3-Labeled Pamoic acid Derivatives - American Chemical Society

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Bioconjugate Chem. 2007, 18, 1924–1934

Necrosis Avidity of 99mTc(CO)3-Labeled Pamoic acid Derivatives: Synthesis and Preliminary Biological Evaluation in Animal Models of Necrosis Humphrey Fonge,† Satish K. Chitneni,† Jin Lixin,† Kathleen Vunckx,‡ Kristof Prinsen,† Johan Nuyts,‡ Luc Mortelmans,† Guy Bormans,† Yicheng Ni,§ and Alfons Verbruggen*,† Laboratory of Radiopharmacy, Faculty of Pharmaceutical Sciences, K.U. Leuven, Herestraat 49, Box 821, BE-3000 Leuven, Belgium, Departments of Nuclear Medicine and Radiology, University Hospital Gasthuisberg, Herestraat 49, BE-3000 Leuven, Belgium. Received June 25, 2007; Revised Manuscript Received August 25, 2007

In a search for an infarct avid tracer agent with improved properties, we have observed that bis-DTPA derivatives of pamoic acid have a high avidity for necrotic tissue. Here, we report the synthesis, radiolabeling, and preliminary evaluation in normal mice and rats with hepatic infarction of the 99mTc-tricarbonyl complexes of N,N′-bis(diethylenetriaminopentaacetato)-4,4′-methylene bis(2-hydroxy-3-naphthoic hydrazide) (99mTc(CO)3-bis-DTPA-pamoate) and [N-(5aminopentyl)pyridin-2-yl-methylamino]methylacetato-4,4′-methylene-2-hydroxy-3-napthalenecarboxamide(2′-hydroxy-3′-naphthoic acid methyl ester) (99mTc(CO)3-12). Radiolabeling with 99mTc(CO)3+ was achieved with a radiochemical yield of over 95% for both tracer agents. In normal mice, the polar 99mTc(CO)3-bis-DTPA-pamoate was cleared from plasma via both the liver and the kidneys, while the more lipophilic 99mTc(CO)3-12 was rapidly cleared via the liver. Blood clearance in mice was faster for 99mTc(CO)3-12 (0.1% injected dose per gram at 4 h postinjection) than for 99mTc(CO)3-bis-DTPA-pamoate (9.3% injected dose per gram at 4 h postinjection). Affinity and specificity of the tracers for necrotic tissue was studied in rats with hepatic infarction and ethanol-induced necrosis of the liver or muscles. Activity ratios of infarct to viable liver tissue of 99mTc(CO)3-bis-DTPA-pamoate quantified by autoradiography of tissue slices ranged from 4 to 18, depending on the necrosis model and time postinjection of the tracer. Infarcts were also visualized in ViVo by 99mTc(CO)3-bis-DTPA-pamoate planar gamma imaging. After injection of 99mTc(CO)3-bisDTPA-pamoate, in ViVo and ex ViVo images correlated well with histochemical staining with triphenyltetrazolium chloride and hematoxylin and eosin. 99mTc(CO)3-12 on the other hand showed no uptake in necrotic tissue. Stability of the tracers was determined in Vitro after storage at room temperature and by histidine challenge experiments, and in ViVo in mouse plasma and in urine (for 99mTc(CO)3-bis-DTPA-pamoate). 99mTc(CO)3-bis-DTPA-pamoate was unstable in Vitro to histidine challenge, while 99mTc(CO)3-12 was 98% stable in Vitro in the same conditions. Both tracers showed good in ViVo stability. 99mTc(CO)3-bis-DTPA-pamoate shows high specificity for necrotic tissue and merits further evaluation as a necrosis avid imaging agent. 99mTc(CO)3-12 is not useful for visualization of necrotic tissue.

INTRODUCTION Cell death occurs by two distinct mechanisms: a disorganized nonphysiologic mechanism called necrosis and a well-orchestrated physiologic mechanism termed apoptosis (1–3). Both forms of cell death have been implicated in many diseases, notably, acute myocardial infarction (AMI), even though it is not very certain which form of death is predominant in the case of AMI (4, 5). Therapies aimed at restoring reperfusion to the infarcted organ most often aggravate the injury by shifting the cell towards death by apoptosis (4, 6, 7). Imaging of apoptosis has become a reality with the discovery that annexin-V binds specifically and with nanomolar affinity (Kd ) 7 nM) to externalized phosphatidylserine (PS) (8–11). Targeting of necrosis in AMI has been challenging, given the fact that a multitude of compounds are suddenly released by the dying cells following disintegration of the sarcolemma. A few necrosis avid tracer agents have been in use in nuclear medicine with none having ideal imaging characteristics (12). * Author for correspondence: Prof. Alfons Verbruggen, Laboratory of Radiopharmacy, Faculty of Pharmaceutical Sciences, K.U. Leuven, Herestraat 49, Onderwijs & Navorsing 2, Box 821, B-3000 Leuven, Belgium. Tel: +32-16-33.04.46. Fax: +32-16-33.04.49. E-mail: [email protected]. † Laboratory of Radiopharmacy. ‡ Department of Nuclear Medicine, University Hospital Gasthuisberg. § Department of Radiology, University Hospital Gasthuisberg.

Among these tracers, 99mTc-glucarate looks the most promising but presents two limitations to its potential clinical applications for imaging AMI (13): (1) it is rapidly washed out from the infarcted myocardium and (2) its localization is limited to the early hours of the acute event, thus limiting its potential usefulness to the acute phase of AMI. In preliminary experiments, we have found that bis-gadolinium N,N′-bis(diethylenetriaminopentaacetato)-4,4′-methylene bis(2-hydroxy-3-naphthoic hydrazide (bisGd bis-DTPA-pamoate) specifically localizes in necrotic tissue in different animal models of necrosis (14, 15). Radiolabeled derivatives of this class of compounds could find more clinical applications in nuclear medicine, e.g., when radiolabeled with technetium-99m, which has almost ideal physical properties for planar and single photon emission computed tomography (SPECT). Classical labeling of diethylene triamine pentaacetate (DTPA) derivatized biomolecules with technetium-99m (99mTc), i.e., addition of 99m Tc-pertechnetate in the presence of stannous ions, requires a rather large amount of DTPA (5–10 mg) and often results in compounds with rather low stability due to reoxidation of technetium to pertechnetate. Technetium-99m tricarbonyl labeling of biomolecules has the advantage of ease of preparation, efficient complex formation, and inertness of the complex due to the d6 metal core (16, 17). Therefore, we have studied the synthesis and labeling of bis-DTPA pamoate with a 99mTc(CO)3+ core (Figure 1A) and report here a preliminary evaluation of 99mTc(CO)3-bis-DTPA-pamoate in rats with rep-

10.1021/bc700236j CCC: $37.00  2007 American Chemical Society Published on Web 10/12/2007

Necrosis Avidity of

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Tc(CO)3-Labeled Pamoic Acid Derivatives

Figure 1. Proposed structures of 99mTc(CO)3-bis-DTPA-pamoate (A) and 99mTc(CO)3-[N-(5-aminopentyl)pyridin-2-yl-methylamino]methylacetato-4,4′-methylene-2-hydroxy-3-napthalenecarboxamide-(2′-hydroxy3′-naphthoic acid methyl ester) (99mTc(CO)3-12) (B).

erfused hepatic infarction and ethanol-induced hepatic or muscular necrosis. Necrosis specificity was assessed in ViVo by planar gamma scintigraphy and ex ViVo by gamma counting and autoradiography and was compared to TTC and H&E staining techniques at different time points postinjection (p.i.). However, DTPA is not an ideal bifunctional chelating agent for labeling a bioactive compound with a 99mTc-tricarbonyl core. For this reason, we have derivatized pamoic acid also via a hydrazine spacer with [N-(5-aminopentyl)pyridyl-2-yl-amino] methyl acetate (18), which is considered an ideal bifunctional chelator for labeling biomolecules with 99mTc(CO)3+ because of the relatively fast complexation and very high thermodynamic and kinetic stability of a Tc(CO)3 complex with this tridentate system (17, 18). The stability and biological characteristics of the resulting 99mTc(CO)3+ complex named 99mTc(CO)3-12 (Figure 1B) have been compared with those of 99mTc(CO)3bis-DTPA pamoate.

EXPERIMENTAL PROCEDURE Reagents. 2-Hydroxy-3-naphthoic acid, N-ethyldiisopropylamine, N,N′-dicyclohexylcarbodiimde, N-hydroxysuccinimide, N-Boc-1,5-diaminopentane, pyridine, and hydrazine monohydrate were obtained from Sigma Aldrich (Steinheim, Germany); paraformaldehyde and all solvents were obtained from Acros Organics (Geel, Belgium) and were used as purchased. Generator eluate containing Na99mTcO4 was obtained from an Ultratechnekow generator (Tyco Healthcare, Petten, The Netherlands). The Isolink kit used for preparing 99mTc(CO)3+ was a generous gift from Tyco Healthcare. Instruments and Methods. 1H NMR spectra were acquired with a Gemini 300 MHz spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are reported in parts per million (ppm) relative to TMS (δ ) 0). Coupling constants are reported in hertz (Hz). Normal-phase column chromatography was performed using silica gel (silica 63–200, 60 Å, MP Biomedicals, Eschwege, Germany). Thin layer chromatography (TLC) was performed on normal-phase 0.2-mm-thick silica gel 60 plates with UV fluorescence (Macherey-Nagel, Düren, Germany), and spots were visualized with UV light (Spectroline, Westbury, NY). TLC plates were developed using one of the following

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mobile-phase systems: system A, EtOAc/MeOH 70/30 v/v; system B, CH2Cl2/hexane 50/50 v/v; system C, CHCl3/hexane 5/95 v/v. High-pressure liquid chromatography (HPLC) analysis was performed on a LaChrom Elite HPLC system (Hitachi, Darmstadt, Germany) using an XTerra RPC18 column (5 µm, 4.6 mm × 250 mm; Waters, Milford, USA). The compounds were eluted from the column using mixtures of 0.05 M ammonium acetate buffer pH 6.8 (solvent A) and acetonitrile (solvent B): 0–8 min, linear gradient from 100% A to 60% A; 8–15 min, 60% A; 15–25 min, linear gradient to 100% B, at a flow rate of 1 mL/min over 30 min. The column effluent was monitored using a UV detector set at 254 nm, and the output signal was acquired on a Laura Lite system (Lablogic, Sheffield, UK). For analysis of radiolabeled compounds, the HPLC eluate was led over a 3 in NaI(Tl) scintillation detector connected to a single-channel analyzer. Radioactivity counting for biodistribution studies was done using an automated system with a gamma counter (3 in NaI(Tl) well crystal) coupled to a multichannel analyzer in a sample changer (Wallac, 1480 Wizard 3”, Turku, Finland). The results were corrected for background radiation and physical decay during counting. Accurate mass measurement was performed by coinfusion with a 10 µg/mL solution of a reference as an internal calibration standard on a time-of-flight mass spectrometer (LCT, Micromass, Manchester, UK) equipped with an electrospray ionization (ESI) interface, operated in positive (ES+) or negative (ES-) mode. Samples were infused in acetonitrile/water using a Harvard 22 syringe pump (Harvard instruments, Massachusetts). Acquisition and processing of data was done using Masslynx software (Micromass, version 3.5). All animal experiments were conducted with the approval of the institutional ethical committee for conduct of experiments on animals.

CHEMISTRY 2-Hydroxy-3-methylnaphthoate (2). To a solution of 2-hydroxy-3-naphthoic acid (19.2 g, 0.094 mol) in acetone (250 mL) was added K2CO3 (13 g, 0.1 mol), water (25 mL), and then with stirring over a period of 3.5 h a solution of dimethyl sulfate (11.1 mL, 0.12 mol). The solution was further stirred at room temperature (RT) until all starting material was esterified as judged by TLC (system C, ester (2) Rf ) 0.9). Undissolved K2CO3 was filtered off and washed with acetone. The combined filtrates were evaporated in Vacuo and the residue was dissolved in CH2Cl2 and dried over sodium sulfate. The filtrate was concentrated, methanol was added to the warm solution, and the precipitate was filtered off. The resulting yellow powder was purified using column chromatography on silica gel eluted with CH2Cl2. Yield 17.8 g (87%). Accurate mass [C12H9O3H]-: theoretical 201.0557 Da, found 201.0559 Da. 1H NMR (CDCl3, 300 Hz) δ 8.46 (s, 1H, 11-CH), 7.78 (d, 1H, 3J ) 8.3 Hz, 6-CH), 7.67 (d, 1H, 3J ) 8.4 Hz, 9-CH), 7.48 (t, 1H, 3J ) 7.4 Hz, 7-CH), 7.29 (t, 1H, 3J ) 9.1 Hz, 8-CH), 4.0 (s, 3H, CH3). 4,4′-Methylene-bis(2-hydroxy-3-methylnaphthoate) (Pamoic Acid Bis-Methyl Ester) (3). To a solution of compound 2 (10.1 g, 46 mmol) in a mixture of acetic acid (80 mL) and sulfuric acid (0.5 mL), paraformaldehyde (2 g, 66.6 mmol) was added, and the mixture was stirred overnight at RT. The yellow precipitate was collected and filtered off, washed with acetic acid and water, and dried. TLC: Rf ) 0.95 (system C). Yield 9.46 g (97%). Accurate mass [C25H19O6-H]-: theoretical 415.1187 Da, found 415.1208 Da. 1H NMR (CDCl3, 300 Hz) δ 8.38.(s, 2H, 11,11′CH), 8.23 (d, 2H, 3J ) 8.7 Hz, 9,9′-CH), 7.67 (d, 2H, 3J ) 8.1 Hz, 6,6′-CH), 7.35 (t, 2H, 3J ) 7.5 Hz, 7,7′-CH), 7.18 (t, 2H, 3 J ) 7.1 Hz, 8,8′-CH), 4.95 (s, 2H, 4,4′-CH2), 4.0 (s, 6H, 2 × CH3).

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Fonge et al.

Scheme 1a

a (i) (CH3)2SO4, K2CO3, RT overnight; (ii) paraformaldehyde, CH3COOH, H2SO4, RT; (iii) NH2NH2, reflux (80 °C), 2 h; (iv) DTPA monoanhydride monoethyl ester, DIEA, followed by hydrolysis (NaOH); (v) 99mTc(CO)3+, pH 11, 70 °C, 20 min.

4,4′-Methylene-bis(2-hydroxy-3-naphthoic hydrazide) (4). A mixture of compound 3 (5.25 g, 12.6 mmol), pyridine (100 mL), methanol (50 mL), and hydrazine monohydrate (16 mL, 1.24 mol) was refluxed for 2 h. Solvents and excess hydrazine were removed azeotropically under reduced pressure with water. The suspension of the residue in methanol was stirred overnight. The hydrazide was collected by filtration and washed with methanol followed by drying over P2O5. Yield 5.05 g (97%). TLC: Rf ) 0 (system B), Rf ) 0.6 (system A). Accurate mass [C23H19N4O4-H]-: theoretical 415.1412 Da, found 415.1451 Da. 1H NMR (DMSO-d6, 300 Hz) δ 10.62 (s, 2H, 2 × CONH), 8.38 (s, 2H, 11,11′-CH), 8.14 (d, 2H, 3J ) 8.6 Hz, 9,9′-CH), 7.68 (d, 2H, 3J ) 8.0 Hz, 6,6′-CH), 7.31 (t, 2H, 3J ) 7.4 Hz, 7,7′-CH), 7.19 (t, 2H, 3J ) 7.1 Hz, 8,8′CH), 4.99 (s, 4H, NH2), 4.78 (s, 2H, 4,4′-CH2). N,N′-Bis(diethylenetriamino pentaacetato)-4,4′-methylene Bis(2-hydroxy-3-naphthoic hydrazide) Sodium Salt (BisDTPA-Pamoate) (5). To a stirred suspension of diethylenetriamine pentaacetic acid monoethyl ester monoanhydride (15) (3.4 g, 8.45 mmol) in dry DMF (50 mL), N,N-diisopropylethylamine (3.7 mL, 21 mmol) and compound 4 (1.5 g, 3.6 mmol) were added and the mixture was stirred at RT for 5 h. Solvents were evaporated under reduced pressure, the oily residue was dissolved in water, and the pH was adjusted to >12 using concentrated NaOH solution. After hydrolysis of the ethyl esters (as judged by HPLC), the volume was reduced at reduced pressure at 60 °C and the pH adjusted to 7.4 with 1 M HCl. The reaction product was purified by reversed-phase adsorption chromatography on silica C-18 stationary phase using a water– acetonitrile gradient. Fractions containing compound 5 were pooled, and the solvents were removed in vacuo. Methanol was added to induce precipitation of compound 5, and the precipitate was collected by filtration and dried over P2O5. Accurate mass [C51H60N10O22-2H]2-/2: theoretical 582.1947 Da, found 582.1916 Da. 1H NMR (DMSO-d6) δ 8.3 (d, 2H, Ar-H), 8.0 (d, 2H, ArH), 7.5 (m, 6H, Ar-H), 4.6 (s, 2H, methylene-H), 4.0 (t, 4H, 2 × CH2), 2.9–3.4 (m, 36H, methylene of DTPA). 4,4′-Methylene-bis(2-hydroxy-3-naphthalene carboxylic acid) Monomethyl Ester (Pamoic Acid Monomethyl Ester) (6). To a solution of compound 1 (2-hydroxy-3-naphthoic acid, 2 g, 9.8 mmol) and 2 (2-hydroxy-3-methylnaphthoate, 2 g, 9.2

mmol) in a mixture of acetic acid (30 mL) and sulfuric acid (0.2 mL), paraformaldehyde (0.42 g, 14 mmol) was added and stirring was continued for 9 h at RT. The yellow precipitate was collected by filtration, washed successively with acetic acid and water, and dried. The precipitate was then dissolved in CHCl3/hexane (1/3 v/v) and purified by column chromatography on silica gel eluted with a gradient from 100% hexane to 100% CHCl3. The first fraction consisted of pamoic acid bis-methyl ester. Pamoic acid monomethyl ester (purity >80%) was eluted together with pamoic acid using CHCl3/MeOH 95/5 v/v. Pure pamoic acid monomethyl ester was obtained by a second purification on silica gel eluted with CHCl3/MeOH 95/5 v/v. The fractions containing pure pamoic acid monomethyl ester (>95%) were evaporated to give 1.55 g (41%) of a yellow powder. TLC: Rf ) 0.6 (system A). Accurate mass [C24H17O6H]-: theoretical 401.1031 Da, found 401.1058 Da. 1H NMR (DMSO-d6, 300 Hz), δ 11.23 (s, 1H, -COOH), 8.49 (s, 2H, 11,11′-CH), 8.12 (t, 2H, 3J ) 9.5 Hz, 7,7′-CH), 7.9 (d, 2H, 3J ) 7.9 Hz, 6,6′-CH), 7.43–7.36 (m, 2H, 9.9′-CH), 7.28–7.21 (m, 2H, 8,8′-CH), 4.9 (s, 2H, 4,4′-CH2), 4.0 (s, 3H, CH3). Succinimidyl Pamoic Acid Monomethyl Ester (7). To a solution of compound 6 (0.5 g, 1.24 mmol) and N-hydroxysuccinimide (0.143 g, 1.25 mmol) in tetrahydrofuran (THF) (50 mL), dicyclohexylcarbodiimide (DCC, 0.25 g, 1.25 mmol, in 10 mL THF) was added dropwise at 0 °C. The reaction mixture was further stirred at RT overnight, after which it was filtered and washed with THF. The filtrate was evaporated to yield a yellow powder. Yield 85%. TLC: Rf ) 0.85 (system A). Accurate mass [C28H20NO8-H]-: theoretical 498.1194 Da, found 498.1254 Da. The bifunctional chelator (BFC) [N-(5-aminopentyl)pyridin2-yl-methylamino]methyl acetate (10) was synthesized in three steps (8, 9, 10; Scheme 2A) as previously reported (18). [N-(5-Aminopentyl)pyridin-2-yl-methylamino]methylacetato-4, 4′-methylene-2-hydroxy-3-napthalenecarboxamide-(2′-hydroxy3′-naphthoic acid methyl ester) (11). N,N′ Diisopropylethylamine (171 µL, 1 mmol) was added to a solution of compound 7 (0.32 g, 0.64 mmol) in a mixture dry THF-DMF (1/1 v/v, 20 mL), followed by the dropwise addition of a solution of compound 10 (0.2 g, 0.8 mmol) in dry THF-DMF (1/1 v/v)

Necrosis Avidity of

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Scheme 2a

a A. (i) (1) 0 °C; (2) NaBH4, RT; (ii) bromomethyl acetate, TEA, RT; (iii) TFA. B. (i) Paraformaldehyde, CH3COOH, H2SO4, RT; (ii) N-hydroxysuccinimide, DCC, RT; (iii) bifunctional chelator (10), DIEA, RT; (iv) 1 M NaOH; (v) 99mTc(CO)3+, pH 7.4, 70 °C, 15 min.

under stirring at RT, and stirring was continued for 15 h. The solvents were evaporated and the crude product was purified by column chromatography on silica gel. Lipophilic impurities were washed off with ethyl acetate, and pure compound 11 was eluted with ethyl acetate–methanol (85/15 v/v). The solvents were evaporated to obtain the title compound as a viscous yellow oil (0.25 g, 60%). Its purity was verified by RP-HPLC on an XTerra C18 column (5 µm, 4.6 mm × 250 mm) using isocratic elution with acetonitrile–0.05 M NH4OAc buffer (pH 6.8) (60/ 40 v/v) at a flow rate of 1 mL/min. The purity was >98%. TLC: Rf ) 0.8 (system A). Accurate mass [C38H38N3O7-H]-: theoretical 648.2715 Da, found 648.2661 Da. 1H NMR (CDCl3) δ 8.45 (1H, d, 3J ) 4.8, 24-CH), 8.40 (s, 1H, 11′-CH), 8.3 (d, 1H, 3J ) 8.7, 9′-CH), 8.2 (d, 1H, 3J ) 8.7, 9-CH), 8.0 (s, 1H, 11CH), 7.7 (d, 1H, 3J ) 8.1, 6′-CH), 7.5 (d, 1H, 3J ) 8.2, 6-CH), 7.4 (d, 2H, 21,23-CH), 7.38–7.28 (m, 2H, 7,7′-CH), 7.2–7.1 (m, 2H, 8,8′-CH), 4.9 (s, 2H, 4,4′-CH2), 4.0 (s, 3H, -CH3), 3.9 (s, 2H, 19-CH2), 3.7 (s, 3H, -CH3), 3.5 (m, 2H, 12-CH), 3.4 (s, 2H, 18-CH2), 2.7 (t, 2H, 16-CH2), 1.3 (m, 6H, 13,14,15-CH2).

[N-(5-Aminopentyl)pyridin-2-yl-methylamino]acetato-4,4′methylene-2-hydroxy-3-napthalenecarboxamide-(2′-hydroxy3′-naphthoic acid) (12). To a solution of compound 11 (0.1 g, 0.15 mmol) in acetonitrile (10 mL) 1 M NaOH (10 mL) was added. The mixture was stirred at RT for 1 h after which the solution was neutralized with 1 M HCl. The solvents were evaporated, and the crude product was purified using column chromatography on silica gel eluted with ethyl acetate–methanol (70/30 v/v). TLC: Rf ) 0.1 (system A). Accurate mass [C36H34N3O7-H]-: theoretical 620.2402 Da, found 620.2393 Da.

RADIOCHEMISTRY 99m Tc(CO)3(H2O)3+ Precursor. The technetium-99m tricarbonyl precursor was prepared using an Isolink kit (19) by adding 1.1–2.5 GBq of Na99mTcO4 in 1 mL saline to the kit followed by heating at 100 °C for 20 min. After cooling, the pH was adjusted as desired with 1 M HCl. Alternatively, homemade 99m Tc(CO)3(H2O)3+ was prepared by bubbling CO gas through a 10 mL sealed vial containing Na2CO3 (4.5 mg), Na/K tartrate


(20 mg), and NaBH4 (25 mg) and adding 1.1–2.5 GBq of Na99mTcO4 in 1 mL saline followed by heating at 100 °C for 20 min. The vial was cooled to RT followed by neutralization with 1 M HCl. The precursor solution was then adjusted to the desired pH with 1 M NaOH before use. The yield of the precursor was 99% using the Isolink kit and 90% using the homemade method. 99m Tc(CO)3-bis-DTPA-pamoate. To a 10 mL labeling vial was added 1 mg of bis-DTPA pamoate (5) dissolved in 0.2 mL of 0.5 M phosphate buffer pH 11. The vial was purged with nitrogen, and 0.2–1.1 GBq of 99mTc(CO)3(H2O)3+ precursor (0.2–0.5 mL, pH 11) was added. The final pH of the labeling mixture was 11. The mixture was heated at 70 °C for 20 min followed by cooling to RT. The crude labeling reaction mixture was analyzed and purified by RP-HPLC as described above. As the radiolabeled compound was not separated from the ligand bis-DTPA pamoate (5) by the HPLC purification, the specific activity of 99mTc(CO)3-bis-DTPA-pamoate was rather low (1 GBq/µmol) if the nonradioactive precursor was taken into account. 99m Tc(CO)3-12. To a 10 mL labeling vial containing 0.5 mg of compound 11 dissolved in 0.5 M phosphate buffer pH 7.4 under nitrogen was added 0.2–1.1 GBq 99mTc(CO)3(H2O)3+ (0.2–0.5 mL, pH 7). The mixture was heated at 70 °C for 15 min followed by cooling and HPLC purification. In this case, the radiolabeled compound was well-separated from the ligand 12 by the HPLC purification; so the specific activity of 99m Tc(CO)3-12 was identical to that of the 99mTc-pertechnetate eluate, i.e., about 19 350 GBq/µmol. Octanol/Buffer Partition Coefficient Determination. To 50 µL (74 kBq) of a solution of HPLC-purified radiolabeled compound in a test vial was added 2 mL of 1-octanol and 2 mL of 0.025 M phosphate buffer pH 7.4. The vial was vortexed at room temperature for 2 min and then centrifuged (Centrifuge 4226, Analis, Gent, Belgium) at 1700 g for 10 min. Approximately 50 µL of the 1-octanol phase and 500 µL of the phosphate buffer phase were pipetted and weighed into separate tared test tubes with adequate care to avoid cross contamination between the phases. The volume of fluid pipetted was calculated by dividing the net weight of the fluid by its density. The radioactivity of the test tubes was counted using a 3 in NaI(Tl) scintillation detector mounted in a sample changer. Corrections were made for background radiation and physical decay during counting. The octanol/buffer partition coefficient P was calculated as P ) cpm/mL in octanol / cpm/mL in buffer where cpm ) counts per min. In Vitro and in ViWo Stability of Tracers. In Vitro stability of 99mTc(CO)3-bis-DTPA-pamoate and 99mTc(CO)3-12 at RT was studied by HPLC analysis of the HPLC-purified tracers over a 24 h period and by histidine challenge experiments. 200 µL aliquots (90 MBq) of solutions of the HPLC-purified 99mTc complexes were incubated with 0.5 mL of a 0.02 M solution of histidine at 37 °C, and the mixtures were analyzed over a 24 h period by RP-HPLC. In ViVo stability of 99mTc(CO)3-bis-DTPA-pamoate was studied in normal mice by determination of the relative amounts of the parent tracer and metabolites in mouse plasma and urine, while in ViVo stability of 99mTc(CO)3-12 was only evaluated in mouse plasma since renal excretion was minimal. After tail vein injection of 2.6 MBq of the tracer into mice, the animals were sacrificed by decapitation at 30 min, 4 h, or 24 h postinjection (one mouse per time point). For urine analysis, urine samples were directly injected onto an Oasis HLB column (hydrophilic– lipophilic balanced; 4.6 mm × 20 mm, Waters) in series with a RP-HPLC column (XTerra RPC18, 5 µm, 4.6 mm × 250 mm; Waters, Milford, USA). Blood was collected into a BD vacutainer (containing 7.2 mg K2EDTA; Beckton Dickinson,

Fonge et al.

Franklin Lakes, USA) and subsequently transferred to a 1.5 mL Eppendorf tube. The blood samples were then centrifuged at 3000 rpm (1837 g) for 5 min to separate plasma. The supernatant plasma sample was injected onto an Oasis column that was preconditioned by successive washings with acetonitrile and water. The proteins of the biological matrix were washed from the Oasis column with 6 mL of water, which was collected in 6 1-mL fractions. The outlet of the Oasis column was then connected to an analytical RP-HPLC column, and both columns in series were then eluted using slightly modified HPLC conditions as compared to the analysis of the intact tracers [0.05 M ammonium acetate buffer (pH 6.8) (solvent A) and acetonitrile (solvent B): 0–8 min, linear gradient from 100% A to 50% A; 8–15 min, 50% A; 15–25 min, linear gradient to 100% B, at a flow rate of 1 mL/min over 30 min]. The HPLC eluate was collected in 35 1-mL fractions, and their radioactivity was measured using a gamma counter. Biodistribution in Normal Mice. Mice were anesthetized with isoflurane (2%) in oxygen at a flow rate of 1 L/min and then injected via a tail vein with 74 kBq of either 99mTc(CO)3bis-DTPA-pamoate in 0.1 mL of saline or 99mTc(CO)3-12 in 0.1 mL of water/poly(ethylene glycol) 400 (PEG 400) 80/20 v/v. They were sacrificed by decapitation under anesthesia at 30 min, 4 h, or 24 h p.i. (n ) 4 mice per time point). The organs were dissected and weighed in tared tubes, and radioactivity in all organs was counted in a gamma counter. Corrections were made for background radiation and physical decay during counting. Activity in the organs was expressed as % injected dose (I.D.)/organ and % I.D./g of organ. Activity in blood was calculated on the assumption that blood constitutes 7% of total body weight. Rat Model of Reperfused Hepatic Infarction. Six adult Wistar rats weighing 350–450 g were anesthetized with i.p. injection of pentobarbital at a dose of 40 mg/kg. Under laparotomy, reperfused hepatic infarction was induced by clamping of the hilum of the right liver lobe for 3 h. After reperfusion by declamping hepatic inflow, the abdominal cavity was closed with two layered sutures, and the animals were allowed to recover for at least 8 h (20). A solution containing 18.5–74 MBq of 99mTc(CO)3-bis-DTPA-pamoate in 0.5 mL saline was injected via a tail vein, and the rats were sacrificed under anesthesia at 4 h (n ) 3) or 24 h (n ) 3) postinjection, followed by ex ViVo studies. Rat model of ethanol induced liver or muscular necrosis. Eight adult Wistar rats (body weight 350–450 g) were anesthetized as described above. Under laparotomy necrosis of the liver was induced by slow infusion of 0.2 mL ethanol into the left liver lobe (21). The abdominal cavity was closed and the animals were allowed to recover for 8–12 h. A solution containing 18.5–74 MBq of 99mTc(CO)3-bis-DTPA-pamoate or 99m Tc(CO)3-12 in 0.5 mL water/PEG 400 80/20 V/V was injected via a tail vein and the rats were sacrificed under anesthesia at 4 h postinjection (n ) 4 per tracer) followed by ex ViVo studies. Under anesthesia, muscular necrosis was induced in four adult Wistar rats by gradual infusion of 0.2 mL ethanol into the right hind limb. The left hind limb was injected with saline and served as control. The animals were injected via a tail vein with 18.5–74 MBq of 99mTc(CO)3-bis-DTPA-pamoate in 0.5 mL saline and then sacrificed under anesthesia at 5.5 h p.i.. The muscles from the treated and control limbs were then subjected to ex ViVo studies. Ex ViWo Evaluation of Tracer Uptake. For ex ViVo studies, the necrotic tissues (liver and muscle) and healthy control tissues were thoroughly washed with saline (4 °C) to remove blood pool activity. The tissues were then stained separately in TTC solution (1% solution in normal saline) at 37 °C for

Necrosis Avidity of

99m



Table 1. Biodistribution in Normal Mice (n g 3 per time point) and in Rats with Reperfused Hepatic Infarction (n ) 3 per time point) of 99m Tc(CO3)-bis-DTPA-Pamoatea 99m

Tc(CO)3-bis-DTPA-pamoate expressed as % injected dose/g ( SD

normal mice

rats with reperfused hepatic infarction

organ

30 min

4h

24 h

4h

24 h

kidneys viable liver lobe necrotic liver lobe spleen + pancreas lungs heart cerebrum cerebellum blood

17.6 ( 7.4 4.5 ( 0.6 NA 2.0 ( 0.1 8.3 ( 1.8 3.8 ( 0.9 0.4 ( 0.1 0.6 ( 0.1 14.3 ( 2.0

25.1 ( 5.1 4.8 ( 0.3 NA 1.6 ( 0.1 5.3 ( 1.4 2.8 ( 0.7 0.3 ( 0.1 0.4 ( 0.1 9.3 ( 1.1

8.6 ( 1.2 3.6 ( 0.2 NA 1.0 ( 0.1 1.9 ( 0.0 1.2 ( 0.0 0.1 ( 0.0 0.1 ( 0.0 2.3 ( 0.4

6.1 ( 2.8 0.7 ( 0.1 1.2 ( 0.2 0.6 ( 0.1 0.8 ( 0.1 0.7 ( 0.1 ND ND 2.4 ( 0.7

6.5 ( 1.6 0.6 ( 0.1 2.1 ( 0.5 0.3 ( 0.0 0.5 ( 0.1 0.3 ( 0.0 ND ND 0.8 ( 0.1

% injected dose ( SD 15.9 ( 5.6 5.9 ( 0.9

b

urine intestines + fecesb

18.4 ( 1.1 7.9 ( 0.4

a NA: not applicable. ND: not determined. SD: standard deviation. bladder and intestines/feces.

b

Tc-12 expressed as % injected dose/g ( SD

organ

30 min

4h

kidneys liver spleen + pancreas brain lungs heart blood

1.5 ( 0.5 24.4 ( 3.0 0.6 ( 0.3 0.0 ( 0.0 2.4 ( 0.7 0.3 ( 0.1 0.2 ( 0.0

0.3 ( 0.1 2.1 ( 0.5 0.3 ( 0.1 0.0 ( 0.0 0.8 ( 0.2 0.1 ( 0.0 0.1 ( 0.0

% injected dose ( SD 0.4 ( 0.3 51.1 ( 4.8

b

urine intestines + fecesb a

5.0 ( 0.5 4.3 ( 0.8

14.8 ( 0.3 9.4 ( 2.2

Values are shown as % of injected dose due to variability in the content of the

Table 2. Biodistribution in Normal Mice (n ) 4) of 99mTc(CO)3-[N(5-aminopentyl)pyridin-2-yl-methylamino]methylacetato-4,4′-methylene-2-hydroxy-3-napthalenecarboxamide-(2′-hydroxy-3′-naphthoic acid methyl ester) (99mTc(CO)3-12)a 99m

34.6 ( 3.7 24.5 ( 3.1

0.9 ( 0.3 93.5 ( 2.3

b

SD: Standard deviation. Values are shown as % of injected dose due to variability of the content of the bladder and intestines/feces.

15 min. Guided by TTC, the necrotic and viable tissue areas were identified, separated, and weighed; and radioactivity in the tissues was immediately counted in a 3 in NaI(Tl) scintillation detector mounted in a sample changer (Wallac Wizard). Corrections were made for background radiation and physical decay during counting, and activity was expressed as % I.D./g tissue. Digital photographs were taken prior to and after staining. The tissues were then quickly frozen in isopentane–liquid nitrogen and cut with a cryotome (Microm HM 550, Walldorf, Germany) into 5–50-µm-thick serial sections mounted on slides. Autoradiograms were obtained from these sections by exposing the slides for 2 days to a high-performance phosphor screen (super resolution screen; Canberra-Packard, Meriden, CT). The images were analyzed with Optiquant software (Canberra-Packard), and the radioactivity concentration in the autoradiograms was expressed in digital light units (DLU)/mm2. Relative tracer concentrations in the necrotic and viable sections of the tissues were estimated by regions of interest (ROIs) analysis of the necrotic and the viable regions of 30 and 50-µm-thick sections of the autoradiograms of all the slices. After exposure for autoradiography, the samples were stained with H&E using a conventional procedure. The stained slices were digitally photographed and contrast enhanced with Image J software (NIH, Bethesda, MD). The H&E stained slices were used to examine microscopic evidence of tissue necrosis.

In ViWo Detection of Hepatic Infarction by 99mTc(CO)3bis-DTPA-pamoate Planar Scintigraphy. Three additional rats with reperfused hepatic infarction and two control rats were used to explore the feasibility of in ViVo detection of necrosis by 99m Tc(CO)3-bis-DTPA-pamoate planar gamma scintigraphy. Rats with reperfused hepatic infarction and a control rat were injected with 53 or 85 MBq of 99mTc(CO)3-bis-DTPA-pamoate followed by 20 min planar imaging at 7 or 23 h p.i. using a parallel hole collimator of a dual-head gamma camera (E-Cam, Siemens Medical Systems, Hoffman Estates, IL) equipped with a pinhole and a parallel hole collimator. Planar imaging was immediately followed by a computed tomography scan (CT, Biograph16, Siemens, Knoxville, TN) for anatomical delineation of the liver.

RESULTS AND DISCUSSION Chemistry. Bis-DTPA-pamoate (5) was synthesized as shown in Scheme 1. Compound 2 was synthesized using dimethyl sulfate in high yield. Coupling of DTPA through one of the terminal carboxylates to pamoic acid bis-hydrazide was realized in good yield using DTPA monoanhydride monoethyl ester (yield 75%). All compounds were characterized by 1H NMR (recorded at 300 Hz). [N-(5-Aminopentyl)pyridin-2-yl-methylamino]acetato-4,4′methylene-2-hydroxy-3-napthalenecarboxamide-(2′-hydroxy-3′naphthoic acid) (12) was synthesized as shown in Scheme 2B. Synthesis of compound 11 started from pamoic acid monomethyl ester followed by activation of the second carboxylic acid using N-hydroxysuccinimide in the presence of DCC as an in situ dehydration reagent with a yield of 60%. Ester hydrolysis of compound 11 with 1 M NaOH provided compound 12 with a yield of 85% (HPLC analysis). Labeling of crude 12 or HPLC-purified 12 resulted in similar labeling yields. In all cases, intermediate and final products were purified on normalphase or reversed-phase silica gel and the purity verified by RP-HPLC to be >95%. Radiochemistry. Labeling of bis-DTPA-pamoate with a 99m Tc(CO)3 core at pH 11 resulted in a main peak (retention time tR ) 13.5 min, 1 GBq/µmol) and a slightly more polar peak. The low specific activity of 99mTc(CO)3-bis-DTPApamoate resulted from a partial coelution of the unlabeled precursor (bis-DTPA-pamoate) with the labeled compound. However, experience from experiments with Gd-labeled pamoate derivatives for MRI, where milligram amounts are used, and from experiments with derivatives of hypericin, both with low and high specific activity, has shown that specific activity


Fonge et al.

Figure 2. RP-HPLC analysis of mouse plasma (A) and urine (B) after intravenous injection of 99mTc(CO)3-bis-DTPA-pamoate at 30 min p.i. and 24 h p.i. (C) RP-HPLC analysis of mouse plasma after intravenous injection of 99mTc(CO)3-12 (B) at 30 min p.i.

Figure 3. (A) Representative autoradiograms (first and third columns) and corresponding H&E stained sections (second and fourth columns) of 30 µm slices of rat liver with reperfused hepatic infarction. Viable liver lobe (upper row) and necrotic liver lobe (bottom row) at 4 h postinjection of 99m Tc(CO)3-bis-DTPA-pamoate and (B) at 24 h postinjection.

is not important with respect to the ability of these agents to localize to a high and sufficient degree in necrotic tissue. A number of isomers could arise from 99mTc(CO)3 labeling of such a bis-DTPA-derivatized compound, including binding of the 99m Tc(CO)3 core between the two DTPA ligands or coordination via two amino nitrogens and a carboxylic oxygen, or via three amino nitrogen atoms besides binding in the way suggested in Figure 1A. The main peak containing 95% of the activity was used for the biological evaluation and is thought to consist of a 99mTc(CO)3 core bonded to two carboxylic oxygens and one amino nitrogen of one of the DTPA chains (Figure 1) analogous to the binding of a 99mTc(CO)3 core to iminodiacetic acid (22). The structure for the 99mTc(CO)3-DTPA moiety as presented

in Figure 1 has also been proposed by a number of other authors (23, 24). Labeling of compound 12 with technetium-99m tricarbonyl was achieved in very good yield (97%) by heating a buffered solution (pH 7.4) of the precursor and the 99mTc-tricarbonyl precursor at 70 °C for 15 min. After efficient HPLC purification, a specific activity of about 19 350 GBq/µmol was obtained. Facile labeling of compound 12 is the result of stabilization by π-electrons of the aromatic nitrogen and d6 electrons of the Tcmetal core (18). We also labeled the BFC (10) as a reference (HPLC tR ) 12.6 min vs 23.5 min for 99mTc(CO)3-12). Octanol/Phosphate Buffer Partition Coefficient. The log P values ((SD) were found to be -1.93 ((0.03) and 2.85

Necrosis Avidity of

99m


((0.13) for 99mTc(CO)3-bis-DTPA-pamoate and 99mTc(CO)312, respectively. Carboxylic acid functions not participating in complex formation account for the hydrophilic nature of 99m Tc(CO)3-bis-DTPA-pamoate, but the hydrophilic character may be different in an in ViVo situation, where there is a possibility of complexation with Ca2+ ions in blood, leading to increased lipophilicity and potential retention. Biodistribution and Stability (in Vitro and in ViWo) of Tracers. Biodistribution results for 99mTc(CO)3-bis-DTPApamoate in normal NMRI mice and in rats with reperfused liver infarction are shown in Table 1 with the results expressed as % injected dose (% I.D.)/g tissue ((SD), except for urine and intestines, where the radioactivity is expressed as % I.D./tissue in view of the variable amount of urine produced and food intake. In normal mice, the tracer was cleared mainly via the kidneys resulting in urine activity of 15.9%, 18.4%, and 34.6% I.D. at 30 min, 4 h, and 24 h p.i., respectively. There was also a significant clearance via the hepatobiliary system resulting in high activity in intestines and feces. Plasma clearance was slow with 6.4% I.D. remaining in blood at 24 h p.i.. Radioactivity in mice carcass was 28.6% and 17.1% I.D. at 30 min p.i. and 24 h p.i., respectively. The reason for the slightly higher carcass activity can be attributed to the slow blood clearance and retention of tracer in muscles. As expected, there was negligible brain uptake of the tracer. Guided by TTC staining, the necrotic lobes of rats with reperfused liver infarction (TTC negative) were separated from the viable lobes (TTC positive). 99mTc(CO)3-bis-DTPA-pamoate showed higher uptake in liver with reperfused infarction than in normal liver at 4 h and 24 h p.i. At 4 h p.i., the radioactivity concentration was 1.7 times higher in necrotic liver tissue than in normal liver, and this ratio increased to 3.5 at 24 h p.i. Apart from this, biodistribution in rats was similar to that in mice. In rats with ethanol-induced liver necrosis, radioactivity per gram tissue at 4 h p.i. was 1.3 times higher in necrotic liver tissue as compared to normal liver (biodistribution data not shown). The actual ratio was higher, as it appeared impossible to dissect purely necrotic tissue, which always was mixed with some healthy tissue. Therefore, uptake in different areas was more accurately quantified by autoradiography. Biodistribution results of 99mTc(CO)3-12 in normal mice are shown in Table 2. The tracer agent showed more rapid clearance from the blood circulation than the more polar 99mTc(CO)3bis-DTPA-pamoate. Activity of 99mTc(CO)3-12 in blood was 0.6% and 0.2% I.D at 30 min and 4 h p.i., respectively. The tracer was cleared mainly via the hepatobiliary pathway, resulting in high radioactivity in intestines and feces. Uptake in carcass was negligible at 30 min and 4 h p.i.. Surprisingly, 99m Tc(CO)3-12 did not show any preferential uptake in the necrotic cells in rat studies, both by activity counting (necrotic to viable tissue radioactivity ratio was 0.9 at 4 h p.i.) and by autoradiography (results not shown). The very high lipophilicity of 99mTc(CO)3-12 is without doubt the reason for its prominent and fast clearance via liver and intestines and can explain why it is cleared more rapidly from plasma than 99mTc(CO)3-bis-DTPA-pamoate. At this moment, however, no further conclusions can be drawn with respect to a potential relationship between the observed lipophilicity of the complexes and the uptake in necrotic tissue. Both hydrophilic agents (e.g., 99mTc-glucarate (25) and 99mTc(CO)3-bis-DTPApamoate) and very lipophilic agents (derivatives of hypericin) (20, 21) have shown high avidity for necrotic tissue, so necrosis avidity does not seem to be primarily associated with the lipophilicity of a compound). It is unclear why 99mTc(CO)3bis-DTPA-pamoate has a higher uptake in necrotic tissue as compared to 99mTc-12. One of the possibilities is that the rapid blood clearance of 99mTc(CO)3-12 does not allow for time


Figure 4. (A) TTC stained muscle after ethanol injection (pale stained, TTC negative area) and adjacent viable muscle tissue (deep red). (B) Autoradiogram of necrotic muscle shows high uptake of 99mTc(CO)3bis-DTPA-pamoate (5.5 h postinjection) in necrotic area with a necrotic to viable tissue ratio of 18.

Figure 5. (A) TTC stained liver lobes of rat with ethanol-induced liver necrosis and (B) corresponding autoradiogram of 50 µm slice of the necrotic and viable liver lobes after intravenous injection of 99mTc(CO)3bis-DTPA-pamoate.

needed for efficient tracer perfusion into the necrotic lesions. On the other hand, the structural differences and the different overall charge and lipophilicity of both tracer agents may contribute to the clearly different biological properties and uptake in necrotic tissue. For both tracers, more than 98% of intact tracer was recovered after incubation of the radiolabeled compound for 24 h at room temperature. In challenge experiments with histidine, known to be one of the most potent tridentate chelators for binding of a 99mTc(CO)3 core and often used in tricarbonyl organometallic chemistry to investigate the in Vitro stability of 99mTc(CO)3 complexes (18), 99mTc(CO)3-bis-DTPA-pamoate was less than 50% stable after 24 h in the presence of 0.01 M histidine (>20fold excess of histidine) at 37 °C, while 99mTc(CO)3-12 was 98% stable under the same conditions. Results of HPLC analysis of mouse plasma and urine after intravenous injection of 99m Tc(CO)3-bis-DTPA-pamoate are shown in Figure 2A,B, respectively. 99mTc(CO)3-bis-DTPA-pamoate showed good stability in plasma and in urine: 98% and 90% of intact tracer were recovered from plasma at 30 min and 24 h p.i., respectively (Figure 2A), while 98% of intact tracer was recovered in urine at 30 min and 4 h p.i. (Figure 2B). This indicates that the in ViVo stability is sufficient for its use in ViVo despite its relative instability in histidine-challenging experiments. Less than 10% of lipophilic metabolites were found at 24 h p.i. Such metabolites could result from in ViVo transchelation of the radiometal to more stable and more lipophilic complexes with plasma proteins.


Fonge et al.

Figure 6. (A) In ViVo planar image of a control rat and a rat with reperfused hepatic infarction at 7 h postinjection of 99mTc(CO)3-bis-DTPApamoate. (B,C) Autoradiograms of 30 µm liver slices of the same rat after sacrifice (B, top row ) necrotic liver and bottom row ) viable liver) and scanned photographs of the same slices after H&E staining (C) staining show good correlation with the in ViVo planar image. 99m

Tc(CO)3-12 was stable in histidine challenge experiments, as 98% of the intact tracer was recovered after 24 h incubation in the presence of excess histidine at 37 °C. The tracer was also stable in ViVo in mice. At 30 min, there was 97% of intact tracer in plasma (Figure 2C). Plasma stability was not evaluated at later time points, since activity in blood was negligible. For both tracers, however, there was minimal reoxidation to TcO4at all time points examined. Ex ViWo and in ViWo Specificity for Necrotic Tissue. In all rat models, TTC staining confirmed the presence of necrosis. TTC is an avid dye for mitochondrial dehydrogenase activity, and its absence results in pale staining of necrotic tissues by TTC (TTC negative), whereas viable tissues with enzymatic activity would reduce TTC into deep red insoluble formazan (TTC positive) (26). Autoradiograms and photographs of the corresponding H&E stained slices of liver of rats with reperfused liver infarction and sacrificed at 4 h or 24 h p.i are shown in Figure 3A,B, respectively. Necrotic to viable tissue activity ratios of 99mTc(CO)3-bis-DTPA-pamoate in rats with reperfused liver infarction were 7 and 9 at 4 h and 24 h p.i., respectively, as determined by quantitative autoradiography. The contrast ratios at 4 h and 24 h did not differ much because of the rather slow clearance of 99mTc(CO)3-bis-DTPA-pamoate from liver. Since the tracer is also excreted via the liver, the specificity was further explored in rats with ethanol-induced muscular necrosis (Figure 4). Following infusion of 0.2 mL ethanol (right hind limb) or 0.2 mL saline (left hind limb, control), 99mTc(CO)3-bis-DTPApamoate was administered via i.v.. injection to the rats (n ) 4) which were sacrificed at 5.5 h p.i.. A TTC negative area was visible in the treated limb (Figure 4A) but not in the control limb (data not shown). Necrotic to viable tissue activity ratios were 15–18 on autoradiography (Figure 4B). The lower necrotic to viable tissue ratio (4:1) observed in the case of ethanolinduced liver necrosis (Figure 5B) could be due to a complete collapse of vasculature following ethanol infusion leading to severe perfusion defects and consequently restricted accessibility of the tracer to necrotic tissue. In ViVo planar imaging was performed in rats with reperfused hepatic infarction at 7 h and 23 h p.i. of 99mTc(CO)3-bis-DTPApamoate. Figure 6A shows representative in ViVo planar images obtained at 7 h p.i. from a control rat and a rat with hepatic infarction, respectively. Corresponding autoradiograms of 30 µm liver slices of the same rat after sacrifice are shown in Figure 6B (top row ) necrotic liver and bottom row ) viable liver) and scanned photographs of the same slices after H&E staining

in Figure 6C. As can be seen from these images, there was a good match between in ViVo and ex ViVo images with high tracer uptake in necrotic liver lobe (hot spots on planar images and autoradiograms). This high uptake was confirmed by gamma counting of dissected liver tissue. For an infarct avid agent to be clinically useful, it should show rapid localization in the infarcted tissue, high avidity and high specificity for necrotic tissue, and a reasonable duration of scan positivity. In the case of AMI, it is important that an infarct avid agent localizes early enough within the time window for thrombolytic therapy (usually

3-Labeled Pamoic acid Derivatives - American Chemical Society

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