178-185. (10) Inui, K., Saito, H., Takano, M., Okano, T., Kitazawa, S., and Hori, R. (1984) Enzyme activities and sodium-dependent active D-glucose trasport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK1). Biochim. Biophys. Acta 769, 514-518. (11) Hori, R., Tomita, Y., Katsura, T., Yasuhara, M., Inui, K., and Takano, M. (1993) Transport of bestatin in rat renal brush-border membrane vesicles. Biochem. Pharmacol. 45, 1763-1768. (12) Biber, J., Stieger, B., Haase, W., and Murer, H. (1981) A high yield preparation for rat kidney brush border membranes different behaviour of lysosomal markers. Biochim. Biophys. Acta 647, 169-176. (13) Wakisaka, K., Arano, Y., Uezono, T., Akizawa, H., Ono, M., Kawai, K., Ohmomo, Y., Nakayama, M., and Saji, H. (1997) A novel radioiodination reagent for protein radiopharmaceuticals with L-lysine as a plasma-stable metabolizable

1616 Bioconjugate Chem., Vol. 16, No. 6, 2005 linkage to liberate m-iodohippuric acid after lysosomal proteolysis. J. Med. Chem. 40, 2643-2652. (14) Glossmann, H., and Neville, D. M. (1972) γ-Glutamyltransferase in kidney brush border membranes. FEBS Lett. 19, 340-344. (15) Kramers, M. T., and Robinson, G. B. (1979) Studies on the structure of the rabbit kidney brush border. Eur. J. Biochem. 99, 345-351. (16) Hooper, N. M., Low, M. G., and Turner, A. J. (1987) Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C. Biochem. J. 244, 465-469. (17) Wallner, S. J., and Walker, J. E. (1975) Glycosidases in cell wall-degrading extracts of ripening tomato fruits. Plant Physiol. 55, 94-98. (18) Skidgel, R. A., Davis, R. M., and Tan, F. (1989) Human carboxypeptidase M. J. Biol. Chem. 264, 2236-2241.

Fujioka et al. (19) Skidgel, R. A. (1988) Basic carboxypeptidases: Regulators of peptide hormone activity. Trends Pharmcol. Sci. 91, 299304. (20) Deddish, P. A., Skidgel, R. A., and Erdos, E. G. (1989) Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase). Biochem. J. 261, 289-291. (21) Patterson, E. K., Gatmaitan, J. S., and Hayman, S. (1975) The effect of Mn2+ and Co2+ on the activities of a zinc metallodipeptidase from a mouse ascites tumor. Biochemistry 14, 4261-4266. (22) Salgado, H. C., Carretero, O. A., Scicli, A. G., and Murray, R. D. (1986) Effect of DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid on the blood pressure response to vasoactive substances. J. Pharmacol. Exp. Ther. 237, 204-208.

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