Preparation, 99mTc-Labeling, and in Vitro Characterization of HYNIC

Bioconjugate Chem. 1999, 10, 431−438

431

Preparation, 99mTc-Labeling, and in Vitro Characterization of HYNIC and N3S Modified RC-160 and [Tyr3]Octreotide Clemens Decristoforo and Stephen J. Mather* Nuclear Medicine Research Laboratory, St. Bartholomews hospital, West Smithfield, London EC1 7BE, U.K. Received October 5, 1998; Revised Manuscript Received February 1, 1999

The synthesis of conjugates of two somatostatin analogues, RC-160 and [Tyr3]octreotide with different bifunctional chelators for labeling with Tc-99m, is described. Conjugates with hydrazinonicotinamide (HYNIC) and two N3S compounds (benzoyl MAG3 and a N3S adipate derivative) were prepared on a small scale with high purity allowing evaluation of different bifunctional chelators on the same peptide without extensive peptide synthesis. High in vitro stability and retained binding affinity was found for all conjugates except for the N3S adipate. Peptide conjugates could be labeled at high specific activities (>1 Ci/µmol) with 99mTc, and different coligands were explored for the HYNIC conjugates. The resulting radiolabeled complexes were highly stable and showed binding affinity to somatostatin receptors in the nanomolar range. Varying labeling yield, stability, lipophilicity, and isomerism were found for different coligands used for labeling HYNIC conjugates, with lower lipophilicity, higher stability, and fewer coordination isomers for EDDA and tricine/nicotinic acid as ternary coligand compared to tricine. In particular, HYNIC complexes showed promising results for further in vivo evaluation.

INTRODUCTION

The radiolabeled somatostatin analogue 111In-DTPAD-Phe1-octreotide (Octreoscan) has found a wide clinical application for imaging a range of tumors, including neuroendocrine cancer, carcinoid, and lymphoma (1-4) but one of the, at least, theoretical disadvantages of this compound is its restricted reactivity to receptor-subtypes SSR2 and SSR5. Recently, it has been demonstrated that another somatostatin analogue (RC-160 or vapreotide) has affinity for a different set of somatostatin receptor subtypes which may increase its usefulness for targeting more common tumors such as those of breast, ovary, exocrine pancreas, prostate, and colon (5-7). In further contrast to octreotide, RC-160 can also pass the bloodbrain barrier and might therefore be used for imaging brain tumors (8). RC-160 has previously been radiolabeled with 123I (9), 111In (10), and recently 188Re (11), and high affinity to somatostatin receptors in vivo has been proven. Although the radiolabel of choice for tumor imaging would be technetium-99m, experience with suitable labeling methods is not yet as great as for other radionuclides [for review, see Liu et al. (12)]. The most widely used method for labeling such small highly specific peptides is by conjugation of bifunctional chelators (BFCs) to the peptide. The number of BFCs suitable for labeling with technetium-99m at the high specific activities necessary to permit the use of pharmacologically highly potent peptides in nontoxic doses is limited, but, in recent years, attempts have been made to label various somatostatin analogues with 99mTc using a number of different BFCs including diamidedithiols, triamidemonothiols and triaminemonothiols (13), propyleneamine oxime (14), tetramine (15), and hydrazinonicotinamide (HYNIC) (16). Such methods are generally considered * To whom correspondence should be addressed. Phone: 44171-601-7153.Fax: 44-171-796-3907.E-mail: [email protected].

more appropriate than the alternative approach of direct labeling following reduction of the disulfide bridge (17, 18), which forms 99mTc complexes of uncertain structure and raises concerns with regard to the retention of specific binding to somatostatin receptors. The aim of this study was to perform a comparison of some of the BFCs previously described, in order that some recommendations can be made on the most suitable system for technetium labeling of these small peptides. This report describes the small-scale preparation of conjugates of RC-160 and [Tyr3]octreotide (TOC) with different BFCs, based on the triamidemonothiol and HYNIC cores. Results from 99mTc-labeling experiments using different coligand systems and in vitro evaluations of the resulting 99mTc complexes are reported and provide data for direct comparison of different BFCs. EXPERIMENTAL PROCEDURES

Materials. Reagents were purchased from AldrichSigma Chemical Co. except where otherwise stated and used as they were received. 6-BOC-hydrazinopyridine-3-carboxylic acid (BOC-HYNIC) was synthesized and characterized according to Abrams et al. (19). 2,3,5,6,-Tetrafluorothiophenyl-S-(1-ethoxyethyl)mercaptoacetamidoadipoylglycylglycine [N3S-adipate (20)] was a gift from Alan R. Fritzberg (NeoRx Corporation, Seattle, WA). N-Hydroxysuccinimide-S-acetyl-mercaptoacetyltriglycin [S-acetyl-NHS-MAG3 (21)] was a gift from Donald J. Hnatowich (University of Massachusetts, Worcester, MA). S-Benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) was a gift from Ole K. Hjelstuen (Isopharma Radiopharmaceuticals, Oslo, Norway). [Lys5-BOC]RC-160, RC-160, [Tyr3, Lys5-BOC]octreotide, and [Tyr3]octreotide (TOC) were synthesized by Bachem UK Ltd.

10.1021/bc980121c CCC: $18.00 © 1999 American Chemical Society Published on Web 03/31/1999

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Decristoforo and Mather

Figure 1. Structures of peptides and BFCs used. From left to right: RC-160, [Tyr3]octreotide BOC-HYNIC, benzoyl-MAG3, N3Sadipate.

(3-[125I]iodotyrosyll1)Somatostatin-14 ([125I]SST14) was purchased from Amersham Life Science Ltd. (Amersham, U.K.). Na99mTcO4- was obtained from commercial 99Mo/99mTc generator (Nycomed Amersham). Deionized water was obtained from a ELGA Elgastat UHP Water System and was of >18 MΩ quality. INSTRUMENTS AND METHODS

HPLC. A Beckman solvent module 125 with Beckman UV detector 166NM and radiometric detection was used for reversed-phase HPLC analysis and preparation. A Beckman Ultrasphere ODS 5 µm, 4.6 × 250 mm column, flow rates of 1 mL/min, and UV detection at 220 nm were employed together with the following solvent systems. Method 1: acetonitrile/0.1%TFA/water. Gradient: 0-3 min 0% ACN; 3-13 min 0 to 50% ACN; 13-23 min 50% ACN, 23-26 min 50 to 70% ACN, 26-27 min 70 to 0% ACN. Method 2: acetonitrile/0.01 N phosphate buffer, pH 6.2. Gradient as in method 1. Method 3: acetonitrile/ 0.1% TFA/water; 0-3 min 0% ACN; 3-10 min 0 to 40% ACN; 10-20 min 40% ACN; 20-23 min 40 to 70% ACN; 26-27 min 70 to 0% ACN. Method 4: acetonitrile/0.01 N phosphate buffer, pH 6.2; 0-3 min 0% ACN; 3-10 min 0 to 25% ACN; 10-20 min 25% ACN; 20-23 min 25 to 70% ACN; 26-27 min 70 to 0% ACN. Methods 1 and 2 were used for analyses of RC-160 conjugates, methods 3 and 4 for TOC conjugates. TLC. Instant thin-layer chromatography on silica gel (ITLC-SG, Gelman Sciences) was performed using different mobile phases. 2-Butanone was used to determine the amount of free 99mTcO4- (Rf ) 1), and the PBS buffer was used to determine non-peptide-bound 99mTc coligand and 99mTcO4- (Rf ) 1), 50% acetonitrile/water for 99mTc colloid (Rf ) 0). SPE Purification. For purification of the radiolabeled peptide for stability studies, a solid-phase extraction (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 water, the radiolabeled peptide eluted with 80% acetonitrile, and the organic solvent evaporated under vacuum. This method efficiently removed all hydrophilic impurities (99mTcO4-, 99mTc coligand) and 99mTc colloid to a concentration of less than 2% when tested by HPLC or TLC. Synthesis of Peptide Conjugates. Structures of RC160, [Tyr3]octreotide (TOC), and the BFC used are shown in Figure 1. Synthesis of HYNIC-RC-160. A total of 0.3 mg of BOCHYNIC, 2.0 mg of O-(7-azabenzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 5 mg of diisopropyethylamine (DEA) in 300 µL of DMF were

reacted for 15 min. A total of 60 µL of this solution was added to 1.0 mg of Lys-BOC-RC-160 in 20 µL of DMF/5 µL of water and allowed to react for 1 h. To stop the reaction, 1 mL of water was added, and the resulting solution was passed through a C18 SEPPAK cartridge (Waters, Milford, MA) washed with additional 5 mL of water and finally eluted with 1 mL of 100% acetonitrile. The acetonitrile solution was reduced under vacuum to a volume of 100 µL. A total of 300 µL of TFA and 10 µL of thioanisole were added and reacted for 10 min. The solution was evaporated to dryness, and the residue dissolved in 200 µL of 50% ethanol and purified on HPLC using method 1. The peak of HYNIC-RC-160 was collected and stored in the HPLC eluent at -20 °C under nitrogen. Further characterization was performed by HPLC analysis and matrix assisted laser desorption ionization MS on a Lasermat 2000 (Finnigan Mat). Synthesis of S-Acetyl-MAG3-RC-160. A total of 0.3 mg of Lys-BOC RC-160, 1.0 mg of S-acetyl-NHS-MAG3, and 1 mg of DEA were reacted for up to 3 h. SEPPAK purification and deprotection was carried out as described for HYNIC-RC-160. Synthesis of S-Benzoyl-MAG3-RC-160. A total of 2.2 mg of S-benzoyl-MAG3, 1.0 mg of HATU, and 1 mg of DEA in 170 µL of DMF were reacted for 15 min. A total of 40 µL of this solution was added to 0.4 mg of Lys-BOCRC-160 in 20 µL of DMF/5 µL of water and allowed to react for 1 h. SEPPAK purification and deprotection was carried out as described for HYNIC-RC-160. Synthesis of N3S-RC-160. A total of 0.4 mg of N3S adipate, 0.67 mg of Lys-BOC-RC-160, and 0.3 mg of DEA in 150 µL of DMF were incubated for 80 min. SEPPAK purification and deprotection was carried out as described for HYNIC-RC-160. Synthesis of S-Benzoyl-MAG3- and HYNIC-TOC. Conjugates were prepared in the same way as the RC-160 conjugates corrected for molecular weight, purified and analyzed by HPLC method 3. 99m Tc Labeling. [99mTc]HYNIC conjugate. Tricine as coligand: in a rubber sealed vial, 10 µg of HYNIC conjugate was incubated with 0.5 mL of tricine solution (100 mg/mL in 25 mM succinate buffer, pH 5.0), 0.5 mL of 99mTcO4- solution (100-1000 MBq), and 25 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. Ethylendiaminediacetic acid (EDDA) as coligand: 10 µg of HYNIC-RC-160 was incubated with 0.5 mL of solution of EDDA (10 mg/mL, pH 7.0), 0.5 mL of 99mTcO4solution (100-1000 MBq), and 5-10 µL of tin(II) solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) for 60 min at room temperature. Nicotinic acid/tricine as coligands: 10 µg of HYNICRC-160, 0.4 mL of tricine solution (100 mg/mL in 25 mM

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succinate buffer, pH 5.0), 0.1 mL of nicotinic acid (NA) (20 mg/mL in 25 mM succinate buffer pH 5.0), 0.5 mL of 99mTcO - solution (100-500 MBq), and 25 µL of tin(II) 4 solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) were heated at 100 °C for 15 min. [99mTc]MAG3 Conjugate. A total of 10 µg of S-benzoylMAG3 conjugate was heated with 0.5 mL of sodium tartrate (100 mg/mL in 0.1 M phosphate buffer, pH 7.2), 1.5 mL of 99mTcO4- solution (100-1000 MBq), and 40 µL of tin(II) solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) at 100 °C for 15 min. [99mTc]N3S-RC-160. A total of 10 µg of N3S-RC-160, 1.0 mL of glucoheptonate solution (40 mg/mL pH 2.5), 0.5 mL of 99mTcO4- solution (100-1000 MBq), and 20 µL of tin(II) solution (10 mg of SnCl2‚2H2O in 10 mL of nitrogen purged 0.1 N HCl) were heated at 80 °C for 20 min. In Vitro Characterization of Radiolabeled Peptides. Stability. The stability of the radiolabeled peptides in aqueous solution was tested by incubation of the SPE purified peptide in 0.1 M phosphate buffer and in a solution containing 1000-fold excess cysteine over the peptide at pH 7.4 at room temperature up to 24 h. Cysteine, naturally occurring and a strong chelating compound for technetium, is thought to be responsible for in vivo degradation of 99mTc complexes and is frequently used for in vitro stability studies (22). As a control, the stability of [99mTc]tetraglycine, a technetium complex with a low complex stability (23), was tested in parallel. Stability in fresh human plasma was also measured at 37 °C for up to 4 h followed by precipitation of plasma protein with acetonitrile. Degradation of the 99mTc complex was assessed by TLC and HPLC. Somatostatin Receptor Binding. The binding affinity of peptide conjugates was tested in a competition assay against [125I]SST14 as described elsewhere (24). Mouse pituitary [AtT20 cells, expressing SST receptors subtype 2 and 5 (25)] and rat pancreatic [AR42J cells, expressing SST receptors subtype2 (26)] tumor cell membranes were used as a source for somatostatin receptors and were separated from free radioligand by filtration through glass fiber filters (Whatman GF/C), and IC50 values were calculated following nonlinear regression with Origin software (Microcal Origin 5.0, Northampton MA). The specific binding of the 99mTc-labeled peptides was determined by competition against unmodified RC-160 in a similar assay. Kd values of 99mTc-labeled peptide conjugates were determined in radioligand saturation assays using AR42J cell membranes as receptor source. RESULTS

Synthesis of Peptide Conjugates. HPLC analysis of reaction mixtures of different RC-160 conjugates is shown in Figure 2, preparation of TOC conjugates gave similar results. Quantitative peptide coupling of BOCHYNIC (Figure 2C, peak 3) and S-benzoyl-MAG3, using HATU, as well as of the N3S adipate active STFP ester was achieved within 1 h. For the N-hydroxysuccinimideactivated S-acetyl-MAG3, no quantitative reaction could be achieved in DMF even at high molar excess of the activated ester over the peptide. Deprotection of the peptide conjugates with TFA resulted in one major HPLC-peak for HYNIC-RC-160 (Figure 2D, peak 4) and S-benzoyl-MAG3 (Figure 2F, peak 5). Overall yields were >50% for both these BFCs. In the case of S-acetyl-MAG3, multiple peaks were observed, indicating instability of the conjugate under acidic conditions (Figure 2E) and no pure peptide conjugate could be isolated. The N3S-RC160 deprotection solution also showed several byproducts

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Figure 2. HPLC traces (UV 220 nm, method 1) from the synthesis of RC-160 conjugates. (A) Lys-BOC-RC-160 standard, (B) RC-160 standard, (C) reaction solution of BOC-HYNIC-RC160 before deprotection, (D) deprotection solution of HYNICRC-160, (E) deprotection solution of S-acetyl-MAG3-RC-160, (F) deprotection solution of S-benzoyl-MAG3-RC-160, (G) deprotection solution of N3S-RC-160. Peaks marked with numbers: 1, Lys-BOC-RC-160; 2, RC-160; 3, BOC-protected-HYNIC-RC-160; 4, HYNIC-RC-160 (deprotected); 5, S-benzoyl-MAG3-RC-160; 6, N3S-RC-160. Table 1. Molecular Weight of Peptide Conjugates Determined by Laser Desorption MS Compared with the Theoretical Value mass

calculated

found

HYNIC-RC-160 S-benzoyl-MAG3-RC-160 N3S-RC-160 HYNIC-TOC S-benzoyl-MAG3-TOC

1264.5 1479.6 1533.6 1170.5 1383.5

1265.0 1479.4 1462.4 1170.0 1383.4

(Figure 2G), and the overall yield was 90%) was achieved for most conjugates and coligand systems. Lipophilic impurities in varying amounts were found in the MAG3 conjugate-labeling mixture (Figure 3D). Labeling HYNIC-RC-160 with EDDA as coligand at room-temperature resulted in an average yield of 69% (Figure 3C), for HYNIC-TOC 63.9%. Labeling yield for the EDDA compounds was independent of pH between 4 and 7.2, and no significant increase was observed by heating the labeling mixture. The optimal EDDA concentration was found to be 5 mg/mL, but the labeling yield was mainly influenced by the concentration of stannous ions in the labeling solution. A rapid decline in labeling yields for HYNIC-RC-160 with increasing amounts of stannous ions was observed (Figure 4) when EDDA was used as coligand. Using pyridine/tricine as ternary ligands, labeling of the peptide conjugate was quantitative, but residues of the binary tricine complex were found when labeling was performed at room temperature. Quantitative labeling to the ternary ligand was only achieved by heating at 100 °C for 15 min. 99mTc-labeled TOC conjugates showed considerably lower retention times on HPLC compared to their RC160 counterparts, indicating the considerably lower lipophilicity of this peptide. HPLC profiles were dependent on the pH of the aqueous solvent employed. When water/ 0.1% TFA HPLC systems were used, retention times were very similar for all 99mTc-labeled conjugates of the same peptide (see Figure 3, left columns). Using a phosphate buffer at pH 6.2, a better distinction between different labeled peptide conjugates was achieved (see Figure 3, right column). Retention times were in the following order: EDDA-HYNIC < tricine/NA-HYNIC < N3S < MAG3 < tricine-HYNIC (main peaks) for both RC-

160 and TOC conjugates. Multiple peaks were found for tricine as coligands (Figure 3A) and two main peaks for tricine/NA as ternary coligands for HYNIC conjugates (Figure 3B). Except for the N3S conjugate, this HPLC method allowed efficient separation of the 99mTc complex from the cold conjugate to yield carrier free Tc complexes that could be used for radioligand binding assays. Stability of 99mTc-labeled Peptide Conjugates. 99mTc-labeled peptide conjugates were stable in aqueous solutions for 24 h, except for tricine-HYNIC conjugates, which, when excess tricine was removed by SEPPAK purification, showed a radiochemical purity of less than 80% after 24 h. Challenge experiments with 1000-fold molar excess of cysteine showed no significant degree of transchelation for N3S and HYNIC conjugates while, for the MAG3 conjugates, 20% transchelation after 24 h was observed. Results of cysteine challenge experiments are shown for RC-160 in Figure 5, the decrease in the radiochemical purity over time can be related to the transchelation to cysteine. All compounds were stable to 4 h incubation in human plasma, although varying amounts of labeled peptide were protein bound and were precipitated with plasma proteins by acetonitrile, higher amounts for RC-160 compared to TOC. Receptor Binding. The cold peptide conjugates all showed a high binding affinity on AtT20 and AR42J cell membranes. Competition curves for RC-160 conjugates against [125I]SST14 are shown in Figure 6. For the RC160 itself, an IC50 value of 0.38 nM was calculated while, for S-benzoyl-MAG3-RC-160, binding affinity was almost identical with an IC50 of 0.36 nM. HYNIC-RC-160 and N3S-RC-160 showed a slightly lower binding affinity with IC50 values of 1.51 and 1.73 nM, respectively (Figure 6). HYNIC-TOC showed an IC50 value of 1.55 nM and S-benzoyl-MAG3-RC-160 of 0.15 nM compared to 0.39 nM for unmodified TOC. All 99mTc-labeled conjugates tested showed high specific binding to somatostatin receptors, with calculated Kd values in the nanomolar range, varying from 0.93 to 2.65 nM. Binding affinities were similar for RC-160 and TOC derivatives. Kd values determined on AR42J cell membranes are listed in Table 3. Saturation curves for the 99mTc-labeled EDDA-HYNIC peptide conjugates are shown as examples in Figure 7. DISCUSSION

Preparation of Conjugates. RC-160 and [Tyr3]octreotide not only have potential clinical utility in their own right but they also represent model compounds which make them ideal for comparison of conjugation and radiolabeling strategies. Both are small octapeptides cyclized through a disulfide bridge and both contain a critical lysine residue, which is essential to preserve in order to retain receptor affinity. The use of a BFC approach for preparation of conjugates for 99mTc labeling thus requires protection of the lysine-epsilon amino group with a cleavable group like BOC, and conjugation and labeling conditions must be chosen which do not put the disulfide at risk. In addition, many BFCs additionally contain thiols for chelating 99mTc that also require protection. In this case, the number of protection strategies is very limited. The thiol-protecting group needs to be stable against the slightly alkaline conditions used in the conjugation reaction and also strong acidic conditions during the cleavage of the BOC-amino protection. At the same time, however, the thiol-protecting group must be readily cleaved during the labeling reaction in order to bind 99mTc. We tested three N3S-BFCs with different

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Figure 3. Radiochromatograms of labeling mixtures of different radiolabeled RC-160 conjugates, analyzed by HPLC method 1 (left column) and method 2 (right column). (A) Tricine-HYNIC-RC-160, (B) tricine/NA-HYNIC-RC-160, (C) EDDA-HYNIC-RC-160, (D) MAG3-RC-160, (E) N3S-RC-160.

Figure 4. Dependence of labeling yields for EDDA-HYNICRC-160 on stannous ion concentration. Conditions for labeling: 10 µg of peptide, 100 MBq 99mTc.

Figure 5. Cysteine challenge of 99mTc-labeled RC160 conjugates in comparison with [99mTc]tetraglycine: Radiochemical purity (%) vs time during challenge with 1000-fold excess of cystein over peptide.

protection groups on the thiol (benzoyl-MAG3, acetylNHS-MAG3, and ethylethoxy-N3S adipate). Only the benzoyl group remained stable during the coupling reaction, and both acetyl and ethylethoxy groups were cleaved, mainly during the TFA deprotection reaction. Using HATU for in situ activation of the free carboxylic acid in the BFC molecule, quantitative coupling yields of the BFC to the peptide and high overall yields on a very small scale (less than 1 mg of peptide) were achieved. Surprisingly, with the N-hydroxysuccinimideactivated ester (NHS-MAG3), which is a widely used system for activating the BFC, even at a high excess of BFC over the peptide, residues of the uncoupled peptide remained which would be likely to cause a problem in the preparation of high-specific activity preparations.

99m Tc Labeling and Stability. Since radiolabeling with technetium requires the use of a reductant, in this case stannous ion, labeling was performed at room temperature, whenever possible, to limit the possibility of a reduction of the disulfide bridge. Although our principle aim was not to achieve the quantitative levels of labeling required for clinical use, sufficiently high labeling efficiencies for in vitro and in vivo evaluations of all the prepared 99mTc-labeled peptide conjugates were achieved. High specific activity labeling, greater than 1 Ci/mmol could be achieved in all cases. Radiolabeling of HYNIC conjugates requires the use of additional coligands to complete the technetium coordination sphere and our results of labeling using aminocarboxylates as coligands was basically in concordance with the findings

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Figure 6. Competition curves of different RC-160 conjugates and calculated IC50 values, determined on AtT20-cell membranes, radioligand: [125I]SST14 (data points: bound ( sd).

Figure 7. Saturation assays of 99mTc-labeled EDDA-HYNIC conjugates with respective Scatchard plot, left RC-160 (A), right [Tyr3]octreotide (B).

of Liu et al. (27), who also addressed the problem of specific activity in more detail. Labeled HYNIC conjugates with varying coligands could better be distinguished on HPLC using a phosphate buffer, pH 6.2, gradient than with TFA. For tricine as coligand, a lower in vitro stability (with degradation of the complex especially after removal of excess tricine) and a greater number of isomers were found compared to EDDA. EDDA gave consistently lower labeling yields, but interestingly, we found that the yields were mainly dependent on the amount of stannous ions added and less on the EDDA

Decristoforo and Mather

concentration, pH, or temperature. Although not explored, this presumably relates to differences in the oxidation state of the technetium in the EDDA complex at different stannous ion concentrations. The high number of isomers and the relatively low stability of 99mTc-labeled HYNIC modified peptides with tricine as coligand can be overcome by adding a ternary ligand to the labeling solution. We preferred not to use the well characterized triphenylphosphines as described by Edwards et al. (28) since their strong reducing properties might affect the disulfide bond. We therefore chose pyridines such as nicotinic acid which were recently described as possible ternary ligands for the tricineHYNIC system (29). Nicotinic acid was compared with other pyridines such as pyridinesulfonic acid or pyridineacetic acid but did not show significantly different properties. To achieve quantitative labeling, heating at 100 °C for 15 min was necessary as ternary ligand complexes were not quantitatively obtained at room temperature. These complexes had a higher stability and lower retention time on HPLC, indicating lower lipophilicity. The number of isomers also was reduced to two compared to more than seven for the tricine binary complex. 99mTc labeling of the benzoyl-MAG3 conjugates at room temperature failed to achieve complexation, even at high pH [as previously described for MAG3 labeling (30)]. Efficient labeling required heating at 100 ˚C to remove the protection group, but even these conditions resulted in the presence of lipophilic impurities possibly due to insufficient deprotection and/or degradation of the peptide conjugate yielding mixed complexes as described for MAG3 itself (30-32). There was also concern that boiling the peptides in the presence of stannous ion might reduce the disulfide bridge. However, radioligand binding assays showed that the [99mTc]MAG3 conjugates retain high binding affinity despite this treatment and no difference in retention times or conjugate stability was observed. Studies on tricine/NA ternary ligand systems comparing ternary complexes obtained at room temperature and after heating at 100 °C also showed no difference in the binding affinity of both complexes. These studies prove the stability of the disulfide bridge against reduction by stannous ions even at elevated temperature. Receptor Binding and Tumor Targeting Properties. In general, all the conjugates prepared and their technetium complexes showed high-affinity specific binding to somatostatin receptors with no relevant differences in binding constants (IC50, Kd values) so far as their likely utility for imaging tumors is concerned. Binding constants of the 99mTc complexes were in the low nanomolar range, which can be considered sufficiently high for imaging SST-receptors in vivo. The compounds thus show promise for future tumortargeting studies initially in animal models and subsequently in patients. Further experiments also have to show the differences of the two investigated peptides described in this study concerning their imaging properties. However, recent publications on 111In-DTPA-RC-160 in comparison with octreotide have shown no advantages of RC-160 in humans (33) also no relevant brain uptake was found for the investigated RC-160 derivative (10). Whether this holds true for the described 99mTc-labeled compounds and whether the difference in receptor specificity might offer advantages for RC-160 derivatives remains to be answered.

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CONCLUSION

This paper describes the preparation of peptide conjugates using a variety of bifunctional chelate systems suitable for 99mTc labeling on a small scale using two model peptides. The methods described can be used to evaluate different labeling techniques without extensive and expensive peptide synthesis. Stable HYNIC binary and tertiary complexes in particular could be prepared easily and in high yield. All 99mTc complexes showed retained binding affinity to somatostatin receptors and further evaluation in vivo will explore their potential for imaging tumors using nuclear medicine techniques. ACKNOWLEDGMENT

The study described was supported by a EC-“Marie Curie Research Training Grant”, proposal E B4001GT963891 and the IAEA Coordinated Research Program “99mTc Labeled Peptides for Imaging of Peripheral Receptors”. Special thanks go to H. R. Maecke and M. Behe for their help in the peptide preparation and for sharing their experience in HYNIC labeling. In addition we would like to thank A. R. Fritzberg, D. J. Hnatowich, and O. K. Hjelstuen for the supply of bifunctional chelators. LITERATURE CITED Eising, E. G., Bier, D., Knust, E. J., and Reiners, C. (1996) Somatostatin-receptor scintigraphy. Methods, indications, results. Radiologe 36, 81-88. Lamberts, S. W., Chayvialle, J. A., and Krenning, E. P. (1993) The visualization of gastroenteropancreatic endocrine tumors. Digestion 54 (Suppl. 1), 92-97. Vekemans, M. C., Urbain, J. L., and Charkes, D. (1995) Advances in radio-imaging of neuroendocrine tumors. Curr. Opin. Oncol. 7, 63-67. Krenning, E. P., Kwekkeboom, D. J., Bakker, W. H., Breeman, W. A., Kooij, P. P., Oei, H. Y., van Hangen, M., Postema, P. T., de Jong M., and Reubi J. C. (1993) Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe] and [123I-Tyr]octreotide: The Rotterdam experience with more than 1000 patients. Eur. J. Nucl. Med. 20, 716-731. Srkalovic, G., Cai, R. Z., and Schally, A. V. (1990) Evaluation of receptors for somatostatin in various tumors using different analogues. J. Clin. Endocrinol. Metab. 70, 661-669. Liebow, C., Reilly, C., Serrano, M., and Schally, A. V. (1989) Somatostain analogues inhibit growth of pancreatic cancer by stimulating tyrosine phosphatase. Proc. Natl. Acad. Sci. U.S.A. 86, 2003-2007. Pinski, J., Milovanovic, T. Y., Yano, T., Hamaoui, A., Radulovic, S., Cai, R. Z., and Schally, A. V. (1992) Biological activity and receptor binding characteristics to various human tumors of acetylated somatostatin receptors. Proc. Soc. Exp. Biol. Med. 200, 49-56. Banks, W. A., Schally, A. V., Barrera, C. M., Fasold, M. B., Durham, D. A., Csernus, V. J., and Kastin, A. J. (1990) Permeability of the murine blood brain barrier to some octapeptide analogues of somatostatin. Proc. Natl. Acad. Sci. U.S.A. 87, 6762-6766. Breeman, W. A. P., Hofland, L. J., Bakker, W. H., van der Pluijm, M., van Koetsveld, P. M., de Jong, M., Setyono-Han, B., Kwekkeboom, D. J., Visser, T. J., Lamberts, S. W. J., and Krenning, E. P. (1993) Radioiodinated somatostatin analogue RC-160: preparation, biological activity, in vivo application in rats and comparison with [123I-Tyr3]octreotide. Eur. J. Nucl. Med. 20, 1089-1094. Breeman, W. A. P., Hofland, L. J., Pluijm, M.van-der, van Koetsveld, P. M., de Jong, M., Setyono-Han, B., Bakker, W. H., Kweekeboom, D. J., Visser, T. J., Lamberts, S. W. J., and Krenning, E. P. (1994) A new radiolabeled somatostatin analogue [111In-DTPA-D-Phe1]RC-160: preparation, biological activity, receptor scintigraphy in rats and comparison with [ 111In-DTPA-D-Phe1]octreotide. Eur. J. Nucl. Med. 21, 328335.

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