Bioconjugate Chem. 1999, 10, 489−495
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Technetium-99m-Labeled Medium-Chain Fatty Acid Analogues Metabolized by β-Oxidation: Radiopharmaceutical for Assessing Liver Function Norio Yamamura,† Yasuhiro Magata,‡ Yasushi Arano,† Takayoshi Kawaguchi,† Kazuma Ogawa,† Junji Konishi,‡ and Hideo Saji*,† Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Department of Nuclear Medicine, Graduate School of Medicine, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan. Received December 28, 1998; Revised Manuscript Received March 8, 1999
External imaging of energy production activity of living cells with 99mTc-labeled compounds is a challenging task requiring good design of 99mTc-radiopharmaceuticals. On the basis of our recent findings that 11C- and 123I-labeled medium-chain fatty acids are useful for measuring β-oxidation activity of hepatocytes, we focused on development of 99mTc-labeled medium-chain fatty acid analogues that reflect β-oxidation activity of the liver. In the present study, monoamine-monoamide dithiol (MAMA) ligand and triamido thiol (MAG) ligand were chosen as chelating groups because of the stability and size of their complexes with 99mTc and their ease of synthesis. Each ligand was attached to the ω-position of hexanoic acid (MAMA-HA and MAG-HA, respectively). In biodistribution studies, [99mTc]MAMA-HA showed high initial accumulation in the liver followed by clearance of the radioactivity in the urine. Analysis of the urine revealed [99mTc]MAMA-BA as the sole radiometabolite. Furthermore, when [99mTc]MAMA-HA was incubated with living liver slices, generation of [99mTc]MAMA-BA was observed. However, [99mTc]MAMA-HA remained intact when the compound was incubated with liver slices in the presence of 2-bromooctanoate, an inhibitor of β-oxidation. The findings in this study indicated that [99mTc]MAMA-HA was metabolized by β-oxidation after incorporation into the liver. On the other hand, poor hepatic accumulation was observed after administration of [99mTc]MAG-HA.
INTRODUCTION
In the liver, the parenchymal cells are responsible for the metabolism of many substances, and parenchymal cell function is also related to the reserve capacity of the liver (1-3). Therefore, the development of radiopharmaceuticals which reflect viability of hepatocytes might provide us useful information for predicting prognosis after surgery or determining the efficacy of the treatment. The β-oxidation of fatty acids is an important pathway for the production of energy for activity of hepatocytes (4, 5). In addition to β-oxidation, however, long-chain fatty acids are esterified and retained in the liver to a fairly large extent. On the other hand, medium-chain fatty acids such as octanoate and hexanoate are recognized only as substrates for β-oxidation and are not esterified in the hepatocytes (6-8). Indeed, [1-11C]octanoate was metabolized rapidly to 11CO2 by β-oxidation and cleared quickly from the liver. In addition, the hepatic elimination rate of the radioactivity was significantly delayed in animal models of hepatitis (9). Such characteristics render [1-11C]octanoate useful as a radiopharmaceutical for measuring the viability of hepatocytes by positron emission tomography (PET) imaging. Medium-chain fatty acid analogues labeled with single photon emitters are more desirable for routine clinical studies to estimate the viability of hepatocytes. We * To whom correspondence should be addressed. Phone: +8175-753-4556. Fax: +81-75-753-4568. † Department of Patho-Functional Bioanalysis. ‡ Department of Nuclear Medicine.
recently found that p-[123I]iodophenylenanthic acid ([123I]IPEA) was taken up by the liver following administration and elimination rate of the final radiometabolite, piodohippuric acid, from the liver reflected β-oxidation activities of the hepatocytes. A significant delay in elimination of the radioactivity from the liver was observed in hepatitis models (10). These findings prompted us to further examine the use of 99mTc-labeled mediumchain fatty acid analogues for hepatic studies since 99mTc possesses ideal characteristics for a radiopharmaceutical such as optimal γ-ray energy (141 keV), short half-life (6 h), low-cost, and widespread availability of the 99Mo/ 99mTc generator. Introduction of a chelating group is necessary for labeling of fatty acids with 99mTc based on the idea of bifunctional chelating agent. A variety of chelating groups have been developed for such purposes. Among these groups, two 99mTc chelating groups were chosen as candidates in the present study because of the stability and molecular size of their complexes with 99mTc and ease of synthesis, and each chelating group was attached to the ω-position of hexanoic acid. One was a recently reported monoamine-monoamide dithiol (MAMA) ligand, which forms a stable, neutral, and slightly lipophilic mononuclear 99mTc complex with pentavalent 99mTcO3+ (11-13). The amino nitrogen was the site of attachment to hexanoic acid (MAMA-HA). The other chelating group was a triamide thiol group (N3S) which has been used clinically as a 99mTc complex of mercaptoacetylglycylglycylglycine (MAG3) (14-16). It forms a negatively charged mononuclear 99mTc complex with pentavalent 99mTcO3+. The terminal glycine of MAG3 was altered to 6-amino-
10.1021/bc9801528 CCC: $18.00 © 1999 American Chemical Society Published on Web 04/16/1999
490 Bioconjugate Chem., Vol. 10, No. 3, 1999
hexanoic acid (MAG-HA). The biodistribution and metabolism of the two 99mTc-labeled medium-chain fatty acid analogues were investigated in rats to estimate the molecular design for measuring the viability of hepatocytes. MATERIALS AND METHODS
Materials. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AC-200 spectrometer, and the chemical shifts are 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). Imaging studies were performed using a SPECT2000H-40 (Hitachi Medical Co., 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 using a Cosmosil 5C18-AR 300 column (150 × 4.6 mm; Nacalai Tesque Co., Ltd., Kyoto). TLC analysis was performed with silica plates (Merck Art 5553). All chemicals were of reagent grade and were used as received. Synthesis of Tr-MAMA-HA and Its Analogues. N-[[[2-[(Triphenylmethyl)thio]ethyl]amino]acetyl]-S-(triphenylmethyl)-2-aminoethanethiol (Tr-MAMA) (3). Trityl chloride (27.3 g, 97.9 mmol) and compound 1 (11.1 g, 97.7 mmol) were dissolved in 150 mL of N,N-dimethylformamide (DMF), and the reaction mixture was stirred for 48 h at room temperature. The solvent was removed in vacuo, and the residue was dissolved in ethyl acetate. After cooling the solution on ice, a saturated solution of NaHCO3 was gradually added. The precipitated white crystals were collected and washed with distilled water to afford compound 2 as the crude product. Crude 2 (9.97 g, 28 mmol) in chloroform (ca. 50 mL) was mixed with triethylamine (14 mL), and the mixture was gradually added to a solution of bromoacetyl bromide (2.84 g, 14 mmol) in 40 mL of chloroform at -78 °C. The reaction mixture was stirred at the same temperature for 30 min and warmed to room temperature over a period of 1 h. After stirring at room temperature for 24 h, the mixture was washed with diluted H2SO4 (pH 3), saturated NaHCO3, and brine. The organic layer was dried over anhydrous CaSO4, and the solvent was removed in vacuo. The crude product of compound 3 was obtained after silica gel column chromatography of the oil residue eluted with chloroform. Further purification was performed by silica gel chromatography using ethyl acetate-hexane (60:40) as the eluent to produce 3 (0.95 g, 10.0% from 2) as a pale yellow oil. 1H NMR (CDCl3): δ 7.44-7.15 (overlapped m, 30H), 3.07 (td, 2H), 3.02 (s, 2H), 2.45 (t, 2H), 2.38-2.31 (overlapped m, 4H). FABMS calcd for C44H42N2OS2 (M + H+): m/z 679. Found: 679. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-6-aminohexanoic Acid Methyl Ester (5). Methyl 6-bromohexanoate (4) (160 mg, 0.77 mmol) in 8 mL of acetonitrile was gradually added to a mixed solution of Tr-MAMA (400 mg, 0.59 mmol) and diisopropylethylamine (120 mg, 1.84 mmol) in 2 mL of acetonitrile. After stirring the reaction mixture at room temperature for 48 h, the solvent was removed in vacuo. The oily residue was purified by silica gel chromatography using ethyl acetate-hexane (95:5) as the eluent to provide compound 5 (117 mg, 24.6%) as a yellow oil. 1H NMR (CDCl3): δ 7.43-7.15 (overlapped m, 30H), 3.64 (s, 3H), 3.01 (td, 2H), 2.82 (s, 2H), 2.402.33 (overlapped m, 4H), 2.28-2.20 (overlapped m, 6H),
Yamamura et al.
1.59-1.48 (m, 2H), 1.37-1.19 (overlapped m, 4H). FABMS calcd for C51H54N2O3S2 (M + H+): m/z 807. Found: 807. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-6-aminohexanoic Acid (Tr-MAMA-HA) (6). Compound 6 was obtained quantitatively as a viscous yellow oil by hydrolysis of the methyl ester of 5 in 5% NaOH. 1H NMR (CDCl3): δ 7.407.14 (overlapped m, 30H), 2.99 (td, 2H), 2.87 (s, 2H), 2.40-2.33 (overlapped m, 4H), 2.28-2.20 (overlapped m, 6H), 1.59-1.48 (m, 2H), 1.32-1.21 (overlapped m, 4H). FABMS calcd for C50H52N2O3S2 (M + H+): m/z 793. Found: 793. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-4-aminobutyric Acid (Tr-MAMA-BA). N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-4-aminobutyric acid ethyl ester was synthesized by reaction of Tr-MAMA and ethyl bromobutyrate according to the procedure described above with 3.25% yield. 1H NMR (CDCl3): δ 7.45-7.15 (overlapped m, 30H), 4.06 (q, 2H), 3.01 (td, 2H), 2.84 (s, 2H), 2.38-2.32 (overlapped m, 4H), 2.29-2.18 (overlapped m, 6H), 1.681.26 (m, 2H), 1.20 (t, 3H). After hydrolysis of the ester bond, Tr-MAMA-BA was obtained. 1H NMR (CDCl3): δ 7.40-7.17 (overlapped m, 30H), 2.99 (td, 2H), 2.87 (s, 2H), 2.36-2.33 (overlapped m, 4H), 2.30-2.23 (overlapped m, 6H), 1.65-1.62 (m, 2H). FABMS calcd for C48H48N2O3S2 (M + Na+): m/z 787. Found: 787. N-[[[[2-[(Triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-[2-[(triphenylmethyl)thio]ethyl]-2-aminoacetic Acid (Tr-MAMA-AA). This compound was synthesized using Tr-MAMA and methyl bromoacetate according to the procedure described above with 17.5% yield. After hydrolysis, N-[[[[2-[(triphenylmethyl)thio]ethyl]amino]carbonyl]methyl]-N-2-[(triphenylmethyl)thio]ethyl]-2-aminoacetic acid (Tr-MAMA-AA) was obtained (72.5%). 1H NMR (CDCl3): δ 7.39-7.14 (overlapped m, 30H), 3.10 (s, 2H), 2.99 (s, 2H), 2.98 (td, 2H), 2.50 (t, 2H), 2.34 (t, 2H), 2.24 (t, 2H). FABMS calcd for C46H44N2O3S2 (M + Na+): m/z 759. Found: 759. Synthesis of MAG-HA. S-Acetylthioglycolic Acid NHydroxysuccinimide Ester (SATA) (10). S-Acetylmercaptoacetic acid (9) was synthesized from mercaptoacetic acid (8) according to the published procedure (17). Compound 9 (5.15 g, 38.5 mmol) and N-hydroxysuccinimide (NHS) (4.45 g, 38.5 mmol) were dissolved in 50 mL of anhydrous tetrahydrofurane (THF), and the reaction mixture was stirred at room temperature for 10 min. After cooling on ice, dicyclohexylcarbodiimide (DCC) (7.95 g, 38.5 mmol) in 5 mL of THF was added and the reaction mixture was stirred overnight. The precipitated dicyclohexylurea was removed by filtration, and the solvent was evaporated to dryness in vacuo (18). The residue was washed with hexane several times to afford compound 10 as white crystals (7.69 g, 86.3%). 1H NMR (CDCl3): δ 3.99 (s, 2H), 2.85 (s, 4H), 2.43 (s, 3H). S-Acetylmercaptoglycylglycylaminohexanoic Acid (Sacetyl MAG-HA) (13). N-Succinimide ester of Boc-glycylglycine (compound 11) was prepared by the reaction of Boc-glycylglycine with NHS in the presence of DCC. Compound 11 (2.37 g, 7.2 mmol) in 30 mL of acetonitrile was slowly added to a solution of 6-aminohexanoic acid (0.79 g, 6 mmol) in 0.2 M NaOH at 50-60 °C, and the reaction mixture was refluxed for 2 h. After cooling, the reaction solution on ice, the pH of the solution was brought to ca. 3 with diluted H2SO4, followed by extraction with ethyl acetate (20 mL × 3). The organic layers were combined and dried over anhydrous CaSO4. Compound 12 (1.41 g, 56.7% from 11) was obtained as a
Technetium-99m-Labeled Fatty Acid Derivatives
viscous oil after removing the solvent in vacuo. Crude 12 (1.3 g, 3.8 mmol) was then treated with 5% anisol in trifluoroacetic acid (TFA) to deprotect the Boc group. After removing TFA in vacuo, the residue was dissolved in 20 mL of water and the solution was neutralized with 1 M NaOH. This solution was then added dropwise to a solution of SATA (1.04 g, 4.5 mmol) in 20 mL of DMF. After stirring the reaction mixture at room temperature for 2 h, the solvent was removed in vacuo to precipitate compound 13 as white crystals (396 mg, 29.1% from 12). 1 H NMR (CDCl3): δ 8.42 (t, 1H), 8.13 (t, 1H), 7.70 (t, 1H), 3.72 (d, 2H), 3.66 (s, 2H), 3.65 (d, 2H), 3.03 (td, 2H), 2.36 (s, 3H), 2.19 (t, 2H), 1.54-1.21 (overlapped m, 6H). FABMS calcd for C14H23N3O6S (M+H+): m/z 362. Found: 362. Radiolabeling. 99mTc complexes of each ligand were prepared by ligand-exchange reaction with 99mTcO4reduced with Sn(II) glucoheptonate (GH). A solution of 99mTcO - eluted from the generator every morning was 4 added to stock freeze-dried powder of Sn(II) GH (made from 4 g of GH and 1.2 mg of SnCl2 in 50 mL of aqueous solution pH 8.6) to produce a final GH concentration of 3-5 mg/mL. After standing for 10 min, the formation of [99mTc]GH was ascertained by TLC developed with acetone. The bistritylated compound 6 (1 mg) was treated with TFA under cation trapping conditions (5% triethylsilane) (19). After removing the solvent under a stream of N2, the residue was neutralized with 1 M NaOH and 0.4 mL of phosphate buffer (0.1 M, pH 8.0). [99mTc]GH (0.4 mL) was then added to the bisthiol compound, and the reaction mixture was left for 1 h at room temperature. [99mTc]MAMA-HA was purified by RP-HPLC eluted with a gradient mobile phase starting from 10% acetonitrile in 0.05 M phosphate buffer (pH 7.0) to 100% acetonitrile in 60 min at a flow rate of 1 mL/min at 30 °C (Rt, 14 min for [99mTc]MAMA-HA, 3 min for [99mTc]GH). 99mTc complexes of MAMA-BA and MAMA-AA were prepared by procedures similar to those described above. 99mTc complex of MAG-HA was also prepared by reaction of [99mTc]GH with S-acetyl MAG-HA as follows. A solution of S-acetyl MAG-HA (3 mg) in 1 mL of phosphate buffer (0.1 M, pH 8.0) was mixed with the same volume of [99mTc]GH, and the reaction mixture was heated at 90-95 °C for 30 min. After cooling, [99mTc]MAG-HA was purified by RP-HPLC eluted with a gradient mobile phase starting from 10% methanol in 0.05 M phosphate buffer (pH 7.0) to 50% methanol in 30 min at a flow rate of 1 mL/min at 30 °C (Rt, 20 min for [99mTc]MAMA-HA, 3 min for [99mTc]GH). The radiochemical yield and purity of 99mTc-labeled fatty acid analogues were determined by RP-HPLC under the conditions described above and TLC developed with acetone. Partition Coefficient Determination. The partition coefficients of [99mTc]MAMA-HA and [99mTc]MAG-HA were measured by mixing 10 µL of each 99mTc-labeled compound with 3 g each of 1-octanol and phosphate buffer (0.1 M, pH 7.4) in a test tube. After vortexing for 1 min × 3 times, the solution was left to stand at room temperature for 20 min. Each tube was then centrifuged at 1000 × g for 5 min. Two weighed samples from the 1-octanol and buffer layers were counted in a γ counter. The partition coefficient was determined by calculating the ratio of counts per minute per gram (cpm/g) of octanol to that of buffer. Cysteine Challenge Assay. To compare the stability of [99mTc]MAMA-HA and [99mTc]MAG-HA, a cysteine challenge assay was performed. Both 99mTc-labeled fatty
Bioconjugate Chem., Vol. 10, No. 3, 1999 491
acid analogues were incubated with 10 µM cysteine, which is the concentration in blood, in phosphate buffer (0.05 M, pH 7.0) at 37 °C. At 5 or 60 min after incubation, the integrity of each was analyzed by RP-HPLC under the conditions used for purification. Animal Studies. Male Wistar rats (200-250 g) were housed for 1 week under a 12 h light/12 h dark cycle and had free access to food and water for subsequent metabolite analyses in vitro, imaging and biodistribution studies. Planar imaging studies of 99mTc-labeled fatty acid analogues were performed as follows. Normal rats were anesthetized with pentobarbital (50 mg/kg i.p.). Dynamic planar scanning (20 s × 30 frames, 1 min × 10 frames, 2 min × 20 frames) was initiated immediately after administration of 99mTc-labeled compounds (15 MBq/0.5 mL) via the tail vein. For biodistribution studies, [99mTc]MAMA-HA (37 kBq in 0.1 mL saline) was administered to normal rats via the tail vein. At appropriate time points after administration, rats were sacrificed by decapitation. Samples of blood and the organs of interest were excised and weighed, and the radioactivity was quantified using a NaI (Tl) γ scintillation counter (Packard Auto-Gamma 500, Packard Inst. Co.). The results are expressed as percent injected dose per gram of blood or organs after decay correction. Determination of Radiometabolites in Vivo. At 60 min postinjection of [99mTc]MAMA-HA, urine samples were obtained from the bladder. After filtration of the urine samples through a 10 kDa cutoff ultrafiltration membrane (Molcut II LGC; NIHON MILLIPORE Co, Yonezawa, Japan), the filtrate was analyzed by RP-HPLC eluted with a gradient mobile phase starting from 10% methanol in 0.05 M phosphate buffer (pH 7.0) to 100% methanol in 60 min at a flow rate of 1 mL/min at 30 °C. In Vitro Metabolite Studies. Male Wistar rats were anesthetized with pentobarbital (50 mg/kg i.p.), and the rat liver was perfused via the portal vein with O2/CO2 (95/5)-saturated cold 20 mM Tris-HCl buffer (pH 7.4) containing 100 mM KCl, 5 mM MgCl2, and 5 mM KH2PO4. The rat liver was immediately excised and sliced at a thickness of 300 µm. After weighing, the slices were incubated for 5 min at 37 °C in the presence or absence of 100 µM 2-bromooctanoate which inhibits β-oxidation of long and medium-chain fatty acids (20, 21). Then, [99mTc]MAMA-HA (3.7 MBq) was added, and the solution was shaken 60 times/min under bubbling O2/CO2 (95/5) at 37 °C for 60 min. After addition of 5 mL of acetonitrile, the solution was homogenized and centrifuged at 1000g for 20 min to precipitate protein. After removing the organic solvent in vacuo, the residue was filtered through polycarbonate membrane with a pore diameter of 0.22 µm (Hospital/pharmacy Milles GV, Millipore), and the filtrate was analyzed by RP-HPLC as described above. The integrity of the hepatocytes in the slices was determined by measuring lactate dehydrogenase (LDH) activity leaked into the buffer at the end of the incubation. There was no significant difference in LDH activity in the presence or absence of 2-bromooctanoate (3285 ( 367 IU/L vs 3175 ( 478 IU/L, respectively). Statistical Analyses. Data were analyzed using unpaired two-tailed alternate Welch t-tests. A value of p < 0.05 was considered significant. RESULTS
Synthesis of Fatty Acid Analogues. The bistritylated derivative of MAMA-HA (6) was synthesized ac-
492 Bioconjugate Chem., Vol. 10, No. 3, 1999 Scheme 1
Yamamura et al. Table 1. Partition Coefficients of Technecium-99m-Labeled Compounds PCa 1.06 ( 0.10 0.89 ( 0.03b
[99mTc]MAMA-HA [99mTc]MAG-HA
a PC, partition coefficient, octanol/phosphate buffer, pH 7.4. < 0.05.
b
P
Table 2. Stability of [99mTc]MAG-HA and [99mTc]MAMA-HA [99mTc]MAG-HAb [99mTc]MAMA-HAb
5 mina
60 mina
87.6 (81.2-100) 97.2 (96.3-97.9)
79.8 (76.9-83.1) 87.1 (83.7-93.8)
a Values are mean (range) percentages of intact 99mTc-labeled analogues for three experiments. b Each fatty acid analogue labeled with 99mTc was incubated with 10 µM cysteine in 0.05 M phosphate buffer (pH 7.0) at 37 °C.
Table 3. Biodistribution of [99mTc]MAMA-HA in Normal Ratsa
Scheme 2
tissue blood liver kidney spleen pancreas lung heart intestine a
cording to the procedure outlined in Scheme 1. After protecting the thiol group of cysteamine chloride with trityl chloride, the resulting compound 2 was reacted with bromoacetyl bromide to prepare 3. The amine group of 3 was then alkylated with 6-bromohexanoate to produce 5. After hydrolysis of the ester group, 6 was obtained as a yellow oil with an overall yield of 2.26%. The synthetic procedure for S-acetyl MAG-HA (13) is outlined in Scheme 2. The active ester of 11 was reacted with the amine group of 6-aminohexanoic acid to provide 12. S-Acetyl MAG-HA was obtained as white crystals by the reaction of SATA (10) with 12 after deprotecting the Boc group of 12. The overall yield was 3.70%. Radiolabeling. [99mTc]MAMA-HA and [99mTc]MAGHA were obtained in radiochemical yields of 77% and 87%, respectively. After RP-HPLC purification, both 99m Tc-labeled fatty acid analogues showed radiochemical purities of over 99% as determined with RP-HPLC and
5 min
20 min
60 min
0.30 (0.28-0.32) 10.59 (9.78-11.24) 8.16 (6.69-9.52) 0.06 (0.05-0.06) 0.13 (0.07-0.22) 0.15 (0.14-0.16) 0.15 (0.13-0.16) 0.17 (0.16-0.18)
0.21 (0.17-0.26) 4.30 (3.61-5.60) 5.80 (4.87-6.57) 0.06 (0.04-0.08) 0.08 (0.06-0.10) 0.14 (0.08-0.21) 0.11 (0.08-0.12) 0.50 (0.29-0.66)
0.16 (0.10-0.26) 2.61 (1.93-3.60) 5.25 (3.09-8.13) 0.05 (0.01-0.11) 0.09 (0.04-0.18) 0.12 (0.07-0.19) 0.07 (0.04-0.12) 1.38 (1.37-1.39)
Values are means (range) as % dose/gram tissue for three rats.
TLC. No changes were observed in the radiochemical purities with the two 99mTc-labeled fatty acids after standing for 24 h at room temperature. On the other hand, the derivatives prepared for metabolite analysis, [99mTc]MAMA-BA and [99mTc]MAMA-AA, were obtained in radiochemical yields of 37.4 and 59.7%, respectively; both radiochemical purities were also over 99% after RPHPLC purification. Partition Coefficient. Table 1 shows partition coefficients of [99mTc]MAMA-HA and [99mTc]MAG-HA at pH 7.4. [99mTc]MAMA-HA showed a slightly but significantly higher partition coefficient than [99mTc]MAG-HA. Cysteine Challenge Assay. Table 2 shows the stability of [99mTc]MAMA-HA and [99mTc]MAG-HA. After 60 min of incubation, 88% of [99mTc]MAMA-HA and 80% of [99mTc]MAG-HA remained intact, indicating that [99mTc]MAMA-HA was more stable than [99mTc]MAG-HA. In Vivo Studies. [99mTc]MAMA-HA showed rapid accumulation of the radioactivity in the liver immediately after administration (Figure 1A). At 60 min postinjection, most of the radioactivity was observed in the kidney and bladder due to its rapid elimination from the liver (Figure 1B). On the other hand, the majority of radioactivity was immediately excreted from the kidney to the bladder after administration of [99mTc]MAG-HA (Figure 1, panels C and D). Similarly, [99mTc]MAMA-BA showed rapid excretion of the radioactivity from the kidney into the urine with little radioactivity in the liver (Figure 1E). At 60
Technetium-99m-Labeled Fatty Acid Derivatives
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Figure 2. Metabolite analysis of urine samples after administration of [99mTc]MAMA-HA in rats. Proposed structures of a series of [99mTc]MAMA derivatives are shown. The parental [99mTc]MAMA-HA (c) and analytical controls, [99mTc]MAMA-BA (b) and [99mTc]MAMA-AA (a), were eluted at retention times of 23, 17, and 16 min, respectively (A). Urine samples showed two radioactivity peaks with a major peak eluted at a retention time of 17 min (B). RP-HPLC analysis of the urine samples coinjected with [99mTc]MAMA-BA showed a single radioactivity peak at a retention time of 17 min in addition to the peak of [99mTc]MAMA-HA (C). Cochromatographic analysis of the urine samples with [99mTc]MAMA-AA showed two peaks well-separated in addition to the peak of [99mTc]MAMA-HA (D).
Figure 1. Planar images of 99mTc-labeled fatty acid analogues. 99mTc-labeled compounds (15 MBq each) were administered to normal rats via the tail vein. Dynamic scanning was initiated immediately after administration of 99mTc-labeled compounds. (A) [99mTc]MAMA-HA, 4-6 min, (B) [99mTc]MAMA-HA, 58-60 min, (C) [99mTc]MAG-HA, 4-6 min, (D) [99mTc]MAG-HA, 5860 min, (E) [99mTc]MAMA-BA, 4-6 min, (F) [99mTc]MAMA-BA, 58-60 min. L, K, and B represent liver, kidney, and bladder, respectively.
min postinjection, radioactivity was observed in the bladder (Figure 1F). Table 3 summarizes the biodistribution of the radioactivity after administration of [99mTc]MAMA-HA to normal rats. [99mTc]MAMA-HA in the liver showed high initial uptake of radioactivity (10.59 % dose/g at 5 min after injection) with low radioactivity levels in the blood for imaging diagnosis. With time, the radioactivity in the liver decreased with only a slight increase in that in the intestine. On the other hand, although the highest radioactivity level was observed in the kidney at 5 min postinjection, high radioactivity levels were still observed at 20 and 60 min postinjection.
The proposed structures of the mother compound [99mTc]MAMA-HA and its expected radiometabolites, [99mTc]MAMA-BA and [99mTc]MAMA-AA, are shown in Figure 2. Figure 2A shows typical radiochromatograms of [99mTc]MAMA-HA, [99mTc]MAMA-BA, and [99mTc]MAMA-AA. The urine samples showed two well-separated radioactivity peaks at retention times of 17 and 23 min with the former peak representing 82.9 ( 9.0% of the radioactivity in the urine (Figure 2B). Cochromatographic analysis of the urine samples with [99mTc]MAMABA indicated two radioactivity peaks at retention times of 17 and 23 min on RP-HPLC (Figure 2C). However, three radioactivity peaks were detected on RP-HPLC at retention times of 16, 17, and 23 min when the urine sample were cochromatographed with [99mTc]MAMA-AA (Figure 2D). In Vitro Metabolite Studies. More than 90% of the radioactivity in the liver homogenate was extracted with acetonitrile. RP-HPLC analysis of the acetonitrileextracted fraction depicted two radioactivity peaks at retention times of 17 and 23 min when the slices were incubated with [99mTc]MAMA-HA in the absence of 2-bromooctanoate (Figure 3A) with the former fraction representing 21.2 ( 3.0% of the radioactivity in the supernatant. However, the supernatant showed a single radioactivity peak at a retention time of 23 min (Figure 3B) when incubated with [99mTc]MAMA-HA in the presence of 2-bromooctanote.
494 Bioconjugate Chem., Vol. 10, No. 3, 1999
Figure 3. Metabolite analysis of [99mTc]MAMA-HA in liver slices. The liver slices were incubated with [99mTc]MAMA-HA for 60 min at 37 °C. Two radioactivity peaks were observed, the former of which had a retention time similar to that of [99mTc]MAMA-BA (A). However, RP-HPLC showed a single radioactivity peak with a retention time similar to that of parental [99mTc]MAMA-HA in the presence of 2-bromooctanoate which inhibits 3-ketothiolase, the last enzyme of the fatty acid oxidation pathway (B). DISCUSSION
External imaging of energy production levels of living cells with 99mTc-labeled compounds is a challenging task for the development of 99mTc-radiopharmaceuticals. Indeed, efforts have been made to develop 99mTc-labeled long-chain fatty acid analogues for myocardial imaging studies. However, their application has been hampered by poor accumulation in the heart, and it seemed that these 99mTc-labeled fatty acid analogues were not recognized as substrates for β-oxidation (22, 23). β-Oxidation is the main pathway providing energy in the liver. On the basis of our recent findings that 11Cand 123I-labeled medium-chain fatty acids were useful for measuring β-oxidation activity of hepatocytes (9, 10), we designed two 99mTc-labeled medium-chain fatty acid analogues as prototype compounds and evaluated their potential as substrates for β-oxidation in the liver. In biodistribution studies in rats, [99mTc]MAMA-HA showed rapid accumulation in the liver following injection. [99mTc]MAMA-HA also exhibited rapid elimination of the radioactivity from the liver into urine. RP-HPLC analysis of urine samples obtained at 60 min postinjection showed that the major radioactivity peak had a retention time identical to that of [99mTc]MAMA-BA. This was supported by cochromatographic analyses of the urine samples with [99mTc]MAMA-BA or [99mTc]MAMAAA. In addition, [99mTc]MAMA-BA showed rapid excretion of the radioactivity into urine via the kidney with little accumulation in the liver, indicating that redistribution of [99mTc]MAMA-BA generated in the body to the liver was negligible. These findings suggested that [99mTc]MAMA-HA would be metabolized to [99mTc]MAMA-BA in the liver via β-oxidation, and the radiometabolite was excreted from the liver into the urine. The metabolism of [99mTc]MAMA-HA to [99mTc]MAMABA by β-oxidation in the liver was also indicated by in vitro studies using living slices of rat liver. [99mTc]MAMABA was observed as the sole radiometabolite after incubation (Figure 3). However, generation of [99mTc]MAMA-BA was completely inhibited when [99mTc]MAMAHA was incubated with the liver slices in the presence of 2-bromooctanote, an inhibitor of 3-ketothiolase, the last enzyme involved in long- and medium-chain fatty acid oxidation in the mitochondria. These findings implied that after uptake by the liver, [99mTc]MAMA-HA was
Yamamura et al.
recognized as a substrate for β-oxidation and metabolized to [99mTc]MAMA-BA. The final radiometabolite of [99mTc]MAMA-HA was not [99mTc]MAMA-AA but [99mTc]MAMA-BA as demonstrated by cochromatographic analyses. Since [99mTc]MAMA-HA possesses a 6-carbon chain, [99mTc]MAMA-AA was expected to be the final radiometabolite after β-oxidation. Indeed, our recent study indicated that the radioiodinated medium-chain fatty acid analogue [123I]IPEA was metabolized to iodohippuric acid via iodobenzoate. This suggested that, unlike iodobenzene, the presence of [99mTc]MAMA chelate might have hindered further recognition of [99mTc]MAMA-BA by the enzymes that are involved in β-oxidation. In contrast to [99mTc]MAMA-HA, [99mTc]MAG-HA showed little accumulation in the liver after administration. Although [99mTc]MAG-HA was less stable than [99mTc]MAMA-HA, about 80% of [99mTc]MAG-HA was intact even after 60 min of incubation. Since the radioactivity in rats following [99mTc]MAG-HA injection was rapidly eliminate from blood to urine as shown in Figure 1, panels C and D, the instability of [99mTc]MAG-HA in the blood was considered not to be a major factor responsible for poor liver uptake. On the other hand, [99mTc]MAG-HA showed a lower partition coefficient than [99mTc]MAMA-HA. [99mTc]MAG-HA had a retention time similar to that of [99mTc]MAMA-BA, a radiometabolite of [99mTc]MAMA-HA, on RP-HPLC under the same conditions. Moreover, [99mTc]MAMA-BA and [99mTc]MAG-HA showed similar biodistributions. These findings suggested that the low lipophilicity of [99mTc]MAG-HA was responsible for its poor hepatic accumulation although negatively charged 99mTc-MAG chelate may also be involved. In conclusion, [99mTc]MAMA-HA was metabolized by β-oxidation to [99mTc]MAMA-BA after uptake into the liver. Thus, [99mTc]MAMA-HA is the first 99mTc-labeled compound recognized as a substrate for energy production in living cells. Appropriate lipophilicity of [99mTc]MAMA chelate would be useful to facilitate rapid elimination of the final radiometabolite, [99mTc]MAMA-BA, from the liver into urine without being retained in the hepatocytes. Since the procedure for synthesis of MAMAHA was applicable to fatty acids with different chain lengths, further derivation of this compound may lead to development of 99mTc-radiopharmaceuticals more appropriate for visualization of energy production levels in the liver. LITERATURE CITED (1) Kudo, M., Todo, A., Ikekubo, K., Hino, M., Ito, H., Yamaguchi, H., Saiki, Y., Yamamoto, K., Yonekura, Y., and Torizuka, K., et al. (1987) Estimation of hepatic functional reserve by asialoglycoprotein receptor-binding, radiolabeled synthetic ligand Tc-99m-galactosyl-neoglycoalbuminspreclinical and clinical studies. Kaku Igaku (Jpn. J. Nucl. Med.) 24, 16531662. (2) Kamiike, W., Burdelski, M., Steinhoff, G., Ringe, B., Lauchart, W., and Pichlmayr, R. (1988) Adenine nucleotide metabolism and its relation to organ viability in human liver transplantation. Transplantation 45, 138-143. (3) Lanir, A., Jenkins, R. L., Caldwell, C., Lee, R. G., Khettry, U., and Clouse, M. E. (1988) Hepatic transplantation survival: correlation with adenine nucleotide level in donor liver. Hepatology 8, 471-475. (4) Martin, D. B. (1976) Metabolism and energy mechanisms. Pathophysiology (E. D. Frohlich, Ed.) pp 365-383, Lippincott, Philadelphia. (5) Zakim, D. (1982) Metabolism of glucose and fatty acids by the liver. Hepatology (D. Zakim, and T. D. Boyer, Eds.) pp 76-109, Saunders, Philadelphia.
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