The labelling of MTX was performed by ligand exchange from a ^99mTc(v)O-gluconate precursor. Sodium gluconate acts as an intermediate exchange ligand for ^99mTc, with stannous chloride as the reducing agent (11). Briefly, a solid mixture of 1 g sodium gluconate, 2 g of sodium bicarbonate and 15 mg of stannous chloride was homogenized and kept under anhydrous conditions. 3 mg of this mixture were dissolved in 1 ml of a sodium pertechnetate solution (Na^99mTcO₄) containing 296 MBq/8 mCi ^99mTc. 10 mg of MTX were added, and the mixture was stirred for 30 min at room temperature. The pH of the final solution was 7.

Radiochemical control of the ^99mTc-MTX complex was carried out with Instant Thin Layer Chromatography-Silica Gel (ITLC-SG) in saline and Whatman 3 mm chromatography paper (PC) in acetone. Briefly, 2 µl of the reaction mixture were applied on Silica Gel (ITLC-SG) and Whatman 3-mm strips. The radiochromatographs were developed in saline (0.9% NaCl) and acetone, respectively, over a distance of 10 cm. After drying, the strips were cut in 1-cm pieces and their radioactivity was counted in a well-type scintillation counter. Furthermore, Reversed-Phase HPLC (RP-HPLC) analysis was performed on an aliquot of the reaction solution, by applying the following linear gradient system: From 0 to 80% solvent B (1–20 min), 80% solvent B (20–23 min), 80 to 0% solvent B (23–25 min) and 0% solvent B (25–30 min), at a 0.8 ml/min flow rate (solvent A: 0.1% Trifluoroacetic Acid (TFA) in H₂O; solvent B: 0.1% TFA in Acetonitrile (AcCN).

In vitro stability of ^99mTc-MTX at room temperature was assessed up to 24 h after preparation of the sample. Plasma stability was carried out in fresh human plasma at 37°C. For preparation of human plasma, a blood sample from healthy donors was collected in heparinised polypropylene tubes and was immediately centrifuged at 2,000 × g for 10 min. The supernatant was collected and used for the stability study. 100 µl (~29.6MBq/0.8mCi) of ^99mTc-MTX was incubated with 900 µl of plasma at 37°C. At 2 and 4 h, 100-µl aliquots were treated with a two-fold excess of ethanol and centrifuged at 1,000 × g for 15 min. The remaining pellet was washed thrice with 1 ml EtOH, and these ethanolic washes were combined with the supernatant and counted in a γ well counter. This activity was compared to the activity in the pellet, to give the percentage of ^99mTc-MTX not bound to proteins. The supernatant was analysed by paper chromatography and ITLC, as described above.

The apparent partition coefficient for ^99mTc-MTX was determined by mixing aliquots of the technetium complex with 1-octanol and phosphate buffer (0.125 M, pH 7.4).

In a centrifuge tube, containing 2 ml of each phase, 100 µl of the ^99mTc complex solution was added, and the mixture was agitated on a Vortex mixer for approximately 1 min and finally centrifuged at 5,000 × g for 5 min. Three samples (0.2 ml each) from each layer were counted in a γ counter. The partition coefficient was calculated as the mean value of each cpm/ml of octanol layer divided by that of the buffer. A sample (1.0 ml) from the octanol layer was subsequently repartitioned in octanol/buffer until constant values were obtained. This was achieved with the third repartition.

All applicable institutional and/or national guidelines for the care and use of animals were followed. These studies were approved by the Ethics Committee of the National Center for Scientific Research of ‘Demokritos’ (Athens, Greece) and animal care and procedures followed are in accordance with institutional guidelines and licenses issued by the Department of Agriculture and Veterinary Policies of the Prefecture of Attiki (Registration nos. EL 25 BIO 022 and EL 25 BIO 021). Mice were housed under constant environmental conditions with 12 h light-dark cycles and had free access to food and water. To induce inflammation, animals were inoculated with 50 µl of pure turpentine oil subcutaneously in the left thigh muscle under slight ether anaesthesia (11,12). All animals developed an oedema 18 to 24 h after turpentine inoculation, which was visible to the naked eye.

Ex vivo animal experiments were performed in Swiss Albino mice with experimentally-induced inflammation (20±2 g, n=3 animals per time-point), by injecting 100 µl (3.7 MBq/0.1 mCi) of the radiotracer via the tail vein. Animals were sacrificed by cardiectomy under slight ether anaesthesia at 2, 4 and 24 h post injection, and the main tissues and organs (blood, heart, liver, stomach, intestines, spleen, lungs, pancreas and bones) were excised, blotted dry and weighed. The inflamed thigh was excised and trimmed of the neighboring subcutaneous tissue. The muscle of the non-inflamed right thigh was also excised, for reasons of comparison. Samples were counted in a gamma counter (NaI gamma counter; Packard, Downers Grove, IL, USA). Standards were prepared from the injected material and were counted each time simultaneously with the tissues excised, allowing for calculations to be corrected for physical decay of the radioisotope. Radiolabeled MTX distribution over time was expressed as injected dose per gram (%ID/g).

The imaging system employed is a compact, Anger-type, γ-ray camera developed at the Center for Gamma-Ray Imaging of the University of Arizona. Details of the system can be found elsewhere (13–15). Briefly, the system comprises a 5 mm thick NaI(Tl) scintillation crystal, a 12 mm thick quartz light guide, a 3×3 array of 1.5 inch diameter photomultiplier tubes (PMTs), and a 40-mm thick lead parallel-hole collimator with hexagonal holes of 1-mm in diameter. The system achieves a spatial resolution of approximately 2.5 mm at the collimator face and degrades linearly with distance. The field-of-view of the camera is 4.5 in × 4.5 in, enough to image a whole mouse without axially moving the camera or the mouse.

A Swiss mouse, with experimentally-induced inflammation for 24 h in the left hind limb area, was anesthetized with an intraperitoneal (IP) injection of ketamine (75 mg/kg) and xylazine (5 mg/kg) and placed on the camera face in the prone position. The animal was injected with 100 µl (25.9 MBq/0.7 µCi) of ^99mTc-MTX. Dynamic planar scintigraphy was performed by collecting 10 consecutive two-minute images, for a total period of 20 min. Two h after tracer injection, additional anaesthesia was applied and a 5-min static image was collected with the animal in the same position. Furthermore, 24 h after injection the animal was euthanized and a 1 h image was acquired.

Hydroxyapatite binding studies were performed in vitro to simulate the binding of the radiotracer under investigation to bone structure. For this purpose, hydroxyapatite (Hap; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was suspended in isotonic saline at 20 mg/ml and then incubated for 24 h at room temperature. The following day, 50 µl (0.444MBq/0.012mCi) of ^99mTc-MTX were added to the Hap fractions. After a 10s vortex, the sample was incubated, under agitation, for 10 min at room temperature and centrifuged. The supernatant was then removed and the Hap fraction was washed twice with saline. The radioactivity of the Hap fraction and the saline washes were then measured with a well-type gamma counter. Control experiments were performed using ^99mTc-MTX, without Hap. ^99mTc-MTX binding to Hap was determined as percent of absorbed onto Hap [Hydroxyapatite binding (%)=(radioactivity of Hap fraction of each sample/total radioactivity)x100].

Radiolabeling of MTX was achieved by the preconjugation approach, via the precursor ^99mTc-gluconate. Radiochemical purity was assessed by paper chromatography (Whatman 3 MM) and ITLC-SG. With ITLC-SG, ^99mTc-MTX and the free pertechnetate ^99mTcO₄-appeared at Rf=0.9–1.0, while ^99mTcO₂ was detected at Rf=0.0–0.1. In acetone the free ^99mTcO₄ had an Rf of 0.9–1.0 while the ^99mTc-MTX and the hydrolysed^99mTcO₂ appeared at Rf=0.0–0.1. The radiochemical purity of ^99mTc-MTX was found to be 95–98%. Under the specific HPLC conditions, there was one product peak of ^99mTc-MTX moving with the solvent front, with a retention time of approximately 20 min, while the retention times of ^99mTcO₄⁻ and ^99mTc-gluconate were <5 min (9).

The stability of ^99mTc-MTX was determined by paper chromatography and ITLC at different time points, as described above. The radiolabeled complex remained stable (up to 90%) at room temperature, for up to 24 h post-labelling. Plasma stability studies showed that 94.9±1.2% of ^99mTc-MTX remained intact at 2 h, dropping to 85.8±2.7% at 4 h post-incubation.

Before performing ex vivo biodistribution studies in mice, the in vitro binding of ^99mTc-MTX was assessed in human plasma. Protein binding in human plasma was found to be 48.1±1.9% at 2 h, remaining practically stable at 4 h post-incubation (49.1±2.2%).

Regarding lipophilicity, the partition coefficient indicated that ^99mTc-MTX had maximum extraction in phosphate-buffered saline (PBS), pH 7.4 (hydrophilic medium), while a negligible amount of activity was observed in octanol (lipophilic medium), thus suggesting that radiolabeled drug was hydrophilic in nature. The logP value was estimated to be −2.28±0.03.

The biodistribution of ^99mTc-MTX was assessed on Swiss Albino mice with experimentally-induced inflammation at 2 and 24 h post injection (Fig. 1). Rapid clearance from blood and predominant excretion via the urinary system was observed for the radiotracer. Uptake in the stomach and spleen was low, providing evidence for the in vivo stability of the tracer. With regard to the other organs, apart from the kidneys, no major uptake was observed in all analysed tissues (≤1.0% ID/g from 2 h post injection). An important observation was the increased uptake of the radiotracer in the inflamed muscle, in comparison to the contralateral normal muscle tissue (0.54±0.11 vs. 0.17±0.10; 0.61±0.12 vs. 0.28±0.22; and 0.14±0.07 vs. 0.05±0.01 at 2, 4 and 24 h post injection, respectively), with the inflammation-to-normal muscle ratio remaining practically stable up to 24 h post injection. Furthermore, an unexpected observation was the significant bone uptake observed, which led to a pronounced differentiation between bone and non-osseous tissue, especially at 24 h post injection (Fig. 2).

A dynamic sequence of γ-ray images of an inflammation-induced mouse injected with 25.9 MBq (0.7 mCi) of ^99mTc-MTX at consecutive time points is presented in Fig. 3. All images are 2-min acquisitions and they are displayed on the same colour scale. The colour intensity represents the magnitude of the deposited radiotracer. The inflammation site, in the left side of the animal, is clearly identified by visual inspection immediately after injection. Liver uptake of the tracer is also observed at a lower concentration than the inflammation site.

Static planar scintigraphic images of the mouse at 2 and 24 h post injection are shown in Fig. 4. Increased uptake of the tracer can be visually identified in the inflammation site as well as the liver 2 h post injection. At 24 h post injection most of the tracer remains at the liver with very small uptake at the inflammation site. However, some uptake at the joints and spine is also observed both at 2 and 24 h after injection.

To assess the uptake of ^99mTc-MTX at the bones and joints, a healthy mouse was injected with 0.7 mCi of ^99mTc-MTX and planar images were acquired 2 and 24 h later. Increased uptake of the tracer was observed at the spine, the knees and the toes of the hind-limbs at both time points (Fig. 5).

Hydroxyapatite binding of ^99mTc-MTX was determined on a 20 mg/ml saline sample of Hap, and was found to be 44.9±1.3%, at 10 min post incubation. In the control experiments, we confirmed that the radioactivity adsorbed to the vials was less than 0.1%.