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Mar 26, 1997 - Our thrombus research program has focused on development of a thrombus imaging agent by labeling a platelet glycoprotein IIb/IIIa (GPII...
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Bioconjugate Chem. 1997, 8, 155−160

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Biological Evaluation of Thrombus Imaging Agents Utilizing Water Soluble Phosphines and Tricine as Coligands When Used To Label a Hydrazinonicotinamide-Modified Cyclic Glycoprotein IIb/IIIa Receptor Antagonist with 99mTc John A. Barrett,* Andrew C. Crocker, David J. Damphousse, Stuart J. Heminway, Shuang Liu,* D. Scott Edwards, Joel L. Lazewatsky, Mikhail Kagan, Theresa J. Mazaika, and Timothy R. Carroll The DuPont Merck Pharmaceutical Company, Radiopharmaceuticals Division, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received October 28, 1996X

A hydrazinonicotinamide-functionalized cyclic glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist [cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotinamido)hexanamide))) (HYNICtide)] was labeled with 99mTc using tricine and a water soluble phosphine [trisodium triphenylphosphine-3,3′,3′′trisulfonate (TPPTS); disodium triphenylphosphine-3,3′-disulfonate (TPPDS); or sodium triphenylphosphine-3-monosulfonate (TPPMS)] as coligands. Three complexes, [99mTc(HYNICtide)(L)(tricine)] (1, L ) TPPTS; 2, L ) TPPDS; 3, L ) TPPMS), were evaluated in the canine arteriovenous shunt (AV shunt) model and canine deep vein thrombosis imaging (DVT) model. All three agents were adequately incorporated into the arterial and venous portions of the growing thrombus (7.8-9.9 and 0.2-3.7% ID/g, respectively) in the canine AV shunt model. In the canine DVT model all three complexes had thrombus uptake that far exceeded the negative control, [99mTc]albumin. The findings indicate similar incorporation into a venous thrombus (% ID/g ) 2.86 ( 0.4, 3.4 ( 0.9, and 3.38 ( 1.1 for complexes 1, 2, and 3, respectively) and similar blood clearance with a t1/2 of approximately 90 min. Gamma camera scintigraphy allowed visualization of deep vein thrombosis in as little as 15 min with the thrombus/muscle ratios being 3.8 ( 0.8, 2.8 ( 0.4, and 3.0 ( 0.8 for complexes 1, 2, and 3, respectively. The visualization of the thrombus improved over time, and the thrombus/muscle ratios were 9.7 ( 1.9, 13.8 ( 3.6, and 9.4 ( 2 for complexes 1, 2, and 3, respectively, at 120 min postinjection. The administration of complexes 1-3 did not alter platelet function, hemodynamics, or the coagulation cascade. Furthermore, complexes 1-3 did not significantly differ in their uptake into the growing thrombus, blood clearance, and target to background ratios. Therefore, all three complexes have the capability to detect rapidly growing venous and arterial thrombi.

INTRODUCTION

Our thrombus research program has focused on development of a thrombus imaging agent by labeling a platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist with 99mTc (1-7). In our previous paper (1), we described the 99mTc-labeling of a platelet GPIIb/IIIa receptor antagonist [[cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotinamido)hexanamide)))) (HYNICtide)] using tricine and water soluble phosphines [trisodium triphenylphosphine-3,3′,3′′-trisulfonate (TPPTS); disodium triphenylphosphine-3,3′-disulfonate (TPPDS); sodium triphenylphosphine-3-monosulfonate (TPPMS)] as coligands. The combination of HYNICtide with tricine and phosphine produces a new and versatile ternary ligand system, which forms technetium complexes, [99mTc(HYNICtide)(L)(tricine)] (Figure 1, 1, L ) TPPTS; 2, L ) TPPDS; 3, L ) TPPMS), in high yield and high specific activity (g20 000 Ci/mmol). It was found that these complexes are formed as equal mixtures of two isomeric forms and are stable for g6 h in both the reaction mixture and dilute solutions. As a continuation of that study, we now present the biological evaluation of complexes 1-3 in the canine arteriovenous (AV) shunt and deep vein thrombosis (DVT) models. * Authors to whom correspondence should be addressed [telephone (508) 671-8696 (S.L.) or (508) 671-8341 (J.A.B.); fax (508) 436-7500]. X Abstract published in Advance ACS Abstracts, February 15, 1997.

S1043-1802(97)00001-3 CCC: $14.00

Deep vein thrombus is the result of a hypercoagulatible state coupled with a period of stasis occurring in a lowshear environment. The end result is the formation of a fibrin-rich thrombus which also contains some platelets and erythrocytes. In contrast, an arterial thrombus is the result of the rupture of an atherosclerotic plaque occurring under high-shear conditions, resulting in the formation of a platelet-rich thrombus (8). The GPIIb/ IIIa complex is expressed on the membrane surface of activated platelets and plays an integral role in platelet aggregation and thrombus formation (9). Initial events in thrombus formation frequently entail the activation of platelets by thrombogenic conditions and their subsequent aggregation (10). Since the GPIIb/IIIa complex is expressed only on the membrane surface of activated platelets, the GPIIb/IIIa receptor makes an excellent target for the development of a thrombus imaging agent. Existing diagnostic modalities are inadequate to diagnose and determine the morphology of the evolving thrombus (11). Thus, the development of agents that will not only detect the location but, in addition, determine the age of the thrombi is a critical unmet need in nuclear diagnostic medicine. EXPERIMENTAL PROCEDURES

Materials. TPPTS was purchased from Aldrich Chemical Co. TPPDS was prepared and purified according to the procedure described in the literature (6). TPPMS was purchased from TCI America, Portland, OR, and was used as received. Na99mTcO4 was obtained from a com© 1997 American Chemical Society

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Figure 1. HYNICtide complexes and three water soluble phosphine coligands.

mercial DuPont Merck 99Mo/99mTc generator, N. Billerica, MA. [99mTc]albumin, canine [125I]fibrinogen, and [111In]oxine kits were purchased from Medi-Physics Inc., Arlington Heights, IL, and were used as directed. Deionized water was obtained from a Millipore MilliQ Water System and was of >18 MΩ quality. Methods. The radio-HPLC method used a HewlettPackard Model 1050 instrument, a reversed phase Vydac C18 column (4.6 mm × 250 mm, 300 Å pore size) at a flow rate of 1 mL/min with the mobile phase starting from 100% A (0.01 M phosphate buffer, pH 6) to 30% B (acetonitrile) at 15 min and 75% B at 25 min. The ITLC method used Gelman Sciences silica gel strips and a 1:1 mixture of acetone and saline as eluant. The 99mTc labeling of the cyclic GPIIb/IIIa HYNICtide was achieved following the previously described procedure (1) with some modification. To a clean 10 mL vial were added 0.5 mL of tricine solution (80 mg/mL in H2O, pH 5), the HYNICtide solution (50 µg/mL in H2O; 0.1 mL for DVT model and 0.2 mL for AV shunt model), 0.2 mL of phosphine coligand solution (5 mg/mL in H2O), 19 mCi of pertechnetate in 0.5 mL of saline, and 25 µL of SnCl2‚2H2O solution (1 mg/mL in 0.1 N HCl). The pH was adjusted to 4-5. The reaction mixture was heated in a water bath at 80 °C for 30 min and was then analyzed by radio-HPLC and ITLC. The radiochemical purity (RCP) for complexes 1-3 was g90% with no detectable [99mTc]colloid. Doses for biological evaluation were prepared by dilution with 2.0% tricine solution in saline to the required concentration: 0.3 mCi/mL for the AV shunt model and 1.5 mCi/mL for the DVT. The diluted solutions were reanalyzed by radio-HPLC before the animal study.

Barrett et al.

Canine Arteriovenous Shunt Methodology. Adult beagle dogs of either sex (9-13 kg) were anesthetized with pentobarbital sodium (35 mg/kg iv) and ventilated with room air via an endotracheal tube (12 strokes/min, 25 mL/kg). For arterial pressure determination, the left carotid artery was cannulated with a saline-filled polyethylene catheter (PE-240) and connected to a Statham pressure transducer (Model P23ID, Gould Co., Oxnard, CA). Mean arterial blood pressure was determined via damping the pulsatile pressure signal. Heart rate was monitored using a cardiotachometer (Grass Instrument Inc., Quincy, MA) triggered from a lead II electrocardiogram generated by limb leads. A jugular vein was cannulated (PE-240) for drug administration. Both femoral arteries and femoral veins were cannulated with silicon-treated (Sigmacote, Sigma Chemical Co., St. Louis, MO), saline-filled polyethylene tubing (PE-200) and connected with a 5 cm section of silicon-treated tubing (PE-240) to form extra corporeal arteriovenous (AV) shunts. Shunt patency was monitored using a Doppler flow system (Model VF-1, Crystal Biotech Inc., Hopkinton, MA) and flow probe (2-3.5 mm, Crystal Biotech) placed proximal to the locus of the shunt. All parameters were monitored continuously on a Model 7D polygraph recorder (Grass Instrument Inc., Quincy, MA) at a paper speed of 10 mm/min or 10 mm/s. On completion of a 15 min postsurgical stabilization period, an occlusive thrombus was formed by the introduction of a thrombogenic surface (4-0 braided silk thread, 5 cm in length, Ethicon Inc., Somerville, NJ) into one shunt with the other serving as a control. A 1 h shunt period was employed with the test agent (∼3.0 mCi in 10 mL) administered as an infusion over 5 min, beginning 5 min before insertion of the first thrombogenic surface. The thrombus formed was comprised of a platelet-rich component on the thrombogenic surface and a fibrin-rich tail. At the end of the 1 h shunt period, the silk was carefully removed, the portions separated and weighed. The percent incorporation was determined via well counting (LKB Model 1282, Wallac Inc., Gaithersburg, MD). Thrombus weight was calculated by subtracting the weight of the silk prior to placement from the total weight of the silk on removal from the shunt. Arterial blood was withdrawn prior to infusion and every 30 min thereafter for determination of blood clearance, whole blood collagen-induced platelet aggregation, prothrombin time (PT), activated partial thromboplastin time (APTT), and platelet count. Template bleeding time was also performed prior to infusion and every 30 min thereafter. [111In]Platelets Preparation. The isolation and 111In-labeling of platelets were performed according to the literature method (12) with some modification. Canine arterial blood (40 mL) was withdrawn in 2.5% ACD solution (2.5 g of trisodium citrate, 1.4 g of citric acid, and 2.0 g of dextrose in 100 mL of H2O, pH 4.5). An additional 20 mL of blood was drawn in 3.8% sodium citrate solution, which serves as control. The samples were centrifuged at 1400 rpm for 15 min to form plateletrich plasma (PRP) and platelet-poor plasma (PPP). The ACD-PRP was pelleted (2800 rpm for 15 min) and washed twice with ACD. The platelet pellet was resuspended in ACD (2 mL), and 60-100 µCi of [111In]Oxine was added. The suspension was incubated at 37 °C for 5 min, followed by the addition of PPP (5 mL). The suspension was centrifuged at 2800 rpm for 10 min. The supernatant and platelet pellet were counted to assess labeling efficiency (>80% in all cases). The platelet pellet was resuspended in PPP (5 mL) and platelet viability determined by comparing collagen-induced platelet ag-

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Figure 2. Thrombus uptakes for [125I]fibrinogen, [111In]platelets, [99mTc]albumin, and complexes 1-3 under both venous and arterial conditions in the canine AV shunt model. All values are expressed as the mean ( SEM; N ) 10 for [111In]platelets and [125In]fibrinogen (except for venous conditions, where N ) 5), N ) 4 for albumin, N ) 6 for complex 1, N ) 3 for complex 2, and N ) 2 for complex 3.

gregation using nonlabeled and 111In-labeled platelets. Only those [111In]platelet preparations that were within 25% of control were injected into the animal. Canine Deep Vein Thrombosis Methodology. This model incorporates the triad of events (hypercoagulatible state, period of stasis, low-shear environment) essential for the formation of a venous fibrin-rich actively growing thrombus. Adult beagle dogs of either sex (9-13 kg) were anesthetized with pentobarbital sodium (35 mg/kg iv) and ventilated with room air via an endotracheal tube (12 strokes/min, 25 mL/kg). For arterial pressure determination, the right femoral artery was cannulated with a saline-filled polyethylene catheter connected to a pressure transducer (Model P23ID, Gould Co.). Heart rate was monitored using a cardiotachometer (Grass Instrument Inc.) triggered from a lead II electrocardiogram generated by limb leads. The right femoral vein was cannulated for drug administration. For the induction of a venous thrombus, a 5 cm segment of both jugular veins was isolated and circumscribed with silk suture. A balloon catheter (3-4 F, Baxter Co., McGraw Park, IL) was advanced from the facial vein into the jugular vein. A microthermister probe (Physitemp Co., Clifton, NJ) was placed on the vessel, which serves as an indirect measure of venous flow. A period of stasis and hypercoagulatibility was induced by inflating the balloon and the local administration of 5 units of thrombin (American Diagnosticia, Greenwich, CT) into the occluded segment. Fifteen minutes later, the balloon was deflated and flow reestablished as verified by the microthermister probe. The test agent was administered over 5 min beginning at reflow. Serial images were acquired using a gamma camera (Digital Dyna Camera, Picker International, Cleveland, OH) every 5 min for 2 h and region of interest and target to background ratios calculated. Arterial blood was withdrawn prior to administration and every 30 min thereafter for determination of blood clearance, hematology, platelet function, and coagulation status. At the end of the protocol the animal was euthanized with an overdose of pentobarbital and the vessel excised. The thrombus was removed and weighed, and the amount of

incorporation was determined via a gamma well counter (LKB 1282, Wallac Inc., Gaithersburg, MD). Hematologic Studies. Platelet, WBC, and RBC counts and hematocrit determinations were performed on whole blood collected in 2 mg/mL disodium EDTA using a Sysmex K1000 (TEA Medical Electronics Co., Los Alamitos, CA). Template bleeding time was assessed via an incision in the lower lip (Surgicutt, Baxter Co.) and the time to formation of a clot monitored. Whole blood platelet aggregation was measured using a lumiaggregometer (Chrono-Log Co., Havertown, PA) by recording the change in impedance (platelet aggregation). Blood samples were collected in 10 mM sodium citrate and diluted 50% with saline supplemented with 0.5 mM Ca. Aggregation was induced with collagen (5 µg/mL, ChronoLog Co.), and the changes in impedance were recorded over 6 min. APTT and PT were monitored using a microsample coagulation analyzer (MCA-210, BIO/DATA Co., Horsham, PA). Data Analysis. All values are expressed as the mean ( SEM. In the DVT studies the target (thrombus) uptake was calculated by drawing a 4 × 4 pixel area in the region interest and determining the average intensity. Background values were determined in a similar manner. Blood background was calculated using the jugular vein just distal to the locus of the thrombus. Statistical analysis consisted of a one-way analysis of variance and, when appropriate, Student’s paired t-test, two-tailed probability for assessing differences within treatment and a Newman-Keuls test for assessing differences between means of treatment groups. Differences were considered significant at P e 0.05. RESULTS AND DISCUSSION

In this study, we used a canine AV shunt model and a canine DVT imaging model to evaluate three platelet GPIIb/IIIa antagonists, [99mTc(HYNICtide)(L)(tricine)] (1, L ) TPPTS; 2, L ) TPPDS; 3, L ) TPPMS), for their potential use in the detection of rapidly growing arterial and venous thrombi. The thrombus formed in the AV shunt model was comprised of a platelet-rich head

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Barrett et al.

Figure 3. Blood clearance of [125I]fibrinogen, [111In]platelets, [99mTc]albumin, and complexes 1-3, expressed as a percentage of the end of infusion value in the canine AV shunt model. All values are expressed as the mean ( SEM; N ) 10 for [111In]platelets and [125I]fibrinogen (except for venous conditions, where N ) 5), N ) 4 for [99mTc]albumin, N ) 6 for complex 1, N ) 3 for complex 2, and N ) 2 for complex 3.

(arterial conditions) and a fibrin-rich tail (venous conditions), which allowed the rapid assessment of an agent under both arterial and venous conditions. To validate this model, the amount of incorporation of [111In]platelets (58 µCi), [125I]fibrinogen (106 µCi), and [99mTc]albumin (153 µCi) was assessed via well counting. [111In]platelets and [125I]fibrinogen were adequately incorporated into the growing thrombus with [111In]platelets favoring the platelet-rich head (5.63 ( 0.6% ID/g) with lesser amounts of incorporation observed in the fibrin-rich tail (0.17 ( 0.02% ID/g). Similar amounts of [125I]fibrinogen were incorporated under both the arterial and venous conditions (0.11 ( 0.02 vs 0.07 ( 0.02% ID/g, respectively), while little incorporation was observed with [99mTc]albumin (arterial, 0.05 ( 0.01% ID/g; venous, 0.04 ( 0.001% ID/g) (Figure 2). The administration of [111In]platelets, [125I]fibrinogen, and [99mTc]albumin did not alter any of the parameters studied (Table 1). Thus, the deposition on the thrombogenic surface mimics that of arterial conditions, i.e. high-shear, platelet-rich thrombus, and the platelet-poor tail mimics that of a venous thrombus, i.e. platelet-poor, low-shear environment.

Figure 4. Biodistribution of complexes 1-3. All values are expressed as the mean ( SEM; N ) 6 for complex 1, N ) 3 for complex 2, and N ) 2 for complex 3.

Complexes 1-3 were assessed in the canine AV shunt model, and all show incorporation into a growing thrombus in both the platelet-rich arterial portion and the venous platelet-poor tail (Figure 2). The greatest uptake of complexes 1-3 was observed under arterial conditions, which reflects the platelet-rich environment. Similar uptakes between complexes 1, 2, and 3 were seen under arterial conditions (arterial range, 7.78-9.93% ID/g), and all have significantly greater uptake than [125I]fibrinogen and [99mTc]albumin. Complex 1 also exhibited significantly greater uptake under arterial conditions than [111In]platelets (P e 0.05). Venous conditions, as expected, demonstrated less uptake in the platelet-poor tail, with all three complexes having greater uptake than [125I]fibrinogen and [99mTc]albumin (P e 0.05). Complex 3 appeared to have greater uptake; however, this apparent difference was not statistically significant due to interanimal variability. The uptake of complex 3 ranged from 1.2 to 6.2% ID/g. Complexes 1-3 share similar blood clearance with a half-life of about 90 min (Figure 3). The biodistribution data demonstrated that complexes 1 and 2 are preferentially renally excreted, while complex 3 was preferentially excreted via the hepatobilliary route (Figure 4). [99mTc]albumin was renally cleared, while [125I]fibrinogen demonstrated a mixed hepatobil-

Table 1. Summary of the Hematological and Hemodynamic Effects of [99mTc]Albumin and Complexes 1-3 at 1 mCi/kg iv in the Canine DVT Model and [111In]Platelets/[125I]Fibrinogen at 150 µCi/kg iv in the Canine AV Shunt Model parametera treatment [111In]platelets/[125I]fibrinogen control EOI 1 h postinfusion [99Tc]albumin control EOI 1 h postinfusion complex 1 control EOI 1 h postinfusion complex 2 control EOI 1 h postinfusion complex 3 control EOI 1 h postinfusion

heart rate (bpm)

mean arterial pressure (mmHg)

APTT (s)

platelet count (×10E3)

aggregation (ohms)

135 ( 5 128 ( 5 124 ( 5

113 ( 8 113 ( 7 112 ( 7

17 ( 1 21 ( 2 17 ( 1

272 ( 17 264 ( 17 264 ( 19

27 ( 3 29 ( 2 29 ( 2

150 ( 6 150 ( 5 147 ( 7

124 ( 8 124 ( 8 124 ( 10

15 ( 0 15 ( 1 15 ( 1

281 ( 8 289 ( 11 284 ( 17

24 ( 1 27 ( 3 23 ( 2

139 ( 5 139 ( 4 138 ( 6

118 ( 7 111 ( 6 109 ( 1

16 ( 1 16 ( 2 15 ( 3

278 ( 12 291 ( 9 287 ( 4

21 ( 2 22 ( 1 23 ( 2

121 ( 8 109 ( 1 113 ( 6

88 ( 15 78 ( 13 84 ( 10

16 ( 1 14 ( 1 14 ( 1

267 ( 20 225 ( 13 264 ( 21

22 ( 2 20 ( 4 26 ( 1

164 ( 7 163 ( 7 166 ( 11

94 ( 8 97 ( 11 103 ( 8

16 ( 1 16 ( 1 15 ( 1

281 ( 37 279 ( 37 281 ( 45

22 ( 3 19 ( 2 22 ( 3

a All values are expressed as the mean ( SEM; N ) 10 for [111In]platelets and [125I]fibrinogen, N ) 4 for [99Tc]albumin and complex 1, N ) 3 for complex 2, and N ) 2 for complex 3.

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Figure 5. Canine DVT thrombus uptake at 120 min (left) and thrombus/blood ratio (right) for [125I]fibrinogen, [111In]platelets, [99mTc]albumin, and complexes 1-3 under both venous and arterial conditions. The thrombus uptake and target/background ratios for all three complexes were significantly greater (P < 0.05) than for [99mTc]albumin. There were no significant differences between complexes 1, 2, and 3 (P < 0.5). Target to background ratios were calculated on the basis of an average 4 × 4 pixel area in the region of interest. All values are expressed as the mean ( SEM; N ) 6 for [99mTc]albumin, N ) 5 for complex 1, N ) 4 for complex 2, and N ) 4 for complex 3.

Figure 6. Representative DVT images of complexes 1-3 at 15, 60, and 120 min postinfusion. The bar to the right of the images indicates the scale from 0 (white) to 506 (greatest/black). The images have not been filtered. Arrows indicate presence of thrombus in jugular veins.

lary/renal excretion pattern. In contrast, [111In]platelets were sequestered in the spleen. Complexes 1-3 and [99mT]albumin have been evaluated in the canine deep vein thrombosis imaging model (DVT). The sample data from the LKB gamma well counter show no significant difference between the three complexes in respect to % ID/g, although all three are significantly greater than [99mTc]albumin (P e 0.05, Figure 5). All three complexes demonstrated similar rates of clearance from the circulating blood.

Representative unfiltered images of the three complexes in the DVT imaging model at 15, 60, and 120 min postinfusion are shown in Figure 6. The scale to the right of the images represents a fixed scale that increases to the hottest or most dense pixel represented by 506 (black). The region of interest (ROI) data acquired from the images are shown in Figure 6 as target to background ratios. Complexes 1, 2, and 3 were rapidly incorporated into the growing thrombus, which can be easily visualized by 15 min with the thrombus/muscle ratios of 3.8 ( 0.8,

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2.8 ( 0.4, and 3.0 ( 0.8, respectively. Increased uptake over time was also observed with exceptional imaging at 60 min. All three complexes are significantly greater than [99mTc]albumin with respect to ROI data (P e 0.05). Hematologic and hemodynamic data were unaffected by the administration of the complexes and remained in normal range throughout the DVT study (Table 1). The present study is in agreement with the work of Oster et al. (14), who demonstrated that the 7E3 antibody, directed against the the platelet GPIIb/IIIa receptor, was capable of detecting both arterial and venous thrombi. More recently, a series of disintegrins derived from snake venom demonstrated limited utility in detecting venous thrombi (15). In addition, a 99mTc-labeled peptide (P280) that targets the platelet GPIIb/IIIa site has been shown to detect growing venous thrombi in man (16). Thus, on the basis of these data the targeting of the platelet GPIIb/ IIIa receptor is a viable approach for detecting thromboembolic events throughout the body. In summay, complexes 1-3 show remarkably similar incorporation into a growing thrombus and blood clearance with a t1/2 of 90 min. None of the compounds affected hemodynamics or hematological values, which was consistent with the subtherapeutic levels administered. In addition, complexes 1-3 exhibit similar abilities to detect a growing thrombus within 15 min postinjection. Further, complexes 1-3 did not significantly differ in their uptake into the growing thrombus, blood clearance, and target to background ratio. Therefore, it is concluded that all three agents are able to detect rapidly growing venous and arterial thrombi. ACKNOWLEDGMENT

Acknowledgment is made to P. R. Damphousse for the synthesis of TPPDS and to Dr. T. D. Harris, Dr. M. Rajopadhye, Dr. D. Glowacka, J. P. Bourque, P. R. Damphousse, and K. Yu for the synthesis of the cyclic GPIIb/IIIa HYNICtide, cyclo[D-Val-NMeArg-Gly-AspMamb(5-(6-(6-hydrazinonicotinamido)hexanamide)))]. LITERATURE CITED (1) Edwards, D. S., Liu, S., Barrett, J. A., Harris, A. R., Looby, R. J., Ziegler, M. C., Heminway, S. J., and Carroll, T. R. (1997) A new and versatile ternary ligand system for technetium radiopharmaceuticals: water soluble phosphines and tricine as coligands in labeling a hydrazino nicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjugate Chem. 8, 146-154. (2) Barrett, J. A., Heminway, S. J., Damphousse, D. J., Thomas, J. R., Looby, R. J., Edwards, D. S., Harris, T. D., Rajopadhye, M., Liu, S., and Carroll, T. R. (1994) Platelet GP IIb/IIIa antagonists in the canine arteriovenous shunt: potential thrombus imaging agents. J. Nucl. Med. 35, 52P (Abstract 202). (3) Harris, T. D., Rajopadhye, M., Damphousse, P. R., Glowacka, D., Yu, K., Bourque, J., Barrett, J. A., Damphousse, D.

Barrett et al. J., Heminway, S. J., Lazewatsky, J., Mazaika, T., and Carroll, T. R. (1996) Tc-99m-labeled fibrinogen receptor antagonists: Design and synthesis of cyclic RGD peptides for the detection of thrombi. Bioorg. Med. Chem. Lett. 6, 1741-1746. (4) Barrett, J. A., Bresnick, M., Crocker, A., Damphousse, D. J., Hampson, J. R., Heminway, S. J., Mazaika, T. J., Kagan, M., Lazewatsky, J., Edwards, D. S., Liu, S., Harris, T. D., Rajopadhye, M., and Carroll, T. R. (1995) RP-431: a potential thrombus imaging agent. J. Nucl. Med. 36, 16P (Abstract 55). (5) Liu, S., Edwards, D. S., Looby, R. J., Harris, A. R., Poirier, M. J., Barrett, J. A., and Heminway, S. J. (1996) Labeling a hydrazino nicotinamide-modified cyclic IIb/IIIa receptor antagonist with 99mTc using aminocarboxylates as coligands. Bioconjugate Chem. 7, 63-71. (6) Liu, S., Edwards, D. S., Looby, R. J., Harris, A. R., Poirier, M. J., Rajopadhye, M., and Bourque, J. P. (1996) Labeling cyclic IIb/IIIa receptor antagonists with 99mTc by the preformed chelate approach. Bioconjugate Chem. 7, 196-202. (7) Barrett, J. A., Damphousse, D. J., Heminway, S. J., Liu, S., Edwards, D. S., Looby, R. J., and Carroll, T. R. (1996) Biological evaluation of 99mTc-labeled cyclic GPIIb/IIIa receptor antagonists in the canine arteriovenous shunt and deep vein thrombosis models: effects of chelators on biological properties of 99mTc-chelator-peptide conjugates. Bioconjugate Chem. 7, 203-208. (8) Knight, L. C. (1990) Radiopharmaceuticals for thrombus detection. Semin. Nucl. Med. 20, 52-67. (9) Fuster, V., Stein, B., Badimon, L., and Chesebro, J. (1988) Antithrombotic therapy after myocardial reperfusion in acute myocardial infarction. J. Am. Col. Cardiol. 12, 78A-84A. (10) Plow, E. F., Marguerie, G., and Ginsberg, M. (1987) Fibrinogen, fibrinogen receptors and the peptides that inhibit these interactions. Biochem. Pharmacol. 36, 4035-4041. (11) Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F. (1985) Changes in the platelet membrane glycoprotein IIb/ IIIa complex during platelet activation. J. Biol. Chem. 260, 11107-11110. (12) Thakur, M. L., Welch, M. J., Joist, J. H., and Coleman, R. E. (1976) In-111 labeled platlets: studies on preparation and evaluation of in vitro and in vivo function. Thrombus Res. 9, 345-357. (13) Haskel, E., Adams, S., Feigen, L., Saffitz, J., Gorczynski, R., Sobel, D., and Abendschein, D. (1989) Prevention of reoccluding platelet-rich thrombi in canine femoral arteries with a novel peptide antagonist of platelet glycoprotein IIb/ IIIa receptors. Circulation 80, 1775-1782. (14) Oster, Z., Srivastava, S., Som, P., Meinken, G., Scudder, L., Yamamoto, K., Atkins, H., Brill, A., and Coller, B. (1985) Thrombus radioimmunoscintigraphy: An approach using monoclonal antiplatelet antibody. Proc. Natl. Acad. Sci. U.S.A. 82, 3465-3468. (15) Knight, L., Maurer, A., and Romano, J. (1996) Comparison of iodine-123-disintegrins for imaging thrombi and emboli in a canine model. J. Nucl. Med. 37, 476-482. (16) Muto, P., Lastoria, S., Varrella, P., Vergara, E., Salvatore, M., Morgano, G., Lister-James, J., Bernardy, J., Dean, R., Wencker, D., and Borer, J. (1995) Detecting deep venous thrombosis with technetium-99m-labeled synthetic peptide P280. J. Nucl. Med. 36, 1384-1391.

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