99mTc-Labeling and in Vivo Studies of a Bombesin Analogue with a

Feb 9, 1999 - 99mTc-Labeling and in Vivo Studies of a Bombesin Analogue with a Novel Water-Soluble Dithiadiphosphine-Based Bifunctional Chelating ...
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Bioconjugate Chem. 1999, 10, 254−260

99mTc-Labeling

and in Vivo Studies of a Bombesin Analogue with a Novel Water-Soluble Dithiadiphosphine-Based Bifunctional Chelating Agent

Srinivasa R. Karra,† Roger Schibli,†,‡ Hariprasad Gali,†,‡ Kattesh V. Katti,† Timothy J. Hoffman,†,§,| Chris Higginbotham,| Gary L. Sieckman,| and Wynn A. Volkert*,†,| Department of Radiology, Department of Chemistry, and Department of Medicine, University of MissourisColumbia, Columbia, Missouri 65211, and H. S. Truman Memorial Veterans Hospital, Columbia, Missouri 65212. Received August 19, 1998; Revised Manuscript Received December 2, 1998

Recent progress in the synthesis of water-soluble phosphine ligand systems and their corresponding 99m Tc complexes prompted the development of a new bifunctional chelating agent (BFCA) based on a tetradentate dithiadiphosphine framework (P2S2-COOH). The detailed synthesis of this new BFCA is described here. The corresponding 99mTc complex, 99mTc-P2S2-COOH, can be formed in >95% yield. To demonstrate the potential of this chelate to efficiently label peptides, 99mTc-P2S2-COOH was coupled to the N-terminal region of the truncated nine-amino acid bombesin analogue, 5-Ava-Gln-Trp-AlaVal-Gly-His-Leu-Met-NH2 [BBN(7-14)], to form 99mTc-P2S2-BBN(7-14). Conjugation to the peptide was performed in borate buffer (pH 8.5) by applying the prelabeling approach in yields of >60%. In competitive binding assays, using Swiss 3T3 mouse fibroblast cells against [125I-Tyr4]bombesin, ReP2S2-BBN(7-14) exhibited an IC50 value of 0.8 ( 0.4 nM. The pharmacokinetic studies of 99mTc-P2S2BBN(7-14) and its ability to target tissue expressing gastrin-releasing peptide (GRP) receptors were performed in normal mice. The 99mTc-P2S2-BBN(7-14) exhibited fast and efficient clearance from the blood pool (0.6 ( 0.1% ID, 4 h postinjection) and excretion through the renal and hepatobiliary pathways (56.4 ( 8.2 and 28.1 ( 7.9% ID, 4 h postinjection, respectively). Significant uptake in the pancreas was observed (pancreatic acini cells express bombesin/GRP receptors), producing pancreas:blood and pancreas:muscle ratios of ca. 22 and 80, respectively, at 4 h postinjection.

INTRODUCTION

Small receptor-avid peptides have attracted considerable interest for use in thrombus, inflammation, and tumor imaging and tumor treatment (1, 2, and references cited therein). A number of malignant tumors overexpress certain types of receptors on their surface which make them potential targets for imaging and therapy with radiolabeled synthetic antagonists and agonists. The 14-amino acid peptide bombesin (BBN) isolated from the skin of the amphibian Bombina and related gastrinreleasing peptides (GRP) exhibit an enhanced response in a variety of tumor tissues, e.g., in small cell lung, prostate, breast, and colon cancer (3-6). Analogues of bombesin with modified structures exhibited a similar or even higher affinity for these receptors (7-9). Today, corresponding synthetic peptides can be readily generated through automated solid phase techniques. For our studies, we produced and used the nine-amino acid bombesin analogue BBN(7-14) as a vehicle to target GRP receptors (Figure 1). The peptide BBN(7-14) has proved in the literature to be a potent GRP agonist. It can be radiolabeled with 123/131I or 105Rh for potential nuclear * To whom correspondence should be addressed: Department of Radiology, 409 Lewis Hall, University of Missouri, Columbia, MO 65211. E-mail: [email protected]. Phone: (573) 882-7695. Fax: (573) 882-6129. † Department of Radiology, University of MissourisColumbia. ‡ Department of Chemistry, University of MissourisColumbia. § Department of Medicine, University of MissourisColumbia. | H. S. Truman Memorial Veterans Hospital.

medical applications by virtue of its retention of a high binding affinity for GRP receptors (10, 11). We introduced an additional spacer function in the form of 5-aminopentanoic acid to the N-terminal region of the peptide to avoid interference of the chelating moiety with the receptor binding C-terminus of the peptide. Most of the bifunctional chelating agents reported in the literature for labeling biomolecules with 99mTc and 186/188Re are based on N S or N S frameworks. Some2 2 3 times, harsh reaction conditions (e.g., high temperatures and extended reaction times) are required to form the corresponding complexes with high specific activity or the labeling efficiency is low. Furthermore, these chelate moieties can exhibit often unfavorable physicochemical characteristics (e.g., high lipophilicity), which can play a critical role in maintaining high receptor binding affinity and in tumor uptake and also in the rate and route of clearance of a biomolecule from target and nontargeted tissues. The hydrazinonicotinamide (HYNIC) ligand system in combination with coligands offers today the only important alternative to the NS systems (12, 13). Our group is focused on the development of hydrophilic chelating moieties to achieve efficient clearance of the 99mTc or 188Re conjugate from the blood pool through the kidneys into the urine with low retention of the radioactivity in the liver and the kidney. Initial in vitro and in vivo studies with water-soluble dithiadiphosphine ligand systems (Figure 2, I) and their corresponding technetium and rhenium complexes, synthesized in our laboratory, showed that they are highly soluble and

10.1021/bc980096a CCC: $18.00 © 1999 American Chemical Society Published on Web 02/09/1999

99mTc-Labeling

of a Bombesin Analogue

Bioconjugate Chem., Vol. 10, No. 2, 1999 255 EXPERIMENTAL PROCEDURES

Figure 1. Bombesin analogue BBN(7-14).

Figure 2. Structure of the model, dithiadiphosphine ligand P2S2 (I) and structure of the bifunctional dithiadiphosphine ligand P2S2-COOH (4a) (II) used for the studies.

stable in aqueous solutions (14-16). The excellent water solubility is facilitated by the positive overall charge of the chelate and by the four hydroxymethyl groups on the phosphine functionalities. The combination of sulfur and phosphorus exhibits enhanced σ-donor and good π-accepting properties for stabilizing the metal center. We recently developed the P2S2-COOH (4b) BFCA based on the dithiadihydroxymethylphosphine framework shown in Figure 2 (II). The complex with 99mTc demonstrated excellent in vitro and in vivo stability and the desirable clearance and excretion properties (17). This study describes the synthesis and the radiolabeling of the new BFCA 4b and demonstrates the utility of 4b in labeling small bioactive peptides. The procedure of labeling BBN(7-14) with 99mTc-P2S2-COOH and the in vitro and in vivo behavior of the conjugate M-P2S2BBN(7-14) (M ) 99mTc or Re) are also presented.

Materials. All chemicals were obtained from either Aldrich Chemical Co. or Fisher Scientific. Diethyl (3bromopropyl)phosphonate was synthesized utilizing Arbuzov Reaction and refluxing triethyl phosphite in a 10-fold excess of 1,3-dibromopropane for 1 h and then purified by vacuum distillation. DL-6,8-Dihydrothioctic acid and ReO2(py)4Cl were synthesized according to the literature procedures (18, 19). The Fmoc-protected amino acids were purchased from Nova Biochem. The chemicals and solvents were of reagent grade and were used without further purification. Na[99mTcO4] was eluted from a 99Mo/99mTc generator (Mallinckrodt Medical, Inc.) using 0.9% saline. CF-1 normal mice were purchased from Taconic Labs. Swiss 3T3 mouse fibroblast cell lines were obtained from American Type Culture Collection (ATCC) and maintained and grown in the University of Missouri Cell and Immunology Care facilities. The [125I-Tyr4]bombesin was purchased from DuPont Merck Pharmaceuticals. The peptide was synthesized on a Synergy 432A personal peptide synthesizer from Applied Biosystems. Nuclear magnetic resonance spectra were recorded on a Bruker ARX-300 spectrometer. The 1H and 13C chemical shifts are reported relative to residual solvent protons as a reference, while the 31P shifts are reported relative to an external reference of 85% H3PO4. IR spectra were recorded on a Mattson Galaxy Series FTIR 3000 spectrometer. FAB-mass spectral analyses were performed by the mass spectrometry laboratories at Washington University (St. Louis, MO). HPLC analyses were performed on a Waters 600E system equipped with a Waters 486 tunable absorption detector, a radiometric detector system, and a Waters 746 Data Module integrating recorder. HPLC solvents consisted of H2O containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B). For the radiochemical experiments (analysis and purification), a C-18 Hamilton PRP-1 column (10 µm, 150 mm × 4.1 mm) was used. The HPLC gradient system started with 95% A/5% B from 0 to 2 min followed by a linear gradient from 95% A/5% B to 50% A/50% B from 2 to 17 min. The gradient remained at 50% A/50% B for 9 min before ramping back to 95% A/5% B at 26 min. The flow rate was 1.5 mL/min. For the purification and quality control of the peptide, a C-18 Phenomenex Jupiter column (250 mm, 10 µm) was used. The gradient started with a linear gradient from 95% A/5% B to 30% A/70% B over the course of 25 min. Preparation of Compound 1. A suspension of 60% NaH in mineral oil (6.5 g, 0.16 mol) was placed in a 1 L two-necked flask fitted with a septum and a N2 inlet. The mineral oil was extracted with dry hexane (3 × 25 mL). The remaining NaH was suspended in dry THF (100 mL). Dihydrolipoic acid (10.5 g, 0.05 mol) in THF (15 mL) was added dropwise under N2 via a syringe at 0 °C. The suspension was stirred for 30 min. Diethyl (3-bromopropyl)phosphonate (27.2 g, 0.10 mol) in THF (200 mL) was added slowly while the mixture was constantly being stirred. After 1 h, the excess NaH was quenched with 5 N HCl (50 mL). THF was removed under reduced pressure, and 500 mL of dichloromethane was added to the residue. The organic layer was washed with water (3 × 25 mL) and brine (100 mL) and dried over anhydrous Na2SO4. After removal of the solvent, the crude product was purified on a silica gel column (98:2 dichloromethane/ methanol). The yield was 25.6 g (90%): LR-FAB-MS m/z 565.4 [M + 1]+; IR (NaCl cell) 1724 cm-1 (s, νCdO); 1H NMR (CDCl3) δ 4.17-4.13 (m, 8H), 2.79-2.66 (m, 8H),

256 Bioconjugate Chem., Vol. 10, No. 2, 1999

2.36 (t, 2H), 2.15-1.99 (m, 4H), 1.90-1.3 (m, 8H), 1.36 (t, 12H); 13C NMR (CDCl3) δ 180.4, 61.4 (d, J ) 6.4 Hz), 61.3 (d, J ) 6.4 Hz), 44.5, 36.8, 34.5 (d, J ) 13.8 Hz), 32.3 (d, J ) 18.2 Hz), 30.5 (d, J ) 18.2 Hz), 28.8, 26.4, 25.57, 24.23 (d, J ) 141.4 Hz), 24.18 (d, J ) 141.4 Hz), 22.6 (d, J ) 4.4 Hz), 22.2 (d, J ) 4.4 Hz), 16.2 (d, J ) 6.0 Hz); 31P NMR (CDCl3) δ 31.8 (s), 31.6 (s). Preparation of Compound 2. N-Methylmorpholine (2.3 g, 0.022 mol) and isobutyl chloroformate (3.1 g, 0.022 mol) were added to a solution of 1 (11.3 g, 0.02 mol) in dry THF (100 mL) at -15 °C under N2. After 10 min, a solution of aniline (2.0 g, 0.022 mol) in THF (25 mL) and triethylamine (2.2 g, 0.22 mol) in THF (25 mL) were added at -15 °C. The mixture was allowed to warm to room temperature and stirred for 2 h. The precipitate of N-methylmorpholine hydrochloride was filtered, and the solvent was removed. The crude product was purified on a neutral alumina column (70:30 chloroform/hexane). The yield was 10.9 g (85%): LR-FAB-MS m/z 640.3 [M + 1]+; IR (NaCl cell) 1657 cm-1 (s, νCdO);1H NMR (CDCl3) δ 8.85 (br s, 1H), 7.62 (d, 2H, J ) 7.6 Hz), 7.28 (dd, 2H, J1 ) 12.0 Hz, J2 ) 7.6 Hz), 7.05 (t, 1H, J1 ) J2 ) 7.4 Hz), 4.09 (m, 8H), 2.75-2.52 (m, 8H), 2.4-2.35 (2H), 2.0-1.5 (m, 15H), 1.33 (t, 6H, J1 ) J2 ) 7.1 Hz), 1.32 (t, 6H, J1 ) J2 ) 7.1 Hz); 13C NMR (CDCl3) δ 171.7, 138.7, 128.4, 123.3, 119.5, 61.4 (d, J ) 5.9 Hz), 44.4, 36.9, 34.5, 34.3, 32.2 (d, J ) 17.7 Hz), 30.8 (d, J ) 17.7 Hz), 28.7, 26.0, 24.2 (d, J ) 140.5 Hz), 24.1 (d, J ) 142.6 Hz), 22.7 (d, J ) 4.2 Hz), 22.2 (d, J ) 4.3 Hz), 16.2 (d, J ) 5.9 Hz); 31P NMR (CDCl3) δ 31.5 (s), 31.4 (s). Preparation of Compound 3. Compound 2 (6.4 g, 0.01 mol) was dissolved in dry diethyl ether (100 mL) and the mixture placed in a 500 mL flask fitted with a reflux condenser. The solution was cooled to 0 °C, and 1 M LiAlH4 in ether (15 mL, 0.15 mol) was added dropwise via a syringe under N2. After the mixture was stirred at room temperature for 30 min, the reaction mixture was diluted with diethyl ether (200 mL). Then, a saturated solution of Na2SO4 (25 mL) was added at 0 °C to quench the excess LiAlH4. The organic layer was cannulated and the ether removed under reduced pressure at room temperature to give the crude diphosphine 3, which was used without further purification: 31P NMR (CDCl3) δ -137.5 (s). Preparation of Compound 4a. The crude diphosphine 3 was dissolved in degassed ethanol (15 mL). A solution of 37% formaldehyde (9 g, 0.1 mol) in ethanol (5 mL) and 5 mL of 30% HCl was added, and the mixture was stirred for 10 min under N2 at room temperature and then refluxed for 5 h. The solvents were removed under reduced pressure to give the crude (hydroxymethyl)phosphonium chloride 4a. The crude product was purified with a reverse phase Sep-Pak C-18 column using a water/methanol gradient. The yield was 3.0 g (45%): LR-FAB-MS m/z 573.6 [M - Cl]+; 1H NMR (D2O) δ 4.49 (m, 12H), 2.75-2.5 (m, 7H), 2.45-2.20 (m, 6H), 2.0-1.20 (m, 12H); 13C NMR (D2O) δ 180.0, 51.1 (d, J ) 54.5 Hz), 45.1, 34.8, 34.7, 32.7 (d, J ) 15.9 Hz), 31.2 (d, J ) 15.8 Hz), 29.3, 26.6, 25.2, 22.4 (d, J ) 4.1 Hz), 21.9 (d, J ) 4.1 Hz), 13.7 (d, J ) 41.2 Hz), 13.6 (d, J ) 41.1 Hz); 31P NMR (D2O) δ 28.6 (s). Preparation of Compound 4c. A solution of compound 4a (901 mg, 1.48 mmol) in water (100 mL) was added to a solution of [ReO2(py)4]Cl (py ) pyridine) (200 mg, 0.37 mmol) in water (25 mL), and the reaction mixture was refluxed for 5 h. The reaction solution was concentrated and purified on a reverse phase Sep-Pak C-18 column (35 cm3, 10 g) using a water/methanol gradient to obtain pure compound 4c as a purple viscous

Karra et al.

Figure 3. 99mTc HPLC traces of the products formed during the labeling procedure: (A) 99mTc-P2S2-COOH, (B) 99mTc-P2S2PFP, and (C) 99mTc-P2S2-BBN(7-14). A reverse phase C-18 column was used for the characterization and purification of the compounds.

oily liquid. The yield was 148 mg (55% based on Re): LRFAB-MS m/z 694.9 [M - Cl]+; IR (NaCl cell) 3333 (bs, νO-H), 1724 (s, νCdO), 912 (s, νsOdRedO), 795 cm-1 (s, νasOdRedO); 1H NMR (D2O) δ 4.30 (m, 8H), 3.43 (br s, 4H), 3.20 (br s, 1H), 2.90 (br t, 2H), 2.45 (br m, 5H), 2.19 (br t, 6H), 1.66 (br m, 2H), 1.31 (br m, 4H); 13C NMR (D2O) δ 179.1 (s), 59.4 (s), 58.9 (s), 58.5 (d), 58.0 (d), 49.9 (s), 38.4 (s), 37.2 (s), 34.0 (s), 33.3 (s), 33.1 (s), 30.1 (s), 24.5 (s), 24.2 (s), 20.9 (s), 17.8 (d), 17.5 (d); 31P NMR (D2O) δ -7.9 (d, JPP ) 12 Hz), -8.8 (d, Jpp ) 12 Hz). 99m Tc-Labeling of 4b. For the preparation of the ligand, 5 mg (8.2 µmol) of 4a was dissolved in 1 mL of H2O. One hundred microliters of a 1 M NaHCO3 buffer solution (pH 9) was added to produce 4b. Thirty microliters of the stock solution of 4b was added to 500 µL of Na[99mTcO4] in saline and the mixture vortexed. The technetium was reduced by addition of 10 µL of a degassed, presaturated stannous tartrate solution to form 99mTc-P S -COOH within 10 min at room temperature. 2 2 The formation of the product was confirmed by reverse phase HPLC (>95% yield). The retention time of Na[99mTcO4] was 1.2 min, whereas 99mTc-P2S2-COOH had a retention time of 10.9 min (Figure 3, I). Activation of the Acid Functionality of 99mTcP2S2-COOH. The carboxylic acid group of 99mTc-P2S2COOH was activated with pentafluorophenol (PFP) using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide methiodide (EDC) to form 99mTc-P2S2-PFP ester. Ten milligrams of EDC (0.3 mmol) and 6 mg of PFP (0.3 mmol in 100 µL of acetonitrile) were added to 500 µL of a freshly prepared 99mTc-P2S2-COOH solution in a 10 mL test tube. The reaction mixture was vortexed for 1 min and kept at room temperature for 20 min. The solution was centrifuged for 2 min, and the aqueous layer was separated from the oily drops of PFP containing >80% of the initial activity in the form of 99mTc-P2S2-PFP. The product was separated from the excess PFP by HPLC after dissolving the oily drops in 200 µL of acetonitrile. The activated ester had a retention time of 18.1 min (Figure 3, II), whereas PFP had a retention of 20.4 min (monitored at 254 nm) on the reverse phase HPLC system. The aqueous layer contained 20% of the initial radioactivity in form of 99mTc-P2S2-PFP (ca. 60%) and some unreacted 99mTc-P2S2-COOH (ca. 40%). Coupling of 99mTc-P2S2-PFP Ester to BBN(7-14). The pH of a previously HPLC-purified solution of 99mTc-

99mTc-Labeling

of a Bombesin Analogue

Bioconjugate Chem., Vol. 10, No. 2, 1999 257 Table 1. Biodistribution (% ID per Organ)a of 99mTc-P S -BBN(7-14) in Normal Mice as a Function of 2 2 Time after Intravenous Administration

Figure 4. Competitive binding of peptide Re-P2S2-BBN(7-14) vs [125I-Tyr4]bombesin using Swiss 3T3 mouse fibroblast cells. Competitive binding of bombesin and Neuromedin B vs [125ITyr4]bombesin in the same cell line performed as controls which have IC50 values of 4 and 1000 nM, respectively (22).

P2S2-PFP was adjusted to 8.5 using borate buffer (0.1 M). Approximately 1 mg of BBN(7-14) (0.9 µmol) was added to a 500 µL aliquot of a 99mTc-P2S2-PFP solution and the reaction mixture vortexed for 1 min and incubated for 30 min at room temperature. The reaction was monitored by reverse phase HPLC. The final product [99mTc-P2S2-BBN(7-14)] was separated from excess BBN(7-14) using the standard HPLC gradient. The conjugate exhibited a retention time of 14.6 min (Figure 3, III), whereas the free BBN(7-14) exhibited a retention time of 12.3 min. The coupling yields were usually between 70 and 80% as determined radiometrically. Preparation of the Re Analogue of the Conjugate. The Re-P2S2-BBN(7-14) was synthesized by solid phase peptide synthesis. ReO2-P2S2-COOH was introduced in the last step of the peptide synthesis. After the peptide was cleaved from the resin, the product was purified by reverse phase HPLC. The molecular constitution of ReP2S2-BBN(7-14) was confirmed by FAB mass spectroscopy. The FAB mass spectral analysis confirmed the presence of a [OdRedO]+ core. This is in agreement with the observation made by Smith et al. for similar Re(V) complexes with P2S2 ligands (15). The conjugate exhibited a retention time of 15.2 min (monitored at 350 nm): FABMS m/e (M + 1) calcd 1616.89, found 1616.4. In Vitro Binding Assay of Re-P2S2-BBN(7-14). In vitro BBN-receptor binding capabilities of the labeled peptide were assessed utilizing displacement binding assays conducted at 37 °C in Swiss 3T3 fibroblast cells with a method similar to that designed by Mahmoud et al. (20). The IC50 value was determined for the nonradiolabeled Re-P2S2-BBN(7-14) analogue by analyzing displacement of bound [125I-Tyr4]bombesin (Figure 4). In vitro binding assays were performed under identical conditions using active BBN and Neuromedin B as positive and negative controls, respectively (Figure 4). Biodistribution of 99mTc-P2S2-BBN(7-14) in Normal Mice. Normal CF-1 mice (average weight, 25 g) were used for the biodistribution studies (Tables 1 and 2). The pH of the HPLC-purified 99mTc-P2S2-BBN(7-14) solution was adjusted to physiological conditions using a 0.01 M phosphate buffer (pH 7.4). Aliquots (80-100 µL) of the labeled peptide solution (55-75 kBq) were injected into each animal via the tail vein. Tissues and organs were excised from the sacrificed animals 30 min, 1 h, and 4 h postinjection. The organs and tissue were weighed; the activity was counted in a NaI counter, and the percent injected dose per organ and the percent injected dose per gram were calculated. The % ID in whole blood was estimated assuming a blood volume of 6.5% of the total

organ

0.5 hb

1h

4h

brain bloodc heart lung liver intestines stomach kidneys pancreas urine

0.04 ( 0.02 1.7 ( 0.03 0.1 ( 0.01 0.2 ( 0.03 10.0 ( 1.50 30.8 ( 1.82 0.7 ( 0.33 2.1 ( 0.10 6.8 ( 0.90 36.3 ( 2.80

0.01 ( 0.01 0.9 ( 0.50 0.1 ( 0.02 0.1 ( 0.02 7.4 ( 1.10 33.1 ( 3.95 0.7 ( 0.11 1.7 ( 0.10 4.7 ( 0.53 43.8 ( 2.90

0.03 ( 0.3 0.6 ( 0.07 0.1 ( 0.02 0.1 ( 0.03 5.3 ( 0.80 28.1 ( 7.94 0.4 ( 0.10 1.3 ( 0.15 2.4 ( 0.25 56.5 ( 8.20

a Values represent the mean ( SD (n ) 5) of the percent injected dose per organ. b n ) 4. c The total blood volume is estimated to be 6.5% of the body weight.

Table 2. Biodistribution (% ID per Gram)a of 99mTc-P S -BBN(7-14) in Normal Mice as a Function of 2 2 Time after Intravenous Administration organ

0.5 hb

1h

4h

brain bloodc heart lung liver kidneys pancreas muscle

0.04 ( 0.01 1.0 ( 0.20 0.4 ( 0.03 1.1 ( 0.16 6.9 ( 1.11 6.3 ( 0.17 21.3 ( 2.10 0.3 ( 0.10

0.1 ( 0.1 0.5 ( 0.28 0.4 ( 0.20 0.8 ( 0.23 5.1 ( 0.45 4.8 ( 0.48 17.4 ( 3.40 0.1 ( 0.05

0.11 ( 0.07 0.4 ( 0.06 0.1 ( 0.02 0.4 ( 0.20 4.0 ( 0.86 3.7 ( 0.44 8.5 ( 1.25 0.1 ( 0.02

a Values represent the mean ( SD (n ) 5) of the percent injected dose per gram unless otherwise indicated. b n ) 4. c The total blood volume is estimated to be 6.5% of the body weight.

Table 3. BBN Receptor Blocking Studies in Normal Mice (% ID per Organ)a organ

unblocked

blockedb

organ

unblocked

blockedb

stomach 0.6 ( 0.10 0.3 ( 0.10 liver 9.1 ( 2.40 8.7 ( 1.67 bloodc 1.3 ( 0.60 1.4 ( 0.34 kidneys 2.0 ( 0.20 1.7 ( 0.10 urine 44.4 ( 7.30 55.1 ( 2.60 pancreas 6.5 ( 0.25 0.6 ( 0.33 intestines 27.0 ( 6.90 23.6 ( 4.43 a n ) 5. b One milligram of BBN(7-14) was subcutaneously administered 35 min prior to the injection of 99mTc-P2S2BBN(7-14). c The total blood volume is estimated to be 6.5% of the body weight.

Table 4. BBN Receptor Blocking Studies in Normal Mice (% ID per Gram)a organ

unblocked

blockedb

organ

unblocked

blockedb

bloodc

0.7 ( 0.36 0.2 ( 0.10 6.0 ( 1.10

0.8 ( 0.20 0.1 ( 0.10 5.9 ( 1.10

kidneys pancreas

5.1 ( 0.50 20.2 ( 1.70

4.5 ( 0.40 1.7 ( 1.20

muscle liver

a n ) 5. b One milligram of BBN(7-14) was subcutaneously administered 35 min prior to the injection of 99mTc-P2S2BBN(7-14). c The total blood volume is estimated to be 6.5% of the body weight.

body weight. Receptor blocking studies were also carried out where excess BBN(7-14) was administered to animals for in vivo saturation of GRP receptors prior to injection of 99mTc-P2S2-BBN(7-14) (Tables 3 and 4). In these studies, each animal received a subcutaneous injection of 1 mg of BBN(7-14) dissolved in 100 µL of a mixture of saline/acetonitrile/DMF (90:5:5) 35 min prior to the injection of 99mTc-P2S2-BBN(7-14). Control (unblocked) animals received an injection of 100 µL of the solvent mixture without BBN(7-14) 35 min prior to the injection of 99mTc-P2S2-BBN(7-14). The animals were sacrificed 30 min postinjection and the tissues removed, weighed, and counted as previously described.

258 Bioconjugate Chem., Vol. 10, No. 2, 1999

Karra et al.

Scheme 1. Synthesis of the P2S2-COOH Ligand and Its Complexation with Re(V)

Scheme 2. Procedure for Labeling BBN(7-14) with

99mTc-P

RESULTS AND DISCUSSION

The crucial molecular building block for the bifunctionalization of the P2S2 ligand system is the commercially available DL-R-lipoic acid. The bifunctional ligand 4a was produced in four steps outlined in Scheme 1. The synthetic procedure for forming the P2S2 backbone is identical with that applied for the underivatized P2S2 ligand (Figure 2, I) described by Smith et al. (15). The carboxylic acid was protected with aniline for the reduction step of the phosphonate groups using a procedure described by Barton et al. (21). The (hydroxymethyl)-

2S2-COOH

phosphonium chloride 4a was prepared by formylation of the P-H bonds of 3 in oxygen-free ethanol in the presence of excess aqueous formaldehyde and hydrochloric acid. In the same step, the carboxylic acid function was deprotected. The new compounds, 1, 2, and 4b, were characterized by 1H, 13C, and 31P NMR and IR spectroscopy and mass spectrometry. Compound 3, which is an intermediate, was characterized by 31P NMR spectroscopy only. Ligands 4a,b are highly soluble in water. The formation of the phosphonium salt of the ligand 4 has two advantages.

99mTc-Labeling

of a Bombesin Analogue

(1) Purification of the phosphonium salt 4a is more convenient than that of 4b. (2) Due to the formation of the phosphonium salt, the PIII centers are protected against oxidation, and the ligand can be stored under aerobic conditions for a long period of time. This is an important advantage for a kit formulation in which ligand systems with (hydroxymethyl)phosphine groups are used. 4a can be readily converted into 4b by adding small amounts of a base before complexation with a metal center. It is noteworthy that 4b is oxidatively stable even under high levels of dilution in aqueous media for several hours, which was confirmed in the 31P NMR experiments. The prelabeling approach was selected to radioactively conjugate the peptide, as difficulties were encountered in activating the carboxylic acid in 4a,b. 99mTc-P2S2COOH can be produced either by direct reduction of Na[99mTcO4] with stannous tartrate in the presence of 4b (total ligand concentration in the samples is on the order of 10-5 M) or by the ligand exchange reaction starting with [99mTc]gluconate and 4b at room temperature (17). In both cases, the complex was efficiently produced (>95%) as a single species which was confirmed by HPLC (Figure 3, I). However, we were only able to form the active ester complex 99mTc-P2S2-PFP in good yields in the absence of the trans-chelating gluconate ligand. Therefore, only direct reduction of the 99mTc center in the presence of 4b was employed for these studies. 99mTcP2S2-PFP was produced in good yields (>90%) in a 0.9% saline solution using an excess of EDC and PFP at room temperature. The HPLC profile again showed the formation of a single species (Figure 3, II). 99mTc-P2S2-PFP is remarkably stable under acidic conditions and could be purified and collected by HPLC where only minor reformation of 99mTc-P2S2-COOH was observed 2 h after collection of 99mTc-P2S2-PFP. For the final labeling step of BBN(7-14), the pH of the purified 99mTc-P2S2-PFP solution was adjusted to 8.5 using a 0.1 M borate buffer. An aliquot of 500 µL of the 99mTc-P2S2-PFP solution was added to 1 mg of the peptide and the mixture incubated at room temperature. HPLC profile III, shown in Figure 3, revealed that after 30 min 99mTc-P2S2-BBN(7-14) was formed as a single species in approximately 80% yield. Extension of the incubation time did not lead to the complete formation of the conjugate due to the decomposition of the remaining 99mTc-P2S2-PFP back to 99m Tc-P2S2-COOH. The labeled peptide was separated from unlabeled BBN(7-14) by HPLC and collected into 0.1 M phosphate buffer to achieve physiological pH conditions for use in animal studies. To evaluate the biological activity of the labeled peptide in vitro, the ability of the nonradioactive conjugate ReP2S2-BBN(7-14) to bind to the GRP receptors on Swiss 3T3 mouse fibroblast cells was examined. The cell binding studies were carried out using [125I-Tyr4]bombesin as the competitive BBN analogue. The results of the experiments shown in Figure 4 demonstrated the high binding affinity of Re-P2S2-BBN(7-14). The IC50 value was determined to be 0.8 ( 0.4 nM and is comparable with those of bombesin and its analogues that are considered to have sufficiently high binding affinity for potential use as targeting vectors for GRP receptor-expressing cancers in vivo. Results of the in vivo biodistribution experiments with 99mTc-P S -BBN(7-14) in normal mice are summarized 2 2 in Tables 1 and 2. The conjugate 99mTc-P2S2-BBN(7-14) exhibited rapid clearance from the blood pool (0.6 ( 0.07% ID at 4 h postinjection) and nontargeted tissues. The excretion of the radioactivity from the body occurs in both the renal (56.5 ( 8.20% ID, 4 h postinjection) and

Bioconjugate Chem., Vol. 10, No. 2, 1999 259

the hepatobiliary (liver and intestines, ca. 35% ID, 4 h postinjection) pathways. Only small amounts of activity were retained in the kidneys 4 h postinjection (1.3 ( 0.15% ID). There was some retention of the conjugate in the liver (5.3 ( 0.80% ID, 4 h postinjection). The amount of radioactivity found in the stomach, an indication of an in vivo decomposition of a 99mTc conjugate and the reformation of [99mTcO4]-, was negligible (0.4 ( 0.10% ID, 4 h postinjection). This provides important evidence that the P2S2 ligand system is capable of stabilizing 99mTc even under in vivo conditions. A significant uptake of radioactivity was observed in the pancreas (4.7 ( 0.53 or 17.4 ( 3.40% ID/g, 1 h postinjection), demonstrating the ability of 99mTc-P2S2-BBN(7-14) to target in vivo GRPexpressing cells (acini cells in the pancreas express GRP receptors that are accessible to the blood stream). The pancreas:muscle and pancreas:blood ratios (based on % ID per gram) reached a maximum of 80 and 22, respectively, at 4 h postinjection. Furthermore, receptor blocking studies with BBN(7-14) confirmed the specificity of BBN(7-14) or 99mTc-P2S2-BBN(7-14) toward the GRP receptors. The difference in the uptake of 99mTc-P2S2BBN(7-14) in the pancreas with and without subcutaneous injection of 1 mg of BBN(7-14) is significant (0.6 ( 0.33 and 6.5 ( 0.25% ID, respectively). The uptake of the 99mTc-labeled peptide in all other organs and tissue was not affected by the blocking experiment (Tables 3 and 4). CONCLUSION

The detailed synthesis and characterization of the first water-soluble, bifunctional chelator 4a,b based on a P2S2 framework is presented. Furthermore, the ability of the novel BFCA to form an in vitro and in vivo stable complex with 99mTc and its utility for the radiolabeling of receptoravid peptides are demonstrated. Although the prelabeling procedure with 99mTc-P2S2-COOH is time-consuming, a P2S2 chelating system might, in the future, serve as a potential alternative to the known tetradentate N2S2, N3S, and HYNIC ligand systems. In fact, recent experiments showed that the synthetic precursor 3 (after deprotection of the acid function) can be efficiently attached to the N-terminal function of peptides. The hydroxymethyl functions are subsequently introduced to the [H2P]2S2 peptide conjugate to achieve the favorable in vivo characteristics of the chelating moiety. This enables in the future a more convenient postlabeling approach of a P2S2-functionalized peptide. ACKNOWLEDGMENT

This work was supported by NIH Grant R01-CA72421, DuPont-Merck Pharmaceuticals, and the Departments of Chemistry and Radiology of the University of Missouris Columbia. LITERATURE CITED (1) Liu, S., Edwards, D. S., and Barrett, J. A. (1997) 99mTc Labeling of High Potent Small Peptides. Bioconjugate Chem. 8, 621-636. (2) Lister-James, J., Moyen, B. R., and Dean, T. (1996) Small peptides radiolabeled with 99mTc. Q. J. Nucl. Med. 40, 221233. (3) Moody, T. W., Carney, D. N., Cuttita, F., Quattrocchi, K., and Minna, J. D. (1985) High affinity of receptors for bombesin/GRP-like peptides on human small cell cancer. Life Sci. 37, 105-113. (4) Qin, Y., Ertl, T., Cai, R. Z., Halmos, G., and Schally, A. V. (1994) Inhibitory effect of bombesin receptor antagonist RC-

260 Bioconjugate Chem., Vol. 10, No. 2, 1999 3095 on the growth of human pancreatic cancer cells in vivo and in vitro. Cancer Res. 54, 1035-1041. (5) Pinski, J., Schally, A. V., Halmos, G., Yano, T., Szepeshazi, K., and Groot, K. (1994) Somatostatin analogues and bombesin/gastrin releasing peptide antagonist RC-3095 inhibit the growth of glioblastomas in vitro and in vivo. Cancer Res. 54, 5895-5901. (6) Radulovic, S., Miller, G., and Schally, A. V. (1991) Inhibition of growth of HT-29 human colon cancer xenografs in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095). Cancer Res. 51, 6006-6009. (7) Jensen, R. T., Jones, S. W., Folkers, K., and Gardner, J. D. (1984) A synthetic peptide that is a bombesin receptor antagonist. Nature 309, 61-63. (8) Safavy, A., Khazaeli, M. B., Qin, H., and Buchsbaum, D. J. (1997) Synthesis of Bombesin Analogues for Radiolabeling with Rhenium-188. Cancer Suppl. 80, 2354-2359. (9) Baidoo, K. E., Lin, K.-S., Zhan, Y., Finley, P., Scheffel, U., and Wagner, H. N., Jr. (1998) Design, Synthesis, and Initial Evaluation of High-Affinity Technetium Bombesin Analogues. Bioconjugate Chem. 9, 218-225. (10) Hoffman, T. J., Sieckman, G. L., and Volkert, W. A. (1995) Targeting Small Cell Lung Cancer Using Iodinated Peptide Analogs. J. Labelled Comp. Radiopharm. 37, 321-323. (11) Li, N., Hoffman, T. J., Sieckman, G. L., Ochrymowycz, L. A., Higginbotham, C., Strutman, M., Volkert, W. A., and Ketring, A. R. (1996) In vitro and in vivo characterization of Rh-105-Tetrathiamacrocycle conjugate of a bombesin analogue. J. Nucl. Med. 37 (Suppl.), 61P. (12) Barrett, J. A., Crocker, A. C., Damphousse, D. J., Heminway, S. J., Liu, S., Edwards, S. D., Lazewatsky, J. L., Kagan, M., Mazaika, T. J., and Carroll, T. R. (1997) Biological Evaluation of Thrombus Imaging Agents Utilizing WaterSoluble Phosphines and Tricine as Coligands When Used to Label a Hydrazinonicotinamide-Modified Cyclic Glycoprotein IIb/IIa Receptor Antagonist with 99mTc. Bioconjugate Chem. 8, 155-160. (13) Edwards, S. D., Liu, S., Barrett, J. A., Harris, A. R., Looby, R. J., Ziegler, M. C., Heminway, S. J., and Carroll, T. R. (1997) New and Versatile Ternary Ligand Systems for Technetium Radiopharmaceuticals: Water-Soluble Phosphines and Tricine as Coligands in Labeling a HydrazinonicotinamideModified Cyclic Glycoprotein IIb/IIa Receptor Antagonist with 99mTc. Bioconjugate Chem. 8, 146-154. (14) Smith, C. J., Reddy, V. S., Karra, S. R., Katti, K. V., and Barbour, L. J. (1997) Synthesis and Coordination Chemistry

Karra et al. of the First Water-Soluble Dithio-Bis(phosphine) Ligands [(HOH2C)2P(CH2)2S-X-S(CH2)P(CH2OH)2] (X ) (CH2)3 or C6H4). X-ray Crystal Structure of [Pd[(HOH2C)2P(CH2)2S(CH2)3S(CH2)P(CH2OH)2](Cl)2. Inorg. Chem. 36, 1786-1791. (15) Smith, C. J., Katti, K. V., Volkert, W. A., and Barbour, L. J. (1997) Synthesis and Characterization of Chemically Flexible, Water-Soluble Dithio-Bis(phosphine) Compounds: (HOH2C)2P(CH2)2S(CH2)3S(CH2)2P(CH2OH)2, (HOH2C)2P(CH2)2S(CH2)4S(CH2)2P(CH2OH)2, and (HOH2C)2P(CH2)3S(CH2)3S(CH2)3P(CH2OH)2. Systematic Investigation of the Effect of Chain Length on the Coordination Chemistry of Rhenium(V). X-ray Crystal Structure of [ReO2(HOH2C)2P(CH2)2S(CH2)3S(CH2)2P(CH2OH)2]2(Cl)2, [ReO2(HOH2C)2P(CH2)2S(CH2)4S(CH2)2P(CH2OH)2]2(ReO4)2,and[ReO2(HOH2C)2P(CH2)2S(CH2)3S(CH2)2P(CH2OH)2](Cl). Inorg. Chem. 36, 39283935. (16) Smith, C. J., Li, N., Katti, K. V., Higginbotham, C., and Volkert, W. A. (1997) In vitro and in vivo Characterization of Novel Water-Soluble Dithio-Bisphosphine 99mTc Complexes. J. Nucl. Med. Biol. 24, 685-691. (17) Schibli, R., Karra, S. R., Gali, H., Katti, K. V., Higginbotham, C., and Volkert, W. A. (1998) In vitro and in vivo Characterization of a Novel Bifunctionalized Water-Soluble Dithia-Bisphosphine 99mTc Complex. Radiochim. Acta (In press). (18) Wagner, A. F., Walton, E., Boxer, G. E., Pruss, M. P., Holly, F. W., and Folkers, K. (1956) Properties and derivatives of R-lipoic acid. J. Am. Chem. Soc. 78, 5079-5081. (19) Beard, J. H., Casey, J., and Murmann, R. K. (1965) The preparation and properties of [Re(amine)4O2]+ type ions. Inorg. Chem. 4, 797-803. (20) Mahmoud, S., Stanley, J., Taylor, J., Bogden, A., Moreau, J. P., Coy, D., Avis, I., Cuttitta, F., Mulshine, J. L., and Moody, T. W. (1991) [Psi 13, 14] bombesin analogues inhibit growth of small cell lung cancer in vitro and in vivo. Cancer Res. 51, 1798-1802. (21) Barton, F. H. R., Herve, Y., Potier, P., and Thierry, J. (1988) Manipulation of the carboxylic groups of R-amino acids and peptides using radical chemistry based on esters of N-hydroxy-2-thiopyridone. Tetrahedron 44, 5479-5486. (22) Moody, T. W., Venugopal, R., Hu, V., Gozes, Y., McDermed, J., and Leban, J. J. (1996) BW 1023490: A new GRP receptor antagonist for small-cell lung cancer cells. Peptides 17, 1337-1343.

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