Synthesis, radiolabeling, and biodistribution of putative metabolites of iodoazomycin arabinoside

Nuclear Medicine & Biology, Vol. 27, pp. 61– 68, 2000 Copyright © 2000 Elsevier Science Inc. All rights reserved.

ISSN 0969-8051/00/$–see front matter PII S0969-8051(00)00089-X

Synthesis, Radiolabeling, and Biodistribution of Putative Metabolites of Iodoazomycin Arabinoside Herbert C. Lee,1 Piyush Kumar,1 Alexander J. McEwan,2 Leonard I. Wiebe1 and John R. Mercer1,2 1

FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES, AND 2DEPARTMENT OF RADIOLOGY AND DIAGNOSTIC IMAGING, UNIVERSITY OF ALBERTA, EDMONTON, ALBERTA, CANADA

ABSTRACT. Scintigraphic evaluation of patients with advanced oncological disease showed uptake of radioactivity in the brain following administration of the hypoxic imaging agent 123I-iodoazomycin arabinoside (123I-IAZA). Three proposed metabolites of IAZA—methyl 5-deoxy-5-iodo-D-arabinofuranoside, methyl 2,3-di-O-acetyl-5-deoxy-5-iodo-␣-D-arabinofuranoside, and 1-(5-deoxy-5-iodo-␣-D-arabinofuranosyl)-2-aminoimidazole (IAIA)—were synthesized, radiolabeled with 125I, and investigated in normal and tumor-bearing murine models for their contribution to this unusual phenomenon. The three compounds were readily radiolabeled by melt or solvent exchange procedures. Biodistribution data indicated rapid blood clearance, rapid excretion, and little tissue accumulation in the brain. IAIA showed significant tumor to blood ratios at 4 h (4.3:1) and liver to blood ratios at 24 h (30:1). NUCL MED BIOL 27;1:61– 68, 2000. © 2000 Elsevier Science Inc. All rights reserved. KEY WORDS. Radioiodination, Iodoazomycin arabinoside, Hypoxia, Nitroimidazoles, Tumor imaging, Arabinofuranosides

INTRODUCTION 123

123

[ I] Iodoazomycin arabinoside ([ I]IAZA, 1A), an arabinofuranosyl 2-nitroimidazole nucleoside analog, has been shown to accumulate selectively in hypoxic tumors in animals (13, 14), in certain tumors in cancer patients (5, 19), and in the feet and legs of patients with diabetes mellitus (1). Radiolabeled IAZA has been used as an indication of hypoxia induction in photodynamic therapy-treated Dunning prostate tumors in rats (16). The pharmacokinetics of IAZA have been studied in rats and human volunteers (27) and its potential in detecting brain hypoxia has been evaluated in a rat stroke model (12). A very unexpected observation in the imaging of approximately one third of patients (3 of 10 patients) from the preliminary cancer studies (5, 19) was the presence of radioactivity in brain tissue in images taken 18 –24 h later. This brain radioactivity was significantly higher than activity in surrounding bone and muscle in the head and upper torso. None of the patients had evidence of brain involvement in their disease. The time course of this highly unusual uptake was not consistent with perfusion images and this uptake may represent metabolic trapping of radioactivity in the brains of 123I-IAZA patients. It is not known whether this uptake reflects an altered brain permeability or unusual drug metabolism unique to some of the cancer patients. Although the metabolism of IAZA has not been fully investigated, several reasonable metabolic pathways for IAZA can be proposed based on observations with this

Address correspondence to: John R. Mercer, Ph.D., Faculty of Pharmacy and Pharmaceutical Sciences, 3118 Dentistry-Pharmacy Building, University of Alberta, Edmonton, Alberta, Canada T6G 2N8; e-mail: jmercer@ pharmacy.ualberta.ca. Received 23 July 1999. Accepted 9 October 1999.

compound and the known biochemical transformations of nucleosides and nucleoside analogs. One metabolic route involves the enzymatic cleavage of the N1-glycosidic bond between the 2-nitroimidazole nucleobase and the radioiodinated arabinofuranosyl sugar moiety. Although IAZA has been shown to be resistant to phosphorolytic cleavage of the glycosidic bond in in vitro incubation studies with thymidine phosphorylase (13), chemical or enzymatic cleavage could occur in vivo or in selected tissues. The resulting radioiodinated arabino sugar could then undergo passive or active uptake into brain and other tissues. The second metabolic pathway results from intracellular reduction of the nitro group on the 2-nitroimidazole moiety. Although 2-nitroimidazoles typically undergo a fully reversible reduction step in well oxygenated cells, in hypoxic tissues further irreversible bioreduction to reactive intermediates (nitroso and hydroxylamino) and finally reduction to the amine can occur (23, 26). The 2-aminoimidazole nucleoside 5 is the resulting product from exhaustive bioreduction of the nitro group of IAZA. It is conceivable that after systemic bioreduction, 5 undergoes passive or active uptake into the brain tissues, possibly facilitated by specific nucleoside and/or amine transporters (7), or by a pH-dependent trapping mechanism in which the amino group is protonated by the slightly lower cerebral pH (9). These two metabolic pathways provide a rational basis for the syntheses and radioiodination of methyl 5-deoxy-5-iodo-D-arabinofuranoside (3), methyl 2,3-di-O-acetyl-5deoxy-5-iodo-␣-D-arabinofuranoside (4), and the 2-aminoimidazole nucleoside 1-(5-deoxy-5-iodo-␣-D-arabinofuranosyl)-2-aminoimidazole (iodoaminoimidazole arabinoside [IAIA, 5]). The biodistributions of these proposed metabolites were then evaluated in mice as an indicator of their potential roles in the observed single photon emission computed tomography (SPECT) brain images of [123I]IAZA patients.

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FIG. 1. Syntheses of compounds 3 and 4. MATERIALS AND METHODS All solvents and chemicals were of reagent grade unless otherwise specified. Where dry solvents were required, these were dried by standard methods. Radioiodine (125I) was purchased from Amersham (Quebec, Canada) as no-carrier-added solutions of NaI in 0.1 N NaOH. Analysis by thin layer chromatography (TLC) was carried out on Whatman silica gel MK6F microplates (Clifton, NJ USA). Column chromatography was used for purification of synthetic products using silica gel (70 –230 mesh). 1 H and 13C nuclear magnetic resonance (NMR) were determined on a Brucker AM-300 spectrometer (Milton, Ontario, Canada). Elemental analyses (EA-1108-Elemental Analyzer, Carlo Erba Instruments, Milan, Italy), low- and high-resolution fast atom bombardment (FAB) mass spectrometry (MS; AEI-Kratos MS-9 spectrometer, Chestnut Ridge, NY USA), and low- and high-resolution electrospray ionization mass spectrometry (ESI-MS) (Micromass ZabSpec Hybrid Sector-TOF spectrometer, Manchester, UK) were

provided by the Department of Chemistry, University of Alberta, as a commercial service. Radiolabeled compounds were purified by column chromatography (3 and 4) or Sep-Pak威 cartridge (Waters, Mississauga, Ontario, Canada) (5). A Beckman Gamma 8000 gamma scintillation counter (Beckman Instruments, Mississauga, Ontario, Canada) was used to analyze the radioactive tissue samples obtained from in vivo biodistribution studies.

Chemistry METHYL 5-DEOXY-5-IODO-D-ARABINOFURANOSIDE (3). Two synthetic approaches were employed in the synthesis of the title compound (Fig. 1). The first approach involved the chemical cleavage from diacetylated IAZA (1B) and has been reported briefly elsewhere (10). The title compound (3) was obtained (from 1A) as a syrup in 90% yield (57 mg). The ␣-␤ ratio in 3 was

FIG. 2. Synthesis of compound 5.

Metabolites of Iodoazomycin Arabinoside

63

TABLE 1. Biodistribution of [125I]-3 Following Intravenous Administration in Normal B6D2F1/J Mice Time (hours) Tissue

0.25

Blood Heart

3.29 ⫾ 0.22 1.67 ⫾ 0.17 0.51 ⴞ 0.02b 2.37 ⫾ 0.11 0.72 ⴞ 0.02 2.43 ⫾ 0.37 0.74 ⴞ 0.08 1.40 ⫾ 0.11 0.43 ⴞ 0.02 5.72 ⫾ 0.18 1.74 ⴞ 0.12 2.61 ⫾ 0.23 0.79 ⴞ 0.07 1.99 ⫾ 0.24 0.61 ⴞ 0.11 1.37 ⫾ 0.05 0.42 ⴞ 0.04 1.59 ⫾ 0.08 0.48 ⴞ 0.04 1.04 ⫾ 0.23 0.32 ⴞ 0.08 0.98 ⫾ 0.05 0.30 ⴞ 0.01 1.45 ⫾ 0.11 0.44 ⴞ 0.01 0.28 ⫾ 0.07d 0.73 ⴞ 0.15e

Lungs Liver Spleen Kidney Stomach Gastrointestinal tractc Muscle Skin Bone Brain Carcass Thyroid

a

0.50

1

2

4

1.35 ⫾ 0.09 0.83 ⫾ 0.18 0.63 ⴞ 0.17 1.40 ⫾ 0.17 1.05 ⴞ 0.19 1.40 ⫾ 0.24 1.05 ⴞ 0.22 0.90 ⫾ 0.16 0.68 ⴞ 0.15 2.02 ⫾ 0.39 1.51 ⴞ 0.36 2.70 ⫾ 0.31 2.03 ⴞ 0.35 1.26 ⫾ 0.27 0.95 ⴞ 0.25 0.77 ⫾ 0.21 0.57 ⴞ 0.15 0.94 ⫾ 0.19 0.71 ⴞ 0.18 0.63 ⫾ 0.03 0.47 ⴞ 0.03 0.49 ⫾ 0.12 0.37 ⴞ 0.11 0.94 ⫾ 0.13 0.70 ⴞ 0.12 0.03 ⫾ 0.02 0.14 ⴞ 0.13

1.26 ⫾ 0.13 0.75 ⫾ 0.06 0.60 ⴞ 0.04 1.08 ⫾ 0.10 0.86 ⴞ 0.06 1.10 ⫾ 0.12 0.80 ⴞ 0.04 0.72 ⫾ 0.05 0.57 ⴞ 0.04 1.63 ⫾ 0.22 1.29 ⴞ 0.11 2.88 ⫾ 0.76 2.32 ⴞ 0.69 1.36 ⫾ 0.36 1.06 ⴞ 0.21 0.47 ⫾ 0.07 0.37 ⴞ 0.02 0.80 ⫾ 0.11 0.63 ⴞ 0.08 0.54 ⫾ 0.03 0.44 ⴞ 0.05 0.40 ⫾ 0.06 0.32 ⴞ 0.03 0.79 ⫾ 0.11 0.62 ⴞ 0.07 0.82 ⫾ 0.17 5.41 ⴞ 1.40

0.45 ⫾ 0.12 0.23 ⫾ 0.04 0.52 ⴞ 0.07 0.43 ⫾ 0.10 0.95 ⴞ 0.04 0.35 ⫾ 0.08 0.77 ⴞ 0.07 0.29 ⫾ 0.06 0.66 ⴞ 0.05 0.44 ⫾ 0.14 0.96 ⴞ 0.08 2.41 ⫾ 0.25 5.70 ⴞ 1.72 0.95 ⫾ 0.20 2.18 ⴞ 0.52 0.16 ⫾ 0.03 0.36 ⴞ 0.05 0.32 ⫾ 0.10 0.71 ⴞ 0.09 0.24 ⫾ 0.06 0.54 ⴞ 0.10 0.08 ⫾ 0.02 0.17 ⴞ 0.01 0.44 ⫾ 0.09 0.54 ⴞ 0.22 0.80 ⫾ 0.90 8.55 ⴞ 9.02

0.23 ⫾ 0.07 0.13 ⫾ 0.02 0.58 ⴞ 0.06 0.20 ⫾ 0.05 0.91 ⴞ 0.10 0.18 ⫾ 0.07 0.77 ⴞ 0.04 0.19 ⫾ 0.02 0.94 ⴞ 0.27 0.21 ⫾ 0.06 0.91 ⴞ 0.08 1.28 ⫾ 0.75 5.30 ⴞ 1.47 0.58 ⫾ 0.49 2.21 ⴞ 1.09 0.07 ⫾ 0.01 0.31 ⴞ 0.06 0.16 ⫾ 0.04 0.74 ⴞ 0.12 0.15 ⫾ 0.08 0.65 ⴞ 0.18 0.02 ⫾ 0.003 0.11 ⴞ 0.03 0.23 ⫾ 0.05 1.10 ⴞ 0.35 1.57 ⫾ 0.74 30.1 ⴞ 10.89

The numbers represent the mean ⫾ SD for percent of injected dose per gram of wet tissue for four animals (three animals for 0.25 and 0.50 h). Tissue-to-blood ratios. c Section of intestine. d Percentage of injected dose in thyroid. e Thyroid radioactivity as a percentage of whole-body radioactivity. a

b

approximately 1:1. 1H NMR (CDCl3): ␦ 4.96 (1H, d, J1␣,2 ⫽ 1.7 Hz, C1␣-H), 4.86 (1H, d, J1␤,2 ⫽ 5.0 Hz, C1␤-H), 4.17 (1H, br s, C2␣-H), 4.11 (2H, m, C3␣,␤-H), 4.04 (1H, m, C2␤-H), 3.92– 4.00 (2H, m, C4␣,␤-H), 3.48 (3H, s, OCH3-␤), 3.42 (3H, s, OCH3-␣), 3.30 –3.40 (4H, m, C5␣,␤-H, C5⬘␣,␤-H). 13C NMR (CDCl3): ␦109.0 (C1␣), 102.0 (C1␤), 85.1 (C4␣ and C2␣), 81.6 (C4␤ and C2␤), 80.9 (C3␣), 78.6 (C3␤), 55.6 (OCH3-␤), 55.1 (OCH3-␣), 7.9 (C5␤), 6.5 (C5␣). POSFAB MS m/z (%): 275([M⫹H]⫹, 3), 243(79), 225(44). Anal. Calcd. for C6H11O4I: C, 26.28; H, 4.01; I, 46.35. Found: C, 26.43; H, 4.13; I, 46.06. The second approach involved a two-step synthesis from D-(-)arabinose (6; Scheme 1). The first step of the synthesis was performed according to a published procedure (18). Compound 7 was iodinated using the triphenylphosphine/iodine/pyridine reagent system (14). Briefly, triphenylphosphine (2.88 g, 10.98 mmol) and iodine (1.40 g, 5.49 mmol) were added to a solution of 7 (0.9 g, 5.49 mmol) in dry pyridine (25 mL). The resulting solution was heated at 50°C for 1 h, after which the reaction was quenched with methanol (2 mL) and then evaporated to dryness. The residue was chromatographed on a silica gel column using chloroform-methanol (9:1) (v/v) to give 1.0 g (67%) of 3 as a syrup after evaporation of the solvent. This product was identical by TLC and NMR to that prepared by the first method. METHYL 2,3-DI-O-ACETYL-5-DEOXY-5-IODO-␣-D-ARABINOFURANOSIDE

Compound 7 (5.0 g, 0.030 mol) was chromatographed on a silica gel column (35 mm ⫻ 440 mm) using dichloromethane-

(4).

methanol (19:1) (v/v). The anomerically pure 7␣ was subsequently eluted using the same solvent system to give 1.1 g (22% from 7, 15% from 6) as a syrup, which was used directly. The 1H and 13C NMR data of 7 were consistent with values found in the literature (4, 22). The iodination procedure was similarly performed for 7␣ with subsequent acetylation in pyridine to give 4 as a syrup (0.72 g, 69%). An anomeric mixture of 4 has previously been prepared, but no NMR data were reported (15). 1H NMR (CDCl3): ␦5.09 (1H, d, J1,2 ⫽ 1.5 Hz, C1-H), 4.95 (1H, br s, C2-H), 4.89 (1H, d, J3,2 ⫽ 1.4 Hz, J3,4 ⫽ 5.4 Hz, C3-H), 4.06 (1H, dd, J4,3 ⫽ 4.0 Hz, J4,5 ⫽ 6 Hz, C4-H), 3.52 (1H, dd, J5,5⬘ ⫽ 10.8 Hz, J5,4 ⫽ 4.8 Hz, C5-H), 3.43 (1H, dd, J5⬘,5 ⫽ 10.8 Hz, J5⬘,4 ⫽ 6 Hz, C5⬘-H), 3.41 (3H, s, OCH3), 2.11 (6H, s, COOCH3). 13C NMR (CDCl3): ␦170.2 and 169.7 (2 ⫻ C⫽O), 106.6 (C1), 81.9 (C4), 81.3 (C2), 80.3 (C3), 54.9 (OCH3), 20.7 (2 ⫻ OAc), 5.0 (C5). POSFAB MS m/z (%): 359([M⫹H]⫹, 16), 327(100), 267(46). Anal. Calcd. for C10H15O6I: C, 33.52; H, 4.19; I, 35.47. Found: C, 33.72; H, 4.01; I, 35.74. IAIA (5). The title compound was synthesized in two steps from 1-(␣-D-arabinofuranosyl)-2-nitroimidazole (AZA, 8) as shown in Figure 2 (Fig. 2) using a procedure described previously (11). Briefly, a solution of 8 (100 mg, 0.41 mmol) in 95% ethanol (5 mL) was reduced under 1 atmosphere of hydrogen at 25°C for 2 h in the presence of palladium/carbon (12.2 mg). The mixture was filtered and the filtrate was evaporated to give AIA (9) (90 mg, 88%) as a

H. C. Lee et al.

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TABLE 2. Biodistribution of [125I]-4 Following Intravenous Administration in Normal Balb/c Mice Tissue

0.25

Blood Heart

4.50 ⫾ 0.22 3.14 ⫾ 1.02 0.74 ⴞ 0.09b 3.85 ⫾ 1.34 0.89 ⴞ 0.09 3.99 ⫾ 1.09 0.96 ⴞ 0.18 2.75 ⫾ 0.89 0.64 ⴞ 0.08 7.40 ⫾ 2.25 1.75 ⴞ 0.33 3.07 ⫾ 0.65 0.79 ⴞ 0.30 3.84 ⫾ 1.04 0.96 ⴞ 0.32 2.50 ⫾ 0.64 0.61 ⴞ 0.12 2.64 ⫾ 0.86 0.61 ⴞ 0.10 1.73 ⫾ 0.76 0.42 ⴞ 0.15 2.77 ⫾ 0.86 0.66 ⴞ 0.10 2.50 ⫾ 0.76 0.59 ⴞ 0.08 0.35 ⫾ 0.08d 0.67 ⴞ 0.02e

Lungs Liver Spleen Kidney Stomach Gastrointestinal tractc Muscle Skin Bone Brain Carcass Thyroid

4 a

0.32 ⫾ 0.13 0.15 ⫾ 0.05 0.48 ⴞ 0.08 0.24 ⫾ 0.11 0.76 ⴞ 0.06 0.23 ⫾ 0.11 0.67 ⴞ 0.14 0.19 ⫾ 0.09 0.60 ⴞ 0.13 0.26 ⫾ 0.11 0.80 ⴞ 0.07 1.59 ⫾ 0.60 5.09 ⴞ 0.65 0.40 ⫾ 0.19 1.24 ⴞ 0.18 0.17 ⫾ 0.09 0.50 ⴞ 0.11 0.28 ⫾ 0.12 0.87 ⴞ 0.08 0.18 ⫾ 0.11 0.56 ⴞ 0.22 0.05 ⫾ 0.02 0.16 ⴞ 0.03 0.31 ⫾ 0.13 0.97 ⴞ 0.15 0.66 ⫾ 0.24 11.7 ⴞ 4.20

The numbers represent the mean ⫾ SD for percent of injected dose per gram of wet tissue for five animals (4 h) and four animals (0.25 h). b Tissue-to-blood ratios. c Section of intestine. d Percentage of injected dose in thyroid. e Thyroid radioactivity as a percentage of whole-body radioactivity. a

FIG. 3. Whole-body elimination (■) and blood clearance (䊐) of radioactivity following intravenous administration of [125I]-3 in normal B6D2F1/J mice (N ⴝ 3 or 4). Error bars are ⴞ1 SD. residue that was used directly. Triphenylphosphine (221.5 mg, 0.73 mmol) and iodine (185 mg, 0.73 mmol) were added to a solution of 9 (78 mg, 0.36 mmol) in dry pyridine (2 mL). The resulting solution was stirred at 25°C for 2 h, then concentrated to dryness. The residue was chromatographed on a column containing silica gel using chloroform-methanol (19:1) (v/v) to give 47 mg (40%) of 5 as an oil.

Radioiodination The radioiodination and purification procedures for 3 and 4 have been reported elsewhere (10). The pivalic acid “melt” method (25) was employed and the best radiochemical yield obtained for 3 was 92% and that of 4 was 91%. The final products ([125I]-3 and [125I]-4) had specific activities of 0.35– 0.40 GBq/mmol and the chemical and radiochemical purity of both compounds was greater than 98%. The radioiodination of 5 was achieved by isotope exchange (21) in 2-propanol. In a typical radioiodination reaction, no-carrieradded Na[125I]I supplied as a solution in 10 ␮L of 0.1 N NaOH was diluted to the desired specific concentrations (5.6 –9.2 MBq in 10 –20 ␮L of aqueous NaOH). The solution was concentrated to dryness in a 1.0 mL V-vial under a stream of argon gas. A solution

of 5 (0.5–1.0 mg, 1.5–3.1 ␮mol) in 100 ␮L of methanol was added to the V-vial and the solvent was evaporated. 2-Propanol (100 ␮L) was added and the vial was capped and heated at 88°C for 2–3.5 h. The radiochemical yields averaged approximately 42%. At the end of the reaction the vial was cooled and [125I]-5 was purified on reverse phase Sep-Pak威 cartridges (Waters) using 0.9% saline. Radiochemical purity was determined by TLC (chloroform:methanol:ammonia (85:15:1, v/v). The final product ([125I]-5) had specific activities of 0.5 GBq/mmol (Batch 1) and 1.3 GBq/mmol (Batch 2). Both batches showed greater than 95% chemical and radiochemical purity.

In Vivo Biodistribution Study All animal studies were approved by the local Animal Use Committee and were performed in accordance with the guidelines of the Canadian Council on Animal Care. The murine Balb/c EMT-6 tumor model was used in the in vivo biodistribution study of [125I]-5. This tumor model was prepared by subcutaneous inoculation of murine EMT-6 cell suspensions (0.1 mL, 107 cells/mL) to the left flank of female Balb/c mice (20 –25 g). This tumor is known to have a high hypoxic fraction (20). The tumors reached the desired size (6 – 8 mm diameter) within 14 days. Radiolabeled compounds (stored dry at ⫺20°C) were reconstituted with physiologic saline ([125I]-3, [125I]-5) or 20% ethanol in saline ([125I]-4) just prior to

Metabolites of Iodoazomycin Arabinoside

65

TABLE 3. Biodistribution of [125I]IAIA (5) Following Intravenous Administration in Normal Balb/c Mice Time (hours) Tissue Blood Kidney Gastrointestinal tractc Liver Muscle Bone Lungs Heart Spleen Stomach Brain Carcass Thyroid

0.25

0.5

1

2

0.92 ⫾ 0.10 0.50 ⫾ 0.07 0.32 ⫾ 0.04 2.94 ⫾ 0.40 1.52 ⫾ 0.23 0.79 ⫾ 0.11 3.19 ⴞ 0.07b 3.00 ⴞ 0.10 2.45 ⴞ 0.18 7.29 ⫾ 0.99 5.41 ⫾ 1.76 2.38 ⫾ 0.34 7.99 ⴞ 1.20 10.67 ⴞ 2.79 7.47 ⴞ 1.42 7.78 ⫾ 0.28 4.84 ⫾ 0.62 2.69 ⫾ 0.35 8.55 ⴞ 0.71 9.63 ⴞ 0.82 8.34 ⴞ 0.82 1.01 ⫾ 0.12 0.57 ⫾ 0.12 0.38 ⫾ 0.035 1.10 ⴞ 0.07 1.13 ⴞ 0.15 1.20 ⴞ 0.25 1.15 ⫾ 0.15 0.45 ⫾ 0.04 0.31 ⫾ 0.13 1.27 ⴞ 0.21 0.89 ⴞ 0.04 1.01 ⴞ 0.53 1.86 ⫾ 0.24 0.98 ⫾ 0.05 0.61 ⫾ 0.06 2.02 ⴞ 0.04 1.96 ⴞ 0.18 1.90 ⴞ 0.09 1.38 ⫾ 0.10 0.50 ⫾ 0.02 0.33 ⫾ 0.02 1.51 ⴞ 0.11 1.01 ⴞ 0.11 1.04 ⴞ 0.04 1.82 ⫾ 0.08 0.86 ⫾ 0.14 0.45 ⫾ 0.09 1.99 ⴞ 0.14 1.70 ⴞ 0.09 1.39 ⴞ 0.14 2.90 ⫾ 0.27 2.00 ⫾ 0.64 1.48 ⫾ 0.11 3.17 ⴞ 0.23 4.16 ⴞ 1.85 4.67 ⴞ 0.91 0.14 ⫾ 0.02 0.05 ⫾ 0.04 0.03 ⫾ 0.01 0.16 ⴞ 0.01 0.10 ⴞ 0.06 0.09 ⴞ 0.05 1.53 ⫾ 0.40 1.20 ⫾ 0.05 0.81 ⫾ 0.03 1.64 ⴞ 0.23 2.40 ⴞ 0.22 2.53 ⴞ 0.38 0.39 ⫾ 0.01d 0.11 ⫾ 0.05 0.20 ⫾ 0.09 1.15 ⴞ 0.21e 0.42 ⴞ 0.26 0.90 ⴞ 0.49 a

4

8

24

0.19 ⫾ 0.04 0.15 ⫾ 0.05 0.075 ⫾ 0.028 0.014 ⫾ 0.002 0.34 ⫾ 0.18 0.22 ⫾ 0.04 0.073 ⫾ 0.032 0.018 ⫾ 0.015 1.90 ⴞ 1.06 1.51 ⴞ 0.23 0.94 ⴞ 0.11 1.19 ⴞ 0.91 1.04 ⫾ 0.09 0.36 ⫾ 0.06 0.21 ⫾ 0.07 0.061 ⫾ 0.027 5.68 ⴞ 0.83 2.75 ⴞ 1.05 2.91 ⴞ 0.68 4.34 ⴞ 1.79 1.56 ⫾ 0.13 1.53 ⫾ 0.32 0.56 ⫾ 0.11 0.46 ⫾ 0.07 8.67 ⴞ 2.05 10.62 ⴞ 1.33 8.26 ⴞ 2.18 33.92 ⴞ 4.88 0.20 ⫾ 0.06 0.30 ⫾ 0.06 0.10 ⫾ 0.03 0.048 ⫾ 0.026 1.16 ⴞ 0.49 2.28 ⴞ 0.87 1.36 ⴞ 0.25 3.40 ⴞ 1.35 0.15 ⫾ 0.07 0.41 ⫾ 0.22 0.078 ⫾ 0.012 0.016 ⫾ 0.011 0.91 ⴞ 0.56 2.76 ⴞ 1.63 1.20 ⴞ 0.39 1.33 ⴞ 0.94 0.25 ⫾ 0.03 0.26 ⫾ 0.09 0.056 ⫾ 0.015 0.003 ⫾ 0.003 1.40 ⴞ 0.32 1.79 ⴞ 0.57 0.79 ⴞ 0.13 0.23 ⴞ 0.22 0.08 ⫾ 0.04 0.18 ⫾ 0.05 0.035 ⫾ 0.011 0.003 ⫾ 0.003 0.40 ⴞ 0.13 1.34 ⴞ 0.51 0.50 ⴞ 0.10 0.15 ⴞ 0.11 0.15 ⫾ 0.07 0.13 ⫾ 0.01 0.050 ⫾ 0.036 0.002 ⫾ 0.002 0.74 ⴞ 0.30 0.92 ⴞ 0.22 0.60 ⴞ 0.28 0.086 ⴞ 0.081 0.86 ⫾ 0.23 0.51 ⫾ 0.24 0.34 ⫾ 0.14 0.058 ⫾ 0.007 5.00 ⴞ 2.12 3.25 ⴞ 0.41 4.61 ⴞ 0.84 4.51 ⴞ 1.33 0.01 ⫾ 0.01 0.03 ⫾ 0.01 0.01 ⫾ 0.01 0.002 ⫾ 0.002 0.04 ⴞ 0.04 0.19 ⴞ 0.11 0.10 ⴞ 0.07 0.15 ⴞ 0.15 0.71 ⫾ 0.08 0.45 ⫾ 0.09 0.22 ⫾ 0.08 0.093 ⫾ 0.014 3.86 ⴞ 0.45 3.27 ⴞ 1.02 3.03 ⴞ 0.63 7.01 ⴞ 1.71 0.11 ⫾0.08 0.29 ⫾ 0.18 0.61 ⫾ 0.27 1.04 ⫾ 1.03 0.89 ⴞ 0.88 3.3 ⴞ 2.5 12.1 ⴞ 0.6 27.7 ⴞ 24.5

The numbers represent the mean ⫾ SD for percent of injected dose per gram of wet tissue for three animals. Tissue-to-blood ratio. c Section of intestine. d Percentage of injected dose in thyroid. e Thyroid radioactivity as a percentage of whole-body radioactivity. IAIA ⫽ 1-(5-deoxy-5-iodo-␣-D-arabinofuranosyl)-2-aminoimidazole. a b

injection via the dorsal vein in a single bolus injection. Each mouse received an intravenous bolus injection of 0.10 – 0.15 mL of a solution containing 10 –15 KBq of 125I in 9 –10 ␮g of [125I]-3, [125I]-4, and [125I]-5 (Batch 1), and 48 – 60 KBq of 125I in 15–18 ␮g of [125I]-5 (Batch 2). Mice (three at a time) were sacrificed at various time intervals after injection. In addition to blood, tissues such as brain, liver, kidney, heart, lung, muscle, bone, spleen, stomach, intestine, skin, tumor (for [125I]-5 study), thyroid, and tail were removed surgically following asphyxiation with carbon dioxide and exsanguination by cardiac puncture. Tissue samples were weighed wet and then analyzed for 125I activity in a Beckman gamma scintillation counter. The carcass was also weighed and measured for residual radioactivity. RESULTS AND DISCUSSION The in vivo radioactivity biodistribution data following intravenous injection of [125I]-3 in normal mice (Table 1) show a rapid early distribution of radioactivity throughout the body, with the brain containing a lower concentration of radioactivity than the other organs. At longer time periods, between 2 and 4 h, brain radioactivity (measured as percent injected dose per gram) remained low, indicating a lack of permeation or metabolic trapping of [125I]-3 in the normal mouse brain. Early hepatic radioactivity may represent either drug metabolism within the liver or hepatobiliary elimination of [125I]-3. Because 3 is a methyl ether and not a physiologic sugar, its biotransformation (O-demethylation) would be expected (17).

The initial high kidney radioactivity suggests extensive renal elimination of [125I]-3. Whole-body and blood radioactivity clearance data (Fig. 3) show initial rapid declines in radioactivity that appear to be biphasic. More than 60% of whole-body radioactivity and 95% of blood radioactivity was eliminated by 0.25 h postinjection. This was followed by slower whole-body elimination for the next 2 h paralleling blood clearance. The amount of radioactivity remaining in blood and the whole body at 4 h were 0.26 and 5.13 %ID, respectively, compared with 1.0 and 20 %ID at 4 h for [125I]IAZA (14). Although the sugar analog 3 cleared four to five times faster from whole body and blood than the corresponding 2-nitroimidazole nucleoside, the observed low overall uptake into the brain and brain-to-blood ratios for [125I]-3 (⬍0.5) would not permit brain imaging. The more lipophilic sugar [125I]-4 also showed a rapid early distribution of radioactivity throughout the body with brain radioactivity comparable to the rest of the organs, especially muscle, stomach, and spleen (Table 2). At 0.25 h postinjection, the measured brain-to-blood ratio (0.66) was almost three times higher than [125I]-3 (t ⫽ 6.05, p ⫽ 0.009). At 4 h, however, no difference in brain radioactivity could be inferred either in the percent injected dose per gram values (t ⫽ 2.49, p ⫽ 0.067) or brain-toblood ratios (t ⫽ 2.21, p ⫽ 0.070). The low brain-to-blood ratios (⬍1.0) were clearly insufficient to achieve brain tissue imaging with [125I]-4. At 0.25 h postinjection, radioactivity levels in all organs (%ID/

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TABLE 4. Biodistribution of [125I]IAIA (5) Following Intravenous Administration in Balb/c Mice Bearing EMT-6 Tumors Time (hours) Tissue

0.25

Blood Tumor

1.32 ⫾ 0.13 1.08 ⫾ 0.16 0.81 ⴞ 0.05b 3.92 ⫾ 0.42 2.96 ⴞ 0.13 8.81 ⫾ 0.81 6.67 ⴞ 0.49 13.88 ⫾ 1.67 10.48 ⴞ 0.87 1.18 ⫾ 0.17 0.90 ⴞ 0.16 1.01 ⫾ 0.13 0.77 ⴞ 0.14 2.46 ⫾ 0.22 1.86 ⴞ 0.15 1.44 ⫾ 0.16 1.09 ⴞ 0.10 1.83 ⫾ 0.13 1.38 ⴞ 0.04 2.70 ⫾ 0.52 2.04 ⴞ 0.28 0.06 ⫾ 0.01 0.05 ⴞ 0.01 1.89 ⫾ 0.14 1.44 ⴞ 0.22 0.66 ⫾ 0.46d 0.53 ⴞ 0.14e

Kidney Gastrointestinal tractc Liver Muscle Bone Lungs Heart Spleen Stomach Brain Carcass Thyroid

0.5 a

0.82 ⫾ 0.15 0.95 ⫾ 0.15 1.18 ⴞ 0.10 2.48 ⫾ 0.57 3.04 ⴞ 0.31 6.47 ⫾ 1.77 7.90 ⴞ 1.25 7.87 ⫾ 1.04 9.77 ⴞ 0.45 0.95 ⫾ 0.21 1.17 ⴞ 0.12 0.91 ⫾ 0.06 0.91 ⴞ 0.14 1.53 ⫾ 0.29 1.88 ⴞ 0.10 0.82 ⫾ 0.10 1.02 ⴞ 0.06 1.15 ⫾ 0.21 1.42 ⴞ 0.17 2.34 ⫾ 0.75 2.82 ⴞ 0.38 0.05 ⫾ 0.01 0.07 ⴞ 0.02 1.57 ⫾ 0.18 1.96 ⴞ 0.24 0.38 ⫾ 0.04 1.00 ⴞ 0.01

1

2

0.49 ⫾ 0.09 0.18 ⫾ 0.03 0.76 ⫾ 0.18 0.28 ⫾ 0.09 1.55 ⴞ 0.18 1.56 ⴞ 0.28 1.20 ⫾ 0.31 0.29 ⫾ 0.08 2.41 ⴞ 0.22 1.65 ⴞ 0.19 3.29 ⫾ 1.05 0.81 ⫾ 0.15 6.59 ⴞ 1.08 4.63 ⴞ 0.09 4.25 ⫾ 0.66 1.76 ⫾ 0.26 8.77 ⴞ 0.36 10.15 ⴞ 0.65 0.79 ⫾ 0.20 0.38 ⫾ 0.13 1.60 ⴞ 0.15 2.08 ⴞ 0.40 0.51 ⫾ 0.08 0.21 ⫾ 0.05 1.06 ⴞ 0.20 1.17 ⴞ 0.09 0.82 ⫾ 0.14 0.31 ⫾ 0.04 1.69 ⴞ 0.04 1.79 ⴞ 0.10 0.47 ⫾ 0.07 0.16 ⫾ 0.02 0.98 ⴞ 0.04 0.94 ⴞ 0.04 0.65 ⫾ 0.17 0.19 ⫾ 0.04 1.31 ⴞ 0.13 1.09 ⴞ 0.11 1.89 ⫾ 0.37 0.64 ⫾ 0.14 3.86 ⴞ 0.08 3.68 ⴞ 0.51 0.04 ⫾ 0.01 0.01 ⫾ 0.003 0.07 ⴞ 0.01 0.08 ⴞ 0.005 1.33 ⫾ 0.15 0.80 ⫾ 0.16 2.76 ⴞ 0.21 4.58 ⴞ 0.26 0.34 ⫾ 0.08 0.13 ⫾0.02 1.20 ⴞ 0.24 0.91 ⴞ 0.25

4

8

24

0.19 ⫾ 0.03 0.051 ⫾ 0.001 0.011 ⫾ 0.002 0.83 ⫾ 0.28 0.096 ⫾ 0.032 0.015 ⫾ 0.006 4.34 ⴞ 0.80 2.07 ⴞ 1.09 1.39 ⴞ 0.49 0.20 ⫾ 0.03 0.071 ⫾ 0.017 0.013 ⫾ 0.002 1.09 ⴞ 0.01 1.40 ⴞ 0.28 1.22 ⴞ 0.05 0.86 ⫾ 0.11 0.16 ⫾ 0.05 0.037 ⫾ 0.006 4.65 ⴞ 0.25 3.02 ⴞ 0.42 3.54 ⴞ 1.06 1.09 ⫾ 0.11 0.75 ⫾ 0.05 0.32 ⫾ 0.03 5.92 ⴞ 0.42 14.96 ⴞ 1.91 30.04 ⴞ 3.36 0.30 ⫾ 0.03 0.12 ⫾ 0.03 0.048 ⫾ 0.007 1.62 ⴞ 0.12 2.22 ⴞ 0.32 4.43 ⴞ 0.47 0.17 ⫾ 0.03 0.06 ⫾ 0.02 0.033 ⫾ 0.018 0.90 ⴞ 0.03 1.22 ⴞ 0.13 3.56 ⴞ 2.39 0.22 ⫾ 0.03 0.055 ⫾ 0.010 0.017 ⫾ 0.002 1.16 ⴞ 0.02 1.08 ⴞ 0.04 1.56 ⴞ 0.23 0.12 ⫾ 0.02 0.025 ⫾ 0.010 0.007 ⫾ 0.002 0.63 ⴞ 0.02 0.46 ⴞ 0.12 0.66 ⴞ 0.24 0.14 ⫾ 0.04 0.027 ⫾ 0.007 0.009 ⫾ 0.003 0.76 ⴞ 0.11 0.51 ⴞ 0.06 0.79 ⴞ 0.15 0.90 ⫾ 0.27 0.45 ⫾ 0.23 0.036 ⫾ 0.009 4.79 ⴞ 1.12 8.29 ⴞ 2.84 3.32 ⴞ 0.62 0.02 ⫾ 0.002 0.004 ⫾ 0.003 0.004 ⫾ 0.003 0.10 ⴞ 0.01 0.07 ⴞ 0.05 0.32 ⴞ 0.21 0.68 ⫾ 0.17 0.20 ⫾ 0.04 0.029 ⫾ 0.006 3.62 ⴞ 0.34 3.83 ⴞ 0.37 2.63 ⴞ 0.21 0.35 ⫾ 0.09 0.17 ⫾ 0.18 0.80 ⫾ 0.12 2.6 ⴞ 0.3 4.4 ⴞ 4.9 45.0 ⴞ 8.7

The numbers represent the mean ⫾ SD for percent of injected dose per gram of wet tissue for three animals. Tissue-to-blood ratio. c Section of intestine. d Percentage of injected dose in thyroid. e Thyroid radioactivity as a percentage of whole-body radioactivity. IAIA ⫽ 1-(5-deoxy-5-iodo-␣-D-arabinofuranosyl)-2-aminoimidazole. a b

gram values) were generally higher after doses of [125I]-4 than of [125I]-3. This is likely a reflection of the higher lipophilicity of [125I]-4, which would be expected to facilitate its diffusion across cell membranes, and implies that the compound is not rapidly metabolized by O-demethylation or O-deacetylation reactions. For both sugars, less than 0.4% of the injected dose remained in the blood after 4 h. Early high hepatic and renal tissue-to-blood ratios for both sugars indicate both hepatobilliary and renal clearance. The increase in both thyroid and stomach-to-blood ratios for both sugars reflects in vivo deiodination and subsequent accumulation of free radioiodide (6). Increasing the lipophilicity through the acetylation of the hydroxyl groups on [125I]-3 did not appear to significantly delay renal clearance or significantly increase brain radioactivity. The high level of esterase activity in blood (8) could be responsible for rapid conversion of 4 to 3, thereby accounting for the biodistribution similarities observed in all but the earliest times after injection. The biodistribution data for [125I]-3 and [125I]-4 suggest that the first proposed metabolic pathway, the conversion of [123I]IAZA to radioactive sugars, does not appear to be responsible for the observed brain uptake of radioactivity in [123I]IAZA patients. This conclusion assumes that the metabolism, biodistribution, and pharmacokinetics of these compounds are similar in the mouse model and in humans. There remains the possibility that these compounds are processed in a very different way in humans.

Tables 3 and 4 list the in vivo biodistribution data for [125I]IAIA (5) in normal and tumor-bearing Balb/c mice, respectively. The data indicate a fast early distribution of [125I]-5 throughout the body with the brain containing a lower level of radioactivity relative to the other organs measured. A statistically significant difference in brain radioactivity was observed between tumor-bearing and normal mice at 0.25 h (dose per gram: t ⫽ 4.75, p ⫽ 0.0177; brain-to-blood ratio: t ⫽ 10.4, p ⫽ 0.00189). Other than this early time period, there is no strong evidence to suggest any differences in brain radioactivity between the normal and the tumor-bearing mice (e.g., at 24 h, dose per gram: t ⫽ ⫺0.632, p ⫽ 0.562; brain-to-blood ratio: t ⫽ ⫺0.812, p ⫽ 0.462). The low brain-to-blood ratios (essentially ⬍0.10 in all time periods) appear to preclude any metabolic trapping of [125I]-5 in the brain in either normal or tumor-bearing mice. Specific nucleoside and/or amine transporters or a pHdependent trapping mechanism in neuronal tissues did not appear to be involved in the biodistribution of 5 in this mouse model. Tumor tissue showed a maximum tumor-to-blood ratio for radioactivity of 4.34 at 4 h, representing 0.83 %ID/g tissue. This was higher than in other tissues with the exception of the gastrointestinal tract, liver, and stomach, suggesting that some mechanism of metabolic trapping may be present. This higher tumor radioactivity could be due to slower clearance of radioactivity from tumor tissue relative to blood because this retention of radioactivity was relatively short-lived. At 24 h, blood and tumor radioactivity were

Metabolites of Iodoazomycin Arabinoside

67

within 24 h. The whole-body elimination and blood clearance profiles for [125I]-5 were similar to those reported for [125I]IAZA (14). The relatively high concentration of radioactivity in tumors, between 4 and 8 h, suggests an uptake of [125I]-5 in hypoxic tumor tissues. The nature of this uptake mechanism is not known. A pH-dependent trapping mechanism could occur in regions of low pH in tumors (2). Additionally, the amine could be involved in DNA binding, because heterocyclic and aromatic amines are known to bind to cellular DNA and form DNA adducts by N-hydroxylation (3, 8) or N-acetylation (24). The high liver radioactivity values (liver-to-blood ratios ⬎30 at 24 h) are suggestive of some metabolic trapping in this organ, and further studies are required to determine the nature of this tissue retention and the potential of compound 5 in the evaluation of liver structure and function. As is the case for compounds 3 and 4, biodistribution data for 5 do not support its involvement in brain radioactivity uptake in patients. As noted previously, we cannot rule out the possibility that the compounds are processed differently in the mouse models than in humans. Another important consideration is that all the human cancer patients imaged with 123I-IAZA were receiving or had received therapy for their disease. It is not possible to rule out the possibility that these treatments contributed to the observed brain activity although we were unable to correlate this observation with any specific drug or therapy. This analysis was complicated by the diversity of treatments and the small patient population.

FIG. 4. Whole-body elimination (—⽧—) and blood clearance (—〫—) of radioactivity following intravenous administration of [125I]-5 in normal Balb/c mice (N ⴝ 3) and whole-body elimination (ⴚⴚⴚⴚ■ⴚⴚⴚⴚ) and blood clearance (ⴚⴚⴚⴚ䊐ⴚⴚⴚⴚ) of radioactivity following intravenous administration of [125I]-5 in EMT-6 tumor bearing Balb/c mice (N ⴝ 3). Error bars are ⴞ1 SD.

We thank Ms. Haiyan Xia, Altarex Inc., Edmonton, Alberta, Canada, for supplying EMT-6 cells; Dr. Koh-Ichi Seki and Dr. Kazue Ohkura, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan, for their independent synthesis and 13C NMR analysis of AIA; and Dr. Angelina Morales-Izquierdo, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, for her kind assistance in ESI mass spectrometry. This work was supported in part by Research Initiative Program Grant (RI-14) from the Alberta Cancer Board, Edmonton, Alberta, Canada.

References comparable (0.011 and 0.015 %ID/g, respectively) with a tumorto-blood ratio of approximately 1.40. At the time of maximal tumor to blood ratio (4 h), the liver, gastrointestinal tract, and stomach all contained comparable or somewhat greater concentrations of radioactivity (1.1, 0.9, and 0.86 %ID/g, respectively). Thyroid radioactivity for both groups of mice showed a very similar (increasing) trend. Approximately 45% of total body radioactivity (0.80 %ID) was present in the thyroid at 24 h. The extent of deiodination, as indicated by the thyroid uptake of activity, is similar to that reported for [125I]IAZA (14). Whole-body clearance data for 5 indicate that at 0.25 h, more than 65% and 45% of radioactivity were cleared from whole body in normal and tumor-bearing mice, respectively (Fig. 4). This difference in clearance was statistically significant (t ⫽ ⫺4.89, p ⫽ 0.039). Greater than 97% of injected radioactivity was eliminated within 24 h in both groups. The blood clearance data (Tables 3 and 4) also show statistically significant differences between the two sets of mice at 0.25 h (t ⫽ ⫺8.42, p ⫽ 0.0035). More than 98% of blood radioactivity was eliminated within the first 0.25 h. At other time intervals, no significant blood level differences were observed (e.g., at 8 h, t ⫽ 1.40, p ⫽ 0.296) between the two groups of mice. Greater than 99.98% of the injected radioactivity was cleared from blood

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