Chemicals
[14C]diclofenac sodium was provided by American Radiolabeled Compounds (101 Arc Dr, St Louis, MO 63146, USA) with a specific activity of 172 μCi/mg (50–60 mCi/mmole, 6.4 MBq/mg). Non-labelled diclofenac sodium supplied in house was used to dilute the radiolabeled material.
λ-carrageenan (article n° 22049-25G) was bought from Sigma-Aldrich (Buchs, Switzerland).
Animals
Sixteen male pigmented Long Evans rats, 8 weeks old, weighing 230 ± 25 g, obtained from Charles River (Raleigh, NC, USA) were used. They had free access to rodent chow (Provimi Kliba SA, CH-4303 Kaiseraugst) and tap water during the entire study.
Dose formulation
For dosing, 5.6 mg of cold diclofenac sodium and 4.4366 g of the radioactive solution above (corresponding to 2.5662 mg of [14C]diclofenac sodium) were dissolved in 40.038 g of 0.9% saline. The dose, 2 mg/kg b.w. of [14C]diclofenac sodium which has been reported to be the ED40 in this animal model [17], was given p.o. by gavage at 10 mL/kg in 0.9% saline. The radioactive dose was 4.11 MBq/kg b.w.
Experimental design
Twelve rats were subjected to an acute local inflammatory reaction by subplantar injection with 0.1 mL of 1% w/v of carrageenan in 0.9% saline into the left front footpad pad and the left hind footpad and into the nape of the neck (0.4 ml of the solution and 0.4 ml air given subcutaneously). The other 4 rats (control group) received 0.9% saline into the same sites and at same volumes. The animals were slightly anesthetized with a 3% isoflurane/oxygen mixture before the injection.
One hour after carrageenan or saline injection, all animals received a single 2 mg/kg oral dose of [14C]diclofenac sodium. Blood (50 μL) was sampled from each animal shortly prior to sacrifice by sublingual puncture for QWBA validation purposes.
Liquid scintillation counting (LSC)
Radioactivity in aliquots of the [14C]diclofenac sodium administration solution and of blood was determined by LSC in a Liquid Scintillation System 2500 TR (Packard Instr. Co., Meriden, CT, USA). For quench correction, the external standard ratio method was used. Quench correction curves were established by means of sealed standards (Packard Instr. Co.). Samples were prepared for analysis as described [18]. The limit of detection (LOD) for determination of radioactivity was defined as the 1.8-fold of the total background count.
Quantitative whole-body autoradioluminography (QWBA)
Three treated rats and one control rat were used per time point. They were euthanized with an overdose of isoflurane at 1, 4, 8, and 24 h after dosing and thereafter immediately deep-frozen for approx. 30 min into an n-hexane/dry ice mixture kept at -70°C. The carcasses were rapidly shaven and the paws removed. The carcasses and the paws were then stored at a temperature below -20°C, and all subsequent procedures were performed at temperatures below -20°C to minimize diffusion of radiolabeled material in the tissues/matrices.
Sectioning procedures
The frozen carcasses and paws were embedded in a mold on a microtome stage by adding an ice-cold aqueous solution of 2% sodium carboxymethylcellulose (CMC). The embedding block was frozen for approx. 30 min in an n-hexane/dry ice mixture at -70°C followed by temperature stabilization overnight in a -20°C freezer. The animals were sectioned using a CM 3600 PLC cryomicrotome (Leica Microsystems GmbH, D-Nussloch) according to the method of Ullberg [19]. Several sagittal 40 μm thick whole-body sections were taken at varying depths, based on sectioning of the requisite organs, tissues and body fluids. Similarly, several 40 μm thick sections were taken at varying depths from the paws. A block of 14C radiolabeled standards, prepared in blood and assayed by liquid scintillation counting, was sectioned in the same manner and on the same day as the rats were sectioned.
The sections were dehydrated in the cryomicrotome for 48 h at -30°C. Thereafter, they were labelled using radioactive ink specifying the test compound, radiolabel, protocol number, dose route, animal strain, time of sacrifice and number of section, resulting in a permanent label on each section and phosphor image autoradiogram.
Imaging procedure
The whole-body autoradiograms were obtained by means of the autoradioluminography method [20]. Tissue/matrix and standard sections were placed in direct contact with Fuji BASIII Imaging plates (Fuji Photo Film Co. Ltd., J-Tokyo) for 3 days at room temperature in a lead shielded box in order to minimize the increase of the background signal. The duration of the exposure was chosen to allow detection of approx. 1 dpm/mg. At the end of the exposure, the sections were separated from the plates, and the plates were first kept for 3–5 min in the dark. They were then transferred into a Fuji BAS 2000 TR phosphor-imager (Fuji Photo Film Co. Ltd.) and scanned at a 100 μm scanning step with a 1024 gradation to produce an autoradiogram.
For reporting, selected sections were re-exposed onto SR screens (Perkin Elmer, formerly Packard Instr., Meriden, CN, USA) for 3 days at room temperature and scanned at a 42 μm scanning step in a ®Cyclone (Packard Instr.) phosphor-imager. The image files were processed using the Adobe Photoshop Elements 2.0 software (Adobe Systems Incorporated, San Jose, California, USA).
Determination of the tissue/matrix concentrations of total radiolabeled components
The concentrations of total radiolabeled components in the tissues/matrices were determined by comparative densitometry and digital analysis of the autoradiogram. The radioactivity concentrations were calculated from the curve generated from the calibration samples present on the image plate and were expressed as nanomoles/gram of tissue/matrix [21]. The resulting photo-stimulated light data files were corrected by subtracting the background and processed electronically with the help of a MCID/Elite (Version 6.0) image analyzer (Imaging Research, St. Catherines, Ontario, Canada). The calibration standards were prepared from fresh whole rat blood and a stock solution of a [14C]-labelled compound. Up to seven calibration concentrations were prepared at concentrations of 2–4000 dpm/mg for 14C which encompass the range of concentrations typically observed in a standard whole-body autoradiography study. To assess the actual concentrations in the calibration standard samples, duplicate 100 μL samples of spiked blood were counted for total radioactivity in a liquid scintillation counter. These values were defined as the actual concentrations and used to generate a calibration curve during digital analysis.
Quality control (section thickness homogeneity and reproducibility checks) was performed by analyzing the radioactivity concentration in the liver on each section. In addition, the concentrations of radioactivity as determined by QWBA in blood were compared to the corresponding ones determined by LSC (blood samples collected from each animal shortly prior to sacrifice).
The limit of detection (LOD) was set equal to the mean of background (n = 10) + 3 SD (standard deviations of the individual values). The quantification limit (LOQ) was empirically taken as 3•LOD. The size of the areas used to determine the background value was normalized to that of the blood standards used to establish the calibration curve. Under the conditions of this study, the LOD and LOQ amounted to 0.034 and 0.10 nmol/g, respectively.
Tissue/matrix distribution of radioactivity
The peak concentrations of radioactivity Cmax and time of peak concentration tmax were recorded as observed. The AUC(0-tlast) and t1/2 were calculated for those tissues/matrices where sufficient quantifiable data points were available. The AUC(0-tlast) were calculated using the linear trapezoidal rule. The t1/2 was taken as ln(2)/λz where λz (terminal elimination rate constant) was the slope of the log linear line from the at least 3 last measurable data points. All calculations were performed using the computer program WinNonlin® (Professional 5.0) (Pharsight Corp., Mountain View, CA, USA). The ratios of tissue/matrix to blood Cmax and AUC(0-tlast) values were also reported where possible.
Residual radioactivity in the body at 24 h after dosing
In order to estimate the overall residual radioactivity in the body at 24 h after dosing, the mean concentrations of total radiolabeled components over the entire section of at least 6 sections per rat (one per sectioning level) were determined as described above and converted into percent of the administered dose by taking into account the weight of the animals, the specific activity of the administration formulations, the volume administered and the actual doses.