624
Bioconjugate Chem. 2001, 12, 624−629
99mTc-Labeling
of a Hydrazinonicotinamide-Conjugated Vitronectin Receptor Antagonist Useful for Imaging Tumors
Shuang Liu,* D. Scott Edwards, Marisa C. Ziegler, Anthony R. Harris, Stuart J. Hemingway, and John A. Barrett Medical Imaging Division, DuPont Pharmaceuticals Company,331 Treble Cove Road, North Billerica, Massachusetts 01862 . Received February 5, 2001; Revised Manuscript Received March 29, 2001
This report describes the 99mTc labeling of a HYNIC-conjugated vitronectin receptor antagonist (SQ168 ) [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-AspD-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}). The ternary ligand complex [99mTc(SQ168)(tricine)(TPPTS)] (RP593) was prepared using a non-SnCl2-containing formulation. The corresponding 99Tc analogue, [99Tc]RP593, was also prepared and characterized by HPLC and LC-MS. A HPLC concordance experiment using RP593 and [99Tc]RP593 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data is completely consistent with the 1:1:1:1 composition for Tc:SQ168:tricine:TPPTS and provides direct evidence that the two radiometric peaks in the radio-HPLC chromatogram of RP593 are indeed due to the resolution of diastereomers. In an in vitro receptor binding assay, [99Tc]RP593 was shown to have comparable binding affinity for the vitronectin receptor to that of SQ168 itself.
INTRODUCTION
There is currently a considerable interest in labeling small biomolecules with 99mTc for the development of target specific imaging agents (1-10). For the last several years, we have been using 6-hydrazinonicotinamide (HYNIC) as a bifunctional coupling agent for the 99mTclabeling of small biomolecules (BM), including a GPIIb/ IIIa receptor antagonist for thrombus imaging (11-18), a chemotactic peptide (19), and LTB4 receptor antagonists for imaging infection and inflammation (20-22). It was found that the combination of HYNIC-BM, tricine, and TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate) produces a unique and versatile ternary ligand system which forms technetium complexes, [99mTc(HYNICBM)(tricine)(TPPTS)] with high solution stability and only two detectable isomeric forms. As a continuation of our efforts in developing new target-specific diagnostic radiopharmaceuticals, we now describe the 99mTc-labeling of a vitronectin receptor antagonist (Figure 1: SQ168 ) [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})cyclo{Lys-Arg-Gly-Asp-D-Phe}). The ternary ligand complex [99mTc(SQ168)(tricine)(TPPTS)] (Figure 1: RP593) was prepared using a nonSnCl2-containing formulation (23) and was characterized using a reversed phase HPLC method. The corresponding 99 Tc analogue, [99Tc]RP593, was also prepared and characterized by HPLC and LC-MS. A HPLC concordance experiment using RP593 and [99Tc]RP593 showed that the same complex was prepared at both the tracer and macroscopic levels. In an in vitro receptor binding assay, [99Tc]RP593 was shown to have comparable binding affinity for the vitronectin receptor as SQ168. Apparently, the attachment of the technetium chelate does not have significant effect on the receptor binding affinity. RP593 * To whom correspondence should be addressed: Tel: 978671-8696 (S.L.); FAX: 978-436-7500; e-mail:
[email protected].
has been evaluated in two spontaneous tumor models (cNeu oncomouse and dog) for its potential use as a new radiopharmaceutical for imaging tumors. The biological properties of RP593 have been described in our previous communications (24, 25). EXPERIMENTAL SECTION
Materials. Succinic acid, TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate), and tricine were purchased from Aldrich Chemical Co. TPPTS was purified according to the literature method (26). The cyclic pentapeptide, cyclo(Arg-Gly-Asp-D-Phe-Lys), was prepared according to the literature method (27). Synthesis of SQ168 has been reported as a separate communication (28). Na99mTcO4 was obtained from a commercial DuPont Pharma 99Mo/99mTc generator, N. Billerica, MA. Analytical Methods. HPLC methods used a HP 1100 system, a homemade NaI radiometric detector, a Zorbax C18 column (4.6 mm × 250 mm) at a flow rate of 1 mL/ min. In method 1, the mobile phase was isocratic at 0-18 min using 90% solvent A (95:5 (v:v) ) 0.025 M ammonium acetate buffer (pH ) 6.8):acetonitrile) and 10% solvent B (95:5 (v:v) ) acetonitrile: 0.025 M ammonium acetate buffer, pH ) 6.8), followed by an isocratic wash using 40% solvent A and 60% solvent B at 19-25 min. In method 2, the gradient mobile phase started from 92% solvent A (0.025 M succinate buffer, pH ) 5.0) and 8% solvent B (acetonitrile) to 80% solvent A and 20% solvent B at 25 min. The mobile phase was isocratic at 26-31 min using 60% solvent B. The ITLC method used Gelman Sciences silica gel ITLC paper strips and 1:1 mixture of acetone and saline as eluant. Using this ITLC method, the radiolabeled peptide and [99mTc]pertechnetate migrate while the [99mTc]colloid remains at the origin. LC-MS Methods. LC-MS spectral data were collected using a HP1100 LC/MSD system with an API-electrospray interface. The LC-MS method used a Zorbax C18 column (4.6 mm × 150 mm, 3.5 µm particle size) and a mobile phase gradient starting from 92% solvent A (10 mM ammonium acetate buffer, pH 7.0) and 8% solvent
10.1021/bc010012p CCC: $20.00 © 2001 American Chemical Society Published on Web 06/07/2001
99mTc-Labeling
of a Vitronectin Receptor Antagonist
Bioconjugate Chem., Vol. 12, No. 4, 2001 625
Figure 1. Structures of a HYNIC-conjugated vitronectin receptor antagonist (SQ168) and its ternary ligand technetium complex [99mTc(SQ168)(tricine)(TPPTS)]. Table 1. LMD Parameters Used for Characterization of RP593 detection mode: mass range: gain: fragmentor: gas temperature: drying gas flow: nebullizer pressure: V capillary: radio-detector: UV detector:
positive 600-2000 1.0 15 V 350 °C 13 L/min 60 psig (max.) 4000 V NaI λ ) 280 nm
B (methanol) to 100% B at 23 min at a flow rate of 1 mL/min. The MSD parameters are listed in Table 1. Synthesis of RP593. RP593 was prepared according to the published procedure (13) with a slight modification. To a lyophilized vial containing 5 mg of TPPTS, 6.5 mg of tricine, 40 mg of mannitol, 38.5 mg of disodium succinate hexahydrate, 12.7 mg of succinic acid, and 0.1 mg of pluronic acid (0.1%); pH ) 4.8) was added 0.5 mL of SQ168 solution (40 µg/mL in water). The vial was placed into a lead pig. To the vial was added 1.0 mL of Na[99mTcO4] solution (50 mCi/mL) in saline. The reconstituted vial was heated at 100 °C for 10 min in a leadshielded water bath. After heating, the vial was placed back into the lead pig and allowed to stand at room
temperature for ∼10 min. A sample of the resulting solution was analyzed by the radio-HPLC (method 1) and ITLC. Solution Stability Studies. For solution stability in the kit matrix, RP593 was first prepared, and samples of the resulting reaction were analyzed by HPLC (method 1) at t ) 0, 2, 4, and 6 h postlabeling. For HPLC-purified RP593, the two peaks at 9.3 and 12 min were collected into a 25 mL round-bottom flask. Solvents were removed using a rotary evaporator. The residue was dissolved in saline and samples of the resulting solution were analyzed by HPLC (method 1) at 0, 2, 4, and 6 h postpurification. [99Tc]RP593. To a clean 10 mL vial were added 1.5 mg of SQ168, 84 mg of tricine, 49 mg of TPPTS, 1.2 mg of NH4[99TcO4] in 0.4 mL of H2O, and 1.5 mL of 25 mM succinate buffer (pH ) 5.0). The reaction mixture was heated in a 100 °C water bath for 30 min. After the mixture was cooled to room temperature, the product was separated by HPLC (method 2). The collections of the two peaks due to [99Tc]RP593 were combined, and volatiles were removed under reduced pressure. The residue was then desalted using the same HPLC method using water as mobile phase A. Removal of the solvent gave the product, [99Tc]RP593.
626 Bioconjugate Chem., Vol. 12, No. 4, 2001
Immobilized Human Placental rvβ3 Receptor ELISA Assay. The placental Rvβ3 receptors were isolated according to the published method (29). The assay conditions were developed and validated using [125I]vitronectin. Assay validation included Scatchard format analysis (n ) 3) where receptor number (Bmax) and Kd (affinity) were determined. Three standards (vitronectin, Rvβ3 antibody, LM609, and Rvβ5 antibody, P1F6) have been evaluated for IC50 determination. Briefly, the method involves immobilizing previously isolated receptors in 96 well plates (EIA/RIA, CORNING Costar) and incubating overnight. The plates are then blocked with 1% bovine serum albumin. During this time, compounds to be tested were prepared in 10 nM biotinylated vitronectin ([B]Vn) solution and were dispensed into a reservoir plate. Blocking buffer is removed, wells were washed, and then compound/[B]Vn mixture was pipetted. Incubation is carried out for 30 min at room temperature. The unbound material was removed, and wells were washed. [B]Vn binding was qualified using goat anti-biotin alkaline phosphatase conjugated antibody (GAB). Unbound GAB was removed, wells were washed, and phosphatase enzyme assay was performed. Enzyme product was detected via spectrophotometry (λ ) 405 nm) for subsequent IC50 determination. All concentrations of the tested compound were run in triplicate. Four independent measurements were made for [99Tc]RP593. The IC50 value was calculated by fitting the percent inhibition values to a regression line. RESULTS AND DISCUSSION
Angiogenesis, the formation of new blood vessels, is a requirement for malignant tumor growth and metastasis (30-32). The angiogenic process depends on vascular endothelial cell migration and invasion, regulated by cell adhesion receptors. The integrin Rvβ3 (vitronectin receptor) is such a cell adhesion receptor and interacts with proteins and peptides containing the RGD tripeptide recognition sequence (33-42). In general, the level of expression of Rvβ3 receptor is low on most cell types and is greatly increased in remodeling and growing tissues (43). Many peptide vitronectin receptor antagonists have high binding affinity for the vitronectin receptor and have been shown to be able to inhibit neovascularization, tumor-induced angiogenesis, and tumor growth (40-46). Therefore, these vitronectin receptor antagonists can serve as targeting molecules for diagnosis and radiotherapy of a wide spectrum of tumors. Recently, Kessler and co-workers (47, 48) reported two 125I-labeled cyclic pentapeptides: 3-125I-D-Tyr4-cyclo(RGDyV) and 3-125I-D-Tyr4-cyclo-(RGDyK(SAA1)) (SAA ) sugar amino acid). It was found that 3-125I-D-Tyr4-cyclo(RGDyV) has fast hepatobiliary and renal excretion (47). The tumor/muscle and tumor/blood ratios for melanoma in nude mice were 5.5 and 9.5, respectively, at 60 min postinjection. Substitution of leucine with a SAA-functionalized lysine amino acid residue resulted in improved blood retention time, renal excretion, and better targetto-background ratio. A blocking study using 3 mg/kg of the Rvβ3 selective cyclo(RGDfV) demonstrated that the localization of radioactivity in the tumor is due to Rvβ3 receptor binding (48). For diagnostic purposes 99mTc is more desirable than 125I due to its easy availability, low cost, and ideal nuclear characteristics, which better match the rapid blood clearance and fast tumor accumulation of small peptides. Therefore, a series of RGD-containing peptide conjugates have been prepared for the development of a target-
Liu et al.
Figure 2. A typical radio-HPLC chromatogram of RP593 (method 1).
Figure 3. Solution stability of RP593 in the kit matrix and after HPLC purification.
specific tumor imaging agent (24, 25). In the c-Neu Oncomouse tumor model, RP593 shows high tumor uptake and long tumor residence time (3.4% ID/g at 2 h and 1.5% ID/g at 24 h postinjection) and is excreted predominantly via the renal system. As an extension of these efforts, we present the synthesis and characterization of the ternary ligand 99mTc complex (Figure 1: RP593) of a HYNIC-conjugated vitronectin receptor antagonist (Figure 1: SQ168). Synthesis of RP593. In this study, RP593 was prepared according to the published procedure (23) using a non-SnCl2-containing formulation. The radiolabeling was achieved by adding 1.0 mL of generator eluant (50 mCi of Na[99mTcO4]) and heating at 100 °C for 10 min. The radiochemical purity (RCP) for RP593 is usually g90%. The formation of [99mTc]colloid is minimal (
