Significance of 111In-DTPA chelate in renal radioactivity levels of 111In-DTPA-conjugated peptides

Significance of111In-DTPA chelate in renal radioactivity levels of111In-DTPA-conjugated peptides

Hiromichi Akizawaa,*, Yasushi Aranob, Masaki Mifunea, Akimasa Iwadoc, Yutaka Saitoa,Tomoya Ueharab, Masahiro Onod, Yasushi Fujiokad, Kazuma Ogawad, Yoshiaki Kisoe,

Hideo Sajid

aFaculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700-8530, JapanbFaculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan

cGraduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama 700-8530, JapandGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

eKyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan

Received 21 October 2000; received in revised form 10 January 2001; accepted 31 January 2001

Abstract

Metabolic studies of111In-DTPA-labeled polypeptides and peptides showed that the radiolabeled (poly)peptides generated111In-DTPA-adducts of amino acid that possess long residence times in the lysosomal compartment of the tissues where (poly)peptides accumulated.However, a recent study suggested that metal-chelate-methionine (Met) might possessin vivobehaviors different from metal-chelate adductsof other amino acids. In this study, to elucidate whether some biological characteristics of Met may accelerate the renal elimination rate of111In-DTPA-adduct of Met into urine,111In-DTPA-Met1-octreotide was synthesized and the renal handling of111In-DTPA-Met wasinvestigated using111In-DTPA-L-Phe1-octreotide (Phe represents phenylalanine), which was reported previously, as a reference. Both111In-DTPA-conjugated octreotide analogs were stable against 3-h incubation in murine serum at 37°C. Both111In-DTPA-octreotideanalogs also showed rapid clearance of the radioactivity from the blood and similar accumulation of the radioactivity in the kidney. Nosignificant differences were observed in the renal radioactivity levels from 10 min to 24 h postinjection between the two. Metabolic studiesindicated that111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide generated111In-DTPA-adducts of Met and Phe, respectively,as the final radiometabolites at similar rates. These findings suggested that the long residence times of the radioactivity in tissues afteradministration of111In-DTPA-labeled peptides and polypeptides would be attributed to inherent characteristics of111In-DTPA chelate.© 2001 Elsevier Science Inc. All rights reserved.

Keywords:111In-DTPA chelate;111In-DTPA-amino acid; Renal radioactivity level;111In-DTPA-peptide

1. Introduction

Indium-111 (111In) constitutes one of the important ra-dionuclides for radiolabeling proteins or peptides for diag-nostic applications in nuclear medicine. Recent advances inmedicinal inorganic chemistry provided a variety of chelat-ing agents for111In radiolabeling of proteins or peptides.Among them, DTPA is still an attractive chelating agent toprepare111In-labeled peptides since DTPA provides111In-labeled peptides with high specific activities. In addition, anintroduction of a111In-DTPA chelate to a low molecular

weight peptide altered pharmacokinetics of the resultingpeptide to urinary excretion, which reduced upper abdomi-nal radioactivity levels that are observed with the parental orradioiodinated peptide [8,12]. Recent development of amonoreactive DTPA derivative provided an easy and effi-cient way to prepare DTPA-conjugated peptides [4,5].However, 111In-DTPA-conjugated peptides showed highand persistent localization of radioactivity in the kidneyafter intravenous administration [12].

Recent metabolic studies of111In-DTPA-conjugatedpolypeptides indicated that when DTPA is attached to lysineresidues of polypeptides,111In-DTPA-adduct of L-lysinewas generated as the final radiometabolite after lysosomalproteolysis of the parental polypeptides in the liver. Thesestudies also showed that the radiometabolite was retained in

* Corresponding author. Tel.:181-86-251-7952; fax:181-86-251-7953.

E-mail address:[email protected] (H. Akizawa).

Nuclear Medicine and Biology 28 (2001) 459–468

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the lysosomes of the tissue for long postinjection intervalsas its intact chelate [6,9,10]. Rogers et al. reported that evenwhen 111In-DTPA is attached to an amine group of N-terminal aspartic acid of an antibody,111In-DTPA-asparticacid was generated as the final radiometabolite and themetabolite showed persistent localization within the tissue[14]. We indicated that111In-DTPA-D-phenylalanine (D-Phe) and111In-DTPA-L-phenylalanine (L-Phe) were gener-ated as the final radiometabolites in the kidney after admin-istration of 111In-DTPA-D-Phe1-octreotide (Fig. 1) and111In-DTPA-L-Phe1-octreotide (Fig. 1), respectively [1].Subcellular distribution studies showed that both radiom-etabolites were present in the lysosomes of renal cells forlong postinjection intervals although the formation rates of111In-DTPA-L-Phe was significantly faster than those of111In-DTPA-D-Phe in the lysosomes. These findings sug-gested that111In-DTPA-adduct of acidic, basic, and aro-matic amino acid would be barely excreted extracellularly.In addition, reabsorption of111In-DTPA-adducts of aminoacid into renal cells would not proceed when they reached tothe lumen of renal tubules from the lysosomes. Such char-acteristics may render111In-DTPA-adducts of amino acidsuitable as radiometabolites of111In-labeled (poly)peptidesfor targeted imaging with low renal radioactivity level whenthe metabolites could be liberated by brush border enzymespresent on the lumen of renal tubules, as proposed recently[3]. The excretion of111In-DTPA-adducts of amino acidsfrom the lumen of renal tubules to urine would facilitateelimination of the radioactivity from the kidney, whereasthe long residence times of111In-DTPA-adducts of aminoacids in lysosomes of tissues would provide high and per-sistent localization of radioactivity in the target tissue evenwhen the parental (poly)peptides are internalized into targetcells.

However, recent study by Wu et al. indicated that whena 67Ga-labeled antibody fragment using 2-(p-isothiocyana-tobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA) as the bifunctional chelating agent was adminis-tered,67Ga-NOTA-Bz-SCN-adduct of methionine (Met) and

67Ga-NOTA-Bz-SCN-adduct of lysine (Lys) were generatedas radiometabolites in the kidney [16]. This study also indi-cated that the former had significantly shorter residence timethan the latter in murine kidney even when considering thegeneration rates of the former might have been faster thanthose of the latter. This suggested that metal-chelate-Met mightpossessin vivobehaviors different from metal-chelates of otheramino acids. Thus, further studies are required to elucidatewhether some biological characteristics of Met may acceleraterenal elimination rate of111In-DTPA-adduct of Met into urinebefore designing radiolabeling reagents that release111In-DTPA-adducts of amino acids by brush border enzymes ofrenal cells.

In the present study, based on our previous findings that111In-DTPA-L-Phe1-octreotide generated111In-DTPA-L-Phe as the major radiometabolite within short postinjectionintervals in the kidney [1],111In-DTPA-Met1-octreotidewas synthesized to estimate an elimination rate of111In-DTPA-Met from the kidney, and111In-DTPA-L-Phe1-oct-reotide, which was reported previously [1], was used as areference to compare elimination rates of the two111In-DTPA-adducts of amino acid from the kidney. The signif-icance of inherent characteristics of111In-DTPA chelate inthe renal retention of111In-DTPA-adduct of amino acids isdiscussed.

2. Materials and methods

2.1. Reagents and chemicals

111InCl3 (121.2 MBq/mL in 0.02 N HCl) was kindlysupplied by Nihon-Medi-Physics (Tokyo, Japan). Nonradio-active indium chloride was purchased from Nacalai Tesque(Kyoto, Japan) as InCl3 z 4H2O. Reversed-phase high-per-formance liquid chromatography (RP-HPLC) was per-formed with a Cosmosil 5C18-MS column (4.63 150 mm,Nacalai Tesque, Kyoto, Japan).111In-DTPA-Met1-oct-reotide was eluted with 0.05 M acetate buffer (pH 5.5) for

Fig. 1. Structures of111In-DTPA-D-Phe1-octreotide,111In-DTPA-L-Phe1-octreotide, and111In-DTPA-Met1-octreotide.

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the first 10 min, followed by a linear gradient of 0 to 55%methanol in 0.05 M acetate buffer (pH 5.5) in 15 min. Thefinal solvent composition was maintained for a further 25min. Cellulose acetate electrophoresis (CAE; Joke Co. Ltd.,Tokyo) was run at an electrostatic field of 0.8 mA/cm inveronal buffer (I 5 0.06, pH 8.6; Nacalai Tesque) for 50 and40 min for 111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide, respectively. Thin layer chromatography(TLC) was developed in a mixture of 10% ammoniumchloride and methanol (1 : 1). 1-tert-Butyl hydrogen 3,6,9-tris(tert-butoxycarbonyl)methyl-3,6,9-triazaundecanedioicacid (mDTPA) was synthesized as reported previously [5].Proton nuclear magnetic resonance (1H-NMR) spectra wererecorded on a Varian VXR200 spectrometer, and the chem-ical shifts are reported in parts per million downfield froman internal tetramethylsilane standard. Fast atom bombard-ment mass spectra (FABMS) were obtained with aVG70-SE (Micromass, Manchester). The matrix-assistedlaser-desorption-ionization time-of-flight mass spectra(MALDI-TOFMS) were obtained with a Voyager-DE RP(PerSeptive Biosystems, Tokyo). Other reagents were ofreagent grade and used as received. To facilitate collectionof urine and feces by 24 h postinjection of111In-DTPA-conjugated peptides, mice were housed in metabolic cages(Metabolica, MM type; Sugiyama-Gen Iriki Co. Ltd.,Tokyo).

2.2. DTPA-Met

DTPA-Met was synthesized using mDTPA. To a solu-tion of L-methioninetert-butyl ester hydrochloride (198 mg,0.819 mmol) in 3 mL of dimethylformamide (DMF),N,N-diisopropylethylamine (0.143 mL, 0.819 mmol) wasadded at 0°C, and the reaction mixture was stirred for 30min at room temperature. After cooling to 0°C, 1-hydroxy-benzotriazole monohydrate (151 mg, 1.117 mmol), 1-ethyl-3-(39-dimethylaminopropyl)carbodiimide hydrochloride(214 mg, 1.117 mmol),N, N-diisopropylethylamine (0.195mL, 1.117 mmol), and a solution of the mDTPA (460 mg,0.745 mmol) in 2 mL of DMF were added successively.After stirring at room temperature for 6 h, the mixture wasextracted with 60 mL of ethyl acetate, and the organic layerwas washed with a 10% citrate (60 mL3 2), a saturatedaqueous solution of NaHCO3 (60 mL 3 2), and a brine (60mL 3 2). After drying over anhydrous sodium sulfate, thesolvent was removedin vacuo.The residue was chromato-graphed on silica gel using a mixture of hexane-ethyl ace-tate (3 : 2) as an eluent to yield DTPA-Met with the fivecarboxylates protected withtert-butyl groups as a pale yel-low oil (272 mg; 45.35%).

Trifluoroacetic acid (TFA, 5.3 mL) was added to theprotected DTPA-Met (64.3 mg, 0.080 mmol) in the pres-ence of thioanisole (0.289 mL, 2.372 mmol),m-cresol(0.167 mL, 1.598 mmol), and 1,2-ethanedithiol (0.402 mL,4.792 mmol) at 0°C. The reaction mixture was stirred at30°C for further 7 h. After removing TFAin vacuo,dry

ether was added to the residue to precipitate the product.This process was repeated three times to obtain pure DTPA-Met as a colorless solid (30.4 mg, 72.56%). FABMS forC19H33N4O11S (MH1): m/z calcd, 525.1867; found,525.1907. 1H-NMR (DMSO-d6): 1.91–2.02 (2H, m,CHCH2CH2), 2.04 (3H, s, CH3), 2.45 (2H, br, CH2S), 2.98(4H, br, CH2CH2), 3.22 (4H, br, CH2CH2), 3.38 (2H, s,NCH2CO), 3.45 (2H, s, NCH2CO), 3.49 (4H, s, NCH2CO),4.00 (2H, s, NCH2CO), 4.34 (1H, q, NHCH), 8.28 (1H, d,NH).

2.3. DTPA-Met1-octreotide

DTPA-Met1-octreotide was synthesized according to theprocedures similar to those used for DTPA-D-Phe1-oct-reotide [4] except for the use of Na-Fmoc-Met in place ofNa-Fmoc-D-Phe and the iodine oxidation in 80% acetic acidin place of 80% methanol. The amino acid ratios after 6 NHCl hydrolysis of DTPA-Met1-octreotide were as follows:Thr 3 1, 0.99; Met3 1, 1.04; Phe3 1, 1.00; Lys3 1, 1.01.MALDI-TOFMS for C59H88N13O19S3 (MH1): m/z calcd,1379.59; found, 1379.46.

2.4. Radiolabeling of DTPA-Met1-octreotide

Lyophilized kits (containing DTPA-Met1-octreotide, 10mg) with trisodium citrate (4.91 mg), citric acid (0.37 mg),inositol (10.0 mg), and gentisic acid (2.0 mg) were preparedusing ultrapure water (Milli Q, Millipore Japan, Tokyo).The 111In labeling of DTPA-conjugated peptides was per-formed by addition of 1 mL solution of111InCl3 (17.3–121.2 MBq) to kits. The final pH was about 4.1. Afterstanding at room temperature for 1 h,111In-DTPA-Met1-octreotide was purified by RP-HPLC to exclude non-pep-tide-bound111In species and unchelated peptides. An ap-propriate amount of DTPA-Met1-octreotide was added tofractions containing111In-labeled peptide, and the solventwas removedin vacuo.A HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)-buffered solution (10mM, pH 7.6) containing 0.1% human serum albumin wasadded to the residue to prepare111In-labeled octreotideanalog containing 0.1mg of the DTPA-peptide in 100mLfor biodistribution studies and 150mL for metabolic studies.The radiochemical purities of111In-DTPA-Met1-octreotideexceeded 95% as determined by RP-HPLC and CAE anal-yses (Fig. 2). Under the analytical conditions,111In-DTPA-Met1-octreotide showed two radioactivity peaks on the RP-HPLC, as reported previously [1,2].

To further characterize the indium-chelated peptide, non-radioactive indium was reacted with DTPA-Met1-octreotideas follows: a solution of InCl3 z 4H2O in 0.05 M acetic acidwas added to a solution of DTPA-Met1-octreotide in 0.05 Macetic acid at a molar ratio of 1:1. RP-HPLC trace (230 nm)

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of the reaction mixture showed two peaks at retention timesidentical to those observed with111In-DTPA-Met1-oct-reotide. In addition, both compounds showed a peak atm/zcalculated for In-DTPA-Met1-octreotide. MALDI-TOFMSfor In C59H85N13O19S3 (MH1): m/zcalcd, 1494.41; found,1494.94 and 1494.97.

2.5. Preparation of111In-DTPA-Met and111In-DTPA

The 111In radiolabeling of DTPA-Met and DTPA wasperformed according to the procedure described previously[1]. Briefly, a solution of sodium acetate (1 M, 10mL) wasmixed with 40mL of a diluted solution of111InCl3 (37–74kBq/mL) in 0.02 N HCl. After the mixture was allowed tostand for 5 min, a 10mL aliquot of DTPA-Met or DTPA (20mM) in 20 mM MES (2-morpholinoethanesulfonic acid)-buffered saline (pH 6.0) was added, and the reaction mix-ture was incubated at room temperature for 1 h.111In-DTPA-Met and111In-DTPA were obtained with over 96%radiochemical yields by RP-HPLC and CAE analyses, andthey were used without further purification.111In-DTPA-Met gave two peaks at retention times of 2.5 and 4.5 min onRP-HPLC and showed a single peak at a migration distance

of about 2 cm to the anode by CAE.111In-DTPA had asingle peak at a retention time of 2.5 min on the RP-HPLCand at a migration distance of 3 cm to the anode by CAEanalyses. To further characterize the two peaks of111In-DTPA-Met, non-radioactive indium was added to a solutionof Met-DTPA in 0.05 M acetic acid at a molar ratio of 1:1.RP-HPLC trace (differential thermal analysis) of the reac-tion mixture showed two peaks at retention times identicalto those of111In-DTPA-Met. Both compounds showed apeak atm/z calculated for In-DTPA-Met. FABMS for InC19H30N4O11S (MH1): m/zcalcd, 637; found, 637 and 637.

2.6. Serum stability of111In-DTPA-Met1-octreotide

A 1.5 mL solution of111In-DTPA-Met1-octreotide (1 ng,45.2–71.7 kBq) was added to freshly prepared murine se-rum (400mL), and the mixture was incubated at 37°C. Afterincubation for 1 and 3 h, a 50mL aliquot of the samples wasdrawn, and the radioactivity was analyzed by CAE. Theradioactivity in the serum was also analyzed by RP-HPLCafter filtering through a 10 kDa cut-off ultrafiltration mem-brane (Microcon-10, Amicon Inc., Beverly, MA).

Fig. 2. CAE (A) and RP-HPLC (B) radiochromatograms of111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide.

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2.7. In vivo studies

Animal studies were performed after approval by ourlocal ethical committee at Okayama University and in ac-cordance with “Interdisciplinary Principles and Guidelinesof the Use of Animals in Research”. Biodistributions ofradioactivity after intravenous administration of111In-DTPA-Met1-octreotide into 6-week-old male ddY normalmice [11] weighing 28–30 g were monitored at 10 and 30min, and 1, 3, 6, and 24 h postinjection. Groups of four tofive mice which received 0.1mg of radiolabeled peptide

were used for the experiments. The organs of interest wereremoved and weighted, and the radioactivity was deter-mined with a well counter (Aloka ARC 2000, Tokyo).

To determine the amounts and routes of the radioactivityexcreted from the body, mice were housed in metaboliccages for 24 h after administration of111In-DTPA-Met1-octreotide, and the urine and feces were collected and theradioactivity was determined.

The radiolabeled species remaining in the kidney at 1, 3,and 24 h postinjection and excreted in the urine by 24 hpostinjection were analyzed according to the procedure de-

Table 1Biodistribution of radioactivity after intravenous administration of111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide in micea

Organ Time after administration

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

111In-DTPA-Met1-octreotide

Blood 3.80c 1.64 0.49 0.14c 0.11c 0.08c

(0.39) (0.51) (0.21) (0.03) (0.00) (0.03)Liver 1.88 1.80 1.16 1.58 1.50 0.81

(0.29) (0.42) (0.17) (0.41) (0.27) (0.36)Kidney 20.72 18.99 18.03 13.69 12.78 4.85

(6.07) (1.83) (3.48) (1.03) (1.75) (2.34)Intestine 0.74 0.50 0.36 0.20c 0.24c 0.22c

(0.11) (0.17) (0.14) (0.03) (0.05) (0.06)Spleen 1.76c 2.29 1.11 2.09 1.55 1.42

(0.39) (0.78) (0.21) (1.03) (0.58) (0.97)Pancreas 1.11c 0.63c 0.20 0.07c 0.03c 0.18

(0.17) (0.17) (0.17) (0.04) (0.02) (0.21)Lung 2.87 1.48d 0.50c 0.14c 0.11c 0.06c

(0.27) (0.39) (0.21) (0.03) (0.03) (0.03)Urineb 62.93

(5.33)Fecesb 25.33

(6.20)

111In-DTPA-L-Phe1-octreotidee

Blood 4.49 2.02 0.53 0.08 0.05 0.03(0.25) (0.38) (0.11) (0.01) (0.01) (0.01)

Liver 2.06 1.85 1.26 1.48 1.50 0.81(0.17) (0.37) (0.13) (0.24) (0.47) (0.13)

Kidney 17.71 18.28 15.89 12.82 9.44 2.83(4.05) (2.99) (3.45) (1.80) (1.80) (1.00)

Intestine 0.91 0.72 0.66 0.67 1.03 0.87(0.05) (0.11) (0.24) (0.10) (0.31) (0.54)

Spleen 3.18 2.23 1.74 1.88 1.31 1.21(0.95) (0.57) (0.62) (0.58) (0.54) (0.82)

Pancreas 1.67 1.04 0.43 0.20 0.14 0.16(0.29) (0.17) (0.03) (0.04) (0.05) (0.01)

Lung 4.74 2.59 0.85 0.47 0.33 0.27(0.63) (0.27) (0.08) (0.16) (0.12) (0.05)

Urineb 57.54(5.64)

Fecesb 22.81(4.08)

a Expressed as a percentage of injected dose per gram. Mean (sd) of four to five animals for each point.b Expressed as a percentage of injected dose (sd). Differences between111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide were analyzed by

the unpaired Mann-Whitney test.c p , 0.05.d p , 0.01.e Results were reported previously (1).

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scribed previously [7]. Briefly,111In-DTPA-Met1-octreotide(0.1 mg, 9.5–12.6 MBq) was administered intravenouslyinto 6-week-old male ddY mice. At 1, 3, and 24 h postin-jection, both kidneys were perfusedin situ with cold 0.1 Mtris-citrate buffer (pH 6.5) containing 0.15 M NaCl, 0.02%sodium azide, 1 TIU/mL aprotinin, 2 mM benzamide-HCl,2 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride,and 5 mM diisopropyl fluorophosphate before the kidneyswere removed. Each tissue sample was placed in a test tubeand subjected to three cycles of freezing (dry ice - acetonebath) and thawing. After the addition of 5 volumes of thesame buffer containing an additional 35 mM ofb-octyl-

glucoside, the sample was homogenized with a Polytronhomogenizer (PT10–35, Kinematica GmgH Littau, Swit-zerland) at full speed with three consecutive 30 s burstsbefore centrifugation at 48,000 g for 20 min at 4°C (HimacCS-120 Centrifuge, Hitachi Co., Tokyo). The supernatantwas separated from the pellets, and the radioactivity wascounted. The kidney supernatant and the urine samples wereanalyzed by CAE and TLC after filtration through a poly-carbonate membrane with a pore diameter of 0.45mm(Myrex, Millipore). The samples were also analyzed byRP-HPLC after filtering through a 10 kDa cut-off ultrafil-tration membrane (Microcon-10, Amicon). CAE and RP-

Fig. 3. CAE radiochromatograms of kidney homogenates at 1 (A), 3 (B) and 24 h (C) postinjection and urine samples (D) excreted by 24 h postinjectionof 111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide.

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HPLC analyses were also performed by co-chromatographywith 111In-DTPA or 111In-DTPA-Met.

3. Results

3.1. Serum stability

When incubated in freshly prepared murine serum at37°C for 3 h, over 96.5% of the initial radioactivity wereobserved as the intact form for both111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide [1] as deter-mined by RP-HPLC. No significant differences were ob-served in serum stability between the two111In-DTPA-conjugated peptides. Similar results were observed by CAEanalyses.

3.2. In vivo study

The biodistributions of radioactivity after administrationof 111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-oc-treotide [1] into mice are summarized in Table 1. Both111In-DTPA-conjugated peptides displayed rapid clearanceof the radioactivity from the blood. Both111In-DTPA-con-jugated peptides also showed similar tissue distributions ofthe radioactivity from 10 min to 24 h postinjection. Nosignificant differences were observed in the renal radioac-tivity levels at all postinjection points and in the radioac-tivity levels of urine and feces excreted by 24 h postinjec-tion of the two111In-labeled peptides.

The supernatants of the kidney homogenates wereextracted with efficiencies of over 90% at all postinjec-tion times. Figure 3 shows CAE radiochromatograms of

Fig. 4. RP-HPLC radiochromatograms of kidney homogenates at 1 (A), 3 (B), and 24 h (C) postinjection and urine samples (D) excreted by 24 h postinjectionof 111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide. Results for111In-DTPA-L-Phe1-octreotide were reported previously [1].

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the kidney homogenates at 1, 3 and 24 h and urine sampleby 24 h following administration of111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide. The kid-ney homogenates of the two111In-DTPA-labeled pep-tides showed a migration of the radioactivity peak fromca. 1.5 cm cathode from the origin to ca. 2 cm anode fromthe origin with postinjection times. On the other hand, themajority of the radioactivity in the urine samples wasobserved at a position identical to that of the intactpeptides. Only a small amount of the radioactivity wasdetected at origin in kidney homogenates at 3 and 24 h

postinjection. Similar results were observed with the two111In-DTPA-labeled peptides by TLC analysis (data notshown). Figure 4 shows RP-HPLC radiochromatogramsof the kidney supernatants at 1, 3, and 24 h postinjectionand the urine samples excreted by 24 h postinjection of111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-oc-treotide [1]. Both111In-DTPA-labeled peptides were de-graded to the final radiometabolitesvia an intermediateradiometabolite at similar rates. At 3 h postinjection, thefinal radiometabolites represented most of the radioactiv-ity in the kidney for the two111In-DTPA-conjugatedpeptides. However, the predominant radiolabeled speciesin the urine had retention times identical to those of theintact peptides. Figure 5 shows RP-HPLC and CAE ra-diochromatograms of kidney homogenates at 24 h postin-jection of 111In-DTPA-Met1-octreotide in the absence orpresence of metabolic standards. The major radioactivityin the kidney after injection of111In-DTPA-Met1-oct-reotide had retention times and migration distances iden-tical to those of111In-DTPA-Met on RP-HPLC and CAEanalyses, respectively, which was confirmed by co-chro-matographic analyses using111In-DTPA-Met and111In-DTPA standards.

Table 2 summarizes the radiolabeled species in the kid-ney and urine after injection of111In-DTPA-Met1-octreotideand 111In-DTPA-L-Phe1-octreotide [1] as determined byRP-HPLC analyses, where the radiolabeled species are clas-sified into four groups; the intact peptide, intermediate spe-cies eluted at the retention times between intact peptide andthe final radiometabolite, the final metabolite (co-elutedwith 111In-DTPA-Met or 111In-DTPA-L-Phe standard), andothers that represented radiometabolites eluted earlier thanthe final radiometabolite. Both111In-DTPA-labeled pep-tides showed that the final metabolite represented about55% of radioactivity in kidney at 1 h postinjection, whereasover 90% of the radioactivity in kidney was present as thefinal metabolite at 3 and 24 h postinjection. About 10% andover 80% of radioactivity in urine existed as the final me-tabolite and the intact peptide, respectively, for both111In-DTPA-conjugated peptides.

4. Discussion

This study was undertaken to elucidate whether somebiological characteristics of Met may accelerate renal elim-ination rate of111In-DTPA-adduct of Met into urine, asobserved with 67Ga-NOTA-Bz-SCN-labeled polypeptide[16]. For selective generation of111In-DTPA-Met from111In-DTPA-(poly)peptides in the kidney,111In-DTPA-Met1-octreotide was synthesized using mDTPA.

When injected into mice, both111In-DTPA-conjugatedpeptides showed the elimination from the blood and theaccumulation to the kidney at similar rates (Table 1). At 3 hpostinjection, only 0.1% injected radioactivity/g was de-tected in the blood for the two peptides. Both111In-DTPA-

Fig. 5. CAE (A) and RP-HPLC (B) radiochromatograms of kidney ho-mogenates at 24 h postinjection of111In-DTPA-Met1-octreotide. The kid-ney homogenates were analyzed in the absence (closed circle, solid line) orin the presence (open circle, broken line) of111In-DTPA-Met. The kidneyhomogenates were also analyzed in the presence of111In-DTPA (opentriangle, solid line).

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conjugated peptides also registered maximum renal radio-activity at 10 to 30 min postinjection. Since both111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotideremained stable in serum for 3 h, both peptides would beincorporated into renal cells as their intact structures afterglomerular filtration, as previously reported [1]. In meta-bolic studies, both111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide [1] generated their final radiom-etabolites, 111In-DTPA-Met and 111In-DTPA-L-Phe, atsimilar rates in the kidney (Fig. 3–5 and Table 2). CAE andTLC analyses also reinforced that transchelation of111In toproteins in the lysosomes was negligible, as also observed inprevious studies [1,6,9,10,14]. Thus, the renal residencetimes of the radioactivity after administration of the two111In-DTPA-conjugated peptides would reflect the resi-dence times of their final radiometabolites,111In-DTPA-Met and111In-DTPA-L-Phe, in the renal cells.

In biodistribution studies, no significant differences wereobserved in renal radioactivity levels between111In-DTPA-Met1-octreotide and111In-DTPA-L-Phe1-octreotide from 10min to 24 h postinjection (Table 1). This indicated that111In-DTPA-Met and 111In-DTPA-L-Phe eliminated fromthe renal cells at similar rates (Table 1), which was contraryto the findings by Wu et al. who observed that67Ga-NOTA-Bz-SCN-eLys was the only radiometabolite in the kidney at24 h postinjection of67Ga-NOTA-Bz-SCN-labeled anti-body fragments [16]. These findings along with the previousstudies using111In-DTPA-conjugated (poly)peptides [1,6,9,10,14] suggested that inherent characteristics of111In-DTPA chelate might be responsible for the long residencetimes of the radioactivity in the kidney or liver after admin-istration of111In-DTPA-conjugated (poly)peptides.

The findings in this and previous studies also impliedthat both111In-DTPA-Met and111In-DTPA-L-Phe would beslowly excreted from the kidney to urine without beingreabsorbed into renal cells when they reached to the lumen

of renal tubules. The long residence times of the radio-metabolites in the kidney also suggested that111In-DTPA-adducts of amino acids might be retained in the lysosomesof target cells where they were generated following inter-nalization of the parental (poly)peptides into target cells.Such characteristics of111In-DTPA-adducts of amino acidsrender DTPA attractive as chelating site of radiolabelingreagents with linkages cleavable by brush border enzymespresent on lumen of renal tubules [3].

The long residence times of111In-DTPA-Met in thekidney also supported rationale behind the application of111In-DTPA radiolabeling procedure to a residualizing labelfor determining the sites and rates of protein catabolisminvivo [13,15]. Prior studies indicated that111In-DTPA-eLys,the most often observed radiometabolite for111In-DTPA-labeled proteins, showed persistent localization in the lyso-somal compartment of tissues where catabolism of proteinsoccurred. Previous studies showed that the radiolabelingprocedure is applicable to proteins with N-terminal asparticacid and phenylalanine [1,14]. This study supported that theradiolabeling procedure is also applicable to proteins evenwhen methionine is present as the N-terminal amino acid.

In conclusion, the findings in this study indicated that111In-DTPA-Met showed residence times in the kidney sim-ilar to other111In-DTPA-adducts of amino acid so far re-ported. These findings suggested that the long residencetimes of 111In-DTPA-adducts of amino acid at sites of(poly)peptide catabolism would be attributed to inherentcharacteristics of111In-DTPA chelate. Such characteristicswould render111In-DTPA applicable as chelating site ofradiolabeling agents for (poly)peptides in diagnostic nuclearmedicine when combined with a cleavable linkage by renalbrush border enzymes. This study also provided furtherrationale for applying111In-DTPA radiolabeling procedureto a residualizing label to determine the sites and rates ofprotein catabolism.

Table 2Radiolabeled species in the kidney and urine after administration of111In-DTPA-Met1-octreotide and111-In-DTPA-L-Phe1-octreotide in mice

Fraction Percentage of radioactivitya

Kidney (1 h) Kidney (3 h) Kidney (24 h) Urine (24 h)111In-DTPA-Met1-octreotide

Intact 10.76 (1.21) 0.20 (0.04) 0.15 (0.10) 80.32 (5.66)Intermediates 29.68 (2.47) 1.81 (0.02) 0.39 (0.11) 6.79 (0.30)Final metabolite 54.90 (4.03) 94.93 (0.14) 91.23 (0.19) 10.75 (4.69)Othersb 2.38 (0.18) 2.45 (0.01) 7.07 (0.01) 0.94 (0.67)

111In-DTPA-L-Phe1-octreotidec

Intact 14.36 (0.26) 0.67 (0.52) 0.15 (0.22) 85.41 (1.83)Intermediates 25.77 (0.62) 3.88 (0.21) 0.00 (0.01) 3.04 (0.26)Final metabolite 57.14 (0.69) 93.65 (0.42) 96.55 (0.03) 9.54 (0.77)Othersb 0.65 (0.04) 0.54 (0.02) 3.17 (0.20) 1.34 (0.22)

a Radiolabeled species in each fraction determined by RP-HPLC analyses. Mean (sd) of three experiments.b Radiolabeled species eluted at earlier retention times than those of111In-DTPA-Met or 111In-DTPA-L-Phe (e.g.111In-DTPA).c Results were reported previously (1).

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Acknowledgments

The authors thank Mr. Shinsaku Nakayama for his tech-nical support. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists from Min-istry of Education, Science, and Culture, Japan.

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