Bioconjugate Chem. 1999, 10, 803−807
803
99mTc-Labeling
of Hydrazones of a Hydrazinonicotinamide Conjugated Cyclic Peptide
D. Scott Edwards, Shuang Liu,* Anthony R. Harris, Michael J. Poirier, and Barbara A. Ewels DuPont Pharmaceuticals Company, Medical Imaging Division, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received February 24, 1999; Revised Manuscript Received April 9, 1999
Eight HYNICtide hydrazones (three with aliphatic substituents and five with aromatic groups) were studied for their potential use as the final intermediate for preparation of RP444, a new radiopharmaceutical under development for imaging thrombosis. The goal of this study is to screen various hydrazones through stability testing and radiolabeling and find those which are able to remain stable without significant degradation in the manufacturing process and at the same time are reactive to produce enough free hydrazine in situ for successful 99mTc-labeling. In an initial screening study, only hydrazones 6 and 8, which contain aliphatic substitients, gave satisfactory (g90%) yields of RP444 using 50 °C and 30 min of heating. However, their solution instability excludes them from being used as commercial reagents. Hydrazones 1 and 4 gave g90% yields when the reaction mixtures were heated at 80 °C for 30 min. Both hydrazone 1 and hydrazone 4 can be used as the final intermediate for preparation of RP444. The combination of 40 mg of tricine, 1-10 mg of TPPTS, 20-40 µg of hydrazone 1 or 4 for 50 mCi of [99mTc]pertechnetate, 20-50 µg of stannous chloride, pH 4.5 ( 0.5, and heating at 80 °C for 30 min gives the best yield for RP444. It is surprising that hydrazones 1 and 4 have both the solution stablity with respect to decomposition and to reaction with aldehydes and ketones and yet are able to hydrolyze in situ to produce enough free HYNICtide for the 99mTc-labeling.
INTRODUCTION
Recently, Abrams and co-workers (1-3) reported the use of organic hydrazines such as hydrazinonicotinamide (HYNIC) in labeling polyclonal IgG with 99mTc for imaging focal sites of infection. This technology has also been used for the 99mTc-labeling of a variety of biologically active molecules. These include chemotactic peptides (47) for infection imaging, somatostatin analogues (8, 9), and antisense DNA oligonucleotides (10-12) for tumor imaging. We are interested in the 99mTc-labeling of glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists for the development of thrombosis imaging agents (13-20). Recently, we labeled an HYNIC-derivatized cyclic peptide [HYNICtide, cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotinamido)hexanamide))))] using tricine and TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate) as coligands (19). The combination of HYNICtide with tricine and phosphine produces a new ternary ligand system which forms stable technetium complexes, [99mTc(HYNICtide)(tricine)(TPPTS)] (RP444) in high yield and high specific activity (g20 000 Ci/mmol). In the canine arteriovenous (AV) and deep vein thrombosis (DVT) models, RP444 has been shown to be able to detect rapidly growing arterial and venous thrombi (20). The phase I clinical trials demonstrated an excellent safety profile and acceptable dosimetry. Preliminary results from phase II trials showed that RP444 can detect DVT within 60 min with 93% sensitivity, 75% specificity, and 82% accuracy (21). * To whom correspondence should be addressed: Phone: 978-671-8696. Fax: 978-436-7500. E-mail: shuang.liu@ dupontpharma.com.
In the manufacturing process, we found that the final intermediate HYNICtide itself is not stable in aqueous solution, particularly under basic conditions, and reacts readily with aldehydes or ketones. The presence of aldehydes and ketones in small quantities is unavoidable in a commercial pharmaceutical manufacturing setting, because they can be extracted from various plastic and rubber materials, and are also found in common disinfectants. In a recent patent application (22), Schwartz et al. disclosed the use of a lower alkyl hydrazone (propylaldehyde hydrazone) to stabilize HYNIC-modified proteins. Although its use can prevent the cross-reaction of the hydrazine with other reactive groups on the proteins, it can also be displaced by other aldehydes, formaldehyde in particular, and ketones to form different hydrazones. This presents a significant problem in maintaining the purity of the final intermediate and, thus, renders lower alkyl hydrazones unattractive as commercial reagents. To prevent the decomposition or degradation of the HYNICtide in the manufacturing process, several stable hydrazones have been synthesized (23, 24) and studied for their solution stability in the presence of formaldehyde. It was found that for a hydrazone to be stable in aqueous solution there must be a conjugated π-system (23, 24). The radiolabeling efficiency of the HYNICtide is dependent on the availability of the free hydrazine group, relative component levels, and reaction conditions. The formation of hydrazones stabilizes HYNICtide and prevents its degradation or decomposition, but it reduces the availability of the free hydrazine group for 99mTc-labeling. To assess the impact of hydrazone formation on the 99mTclabeling of the HYNICtide, we performed a series of radiolabeling studies and explored the factors influencing the 99mTc-labeling kinetics of several stable hydrazones (Figure 1). The results are described in this report.
10.1021/bc990022e CCC: $18.00 © 1999 American Chemical Society Published on Web 07/08/1999
804 Bioconjugate Chem., Vol. 10, No. 5, 1999
Edwards et al.
Figure 2. Synthesis of RP444 from HYNICtide hydrazones.
Figure 1. Hydrazones of the HYNICtide. EXPERIMENTAL SECTION
Materials. TPPTS (trisodium triphenylphosphine3,3′,3′′-trisulfonate) and tricine were purchased from Aldrich Chemical Co. TPPTS was purified according to the literature method (25) before use for the radiolabeling. Na99mTcO4 was obtained from a commercial DuPont Pharma 99Mo/99mTc generator, N. Billerica, MA. The syntheses of hydrazones 1-8 of the HYNICtide with benzaldehyde, 4-(N,N-dimethylamino)benzaldehyde, 4-carboxy-benzaldehyde, sodium 2-formylbenzenesulfonate, 4-pyridinecarboxaldehyde, propylaldehyde, crotonaldehyde, and glyoxylic acid will be reported as a separate communication (24). General Procedure for the 99mTc-Labeling of Hydrazones. To a clean 10 mL vial were added 0.4 mL of tricine solution (100 mg/mL in H2O), 0.2 mL of the hydrazone solution (50 µg/mL in H2O), 0.1 mL of phosphine coligand solution (10 mg/mL in H2O), 0.5 mL of Na99mTcO4 solution (100 mCi/mL in saline), and 20 µL of SnCl2‚2H2O solution (1.0 mg/mL in 0.1 N HCl). The pH was adjusted to 4.5 ( 0.5, if necessary. The reaction mixture was heated at 50 or 80 °C for 30 min. After cooling at room temperature, the reaction mixture was analyzed by radio-HPLC and ITLC. The HPLC method used a heated (50 °C) Zorbax C18 column (4.6 mm × 250 mm, 80 Å pore size) at a flow rate of 1 mL/min. The mobile phase is isocratic from 0 to 14 min using 100% mobile phase A [90:10 (0.025 M phosphate buffer, pH 8):acetonitrile] and from 14 to 25 min using 100% mobile phase B [50:50 (0.025 M phosphate buffer, pH 8):acetonitrile]. 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, RP444 and [99mTc]pertechnetate migrate while the [99mTc]colloid remains at the origin. RESULTS AND DISCUSSION
In the development of a new 99mTc-based radiopharmaceutical, several factors need to be considered. These include biological efficacy (high target uptake, high target-to-background ratio, and favorable pharmacokinetics), high radiochemical purity (RCP g 90%), and high
Figure 3. HPLC chromatogram of RP444 prepared from hydrazone 4.
solution stability (g6 h). In addition, a kit formulation is often required due to the 6 h half-life of 99mTc. A kit contains the final drug intermediate (a chelator-peptide conjugate for peptide-based radiopharmaceuticals), a reducing agent such as stannous chloride, if necessary, and other components such as a bulking agent or a weak transfer ligand. Kits can be purchased and stored for daily preparation. In many cases, the 99mTc-labeling can be accomplished simply by adding [99mTc]pertechnetate to the kit. RP444 contains three different ligands (HYNICtide, TPPTS, and tricine) in the technetium chelate. All of them are considered as active ingredients or final intermediates. Tricine and TPPTS are relatively stable to oxidation and inert to reactions with chemicals often found in commercial pharmaceutical manufacturing settings. The free hydrazine group of the HYNICtide is not stable to oxidation, particularly under basic conditions, and reacts readily with aldehydes or ketones, which are extracted from various plastic and rubber materials. This makes it very difficult to maintain the purity and the stability of the HYNICtide. To solve the problem, several aromatic aldehydes have been used to form stable hydrazones with the HYNICtide. These hydrazones were found to be stable to the challenge of 10-fold excess formaldehyde (23, 24). Hydrazones are used only as protecting groups for the HYNICtide. In the radiolabeling process, the hydrazones must hydrolyze (Figure 2) to produce sufficient quantity of the hydrazine that binds to the technetium via either a Tc-hydrazino or Tc-diazenido bond. However, the hydrolysis does not need to go to completion since there are typically 50-100 equiv of the hydrazone to total Tc in each reaction. All the hydrazones react with [99mTc]pertechnetate in the presence of stannous chloride, excess tricine, and TPPTS coligands to form the same ternary ligand complex RP444 as that prepared from the unprotected HYNICtide. Figure 3 shows a representative radioHPLC chromatogram of RP444 prepared from hydrazone 4. The presence of two radiometric peaks in the radioHPLC chromatogram is due to the resolution of diaster-
99mTc-Labeling
of Hydrazones
Bioconjugate Chem., Vol. 10, No. 5, 1999 805
Table 1. Radiolabeling Yields of RP444 Using Hydrazones at 50 °C
Table 2. Radiolabeling Yields of RP444 Using Hydrazones at 80 °C
hydrazone
RCP (%)a
n
hydrazone
RCP (%)a
n
hydrazone
RCP (%)
n
hydrazone
RCP (%)
n
1 2 3 4
85.7 ( 2.5 79.0 ( 1.0 74.5 ( 0.7 79.7 ( 1.5
4 3 3 5
5 6 7 8
31.3 ( 5.5 94.5 81.0 ( 7.9 91.3 ( 0.6
3 1 3 3
1 (TFA salt) 1 (mesylate salt) 2
94.0 ( 1.4 92.0 ( 1.0 86.0 ( 1.7
4 3 3
3 4 7
79.5 91.0 ( 2.0 83.3 ( 1.5
1 5 3
a
Calculated by subtracting the percent peak area for the [99mTc]colloid determined by ITLC from the percent peak area of RP444 in the radio-HPLC. In all the cases, the [99mTc]colloid was less that 2%.
eomers, which result from the presence of chiral cyclic peptide and the formation of two enantiomers of the technetium chelate (19, 26). The small peak at ∼16 min is from a combination of several radioimpurities. To develop an accurate RCP method for RP444, we intentionally “squeezed” these radioimpurities into a single peak using the chromatographic conditions described in the experimental. Since these radioimpurities are less than 1.0%, no further characterization was performed. The yield of RP444 depends largely on the kinetics of hydrolysis, as well as the total concentration of the unprotected HYNICtide in the reaction mixture. The higher degree of the hydrolysis of the hydrazone, the more free hydrazine groups available for Tc-binding, and the higher the radiolabeling yield of RP444, within certain limits. The goal of this study was to screen various hydrazones through stability testing and radiolabeling and find those which are able to remain stable without significant degradation in the manufacturing process and at the same time are reactive to produce enough free hydrazine in situ for successful 99mTclabeling. There are several factors influencing the hydrolysis of these hydrazones. These include the type of hydrazone, the heating temperature, the reaction time, and the pH of the reaction mixture. The initial labeling study was focused on the screening of hydrazones. The hydrazones screened included three with aliphatic substituents and five with aromatic groups functionalized by electron withdrawing or electron donating substituents. In this study, we fixed the amount of hydrazone (10 µg), tricine (40 mg), TPPTS (1 mg), 99mTcO4- (50 mCi), and stannous chloride (25 µg). The total volume for each radiolabeling kit was ∼1.2 mL. The pH was adjusted to 4.0 ( 0.5. After heating in a 50 °C water bath for 30 min, the reaction mixtures were analyzed by HPLC and ITLC. The RCP of RP444 was corrected for the contribution from [99mTc]colloid. The results are summarized in Table 1. It is obvious that the type of hydrazone has dramatic impact on the yield of RP444. The hydrazones 6 and 8 contain aliphatic substituents and gave highest yield (>90%) of RP444 because they can be easily hydrolyzed in situ to produce the free HYNICtide for the 99mTc-labeling. However, both of them were found to be unstable to the challenge of 10-fold excess formaldehyde (23, 24). Therefore, they were not suitable as commercial reagents for the preparation of RP444. Hydrazone 5 contains a substituted pyridine ring and gave very low yield (∼30%) of RP444 probably because it is too stable in aqueous solution to produce enough free HYNICtide for the 99mTc-labeling. Therefore, it is not suitable to be used as a commercial reagent either. Hydrazones 1-4 contain an aromatic benzene ring with various substituents, while the hydrazone 7 has propenyl substituent (Figure 1). These five hydrazones gave yields between 75 and 85% and were studied in the
a Calculated by subtracting the percent peak area for the [99mTc]colloid determined by ITLC from the percent peak area of RP444 in the radio-HPLC. In all the cases, the [99mTc]colloid was less that 2%.
Figure 4. Plot of RCP of RP444 versus pH. Each vial contains 12.5 µg of hydrazone 4, 3.5 mg of TPPTS, 40 mg of tricine, and 40 µg of SnCl2‚2H2O with pH varying from 2.0 to 7.0.
next radiolabeling experiment using higher heating temperature to see if the yield of RP444 could be improved. In this experiment, all the component levels and reaction conditions remain the same as those used in the screening study except that the reaction mixtures were heated at 80 °C for 30 min. After radiolabeling, the reaction mixtures were analyzed by HPLC and ITLC. The RCP of RP444 was corrected for the contribution from [99mTc]colloid. The results are shown in Table 2. In general, the higher temperature and longer reaction time gave better yield for RP444, probably due to faster hydrolysis at higher temperatures. Hydrazones 1 and 4 gave yields of RP444 higher than 90% using 80 °C and 30 min heating. Different salt forms (trifluoroacetate and mesylate) of hydrazone 1 did not have a detectable effect on the yield of RP444. The remaining three hydrazones did not give significantly higher yields than those obtained at 50 °C. On the basis of these data, it was concluded that hydrazones 1 and 4 can be used as the final intermediate for preparation of RP444 under the radiolabeling conditions descibed in the Experimental Section. It should be noted that these conditions are not optimized. Under more forcing conditions (such as heating at 100 °C), hydrazones 2 and 7 may also give g90% yields. We also explored the effect of varying pH and the component levels on the yield of RP444 using hydrazone 4. The component levels are shown in Figures 4-7. Vials containing these components were reconstituted by adding 0.5 mL of [99mTc]pertechnetate solution (100 mCi/mL). The prior elution time for the generator was 24 ( 2 h, and the eluate age was 0-2 h. The total volume of the reaction mixture was ∼1.5 mL. The stannous chloride concentration was fixed at 40 µg/vial. The pH of the reaction mixture was adjusted using 1.0 N HCl. Reactions were carried out in duplicate by heating reaction mixtures at 80 °C for 30 min. The resulting solutions were analyzed by radio-HPLC and ITLC. The RCP was corrected for the contribution from [99mTc]colloid. pH Effect. The pH of the reaction mixture has a dramatic impact on the RCP of RP444 (Figure 4). At pH
806 Bioconjugate Chem., Vol. 10, No. 5, 1999
Figure 5. Plot of RCP of RP444 versus tricine concentration. Each vial contains 12.5 µg of hydrazone 4, 3.5 mg of TPPTS, 40 µg of SnCl2‚2H2O, and various amounts of tricine with pH 3.5.
Figure 6. Plot of RCP of RP444 versus TPPTS concentration. Each vial contains 12.5 µg of hydrazone 4, 40 mg of tricine, 40 µg of SnCl2‚2H2O, and various amounts of TPPTS with pH 3.5.
Edwards et al.
tide)(tricine)2] to form RP444. Figure 6 shows the effect of TPPTS concentration on the RCP of RP444. If the amount of TPPTS was lower than 1.0 mg/1.5 mL, the yield of RP444 was dramatically reduced. Increasing the amount of TPPTS from 1.0 mg/1.5 mL to 10.0 mg/1.5 mL had no significant effect on the yield of RP444 within the experimental error. Under ideal radiolabeling conditions, the TPPTS concentration could be varied from 1.0 mg/ 1.5 mL to 10 mg/1.5 mL. Hydrazone Concentration. Hydrazone concentration also affects the RCP of RP444. When the hydrazone is in large excess over the total Tc (99mTc and 99Tc) in each reaction vial, hydrolysis of a small percentage of hydrazone should produce enough free HYNICtide for radiolabeling. Thus, it is not surprising that increasing the amount of hydrazone 4 from 10 to 40 µg did not have a significant effect on the yield of RP444 (Figure 7). The optimum concentration for hydrazone 4 is 20-40 µg/1.5 mL for 50 mCi of [99mTc]pertechnetate. It should be noted that the purity of hydrazones also has dramatic impact on the yield of RP444. HYNICtide hydrazones are prepared by conjugation of cyclic peptide with slight excess succinimidyl 6-(2-aldehydehydrazino)nicotinate (23, 24). The excess succinimidyl ester is then converted to 6-(2-aldehyde-hydrazino)nicotinamide for the purpose of easy separation. Hydrazones used in this study often contain a small amount of the HYNIC-amide hydrazone as impurities. For example, hydrazone 1 contains a trace amount of 6-(2-benzaldehydehydrazino)nicotinamide. Since the molecular weight of 6-(2-benzaldehydehydrazino)nicotinamide is only ∼1/5 of that of hydrazone 1, one percent of it will cause at least a 5% decrease in the yield of RP444. Stannous chloride is a reducing agent for [99mTc]pertechnetate. If the stannous chloride level is too low (e5 µg/mL), [99mTc]pertechnetate is often not completely reduced, and the yield of RP444 is low. If a large amount (g100 µg/vial) is used, [99mTc]colloid formation becomes a major problem, and the corrected yield of RP444 is also low. During these radiolabeling studies, we found that the optimum range of stannous chloride is 20-50 µg/mL. CONCLUSION
Figure 7. Plot of RCP of RP444 versus HYNICtide concentration. Each vial contains 3.5 mg of TPPTS, 40 mg of tricine, 40 µg of SnCl2‚2H2O, and various amounts of hydrazone 4 with pH 5.0.
e3.5, the tricine coligand may become protonated, and is not able to stabilize the [99mTc]tricine intermediate. This may lead to decomposition of the [99mTc]tricine intermediate, and formation of a significant amount of [99mTc]pertechnetate. At pH g5.0, the hydrazone remains highly stable in solution. There is not enough hydrolyzed free hydrazine for 99mTc-labeling. Therefore, the yield of RP444 was also low. The ideal pH range for successful radiolabeling is 3.5-5.0. Tricine Concentration. Tricine is both a stabilizing agent for the [99mTc]tricine intermediate and a coligand in the ternary ligand system at the same time. Increasing the amount of tricine from 40 to 60 mg did not have significant impact on the yield of RP444 (Figure 5). At a lower tricine level (e20 mg), [99mTc]colloid formation became significant, and resulted in lower yield for RP444. The optimum concentration for tricine is 40-60 mg/vial. TPPTS Concentration. In the reaction mixture, TPPTS reacts with the binary complex [99mTc(HYNIC-
In conclusion, eight HYNICtide hydrazones (three with aliphatic substituents and five with aromatic groups functionalized by electron withdrawing or electron donating substituents) were screened and studied for their potential use as the final intermediate for preparation of RP444. It was found that using 50 °C and 30 min, heating only hydrazones 6 and 8 gave satisfactory (g90%) yields of RP444. However, their solution instability excludes them from being used as commercial reagents. Hydrazones 1 and 4 gave g90% yields when reaction mixtures were heated at 80 °C for 30 min. The salt forms (trifluoroacetate and mesylate) of hydrazone 1 did not show a detectable effect on the yield of RP444. Both hydrazone 1 and hydrazone 4 can be used as the final intermediate for preparation of RP444. It seems that the combination of 40 mg tricine, 1-10 mg of TPPTS, 1040 µg of hydrazone for 50 mCi of [99mTc]pertechnetate, 20-50 µg of stannous chloride, pH 4.5 ( 0.5, and heating at 80 °C for 30 min gives best yield of RP444. Under more forcing conditions (such as heating at 100 °C for 30 min), hydrazones 2 and 7 may also be used as the final intermediate for RP444 and give g90% yields. Both hydrazones 1 and 4 have demonstrated their solution stability. They do not react with other aldehydes
99mTc-Labeling
of Hydrazones
and ketones (23, 24). It is surprising that both hydrazones are stable enough to withstand a 10-fold excess of formaldehyde, and at the same time are reactive enough to hydrolyze in situ producing free HYNICtide for successful 99mTc-labeling. LITERATURE CITED (1) Schwartz, D. A., Abrams, M. J., Hauser, M. M., Gaul, F. E., Larsen, S. K., Rauh, D., and Zubieta, J. A. (1991) Preparation of hydrazino-modified proteins and their use for the synthesis of 99mTc-protein conjugates. Bioconjugate Chem. 2, 334-336. (2) Abrams, M. J., Juweid, M., tenKate, C. I., Schwartz, D. A., Hauser, M. M., Gaul, F. E., Fuccello, A. J., Rubin, R. H., Strauss, H. W., and Fischman, A. J. (1990) Technetium-99mhuman polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats. J. Nucl. Med. 31, 2022-2028. (3) Larson, S. K., Solomon, H. F., Caldwell, G., and Abrams, M. J. (1995) [99mTc]Tricine: a useful precursor complex for the radiolabeling of hydrazinonicotinate protein conjugates. Bioconjugate Chem. 6, 635-638. (4) Edwards, D. S., Barrett, J. A., Liu, S., Ziegler, M. C., Mazaika, T., Vining, M., Bridger, G., Higgins, J., III, and Abrams, M. J. (1996) A stabilized Tc-99m complex of a chemotactic peptide-HYNIC conjugate for imaging infection. Eur. J. Nucl. Med. 23, 1142 (abstr. Omo440). (5) Babich, J. W., Solomon, H., Pike, M. C., Kroon, D., Graham, W., Abrams, M. J., Tompkins, R. G., Rubin, R. H., and Fischman, A. J. (1993) Technetium-99m-labeled hydrazino nicotinamide derivatized chemotactic peptide analogues for imaging focal sites of bacterial infection. J. Nucl. Med. 34, 1967-1974. (6) Babich, J. W., and Fischman, A. J. (1995) Effect of “coligand” on the biodistribution of 99mTc-labeled hydrazino nicotinic acid derivatized chemotactic peptides. Nucl. Med. Biol. 22, 25-30. (7) Fischman, A. J., Babich, J. W., and Rubin, R. H. (1994) Infection imaging with technetium-99m-labeled chemotactic peptide analogues. Semin. Nucl. Med. 24, 154-168, references therein. (8) Ma¨cke, H. R., and Behe, M. (1996) New octreotide derivatives labelled with technetium-99m. J. Nucl. Med. 37, 29P (abstr. 107). (9) Behe, M., and Ma¨cke, H. R. (1995) New somatostatin analogues labelled with technetium-99m. Eur. J. Nucl. Med. 22, 791 (abstr. 267). (10) Dewanjee, M. K., Ghalfouripour, A. K., Subramanian, M., Hanna, M., Kapadvanjwala, M., Serafini, A. N., Ezuddin, S., Lopez, D., and Sfakianakis, G. N. (1994) Labeling antisense deoxyoligonucleotide with Tc-99m and hybridation with c-myc oncogene mRNA in P388 leukemic cells. J. Labeled Compds Radiopharm. 35, 40-42. (11) Dewanjee, M. K., Abrams, M. J., Wu, S. M., Kapadvanjwala, M., Serafini, A. N., and Sfakianakis, G. N. (1995) Technetium-99m labeled single-strand DNA probe for imaging thrombin in viscera formed during cardiopulmonary bypass. J. Nucl. Med. 36, 16P (abstr. 54). (12) Hnatowich, D. J., Winnard, P., Jr., Virzi, F., Fogarsi, M., Sano, T., Smith, C. L., Cantor, C. L., and Rusckowski, M. (1995) Technetium-99m labeling of DNA oligonucleotides. J. Nucl. Med. 36, 2306-2314. (13) Liu, S., Edwards, D. S., and Barrett, J. A. (1997) 99mTclabeling of highly potent small peptides. Bioconjugate Chem. 8, 621-636. (14) Edwards, D. S., and Liu, S. (1997) 99mTc-labeling of hydrazino nicotinamide (HYNIC) modified highly potent small molecules: problems and solutions. Transition Metal Chem. 22, 425-426.
Bioconjugate Chem., Vol. 10, No. 5, 1999 807 (15) Liu, S., and Edwards, D. S. (1995) New N2S2 diamidedithiol and N3S triamidethiols as bifunctional chelating agents for labeling small peptides with technetium-99m. In Technetium and Rhenium in Chemistry and Nuclear Medicine (M. Nicolini, G. Banoli, and U. Mazzi, Eds.) Vol. 4, pp 383-393, SGEditorali, Padova. (16) Liu, S., Edwards, D. S., Looby, R. J., Harris, A. R., Poirier, M. J., Barrett, J. A., Heminway, S. J., and Carroll, T. R (1996) Labeling a hydrazino nicotinamide-modified cyclic IIb/IIIa receptor antagonist with 99mTc using aminocarboxylates as coligands. Bioconjugate Chem. 7, 63-71. (17) 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: effects of chelators on properties of [99mTc]chelator-peptide conjugate. Bioconjugate Chem. 7, 196-202. (18) 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. (19) Edwards, D. S., Liu, S., Harris, A. R., Looby, R. J., Ziegler, M. C., Heminway, S. J., Barrett, J. A., 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 hydrazinonicotinamidemodified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjugate Chem. 8, 146-154. (20) Barrett, J. A., Crocker, A. C., Damphousse, D. J., Heminway, S. J., Liu, S., Edwards, D. S., Harris, A. R., Looby, R. J., Lazewatsky, J. L., Kagan, M., Mazaika, T. J., and Carroll, T. R. (1997) Biological evaluation of thrombus imaging agents utilizing water soluble phosphines and tricine as coligands to label a hydrazinonicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjuate Chem. 8, 155-160. (21) Line, B. R., Seabold, J. E., Edell, S. L., Weiland, F. L., Heit, J. A., Crane, P. D., Widner, P. L., and Haber, S. J. (1998) Preliminary diagnostic results with DMP444, a new thrombus imaging agent. J. Nucl. Med. 39, 218P (abstr. 969). (22) Schwartz, D. A., Abrams, M. J., Gladomenico, C. M., and Zubieta, J. A. (1993) Certain pyridyl hydrazines and hydrazides useful for protein labeling. U.S. Patent 5,206,370. (23) Sworin, M., Rajopadhye, M., Harris, T. D., Edwards, D. S., Cheesman, E. H., and Liu, S. (1998) Stable hydrazones linked to a peptide moiety as reagents for the preparation of radiopharmaceuticals. U.S. Patent 5,750,088. (24) Harris, T. D., Rajopadhye, M., Sworin, M., and Cheesman, E. H. Synthesis of stable hydrazones of a hydrazinonicotinylmodified peptide for the preparation of 99mTc-labeled radiopharmaceuticals. Bioconjugate Chem. (submitted for publication). (25) Bartik, T., Bartik, B., Hanson, B. E., Glass, T., and Bebout, W. E. (1992) Comments on the synthesis of trisulfonated triphenylphosphine: reaction monitoring by NMR spectroscopy. Inorg. Chem. 31, 2667-2670. (26) Liu, S., Edwards, D. S., Harris, A. R., Heminway, S. J., and Barrett, J. A. (1999) Technetium complexes of a hydrazinonicotinamide-conjugated cyclic peptide and 2-hydrazinopyridine: synthesis and characterization. Inorg. Chem. 38, 1326-1335.
BC990022E
