Bioconjugate Chem. 2002, 13, 881−886
881
99mTc-Labeling
of a Hydrazinonicotinamide-Conjugated LTB4 Receptor Antagonist Useful for Imaging Infection and Inflammation Shuang Liu,* Anthony R. Harris, Neal E. Williams, and D. Scott Edwards Bristol-Myers Squibb Medical Imaging,† 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received September 23, 2001; Revised Manuscript Received January 23, 2002
This report describes the 99mTc labeling of a hydrazinonicotinamide (HYNIC)-conjugated LTB4 receptor antagonist (SG380). The ternary ligand technetium complex [99mTc(SG38)(tricine)(TPPTS)] (RP517) was prepared using a non-SnCl2-containing formulation ((2001) J. Pharm. Sci. 90, 114-123). Unlike other HYNIC-conjugated small biomolecules, SG380 is lipophilic and has low solubility in the kit matrix. Using a combination of a solubilizing agent (Lysolecithin) and a cosolvent (ethanol), we have developed a new formulation for routine preparation of RP517. Using this formulation, RP517 can be prepared in high radiochemical purity (RCP > 90%) and remains stable in the kit matrix for at least 6 h. We also prepared the corresponding 99Tc analogue, [99Tc]RP517. An HPLC concordance experiment using RP517 and [99Tc]RP517 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data are completely consistent with the 1:1:1:1 composition for Tc:SG380:tricine:TPPTS.
INTRODUCTION
Accurate and rapid detection of infectious and inflammatory foci is of great importance for elucidation of the cause of the disease, early prevention of the onset of complications, and rapid implementation of a tailored therapeutic regimen. While in some cases infections are diagnosed on the basis of clinical history, physical examination of the patient, in others it is more difficult because they are asymptomatic or cause nonspecific symptoms. Numerous imaging procedures have been developed for visualization of infectious and inflammatory lesions (1-14). These include ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), and nuclear medicine procedures. Ultrasonography, CT, and MRI provide details of morphological changes, variations in density, and differences in proton content in tissues (13). Nuclear medicine procedures can be used for in vivo characterization of cellular structure and function and for monitoring biological changes in the infectious tissues at the molecular level. Various 99mTc-labeled small biomolecules that bind to receptors on inflammatory cells have been studied as potential new radiopharmaceuticals for scintigraphic imaging of infection and inflammation. These include include 99mTc-labeled chemotactic peptides (15-23), tuftsin receptor antagonists (24-27), antimicrobial peptides (28-30), and a fragment of human platelet factor 4 (31, 32). Although animal studies show that the 99mTc-labeled chemotactic peptides bind to leukocytes and localize at sites of infection, the use of 99mTc-labeled agonists as radiopharmaceuticals suffers a major drawback, severe reduction of peripheral leukocyte count. This problem can be avoided by using antagonist peptides as targeting molecules. Unfortunately, the receptor binding affinity * To whom correspondence should be addressed: Tel: 978671-8696 (S.L.); FAX: 978-436-7500; E-mail: shuang.liu@ bms.com. † Formerly Medical Imaging Division, DuPont Pharmaceuticals Company.
of the antagonists tested to date appear to be much lower than that of the agonists. Leukotriene B4 (LTB4) is a proinflammatory mediator, which is derived from the action of 5-lipoxygenase on arachidonic acid and is synthesized primarily by PMNLs, monocytes, and macrophages. LTB4 is known to stimulate degranulation, aggregation, chemotaxis, and chemokinesis of PMNLs, as well as promote superoxide generation. LTB4 also promotes the adhesion of neutrophils to the vascular endothelium. LTB4 receptors are highly expressed on a number of inflammatory cells such as neutrophils, monocytes, macrophages, and lymphocytes. Numerous LTB4 receptor antagonists have been evaluated for their potential in the treatment of various infectious and inflammatory diseases. In our previous communication (33), we reported a nonSnCl2-containing formulation useful for the 99mTc-labeling of hydrazinonicotinamide-conjugated small biomolecules (HYNIC-BM1). In the non-SnCl2 formulation, each lyophilized vial contains 20 µg of HYNIC-BM, 5 mg of TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate), 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 (Poloxamer-188, 0.1%). HYNIC is the bifunctional coupling agent for 99mTclabeling. Tricine and TPPTS are used as coligands to stabilize the [99mTc]HYNIC core. The combination of HYNIC-BM, tricine, and TPPTS produces a versatile ternary ligand system which forms technetium complexes, [99mTc(HYNIC-BM)(tricine)(TPPTS)], with high solution stability. We have been able to use the non-SnCl2 formulation for the 99mTc-labeling of many small biomolecules, including a GPIIb/IIIa receptor antagonist for thrombus imaging (33-35), a chemotactic peptide (23) for imaging infection and inflammation, and vitronectin receptor antagonists for tumor imaging (36). 1Abbreviations: RCP: radiochemical purity; HYNIC-BM: 6-hydrazinonicotinamide-conjugated biomolecule; TPPTS: trisodium triphenylphosphine-3,3′,3′′-trisulfonate.
10.1021/bc010120b CCC: $22.00 © 2002 American Chemical Society Published on Web 06/28/2002
882 Bioconjugate Chem., Vol. 13, No. 4, 2002
Figure 1. Structures of the HYNIC-conjugated LTB4 receptor antagonist (SG380) and its ternary ligand technetium complex (RP517).
SG380 is an HYNIC-conjugated LTB4 antagonist. Theoretically, we should be able to use the non-SnCl2containing formulation for the 99mTc-labeling of SG380. To our surprise, direct substitution of 20 µg of SG-380 for HYNIC-BM in the non-SnCl2-containing formulation gave very low yields (60-70%) for RP517 even though the radiolabeling conditions are the same as those used for preparation of the 99mTc-labeled GPIIb/IIIa receptor antagonist (33) and vitronectin receptor antagonists (36). The goal of this study is to explore the factors influencing the yield of RP517 and to develop a formulation that provides RCP g 90% for routine preparation of RP517. EXPERIMENTAL SECTION
Materials. N,N-Dimethylacetamide (DMA), L-R-lysophosphatidylcholine (Lysolecithin), Tween-80, mannitol, pluronic acid (Poloxamer-188), succinic acid, TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate), and tricine were purchased from Aldrich or Sigma. Synthesis of SG380 will be published as a separate communication (37). Na99mTcO4 was obtained from a commercial DuPont Pharma 99Mo/99mTc generator, N. Billerica, MA. The generator eluant was collected into a dry and clean 30 mL vial and diluted to the required concentration before use. Analytical Methods. The HPLC method used a HP1090 system, a homemade NaI radiometric detector, and a heated (50 °C) Cosmosil C18 column (4.6 mm × 250 mm) at a flow rate of 1 mL/min. The gradient mobile phase started from 100% solvent A (75:25 0.025 M succinate buffer, pH ) 5.0/acetonitrile) to 30% solvent A and 70% solvent B (acetonitrile) at 35 min. The mobile phase was isocratic at 35-45 min using 30% solvent A and 70% solvent B. 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 B (methanol) to 100% B at 23 min at a flow rate of 1 mL/ min. The ITLC method used Gelman Sciences silica gel ITLC paper strips and a 1:1 mixture of acetone and saline as eluant. Using this ITLC method, RP517 and [99mTc]-
pertechnetate migrate while the [99mTc]colloid remains at the origin. A General Procedure for Radiolabeling. RP517 was prepared according to the published procedure (33) with a slight modification. Briefly, 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 1.0 mL of SG380 solution (20 µg/mL in an appropriate solvent or mixture of solvents). The vial was placed into a lead pig, and to the vial was added 0.5 mL of Na[99mTcO4] solution (100 mCi/ mL) in saline. The reconstituted vial was heated at 100 °C for 10-40 min in a lead-shielded water bath. After being cooled to room temperature, the reaction mixture was analyzed by radio-HPLC and ITLC. The radiochemical purity (RCP) was calculated as the percentage of integrated area for the radiometric peak of RP517 versus the total area. Since the [99mTc]colloid formation is negligible (
