A Tc-99m-Labeled Long Chain Fatty Acid Derivative for Myocardial

Feb 28, 2004 - Sciences, Kyoto University, Kyoto, Japan, Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical Sciences ...
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Bioconjugate Chem. 2004, 15, 389−393

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A Tc-99m-Labeled Long Chain Fatty Acid Derivative for Myocardial Imaging Yasuhiro Magata,*,†,‡ Takayoshi Kawaguchi,† Misa Ukon,† Norio Yamamura,† Tomoya Uehara,| Kazuma Ogawa,† Yasushi Arano,| Takashi Temma,† Takahiro Mukai,§ Eiji Tadamura,§ and Hideo Saji† Laboratory of Genome Bio-Photonics, Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan, Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical Sciences, Chiba University, Japan, and Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Received October 15, 2003; Revised Manuscript Received December 16, 2003

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-aminohexanoic acid ([99mTc]MAMA-HA), a medium chain fatty acid derivative, is metabolized by β-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMAHDA, and performed radiolabeling and biodistribution studies. [99mTc]MAMA-DA and [99mTc]MAMAHDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [99mTc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[125I]iodophenylpentadecanoic acid. Metabolite analysis showed [99mTc]MAMA-HDA was metabolized by β-oxidation in the body. In conclusion, [99mTc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating β-oxidation of fatty acid in the heart.

INTRODUCTION

Radiopharmaceuticals of long chain fatty acid analogues have important roles in the diagnosis of heart disease. C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism (1-4), especially in the differential diagnosis of unstable angina or severe heart ischemia (5). However, an on-site cyclotron is needed to produce positron nuclides such as C-11, and radioiodinated compounds must be obtained from radiopharmaceutical companies. Since heart disease generally requires an urgent examination, it would be useful if an on-site radiopharmceutical could be used for clinical diagnosis. A Tc-99m-labeled radiopharmaceutical is most desirable because this nuclide can be supplied from a generator system whenever needed. Although many Tc-99m-labeled long chain fatty acid derivatives have been reported (6-10), no compound has been put to practical use. * To whom correspondence should be addressed. Tel: +8153-435-2398. Fax: +81-53-435-2398. e-mail: [email protected]. ‡ Hamamatsu University School of Medicine. † Department of Patho-Functional Bioanalysis, Kyoto University. | Chiba University. § Department of Nuclear Medicine and Diagnostic Imaging, Kyoto University.

We have reported that [99mTc]MAMA-HA, a medium chain fatty acid derivative, is metabolized by β-oxidation in the liver and the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group with Tc-99m (11). Based on these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. An appropriate chelating group is needed in the chemical structure to label it with Tc-99m, as mentioned above. The effect of the chelating group on the biodistribution of long chain fatty acid analogues has not been clarified. In this study, some Tc-99m-labeled fatty acid analogues, varying in carbon chain length, were synthesized and the heart uptake of these compounds was compared in vivo. MATERIALS AND METHODS

Materials. Proton nuclear resonance (1H NMR) spectra were recorded on a Bruker AC-200 spectrometer, and the chemical shifts reported in parts per million (ppm) downfield from an internal tetramethylsilane standard. Fast atom bombardment mass spectra (FABMS) were obtained with a JMS-HX/HX 110 A (JEOL Ltd. Tokyo). [99mTc]Pertechnetate (99mTcO4-) was eluted in saline solution on a daily basis from Daiichi Radioisotopes Labs generator (Chiba, Japan). Reversed-phase high performance liquid chromatography (RP-HPLC) was performed

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Figure 1. Synthesis of fatty acid analogues with the TrtMAMA group at the omega position.

using a Cosmosil 5C18-AR 300 column (150 × 4.6 mm; Nacalai Tesque Co., Ltd., Kyoto) eluted with a gradient solvent system (from 10% methanol/5 mM phosphate buffer pH 7.0 to 100% methanol in 60 min) at 1.0 mL/ min. TLC analysis was performed with silica plates (Merck Art 5553) using 100% acetone or chloroform/ methanol/water ) 50/10/1 as developing solvent systems. All chemicals were of reagent grade and were used as received. Synthesis of Fatty Acid Derivatives. Each fatty acid derivative was synthesized according to the method shown in Figure 1. In brief, the amino group of tritylMAMA was alkylated with a ω-bromo-fatty acid analogue and hydrolyzed. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-6-aminohexanoic Acid (Trt-MAMA-HA)(1, n ) 5). Trt-MAMA-HA was synthesized according to a previous report (11). N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-12-aminododecanoic Acid Ethyl Ester (4, n ) 11, R ) Et). 12Bromodecanoic acid (2) was esterified in ethanol bubbled with HCl gas. N-[[[2-[(Triphenylmethyl)thio]ethyl]amino]acetyl]-S-(triphenylmethyl)-2-aminoethanethiol (Trt-MAMA, 250 mg, 0.37 mmol) and diisopropylethylamine (100 µL) were dissolved in 6 mL of DMF. To this mixture was added ethyl 12-bromodecanoate (3, n ) 11, R ) Et) (110 mg, 0.38 mmol) in DMF under stirring. After being heated at 140 °C for 12 h, the mixture was subjected to silica gel column chromatography with ethyl acetate/ hexane ) 4/7 as an eluting solvent. Yield: 150 mg (yellow oil, 43.8%). 1H NMR (CDCl3): δ 7.45-7.15 (overlapped m, 30H), 4.08 (q, 2H), 2.97 (td, 2H), 2.82 (s, 2H), 2.402.23 (overlapped m, 10H), 1.58-1.22 (overlapped, 21H). FABMS calcd for C58H68N2O3S2 (M + H+): m/z 905. Found: 905. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-12-aminododecanoic Acid (Trt-MAMA-DA) (5, n ) 11). Compound 4 was hydrolyzed by sodium hydroxide in methanol. Yield: 99.9%. 1H NMR (CD3OD): δ 7.42-7.16 (overlapped m, 30H), 3.02(t, H), 2.82-2.69 (overlapped m, 8H), 2.33 (t, 2H), 2.23 (t, 2H), 1.60-1.25 (overlapped m, 18H). FABMS calcd for C56H64N2O3S2 (M+ H+): m/z 876. Found: 876. Elemental analysis agreed with the calculated value for C56H64N2O3S2: C; 76.66%, H; 7.37%, N; 3.19%. Found: C; 76.51%, H; 7.41%, N; 2.92%. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-16-aminohexadecanoic Acid Ethyl Ester (9, n ) 15, R ) Et). 16Hydroxyhexadecanoic acid (6) (1.0 g, 3.67 mmol) was added to 50 mL of dry ethanol followed by concentrated sulfonic acid (0.5 mL). The reaction mixture was stirred for 5 h at room temperature. After evaporation of the solvent, 1.1 g of ethyl 16-hydroxyhexadecanoate as white

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crystals (7) (yield > 99%) was obtained. A mixture of 1.0 g of ethyl 16-hydroxyhexadecanoate (7) (3.49 mmol), N-bromosuccinimide (0.62 g, 3.49 mmol), and triphenylphophine (0.92 g, 3.49 mmol) was dissolved in 30 mL of dry chloroform. After being heated for 1 h at 50 °C, the solvent was evaporated in vacuo. The residue was purified by column chromatography with ethyl acetate/ hexane ) 1/3, and ethyl 16-bromohexadecanoate (8) was obtained as a pale yellow oil in a yield of 400 mg (32.8%). The bromo compound (400 mg, 1.10 mmol) was dissolved in 5 mL of DMF, and the DMF solution was added to a mixture of Trt-MAMA (900 mg, 1.32 mmol) and diisopropylethylamine (400 µL) dissolved in 6 mL of DMF. After being heated at 140 °C for 12 h, the mixture was subjected to silica gel column chromatography with ethyl acetate/hexane ) 4/7 as an eluting solvent. Yield: 170 mg (yellow oil, 16.1%). 1H NMR (CDCl3): δ 7.45-7.15 (overlapped m, 30H), 4.08 (q, 2H), 2.97 (td, 2H), 2.82 (s, 2H), 2.40-2.23 (overlapped m, 10H), 1.58-1.22 (overlapped, 29H). FABMS calcd for C62H76N2O3S2 (M+ H+): m/z 961. Found: 961. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-16-aminohexadecanoic Acid (Trt-MAMA-HDA) (10, n ) 15). Compound 9 was hydrolyzed by sodium hydroxide in methanol. Yield: 99.9%. 1H NMR (CDCl3): δ 7.40-7.15 (overlapped m, 30H), 3.30(t, 2H), 2.90 (t, 2H), 2.84 (s, 2H), 2.47-2.22 (overlapped m, 12H), 1.59-1.23 (overlapped m, 26H). FABMS calcd for C62H76N2O3S2 (M + H+): m/z 933. Found: 933. The result of the elemental analysis agreed with the calculated value for C62H76N2O3S2: C; 77.20%, H; 7.79%, N; 3.00%. Found: C; 77.00%, H; 7.76%, N; 2.83%. Tc-99m-Labeling of Fatty Acid Derivatives. Each compound was labeled with Tc-99m by a ligand exchange reaction according to a previously reported method with a slight modification (11). In brief, a bistritylated fatty acid analogue (1 mg) was treated with TFA under cationtrapping conditions (5% triethylsilane) (12). After removal of the solvent under a stream of N2, the residue was solved in 0.5 mL of a 40% methanol/phosphate buffer (0.1 M, pH 8.0). [99mTc]GH (0.5 mL) was added to a solution of a bisthiol compound, and the reaction mixture was heated for 1 h at 90 °C. The labeled compound was extracted with a mixture of ethyl acetate/hexane ) 1/4, and the organic layer was evaporated in vacuo. The radiochemical yield and purity of Tc-99m-labeled fatty acid analogues were determined by RP-HPLC, and the TLC was developed with acetone. Radiolabeling of p-Iodophenylpentadecanoic Acid (IPPA). p-Iodophenylpentadecanoic acid (IPPA) (0.5 mg) was solved in a mixture of acetonitrile 950 µL and water 50 µL. Copper sulfate (4 mM, 20 µL), ammonium chloride (0.15 M, 10 µL), and [125I]NaI were added to the IPPA solution, and the reaction mixture was heated in a closed system for 3 h at 120 °C. [125I]IPPA was purified by RPHPLC eluted with a gradient mobile phase starting from 100% water to 100% methanol for 20 min at a flow rate of 1 mL/min. The retention volumes of IPPA and NaI were 26 mL and 2.5 mL, respectively. The radiochemical yield was 84.7%. Stability of [99mTc]MAMA-Fatty Acids. To evaluate the stability of [99mTc]MAMA-fatty acids, [99mTc]MAMAHA, [99mTc]MAMA-DA, and [99mTc]MAMA-HDA were each incubated in a 40% ethanol/0.1 M phosphate buffer (pH 7.4) at 37 °C. After incubation for 3, 6, and 12 h, the radiochemical purity was analyzed by TLC developed with chloroform/methanol/water ) 50/10/1. Moreover, the stability of these fatty acid compounds in rat plasma was

Tc-99m-Labeled Long Chain Fatty Acid

Figure 2. Stability of Tc-99m-MAMA-fatty acid analogues. Closed diamonds, [99mTc]MAMA-HA; open squares, [99mTc]MAMA-D; closed triangles, [99mTc]MAMA-HDA.

evaluated. Then, 10 µL of labeled compound was added to 1 mL of rat plasma and the mixture was incubated at 37 °C. After incubation for 30 and 60 min, 100 µL of the mixture was sampled and 200 µL of ethanol was added. The mixture was filtered with a 0.45 µm Millipore filter, and the radiochemical purity was analyzed by RP-HPLC. Biodistribution Studies. Male ddY mice (25-30 g) and male Wistar rats (150-200 g) were housed for 1 week under a 12-h light/12-h dark cycle with free access to food and water for subsequent biodistribution studies and metabolic analyses. Animals were fasted before experiments for 12 h. For biodistribution studies, [99mTc]MAMA-HA was solved in saline. [99mTc]MAMA-DA and [99mTc]MAMAHDA were solved in a 40% ethanol solution after being radiolabeled and 100 µL of the solution was added to 1 mL of saline, containing 1% human serum albumin. After passing through a 0.22 µm Millipore filter, the solution (37 kBq in 0.1 mL) was administered to normal mice via the tail vein. At appropriate time points after the administration, mice were sacrificed by decapitation. Samples of blood and organs of interest were excised and weighed, and radioactivity was quantified using a NaI(Tl) gamma scintillation counter (Packard Auto-Gamma 500, Packard Inst. Co., Albertville, MI). The results are expressed as the percent injected dose/gram of blood or organs after decay correction. A mixture of [99mTc]MAMA-HDA and [125I]IPPA was injected into normal rats via the tail vein. At appropriate time points after the administration, rats were sacrificed by decapitation and calculations made as mentioned above. Metabolic Analysis. At 30 min postinjection of [99mTc]MAMA-HDA in normal rats, urine samples were obtained from the bladder. After filtration of the urine samples through a 0.22 µm Millipore filter, the filtrate was analyzed by RP-HPLC eluted with a gradient mobile phase starting from 10% methanol in a 0.05 M phosphate buffer (pH 7.0) to 100% methanol for 60 min at a flow rate of 1 mL/min. RESULTS

Synthesis of Fatty Acid Derivatives. Each fatty acid derivative was synthesized according to the method shown in Figure 1. In brief, the amino group of trt-MAMA was alkylated with a ω-bromo-fatty acid analogue. After hydrolysis of the ester group, trt-MAMA-DA and trtMAMA-HDA were obtained as a yellow oil with overall

Bioconjugate Chem., Vol. 15, No. 2, 2004 391

Figure 3. Comparison of heart/blood ratio of Tc-99m-MAMAHDA and I-125-IPPA in normal rats. Open diamonds, [125I]IPPA; open circles, [99mTc]MAMA-HDA.

Figure 4. Metabolite analysis in urine postinjection of Tc-99mMAMA-HDA in normal rats. Upper chart: radiochromatogram in urine at 30 min postinjection of [99mTc]MAMA-HDA; lower chart: radiochromatogram of authentic labeled compound of [99mTc]MAMA-BA.

yields of 43.8% and 5.3%, respectively (calculated from the starting compound of fatty acid derivative). Tc-99m-Labeling of Fatty Acid Derivatives. Each compound was labeled in a ligand exchange reaction with [99mTc]pertechnetate reduced by Sn(II) glucoheptonate in diluted ethanol solution and purified by HPLC. The retention times of [99mTc]MAMA-DA and [99mTc]MAMAHDA were 43 and 49 min, respectively. The retention time of [99mTc]MAMA-HA was 23 min (11). The radiochemical yields of [99mTc]MAMA-DA and [99mTc]MAMAHDA were 70% and 75%, respectively. The radiochemical purity of both labeled compounds was >99%. Stability of [99mTc]MAMA-Fatty Acids in Vitro. Figure 2 shows the stability of three [99mTc]MAMA-fatty acids, [99mTc]MAMA-HA, [99mTc]MAMA-DA, and [99mTc]MAMA-HDA, in a phosphate-buffered solution and rat plasma in vitro. High stability was observed for all radiochemical compounds in these solutions. Biodistribution Studies. Table 1 shows the biodistribution of Tc-99m-labeled fatty acids in normal mice. A rapid clearance of radioactivity from the blood was observed for all compounds. [99mTc]MAMA-HDA showed

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Table 1. Biodistribution of [99mTc]MAMA-Fatty Acids in Normal Micea time after injection (min) 0.5

a

2

heart blood lung liver

11.22 (0.25) 9.84 (1.11) 7.51 (0.32) 21.07 (1.35)

5.46 (1.15) 3.61 (0.30) 3.53 (2.15) 25.79 (4.72)

heart blood lung liver

5.68 (0.61) 12.35 (0.10) 9.26 (1.26) 15.74 (1.70)

2.83 (0.30) 4.48 (2.72) 4.24 (0.41) 24.63 (2.51)

heart blood lung liver

4.04 (0.72) 8.28 (1.24) 5.42 (1.05) 11.57 (2.81)

1.46 (0.22) 2.66 (0.45) 1.92 (0.27) 26.86 (2.39)

5 MAMA-HDA 2.40 (0.22) 2.91 (0.29) 2.27 (0.51) 25.88 (4.71) MAMA-DA 1.50 (0.09) 3.64 (1.30) 2.72 (0.13) 18.13 (10.18) MAMA-HA 0.70 (0.15) 1.17 (0.13) 0.98 (0.18) 31.83 (0.83)

20

60

0.60 (0.03) 0.78 (0.07) 0.72 (0.30) 18.14 (2.96)

0.32(0.09) 0.37 (0.01) 0.35 (0.05) 13.21 (3.49)

0.82 (0.10) 1.54 (0.14) 1.39 (0.22) 18.20 (3.60)

0.40 (0.09) 0.96 (0.14) 0.79 (0.14) 14.51 (1.74)

0.29 (0.03) 0.47 (0.07) 0.38 (0.07) 35.00 (6.59)

0.19 (0.08) 0.29 (0.02) 0.23 (0.03) 30.95 (4.56)

Each value shows the mean (SD) of % dose/g (n ) 5).

the highest uptake in the mouse heart among the three Tc-99m-labeled fatty acids. Only [99mTc]MAMA-HDA had a heart-to-blood ratio greater than one through to 2 min postinjection of the compound. [99mTc]MAMA-HDA and [125I]IPPA were simultaneously injected into normal rats. The heart-to-blood ratio was 3.6 at 2 min postinjection of [99mTc]MAMA-HDA in normal rats, while it was 6.2 at 2 min postinjection of [125I]IPPA. The kinetics of the clearance of radioactivity from the heart were very similar between [99mTc]MAMAHDA and [125I]IPPA (Figure 3). Metabolic Analysis. Rat urine samples were collected at 30 min postinjection of [99mTc]MAMA-HDA and the metabolites in them were analyzed by RP-HPLC. The lower chart of Figure 4 shows a typical chromatogram of [99mTc]MAMA-BA (11). The retention time of [99mTc]MAMA-BA was 17 min. The major peak in the urine samples was observed at retention time of 17 min on RPHPLC (Figure 4, upper). DISCUSSION

External imaging of energy production levels of living cells with Tc-99m-labeled compounds is a challenging task for the development of Tc-99m-radiopharmaceuticals. Indeed, efforts have been made to develop Tc-99mlabeled long chain fatty acid analogues for myocardial imaging studies. However, their application has been hampered by poor accumulation in the heart, and it appeared that these Tc-99m-labeled fatty acid analogues were not recognized as substrates for β-oxidation (8, 9). In this study, two long chain fatty acid derivatives, including the MAMA group as a chelating group with Tc99m, were synthesized according to the method shown in Figure 1. Their chemical structures were assigned based on instrumental analyses. Tc-99m labeling reactions were carried out according to a previously reported method (11) with a slight modification. MAMA-HA, which is a medium chain fatty acid derivative reported previously, was radiolabeled with Tc-99m in solution because of its hydrophilicity. However, the long chain fatty acid derivatives MAMA-HDA and MAMA-DA were radiolabeled in a 40% ethanol solution because their solubility in solution was poor compared with that of the medium chain fatty acid analogues. Tc-99m-labeled compounds were obtained at a good yield by this method, despite the ethanol-containing condition. This exchange labeling method is useful for Tc-99m labeling of MAMA chelating groups. Moreover, the high stability of each labeled compound is shown in Figure 2.

The myocardium uses long chain fatty acids as its energy source under normal conditions, although it is affected by the blood sugar or nonesterified fatty acid concentration in the serum (13). Biodistribution studies in mice showed that the longer the carbon chain, the higher the heart uptake. Only [99mTc]MAMA-HDA showed a heart-to-blood ratio of radioactivity uptake greater than one in the early phase postinjection of the compound (Table 1). Then, a mixture of [99mTc]MAMA-HDA and [125I]IPPA was injected into normal rats, and the heart uptake of both was compared. Although the uptake of the radioactivity of Tc-99m was almost half the value of that of I-125, the heart-to-blood ratio (%dose/g) of both Tc-99m and I-125 showed similar curves (Figure 3). The heartto-blood ratio of Tc-99m was about 3.6 at 2 min postinjection. Metabolites in urine were evaluated (Figure 4). In this figure, [99mTc]MAMA-BA was observed at 30 min postinjection of [99mTc]MAMA-HDA. This [99mTc]MAMABA was observed after six rounds of β-oxidation. The result indicated that [99mTc]MAMA-HDA was recognized as a fatty acid in the body. The metabolite [99mTc]MAMABA has been observed postinjection of the medium chain fatty acid derivative, [99mTc]MAMA-HA (11). Since long chain fatty acids are metabolized by β-oxidation in the liver as well as in the heart, β-oxidation of the compound in the heart should be evaluated. Although the metabolism of [99mTc]MAMA-HDA is now being evaluated, this compound has been recognized as a long chain fatty acid in vivo. In conclusion, [99mTc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimation of the β-oxidation of fatty acids in the heart. Further investigation with metabolites is now under way. ACKNOWLEDGMENT

A part of this study was supported by Grant in Aide for the Center of Excellence (COE) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. LITERATURE CITED (1) Fujino, T., Ishii, Y., Takeuchi, T., Hirasawa, K., Tateda, K., Kikuchi, K., and Hasebe, N. (2003) Recovery of BMIPP Uptake and Regional Wall Motion in Insulin Resistant Patients Following Angioplasty for Acute Myocardial Infarction. Circ. J. 67, 757-762. (2) Hoshina, M., Shiraishi, H., Igarashi, H., Kikuchi, Y., Ichihashi, K., and Momoi, M. Y. (2003) Efficacy of iodine-123-

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Bioconjugate Chem., Vol. 15, No. 2, 2004 393 99mTc-labeled fatty acid analogues for myocardial imaging. Int. J. Rad. Appl. Instrum. B 18, 215-226. (9) Jones, G. S., Jr., Elmaleh, D. R., Strauss, H. W., and Fischman, A. J. (1994) Synthesis and biodistribution of a new 99mtechnetium fatty acid. Nucl. Med. Biol. 21, 117-123. (10) Jones, G. S., Jr., Elmaleh, D. R., Strauss, H. W., and Fischman, A. J. (1996) 7,10-Bis(2-mercapto-2-methyl)propyl7, 10-diazapalmitic acid: A novel, N2S2 ligand for technetium-99m. Bioorg. Med. Chem. Lett. 6, 2399-2404. (11) Yamamura, N., Magata, Y., Arano, Y., Kawaguchi, T., Ogawa, K., Konishi, J., and Saji, H. (1999) Technetium-99mlabeled medium-chain fatty acid analogues metabolized by β-oxidation: radiopharmaceutical for assessing liver function. Bioconjug. Chem. 10, 489-495. (12) Meltzer, P. C., Blundell, P., Jones, A. G., Mahmood, A., Garada, B., Zimmerman, R. E., Davison, A., Holman, B. L., and Madras, B. K. (1997) A technetium-99m SPECT imaging agent which targets the dopamine transporter in primate brain. J. Med. Chem. 40, 1835-1844. (13) Corbett, J. R. (1999) Fatty acids for myocardial imaging. Semin. Nucl. Med. 29, 237-258.

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