RP463: A Stabilized Technetium-99m Complex of a Hydrazino

DuPont Pharmaceuticals Company, Medical Imaging Division, 331 Treble Cove Road,. North Billerica, Massachusetts 01862. Gary J. Bridger and Michael J...
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Bioconjugate Chem. 1999, 10, 884−891

RP463: A Stabilized Technetium-99m Complex of a Hydrazino Nicotinamide Derivatized Chemotactic Peptide for Infection Imaging D. Scott Edwards, Shuang Liu,* Marisa C. Ziegler, Anthony R. Harris, Andrew C. Crocker, Stuart J. Heminway, and John A. Barrett DuPont Pharmaceuticals Company, Medical Imaging Division, 331 Treble Cove Road, North Billerica, Massachusetts 01862

Gary J. Bridger and Michael J. Abrams AnorMed Inc., 200-20353 64th Avenue, Langley BC V2Y 1N5, Canada

John D. Higgins III McNeil Consumers Products, 7050 Camp Hill Road, Fort Washington, Pennsylvania 19034. Received April 28, 1999; Revised Manuscript Received June 8, 1999

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with 99mTc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with 99mTc produced a ternary ligand complex [99mTc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [99mTc(fMLFK-HYNIC)(tricine)2] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [99mTc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually g90% using 10 µg of fMLFK-HYNIC and 100 mCi of [99mTc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [99Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [99Tc]RP463 showed an IC50 of 2 nM against binding of [3H]fMLF to receptors on PMNs. [99Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC50 value of 0.2 ( 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 ( 0.2 at 15 min postinjection to 7.5 ( 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.

INTRODUCTION

Accurate and rapid localization of infectious and inflammatory foci facilitate elucidation of the cause of disease, and the implementation of a tailored therapeutic regiment. Numerous diagnostic imaging procedures have been developed in the past three decades. Morphological imaging modalities such as X-ray-computed tomography (CT), ultrasound (US), conventional radiography, and magnetic resonance imaging (MRI) rely primarily on focal changes in tissue density to define lesions. In the early stage of an inflammatory lesion, however, lesion localization using these procedures may be difficult because the tissue changes associated with necrosis have not occurred (1). White blood cells (WBCs), particularly polymorphonuclear leukocytes (PMNs) and monocytes, accumulate * To whom correspondence should be addressed. Phone: (978) 671-8696. Fax: (978) 436-7500. E-mail: shuang.liu@ dupontpharma.com.

in high concentrations at the site of infection. Therefore, recent attention has been directed toward radiolabeling small molecules that bind to both circulating granulocytes and leukocytes. These include chemotactic peptides, analogues of N-formyl-methionyl-leucyl-phenylalanine (fMLF) (1-8), small peptides based on platelet factor 4 (9), and tuftsin receptor antagonists (10-12). The chemotactic peptide, fMLF, is a bacterial product. It binds with high affinity to a specific receptor expressed on the surface of WBCs, stimulates chemotaxis of granulocytes, upregulates expression of adhesion molecules, and increases vascular permeability. In 1991, Fischman and co-workers (13) first reported the potential diagnostic use of 111In-labeled chemotactic peptide analogues of fMLF. Since then, they have studied a series of radiolabeled chemotactic peptides as infection/inflammation imaging agents (1, 3-8). These peptides were modified with either HYNIC (for 99mTc) or DTPA (for 111In) at the C-terminus. The 99mTc labeling of HYNIC-modified chemotactic peptides (such as fMLFK-HYNIC) can be achieved

10.1021/bc990049y CCC: $18.00 © 1999 American Chemical Society Published on Web 07/20/1999

RP463: A Stabilized Technetium-99m Complex

using coligands such as glucoheptonate, mannitol, and glucamine (4). It was reported that very high specific activity (g20 000 mCi/µmol) could be achieved using glucoheptonate as the coligand. Different biodistributions were observed using different coligands (4). Recently, van der Laken and co-workers (14) also reported the use of a 99mTc-labeled chemotactic peptide fMLFK-HYNIC for imaging acute infection and sterile inflammation. Animal studies have shown evidence of binding to leukocytes in vivo and localization at the infection site (1, 5-8, 15). Recently, we found that both tricine and glucoheptonate form binary technetium complexes, [99mTc(fMLFKHYNIC)(L)2] (L ) tricine and glucoheptonate), which exist in many isomeric forms and are not stable in solution in the absence of excess coligand (16). Although the biological data from animal studies show that these agents are able to localize at the infection site (14-16), it would still be difficult to develop them for routine clinical use, due to solution instability and the presence of many isomers. In this study, a ternary ligand system was used to prepare the stabilized complex [99mTc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). The goal was to minimize the number of isomeric forms and increase the solution stability of the 99mTc complex. RP463 was prepared, and its solution stability was compared to that of the binary ligand complex, [99mTc(fMLFK-HYNIC)(tricine)2] (RP469). RP463 was evaluated in a rabbit focal infection model for its potential use as an infection imaging agent. EXPERIMENTAL SECTION

Materials. TPPTS (trisodium triphenylphosphine3,3′,3′′-trisulfonate) and tricine were purchased from Aldrich Chemical Co. Na99mTcO4 was obtained from a Technelite 99Mo/99mTc generator, DuPont Pharmaceuticals Co., North Billerica, MA. Deionized water was obtained from a Millipore MilliQ Water System and was of >18 MΩ quality. The peptide fMLFK was synthesized and conjugated to 6-hydrazinonicotinic acid (HYNIC) according to the literature procedure (3). Instruments and Methods. The electrospray mass spectrum of [99Tc]RP463 was obtained using a HP 1100 Series LC/MSD (G1946A) mass spectrometer and 0.1% formic acid/acetonitrile as the mobile phase. The HPLC methods used a Hewlett-Packard model 1050 instrument with a NaI radiometric detector. The ITLC method used Gelman Sciences silica gel strips and a 1:1 mixture of acetone and saline as the eluant. HPLC method 1 used a Zorbax C18 reversed-phase column (4.6 mm × 250 mm, 80 Å pore size) at a flow rate of 1 mL/min. Solvent A contained 90% 25 mM phosphate buffer (pH 8) and 10% acetonitrile while solvent B was a 50:50 (v:v) mixture of acetonitrile and 25 mM phosphate buffer (pH 8). A linear gradient from 0% B to 20% B over 40 min was used. HPLC method 2 used a Zorbax C18 reversed-phase column (4.6 mm × 250 mm, 80 Å pore size) and a Rainin Dynamax UV-vis detector (model UV-C, λ ) 230 nm). The flow rate was 1 mL/min with a gradient from 90% solvent A (0.01 M phosphate buffer, pH 6) and 10% solvent B (acetonitrile) to 70% solvent A and 30% solvent B at 30 min. Synthesis of RP463. Two-Step Labeling Using Stannous Chloride. To a 10 mL vial were added 0.2 mL of fMLFK-HYNIC solution (50 µg/mL in H2O), 0.4 mL, of tricine solution (100 mg/mL in H2O), 0.5 mL of Na99mTcO4 solution (100 mCi/mL in saline), and 25 µL of SnCl2‚2H2O solution (1.0 mg/mL in 0.1 N HCl). The reaction mixture

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was allowed to stand at room temperature for 30 min. The labeling yield for the resulting complex RP469 was usually g90%. To the solution above was added 0.3 mL of TPPTS solution (50 mg/mL in H2O). The reaction mixture was heated at 50 °C for 30 min and then analyzed by radio-HPLC (method 1). ITLC was used to determine the amount of [99mTc]colloid. The product RP463 migrated while the [99mTc]colloid remained at the origin. One-Step Labeling Using Stannous Chloride. To a shielded 10 mL vial was added 0.5 mL of Na99mTcO4 solution (100 mCi/mL in saline), 0.4 mL of tricine solution (100 mg/mL in H2O), 0.2 mL of the fMLFK-HYNIC solution (50 µg/mL in H2O), 0.3 mL of TPPTS solution (50 mg/mL in H2O), and 25 µL of SnCl2‚2H2O solution (1.0 mg/mL in 0.1 N HCl). The reaction mixture was heated at 80 °C for 30 min and then analyzed by radioHPLC (method 1) and ITLC. RP463 prepared by this method was shown by HPLC to be identical to that prepared according to the two-step synthesis. The radiolabeling yield was g90%. RP463 was purified by HPLC (method 1) to remove excess reactants, particularly unlabeled fMLFK-HYNIC. The fraction at 10-12 min was collected into a 25 mL round-bottom flask. Volatiles were removed under reduced pressure. The residue was dissolved in saline at a concentration of 1.5 mCi/mL for animal studies. The resulting solution was reanalyzed by radio-HPLC. One-Step Labeling without Stannous Chloride. To a shielded 10 mL vial were added 0.5 mL of Na99mTcO4 solution (100 mCi/mL in saline), 0.1 mL of tricine solution (100 mg/mL in H2O), 0.2 mL of the fMLFK-HYNIC solution (50 µg/mL in H2O), and 0.2 mL of TPPTS solution (50 mg/mL in H2O). The reaction mixture was heated at 80 °C for 30 min and then analyzed by radioHPLC (method 1). RP463 prepared by this method was shown by HPLC to be identical to that prepared using the stannous formulation. Solution Stability Studies. Complexes RP463 and RP469 were prepared and separated from the kit matrix by HPLC (method 1). Fractions of interest were collected into a 25 mL round-bottom flask. Volatiles in the collected fractions were removed under reduced pressure. The residue was dissolved in saline to give a concentration of 1.5 mCi/mL. The stability of RP463 and RP469 was monitored by HPLC (method 1) over ∼6 h. Synthesis of [99Tc]RP463. To a 10 mL vial were added tricine (41.7 mg, 0.230 mmol), followed by [NH4][99TcO4] (3.7 mg, 0.021 mmol) in water (1 mL), fMLFKHYNIC (16.5 mg, 0.022 mmol) in methanol (1 mL), and TPPTS (50 mg, 0.088 mmol). The vial was crimped and heated in a boiling water bath for 30 min. The product [99Tc]RP463 was separated from the reaction mixture by HPLC (method 2). The product fraction was collected, and volatiles were removed under reduced pressure. Analysis of the product by HPLC (method 2, λ ) 230 nm) showed no residual starting peptide. Receptor Binding Assays. Human PMNs were isolated from heparinized venous blood obtained from healthy donors. Blood samples of 40 mL were mixed with an equal volume of Hanks’ balanced salt solution (HBSS), layered on a Ficoll-Hypaque gradient and centrifuged at 10 °C and 1300 rpm for 30 min. The supernatant was discarded, and the PMN-rich pellet was resuspended in cold HBSS, to which cold 2% dextran solution (in saline) was added to remove residual red blood cells. After 25 min of sedimentation at room temperature, the suspended cells were removed and centrifuged at 10 °C and 1300 rpm for 10 min. The supernatant was discarded,

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and the pellet was resuspended in 10 mL of cold hypotonic lysing buffer for a 5-min lysis of residual RBC. After lysis, cold HBSS (40 mL) was added, and the cells were centrifuged at 10 °C and 1300 rpm for 10 min. This final PMN pellet was washed once and resuspended to the desired cell concentration in phosphate incubation buffer containing 140 mM NaCl, 1.0 mM KH2PO4, 5 mM Na2HPO4, 0.5 mM MgCl2, 0.15 mM CaCl2, and 0.5% BSA (pH 7.4) for fMLF binding assays. For the cytochrome c reduction assays, cells were resuspended in HBSS. fMLF Binding Assay. [3H]fMLF (10 nM), [99Tc]RP463, and 100 µL of PMN solution (8 × 106 PMNs/mL) were added to a 96-well microplate with filters (0.65 µm pore size). The microplate was incubated for 60 min at room temperature with gentle agitation. The microtiter plate was then placed on a filtration system. The wells were washed with incubation buffer three times and dried. The filters were removed from the microplate, placed into scintillation vials, and agitated for 1 h at room temperature in the presence of scintillation fluid. Percentage inhibition of [3H]fMLF binding to PMNs was calculated by dividing the specific binding (total binding -nonspecific binding) obtained in the presence of technetium complex by that obtained in the absence of the technetium complex. IC50 values were calculated by fitting the percentage inhibition values to a regression line. Cytochrome c PMN Free Radical Release Assay. The biological activity of [99Tc]RP463 was determined by measuring superoxide release of PMNs after they are exposed to the technetium complex. Typically, the PMN solution (1 mL, 1 × 106 PMNs/mL) was incubated with cytochalasin B (10 µM) and 100 µL of cytochrome c (40 µM) for 10 min at 37 °C. Then [99Tc]RP463 was added, and the tubes were incubated for 10 min at room temperature. After centrifugation (200 × g, for 10 min at 10 °C), the supernatants were transferred to cuvettes for spectrophotometric analysis to measure reduction of cytochrome c, reflecting the amount of superoxide release by PMNs. Superoxide release in the presence of [99Tc]RP463 was expressed as a percentage of maximal response. EC50 values were calculated by fitting a curve to the data generated. Animal Studies. Absesses were induced in the left thigh muscle of female New Zealand rabbits (2.2-2.8 kg) with 1.5 × 1010 colony forming units of Escherichia coli in 0.5 mL. During the procedure, the rabbits were anesthetized with subcutaneous injection of a 0.5 mL mixture of 0.315 mg/mL fentanyl and 10 mg/mL fluanisone. After 24 h, when swelling of the muscle was apparent, groups of four rabbits were immobilized, and were placed prone on the gamma camera. RP463 was administered (1.0 mCi/kg, i.v.) in the lateral ear vein. Serial images were acquired using a gamma camera (Digital Dyna Camera, Picker International, Cleveland, OH) every 15 min for 4 h. The scintigraphic results were analyzed quantitatively by drawing regions of interest over the abscess, the uninfected contralateral thigh muscle (background), and the whole body. Abscessto-background ratios and percentage residual activity in the abscess (abscess-to-whole body ratio × 100%) were calculated. Arterial blood was withdrawn prior to administration and every 30 min thereafter for determination of blood clearance. At the end of the protocol, the animal was euthanized with an overdose of pentobarbital. Samples of blood, infected thigh muscle, uninfected contralateral thigh muscle, bone, bone marrow, lung, spleen, liver, kidneys, and intestines were collected. The dissected tissues were weighed and counted in a gamma well counter (LKB 1282, Wallac Inc., Gaithersburg, MD).

Edwards et al.

Figure 1. Structures of fMLFK-HYNIC and RP463. Chart 1. Synthesis of RP463

To correct for radioactive decay, injection standards were counted simultaneously. The measured activity in samples was expressed as injected dose per gram (% ID/g). Abscess-to-contralateral muscle ratios and abscess-toblood ratios were calculated. RESULTS

Synthesis of RP463. RP463 was synthesized either in two steps, in which the binary ligand complex RP469 is formed first and then reacts with TPPTS, or in one step by direct reduction of [99mTc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS (Chart 1). The radiolabeling yield for the complex RP463 is usually g90% using 10 µg of fMLFK-HYNIC and 100 mCi of [99mTc]pertechnetate. The formation of [99mTc]colloid is minimal. Since the radiolabeled kit contains excess unlabeled peptide (fMLFKHYNIC), which has been shown to be a strong agonist (3, 14), RP463 has to be purified by HPLC before animal studies. HPLC Characterization RP463 and RP469. RP463 and RP469 were characterized using the same reversedphase HPLC method. Figure 2 shows the chromatograms (method 1) for the binary ligand complex RP469 (top) and the ternary ligand complex RP463 (bottom). There are several radiometric peaks for RP469 at retention times between 20 and 45 min, suggesting that there are many 99mTc-species in solution. The ratios of the peak areas in the radio-HPLC chromatograms change with time, reaction temperature, and pH. Using the same method, the

RP463: A Stabilized Technetium-99m Complex

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Figure 3. Solution stability of HPLC purified RP463 and RP469.

Figure 2. Radio-HPLC chromatograms (method 1) for RP469 (top) and RP463 (bottom).

chromatogram of RP463 showed only one peak at 23 min (bottom in Figure 2). RP463 prepared by the two-step synthesis was found to be exactly the same as that prepared by the one-step synthesis. The technetium chelate moiety is chiral, due to the tricine ligand being pro-chiral, and should be formed in an equal mixture of D and L enantiomers. These enantiomers in combination with the chiral centers on the peptide backbone should result in two diastereomers, DLLLL and LLLLL. Theoretically, these two diastereomers should be separable under ideal conditions. We tried several different reversed-phase HPLC methods under either gradient or isocratic conditions and were unable to resolve these two diastereomers, probably due to the high flexibility of the linear peptide. Solution Stability Studies. The solution stability of HPLC-purified RP463 was examined, and was compared to that of RP469. Both complexes were separated from the kit matrix. Their solution stability in saline (pH ∼6) was monitored by HPLC (method 1) for 6 h. Figure 3 shows the plot of RCP vs time for RP463 and RP469. It is clear that the HPLC-purified RP469 was not stable in solution. The RCP dropped to ∼50% at 6 h post HPLC purification. The ternary ligand complex RP463 remained relatively unchanged in solution. The RCP only dropped slightly from 94% to 91% at 6 h post HPLC purification. [99Tc]463. [99Tc]RP463 was prepared in one step by reacting fMLFK-HYNIC with [NH4][99TcO4], in the presence of excess tricine and TPPTS. It was isolated by HPLC purification (method 2). The HPLC concordance (Figure 4) of RP463 and [99Tc]RP463 showed that the same complexes were prepared at the tracer (99mTc) and macroscopic (99Tc) levels. Electrospray mass spectrometry gave m/z 1476 for [99Tc]RP463 ([C57H72N9O21S4Tc]+) in its acid form. In Vitro Studies. The binding of [3H]fMLF to receptors on PMNs could be completely inhibited by [99Tc]RP463. The concentration of [99Tc]RP463 required for 50% inhibition against [3H]fMLF binding to PMNs (IC50) was 2 nM. Nonspecific binding on average was less than 15%. Like the unlabeled peptide (fMLFK-HYNIC), [99Tc]-

Figure 4. HPLC concordance (method 2) for RP463 (top, NaI radio-detector) and [99Tc]RP463 (bottom, UV detector).

RP463 is also an effective inducer of superoxide release of PMN, with an EC50 value of 0.2 ( 0.2 nM (n ) 3). In Vivo Animal Studies. RP463 was evaluated in rabbits infected with E. coli. After injection of RP463 into infected rabbits, a minimal transient reduction in peripheral leukocyte levels was observed. Immediately after injection, the WBC count decreased to 65% of the initial level and then returned to 90-95% within 30 min postinjection (Figure 5). Similar phenomena were observed for the complex RP469 in both the healthy and the infected rabbit (14). RP463 cleared from the blood rapidly (Figure 6). The biodistribution data (Figure 7) at 4 h postinjection showed that RP463 excreted via both the renal and hepatobillary systems and were consistent with the imaging data. Compared with RP469, RP463 showed comparable renal excretion and less blood residence. Despite fast blood clearance, images clearly showed rapid accumulation of RP463 in the infected site, and the

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Edwards et al. DISCUSSION

Figure 5. White blood cell counts in blood of the infected rabbits after injection of the HPLC purified RP463 and RP469.

Figure 6. Blood clearance of RP463 and RP469 in the infected rabbits. Data expressed as percentage of the injected dose per gram (% ID/g, mean values ( sem).

uptake at the infection site increased with time (Figure 8). The infection foci could be clearly visualized at 2 h postinjection. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 ( 0.2 at 15 min postinjection to 3.5 ( 0.4 at 2 h postinjection and to 7.5 ( 0.4 at 4 h postinjection (Figure 9).

In the development of a new receptor-based 99mTc infection imaging agent, several factors need to be considered to satisfy the clinical requirements. The agent has to demonstrate the biological efficacy, including high specificity and high uptake at the infection site. It should also have favorable pharmacokinetics, show rapid blood clearance, and be renally excreted. A kit formulation is often required due to the 6 h half-life of 99mTc. Injection of the whole or part of the reconstituted kit should not cause any significant biological effect such as neutropenia. The agent should have high radiochemical purity (RCP ) 90%) and high solution stability with a shelf life of preferably 6 h. Finally, the 99mTc labeling should be accomplished in 10-30 min, preferably at room temperature. Development of a specific agent for imaging focal infection remains a major challenge for investigators in nuclear medicine. For a number of years, imaging inflammation and infection has been performed using [67Ga]citrate. But the specificity is low and the exact mechanism of localization is still not well understood. 111In- and 99mTc-labeled nonspecific polyclonal IgGs were also used for detection of infection foci (17-24). However, it usually takes 24 h to obtain diagnostically useful images due to their slow clearance from the blood pool. [99mTc]HMPAO-WBCs (25-27), and [99mTc]albumin colloid-WBCs (28) have specificity for the infected foci since WBCs, particularly PMNLs and monocytes, accumulate at the site of acute infection as part of the inflammatory response induced by bacteria. Imaging using both agents can be completed on the same day, but these procedures may impose significant risks to laboratory personnel and patients, particular with the increasing prevalence of human immunodeficiency virus in the population (29, 30). 99m Tc labeling of biologically active small peptides is of current interest because small peptides are necessary elements in many biological processes and, in some cases, have affinities significantly higher than those of antibodies or antibody fragments (5). Small peptides are easy to synthesize and modify, are less likely to be immunogenic, and can have rapid blood clearance. The faster blood clearance results in adequate T/B ratios earlier so that it is practical to use 99mTc, which is the preferred radionuclide for diagnostic nuclear medicine. Examples

Figure 7. Biodistribution of RP463 versus RP469 in the infected rabbits. Data expressed as percentage of the injected dose per gram (% ID/g, mean values ( sem).

RP463: A Stabilized Technetium-99m Complex

Figure 8. Representative images (anterior view) of rabbits infected with E. coli at 30, 60, 120, and 240 min after injection of the HPLC purified RP463. Arrows indicate location of the infected thigh.

Figure 9. Target-to-background (infected muscle to contralateral muscle) ratios, calculated from rabbits infected with E. coli at 15, 60, 120, 180, and 240 min after injection of HPLC-purified RP463 and RP469.

of 99mTc-labeled small peptides for imaging infection or inflammation include chemotactic peptides (1-8), small peptides based on platelet factor 4 (9), and tuftsin receptor antagonists (10-12). The 99mTc-labeled fMLFK-HYNIC has been studied for infection imaging using several different animal models (3, 4, 6-8, 15). Various coligands such as glucoheptonate, mannitol, and glucamine have been used for the 99mTc labeling. Different biodistributions were observed depending on the coligand (4). However, these studies did not address problems associated with the presence of multiple species of the binary ligand complexes such as RP469 and the solution stability of the 99mTc-labeled peptides. In this study, it has been clearly demonstrated by a reversed-phase HPLC method that the binary ligand

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complex RP469 exists in solution as a mixture of several isomeric forms. Similar results were obtained for the complex [99mTc(fMLFK-HYNIC)(glucoheptonate)2] (16) and for the 99mTc complex of a HYNIC-conjugated cyclic platelet GPIIb/IIIa receptor antagonist (31). In addition, RP469 was found to be unstable in the absence of excess tricine coligand (Figure 3). Thus, it would be difficult to develop RP469 into a product for routine clinical use because of the solution instability and the presence of the many isomeric forms even though animal studies showed that the agent was able to localize at the site of infection (1-8, 14, 15). To minimize the formation of various isomeric forms and increase the solution stability of the 99mTc complex, a ternary ligand system was used to prepare RP463. This new ligand system has been successfully used for the 99m Tc labeling of an HYNIC-conjugated cyclic GPIIb/IIIa antagonist for the development of a thrombus imaging agent, RP444 (32, 33). Using the combination of fMLFKHYNIC, tricine, and TPPTS, RP463 can be synthesized in high yield and high specific activity (>10 000 mCi/µmol fMLFK-HYNIC) without HPLC purification. It was found that RP463 was stable for up to 6 h in solution. Apparently, the addition of TPPTS coligand dramatically reduces the number of isomeric forms and significantly increases the solution stability of 99mTc-labeled peptide. The composition of RP463 and the 1:1:1:1 ratio for Tc: fMLFK-HYNIC:tricine:TPPTS was confirmed by mass spectral data of [99Tc]RP463. In an in vitro binding assay, [99Tc]RP463 was able to effectively compete with [3H]fMLF for receptors on PMNs, with an IC50 value of 2 nM. Like the unlabeled peptide, fMLFK-HYNIC (IC50 ) 3 ( 2 nM; EC50 ) 0.8 ( 0.4 nM) (14), [99Tc]RP463 also induces superoxide release of PMN with an EC50 value of 0.2 ( 0.2 nM. These data clearly show that RP463 is a high affinty agonist of the fMLF receptors on PMNs, and the 99mTc-labeling did not significantly alter the binding affinity even though the molecular weight after radiolabeling is more than doubled. After injection of RP463 in the rabbit, a transient reduction in peripheral leukocyte levels was observed. Since RP463 was purified by HPLC, it should be free of unlabeled fMLFK-HYNIC. The injected dose for the rabbit was 1 mCi/kg. For a generator with 24 h prior elution time, the total amount of Tc (99mTc + 99Tc) in 1 mCi of activity is ∼7 × 10-12 mol. The total amount of technetium complex (RP463 + [99Tc]RP463) injected into each infected rabbit (∼2.5 kg) is ∼25 ng (MW ) 1542 for [99Tc]RP463 in its sodium salt form). A study in primates showed that the minimal amount of peptide causing decrease of white blood cells is 10 ng/kg for fNleLFNleYKDTPA (6). Therefore, it is not surprising that even the HPLC purified RP463 could cause the biological effect at a dose of 1 mCi/kg. The use of 99mTc-labeled highly potent agonists as radiopharmaceuticals suffers a major drawback: severe reduction of peripheral leukocyte count. In general, there are three approaches to avoid this problem. The first approach is to separate the radiolabeled peptide from the excess unlabeled peptide using HPLC. This results in a product almost at its theoretical specific activity but is inconvenient for routine clinical use. The results described in this study clearly show that even the HPLC purified 99mTc-labeled peptide can cause a neutropenic effect if the 99mTc complex itself is a very potent agonist. The second approach involves using less active agonists with similar binding characteristics (15). However, lower binding affinity may result in less accumulation of the radioactivity at the receptor sites. The third approach is

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to use antagonist peptides, which bind to the receptor but do not cause any neutropenic response, as targeting molecules. Unfortunately, the receptor binding affinity of the antagonists tested to date appear to be much lower than that of the agonists (34). Recently, van der Laken and co-workers (14) have demonstrated that accumulation of the 99mTc-labeled fMLFK-HYNIC (RP469) in acute infection and sterile inflammation took place by virtue of binding to receptors on locally present PMNs and monocytes. Both the scintigraphic and biodistribution data showed that RP469 was cleared via both the kidneys and the liver. The high blood activity is probably related to the instability of RP469 and plasma protein binding. This assumption is supported by the fact that imine-N containing heterocycles can act as coligands and form stable ternary ligand complexes with the 99mTc-labeled HYNIC-peptide conjugate (35). The use of TPPTS coligand significantly increases the solution stability and hydrophilicity of the radiolabeled fMLFK-HYNIC. As a result, the excretion route for RP463 is mainly the renal system. The fast accumulation of RP463 at the infection site in combination with its rapid clearance results in continuously improving target-to-background ratios over 4 h. Traditionally, modification of pharmacokinetics of a radiopharmaceutical is achieved either by attaching a pharmacokinetic modifier on the targeting biomolecule or by using a linker between the targeting biomolecule and the technetium chelate. The lipophilicity of the linker depends on pharmacokinetic requirements for the radiopharmaceutical. Results from this study and others (4) clearly show that the coligand of the technetium chelate can also be used for modifying pharmacokinetics and biodistribution of the radiopharmaceutical. As a matter of fact, one of the advantages using HYNIC as BFCA is that various coligands can be used for synthesis of ternary ligand technetium chelates. CONCLUSION

The HYNIC-conjugated chemotactic peptide (fMLFKHYNIC) has been labeled with 99mTc using a ternary ligand system (fMLFK-HYNIC, tricine and TPPTS). The complex [99mTc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463) was prepared in high yield (g90%) and high specific activity (10 000 mCi/µmol fMLFK-HYNIC). The use of TPPTS as a coligand dramatically reduced the number of 99mTc species in the radiolabeled kit and increased the solution stability of the radiolabeled peptide. Compared to the unlabeled fMLFK-HYNIC (IC50 ) 3 ( 2 nM against binding of [3H]fMLF to receptors on PMNs; EC50 ) 0.8 ( 0.4 nM), RP463 remains to be a strong agonist for the chemotactic receptor (IC50 ) 2 nM against binding of [3H]fMLF to receptors on PMNs; EC50 ) 0.2 ( 0.2 nM) even though the molecular weight of RP463 is more than twice that of fMLFK-HYNIC. The localization of RP463 in the infection foci was assessed in the rabbit infection model. Visualization of the infected foci could be as early as 2 h postinjection with the target-to-background ratio increasing from 1.5 ( 0.2 at 15 min postinjection to 7.5 ( 0.4 at 4 h postinjection as a result of a faster blood clearance mainly through the renal system. However, a 35% transient decrease in WBC count was observed during the first 30 min after injection of the HPLC-purified RP463. Because of this limitation, further research in this area should focus on the development of 99mTc-labeled antagonists with similar binding affinity for chemotactic peptide receptor or other receptors on PMNs and monocytes.

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