99mTc-Labeling and in Vitro and in Vivo Evaluation of HYNIC- and

This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling age...
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Bioconjugate Chem. 2004, 15, 864−871

99mTc-Labeling

and in Vitro and in Vivo Evaluation of HYNIC- and (Nr-His)Acetic Acid-Modified [D-Glu1]-Minigastrin E. von Guggenberg,† M. Behe,‡ T. M. Behr,‡ M. Saurer,§ T. Seppi,§ and C. Decristoforo†,* Department of Nuclear Medicine, Leopold-Franzens-University of Innsbruck, Innsbruck, Austria, Department of Nuclear Medicine, Philipps-University of Marburg, Marburg, Germany, and Department of Radiotherapy and Radiooncology, Leopold-Franzens-University of Innsbruck, Innsbruck, Austria . Received December 18, 2003; Revised Manuscript Received March 24, 2004

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (NR-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (NR-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 µg of minigastrin I human (MGh). HYNIC-MG and (NR-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/µmol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with 99mTcEDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/ g) and 99mTc-(NR-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/ g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc(NR-His)Ac-MG showed low tumor uptake in vivo.

INTRODUCTION

Cholecystokinin-2 (CCK-2, formerly CCK-B)/gastrin receptors have recently found increasing interest as targets for nuclear medicine applications. A high incidence of CCK-2 receptors has been found in human samples of medullary thyroid cancer (MTC), small cell lung cancer (SCLC), stromal ovarian cancers (1), and also in vivo in gastrointestinal neuroendocrine tumors, especially if somatostatin receptor scintgraphy is negative (2). Radiolabeled analogues of both CCK (3) and gastrin (4) have been developed. [DTPA-D-Glu1]-minigastrin (DTPA* Corresponding author: Clemens Decristoforo, Department of Nuclear Medicine, Leopold-Franzens-University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. Tel: +43-512-5042671. Fax: +43-512-504-2683. E-mail: Clemens.Decristoforo@ uibk.ac.at. † Department of Nuclear Medicine, Leopold-Franzens-University of Innsbruck. ‡ Philipps-University of Marburg. § Department of Radiotherapy and Radiooncology, LeopoldFranzens-University of Innsbruck.

MG) radiolabeled with 111In has shown promising properties for detection of CCK-2 receptor positive tissue in patients (5) and has been extensively evaluated clinically, radiolabeled with 90Y even for therapeutic purposes in MTC (6). For diagnostic applications, technetium-99m still is the label of choice for routine applications due to its availability, low costs, low radiation dose for the patient, and optimal gamma-energy profile. Labeling approaches of peptides with 99mTc have to fulfill several criteria (7): labeling has to be achieved at high specific activities without interfering with the amino acid sequence responsible for receptor binding, and it has to ensure reasonably hydrophilic properties of the peptide resulting in predominant renal clearance and a high in vivo stability of the 99mTc complex. These criteria limit the numbers of available labeling approaches, and only a few have so far shown applicability in patient studies. Hydrazinonicotinic acid (HYNIC) is a bifunctional coupling agent for 99mTc-labeling of peptides and has been used to radiolabel a number of peptides such as somatostatin analogues (8) or GPIIb/IIIa antagonists (9). In

10.1021/bc0300807 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/02/2004

99mTc-Labeled

Conjugates

Figure 1. Molecular formulas of HYNIC and (NR-His)acetic acid conjugated to [D-Glu]1-minigastrin (MG).

the HYNIC labeling approach, technetium-99m is bound to the hydrazino group, and the other coordination sites are occupied by one or more coligands. A number of suitable coligands has been described, but it is also known that the choice of coligand can influence the stability and lipophilicity of the radiolabeled peptide and in the end the biodistribution (10). Another interesting approach to radiolabel peptides at high specific activities is the use of the technetium-carbonyl-core [Tc(CO)3]. Alberto et al. described the one-step synthesis of the technetium-carbonyl aquaion [99mTc(OH2)3(CO)3]+ (11). This precursor has been shown to have a high affinity for histidine (His) and radiolabels peptides at very high specific activities (12). Further, the denticity of the Tc(CO)3 coordinating ligand can influence the biological behavior of radiolabeled peptides, and tridentate ligands have advantages over bidentate systems (13). One promising tridentate derivative for peptide conjugation is (NRHis)Ac that has shown suitable properties for 99mTc labeling after conjugation to a neurotensin derivative (14). We herein describe the labeling, in vitro characterization including receptor binding, and biodistribution of HYNIC and (NR-His)acetic acid conjugated to [D-Glu]1minigastrin (MG, D-Glu-Glu5-Ala-Tyr-Gly-Trp-Met-AspPheNH2), Figure 1. EXPERIMENTAL PROCEDURES

Materials. Reagents were purchased from AldrichSigma Chemical Co. unless stated otherwise and used as they were received. [HYNIC-D-Glu1]-minigastrin (HYNIC-MG) and [(NR-His)Acetyl-D-Glu1]-minigastrin ((NR-His)Ac-MG) were synthesized by PiChem (Graz, Austria) with a purity of >95% as analyzed by reversed phase HPLC (RP-HPLC) and MS. [125I -Tyr12]-gastrin I was purchased from Perkin-Elmer Life Science (Boston, MA). Na 99mTcO4 was obtained from a commercial 99Mo/ 99mTc generator (ULTRATECHNEKOW, Mallinckrodt, The Netherlands). Analytical Methods. HPLC. A Gynkotek M480 pump with a Spectra Physics Spectra Chrom 100 variable UV detector and Bioscan radiometric detection was used for RP-HPLC analysis. A Macherey & Nagl Nucleosil 120-5 C18 250 × 4.6 mm column, flow rates of 1 mL/min, and UV detection at 220 nm were employed with the following gradients: Method 1: Acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA)/H2O: t: 0-3 min 0% ACN, 3-5 min 0-25% ACN, 5-20 min 25-40% ACN, 20-25 min 40-60% ACN, 2528 min 60-0% ACN, 28-33 min 0% ACN Method 2: 0.05 M Na acetate pH 5.4/ACN: t: 0-3 min: 0% ACN, 3-33 min: 0-100% ACN, 33-35 min: 100% ACN, 35-36 min: 100-0% ACN.

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Purification by Solid-Phase Extraction (SPE). For purification of the radiolabeled peptide for stability studies, a SPE method was used. The radiolabeling mixture was passed through a C18-SEPPAK-mini cartridge (Waters, Milford, MA). The cartridge was washed with 5 mL of saline, and the radiolabeled peptide was eluted with 70% ethanol. This method efficiently removed all hydrophilic, nonpeptide bound impurities (mainly 99m TcO4-, 99mTc coligands). 99mTc-Labeling. HYNIC-MG: Tris(hydroxymethyl)methylglycine (Tricine) as Coligand. In a rubber-sealed vial, 3 µg of HYNIC-MG was incubated with 0.5 mL of Tricine solution (70 mg/mL in water), 0.5 mL of 99mTcO4solution (>200 MBq), and 20 µL of tin(II) solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) for 20 min at room temperature or 75 °C. Ethylenediaminediacetic Acid (EDDA) as Coligand. In a rubber-sealed vial, 10 µg of HYNIC-MG was incubated with 0.5 mL of EDDA solution (20 mg/mL in pH 6-7), 0.5 mL of 99mTcO4- solution (400 MBq), and 20 µL of tin(II) solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) for 30 min at room temperature and for 10 min in a boiling water bath. Tricine/EDDA Exchange Labeling. In a rubber-sealed vial, 20 µg of HYNIC-MG was incubated with 1 mL of EDDA/Tricine solution (20 mg/mL Tricine, 10 mg/mL EDDA in pH 6-7), 1 mL of 99mTcO4- solution (800 MBq), and 20 µL of tin(II) solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) for 10 min in a boiling water bath. (NR-His)Ac-MG. Carbonyl aquaion [99mTc(OH2)3(CO)3]+ was prepared as described previously (15). Briefly, in a rubber-sealed vial 4 mg of Na2CO3, 5.5 mg of NaBH4, and 20 mg of NaK-tartrate were CO-purged for 15 min and reacted with 2.5 mL of 99mTcO4- solution (3-4 GBq) for 30 min at 75 °C. Pressure from the evolving H2 gas was balanced with a 20 mL syringe, and 1 M NaH2PO4 was added for neutralization. Quality control was performed by HPLC method 1. Peptide Labeling. In a rubber-sealed vial 20 µg of (NRHis)Ac-MG was incubated with 0.5 mL of Carbonyl precursor (800 MBq) for 30 min at 75 °C. Radiolabeled peptides were characterized and labeling yields determined by HPLC methods 1 and 2. In Vitro Evaluation of Radiolabeled Peptides. Stability. The stability of the radiolabeled peptides in aqueous solution was tested by incubation of the reaction mixture purified by SPE at a concentration of 200-1000 pmol peptide/mL in phosphate buffer (pH 7.4), in a solution containing 10 000-fold molar excess of cysteine or histidine (pH 7.4) over the peptide and in fresh human plasma at 37 °C up to 24 h. After incubation, plasma samples were precipitated with acetonitrile and centrifuged (1750g, 5 min). Degradation of the 99mTc complexes was assessed by HPLC method 1. For protein binding assessment, the SPE-purified complexes were incubated at a concentration of 20-100 pmol peptide/mL in fresh human plasma at 37 °C and analyzed for up to 6 h by size-exclusion chromatography (MicroSpin G-50 Columns; Sephadex G-50). Protein binding of the 99mTc complex was determined by measuring column and eluate in a gamma counter. For incubation in kidney and liver homogenates, kidneys or liver freshly excised from rat were rapidly rinsed and homogenized in 20 mM HEPES buffer pH 7.3 with an Ultra-Turrax T25 homogenator for 1 min at RT. The radiopeptides were incubated with fresh 30% homogenates at a concentration of 250-500 pmol peptide/mL at 37 °C for up to 2 h. Samples were precipitated with

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acetonitrile, centrifuged (1750g, 5 min), and analyzed by HPLC method 1. Receptor Binding Studies. The binding affinity of peptide conjugates was tested in a competition assay against [125I-Tyr12]-gastrin I. Rat pancreatic tumor (AR42J) cell membranes were used as the source for gastrin receptors. For membrane preparation, cells were homogenized three times for 10 s at 4 °C with an Ultra-Turrax T25 homogenator in 20 mM HEPES buffer pH 7.3/10 mM MgCl2/10 µM bacitracin, and protein concentration was assessed to 50 µg/100 µL (Bradford assay). In a Multiscreen well plate (glass fiber filters; Whatman GF/C), 50 µL of competitor solution of increasing concentrations (0.001-1000 nM in 1% BSA/10 mM MgCl2/10 µM bacitracin), 50 µL of radioligand solution (50 000 cpm in 1% BSA/10 mM MgCl2/10 µM bacitracin), and 100 µL of membrane solution (50 µg protein/tube) were incubated in triplicates for 2 h at RT. Incubation was interrupted by filtration of the medium and rapid rinsing with icecold washing buffer (1 × 200 µL, followed by 1 × 50 µL 15 mM TRIS/139 mM saline pH 7.4), and filters were counted in a gamma-counter. IC50 values were calculated following nonlinear regression with Origin software (Microcal Origin 5.0, Northampton MA). For saturation studies, similar to that described above for the competition assays on membranes, in a Multiscreen well plate, radiolabeled peptide conjugate of increasing concentrations (0.05-20 nM in 1% BSA/10 mM MgCl2/10 µM Bacitracin) and AR42J membrane solution (50 µg protein/tube) were incubated in triplicate for 2 h at RT in a total volume of 200 µL. Nonspecific binding was determined in a parallel series containing 1 µM MGh. Kd values were calculated following nonlinear regression and Scatchard plot (linear regression) with Origin software (Microcal Origin 5.0, Northampton MA). Internalization Studies. For internalization experiments, AR42J cells were seeded at a density of 1 × 106 cells per well in six-well plates (Greiner Labortechnik, Germany) and grown to confluency for 48 h. On the day of the experiment, cells were washed twice with ice-cold internalization medium prepared by RPMI1640 supplemented by 1% (v/v) fetal bovine serum. The cells were supplied with fresh medium (1.2 mL) and incubated with 300 000 cpm of the radiolabeled peptide (150 µL in PBS/ 0.5%BSA buffer, corresponding roughly to 200 fmol of total peptide) and either PBS/0.5%BSA buffer alone (150 µL, total series) or with 10 µM MGh in PBS/0.5%BSA buffer (150 µL, nonspecific series). The cells were incubated at 37 °C in triplicate for each time point of 5, 15, 30 min, 1 and 2 h incubation. Incubation was interrupted by removal of the medium and rapid rinsing with icecold internalization medium two times. Thereafter, the cells were incubated twice at ambient temperature in acid wash buffer (50 mM glycine buffer pH 2.8, 0.1 M NaCl) for 5 min, a period sufficient to remove over 90% of membrane-bound radioligand. The supernatant was collected, and the cells were rinsed with PBS/0.5% BSA (membrane bound radioligand fraction). Finally, cells were lyzed by treatment in 1 N NaOH and collected (internalized radioligand fraction). All fractions were counted in a gamma-counter, and mean specific values were calculated. The internalized fraction was expressed in relation to the total activity added (% of total) as well as in relation to the activity bound to the cells, i.e., internalized plus membrane bound fraction (% of bound). In Vivo Evaluation of Radiolabeled Peptides. Biodistribution. All animal experiments were conducted in compliance with the Austrian animal protection laws and with approval of the Austrian Ministry of

von Guggenberg et al. Scheme 1. 99mTc-Labeling Routes of HYNIC- and (Nr-His)Ac-MGa

a Complex structures of 99mTc-Tricine/HYNIC-MG, 99mTcEDDA/HYNIC-MG, and 99mTc(CO)3-(NR-His)Ac-MG according to refs 16, 17, and 13, respectively.

Science. Normal biodistribution studies were performed in Balb/c mice (Charles River, Germany). On the day of experiment, three mice received the radioactive conjugate (1 MBq/mouse, corresponding to 0.15-0.2 µg of peptide) intravenously injected into the tail vein, with or without coinjection of 50 µg of MGh. They were sacrificed by cervical dislocation 4 h postinjection. Different organs and tissues (blood, lung, heart, stomach, spleen, liver, pancreas, kidneys, muscle, intestine) were removed. The amount of radioactivity was determined with a gammacounter. Results were expressed as percentage of injected dose per gram of tissue (%ID/g). Tumor uptake studies were performed in nu/nu mice (Charles River, Germany). For the induction of tumor xenografts, AR42J cells were subcutaneously injected at a concentration of 10 × 106 cells/mouse, and tumors were allowed to grow until they had reached a size of 0.5-1 mL (10-15 days). On the day of the experiment, the animals were treated as described above. Tumors and other tissues (blood, lung, heart, stomach, spleen, liver, pancreas, kidneys, muscle, intestine) were removed. The amount of radioactivity was determined with a gammacounter. Results were expressed as percentage of injected dose per gram of tissue (%ID/g), and tumor-to-organ, stomach-to-organ, pancreas-to-organ, and kidney-toorgan ratios were calculated. Metabolic Stability. To evaluate the in vivo degradation of the conjugate, urinary excretion products were characterized by RP-HPLC (method 1). RESULTS

Radiolabeling. Radiolabeling of all peptide conjugates could be performed according to Scheme 1 at high specific activities (>70 GBq/µmol) with high yields. In the case of HYNIC-MG, only Tricine as coligand resulted in reasonable labeling yields, whereby two main peaks could be characterized by HPLC. At room temperature a shift to the more hydrophilic species over time was observed; by heating the reaction mixture at 80 °C for 15 min, only this peak was detected. Addition of monodentate ligands such as nicotinic acid to the reaction mixture did not significantly alter the HPLC profile. Therefore, 99mTcHYNIC-MG prepared at higher temperature was chosen for further characterization. EDDA as coligand resulted in very low labeling yields even at elevated temperature;

99mTc-Labeled

Conjugates

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Figure 2. HPLC-radiochromatograms of the radiolabeled peptides (HPLC method 1).

Figure 4. Stability of radiolabeld peptides in rat tissue homogenates: (a) kidney, (b) liver. Table 2. Stability of the Radiolabeled Peptides in Aqueous Solutions 0h

Figure 3. Protein binding over time determined by size exclusion chromatography (MicroSpinTM G-50 columns, Sephadex G-50). Table 1. Retention Times of the Radiolabeled Peptides in Two Different HPLC Gradients retention time (min) HPLC gradient

99mTc-Tricine/

99mTc-EDDA/

99mTc(CO)

HYNIC-MG

HYNIC-MG

(NR-His)Ac-MG

method 1 method 2

13.4 14.3

15.7 14.9

18.3 15.8

3-

however, using an exchange labeling approach from Tricine to EDDA and boiling the reaction mixture for 10 min, a single peak (99mTc-EDDA/HYNIC-MG) with distinct retention time on HPLC to the 99mTc-Tricine/ HYNIC-MG complex was observed with labeling yields >90%. (NR-His)Ac-MG could be radiolabeled by addition of [99mTc(OH2)3(CO)3]+; high labeling yields >90% were achieved only when the concentration of peptide in the reaction solution was kept higher than 20 µM. In Figure 2 are shown typical HPLC-radiochromatograms of reaction mixtures of all three 99mTc-labeled MG derivatives. Retention times were different, utilizing two different HPLC gradients at different pH values (methods 1 and 2) and are summarized in Table 1. Lipophilicity as determined from the RP-HPLC retention behavior was increasing in the order 99mTc-Tricine/HYNIC-MG < 99mTcEDDA/HYNIC-MG < 99mTc(CO)3-(NR-His)Ac-MG. Stability Studies. Plasma protein binding behavior at different time points is presented in Figure 3. At early time points, 99mTc(CO)3-(NR-His)Ac-MG showed considerably higher levels compared to 99mTc-HYNIC conjugates. At later time points, 99mTc-Tricine/HYNIC-MG showed increasing values reaching levels comparable to 99mTc(CO)3-(NR-His)Ac-MG after 6 h incubation. Protein bind-

1h

2h

4h

Intact 99mTc-Tricine/HYNIC-MG (%) phosphate 99.6 98.2 96.3 91.1 serum 98.9 98.8 98.4 96.7 cysteine 95.5 92.6 92.5 88.4 histidine 93.4 90.1 89.3 88.4 Intact 99mTc-EDDA/HYNIC-MG (%) phosphate 99.9 99.4 99.1 99.4 serum 99.7 99.3 96.6 97.4 cysteine 99.0 99.2 98.7 99.1 histidine 99.5 99.2 99.1 99.4 Intact 99mTc(CO)3-(NR-His)Ac-MG (%) phosphate 98.8 99.6 98.7 99.2 serum 98.6 99.6 98.2 98.9 cysteine 98.5 99.8 99.4 99.9 histidine 98.7 99.7 99.9 99.9

24 h 80.8 94.6 81.4 81.4 94.8 90.9 96.4 96.1 96.8 94.4 96.2 97.6

ing values of 99mTc-EDDA/HYNIC-MG were similar to its Tricine counterpart at 1 h with little change over time. Results from stability studies are summarized in Table 2. Incubation in PBS, serum, cysteine, and histidine for up to 24 h of SPE-purified peptides revealed high stability of all 99mTc-labeled peptide conjugates under investigation. Only 99mTc-Tricine/HYNIC-MG showed generally somewhat lower purity levels, especially after 24 h in all incubation solutions (80.8-81.4%) except serum. Incubation in kidney homogenates resulted in a very rapid decrease of activity related to the original peptide peak (Figure 4a). After 30 min incubation, less than 10% intact peptide could be detected for 99mTc(CO)3-(NR-His)Ac-MG and 99mTc-EDDA/HYNIC-MG; for 99mTc-Tricine/ HYNIC-MG, this value was reached only after 60 min incubation. After incubation in liver homogenates (Figure 4b), also a low stability of all three peptide conjugates was found with differences in the rate of decrease, reaching 69% for 99mTc(CO)3-(NR-His)Ac-MG, 32% for 99m Tc-EDDA/HYNIC-MG, and 44% for 99mTc-Tricine/ HYNIC-MG after 120 min incubation time.

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Figure 5. Receptor binding on AR42J membranes: (a) Displacement curves of [125I-Tyr12]-gastrin I using minigastrin I human (IC50 ) 4.58 nM), HYNIC-MG (IC50 ) 15.5 nM) and (NRHis)Ac-MG (IC50 ) 2.45 nM) as competitors (mean ( sd, n ) 3). (b) Saturation curve and Scatchard plot of 99mTc-EDDA/ HYNIC-MG (Kd ) 10.3 nM, Bmax ) 647 fmol/mg protein).

Receptor Binding and Internalization. Both HYNIC-MG and (NR-His)Ac-MG had a high affinity to the gastrin/CCK-2 receptor. Displacement of [125I-Tyr12]gastrin I by the two peptide conjugates in comparison with unmodified MG is shown in Figure 5a. IC50 values for HYNIC-MG were somewhat higher (15.5 nM) compared to (NR-His)Ac-MG (2.45 nM) and MG (4.58 nM). Saturation assay of 99mTc-EDDA/HYNIC-MG revealed high binding affinity of the 99mTc-labeled peptide with a Kd of 10.3 nM and a Bmax of 647 fmol/mg protein (Figure 5b). Internalization behavior on AR42J cells is summarized in Figure 6. All three 99mTc-labeled compounds showed a rapid internalization of cell-bound activity, reaching a plateau of more than 80% internalized fraction after 30 min incubation (Figure 6a). Looking at the percentage internalized activity of the total activity added, we observed differences (Figure 6b). 99mTc(CO)3-(NR-His)AcMG showed the highest percentage of internalized activity (reaching 13.7% after 120 min incubation), followed by 99mTc-EDDA/HYNIC-MG (10.1%) and 99mTc-Tricine/ HYNIC-MG (7.0%). Biodistribution and Tumor Uptake. Biodistribution in Balb/c mice 4 h pi revealed a rapid elimination from most organs and mainly renal excretion (Figure 7). Intestinal activity was minimal, varying from 1.18%ID/g for 99mTc-Tricine/HYNIC-MG to 0.44%ID/g for 99mTcEDDA/HYNIC-MG, indicating low hepatobiliar excretion. However, great differences were observed in kidney uptake. 99mTc(CO)3-(NR-His)Ac-MG showed renal retention of 2.81%ID/g, whereas 99mTc-EDDA/HYNIC-MG and 99m Tc-Tricine/HYNIC-MG accumulated to much higher levels in the kidneys with 127 and 73%ID/g, respectively. In animal urine only degradation products of the 99mTc-

Figure 6. Time dipendent internalization of radiolabeled peptides in AR42J cells expressed as (a) % of total activity, (b) % of bound activity (mean ( sd, n ) 3).

Figure 7. Normal biodistribution of the radiolabeled peptides in Balb/c mice 4 h after injection. Values are expressed as %ID/g ) percentage injected dose/g tissue (means ( sd, n ) 3).

labeled peptide conjugates could be detected by RPHPLC. Similar differences were also observed in a mouse tumor model. Results of biodistribution in nu/nu mice bearing AR42J tumor xenografts 4 h pi is summarized in Table 3. Whole body retention in this tumor model 4 h pi were 23.96 ( 4.02%ID for 99mTc-Tricine/HYNIC-MG, 38.96 ( 2.78%ID for 99mTc-EDDA/HYNIC-MG, and 2.38 ( 0.53%ID for 99mTc(CO)3-(NR-His)Ac-MG. Again, low levels of intestinal activity and accumulation in other organs were detected (