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UTILITY OF WHOLE-BODY AUTORADIOLUMINOGRAPHY IN DRUG DISCOVERY FOR THE QUANTIFICATION OF TRITIUM-LABELED DRUG CANDIDATES

Pfizer Inc., Global Research and Development, Pharmacokinetics Dynamics and Metabolism Development, Groton, Connecticut

(Received March 2, 2004; accepted June 29, 2004)

	Abstract

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Whole-body autoradioluminography (WBAL) has evolved as the preferred method for conducting tissue distribution studies that are required for regulatory filings of a new drug entity (DE) and for projecting tissue dosimetry in human mass balance studies. Four experiments were designed to assess WBAL utility using tritium as early as lead development in the drug discovery process. The objective of experiment 1 was to determine the minimum amount of tritium to administer to rats required for obtaining widespread distribution into most tissues at concentrations greater than quantification limits. Experiments 2, 3, and 4 were conducted to identify a tissue compartment responsible for observed triphasic pharmacokinetics, to characterize the distribution of a [³H]DE into brain tissue, and to compare tissue distribution patterns between two rat strains, respectively. The minimum amount of tritium necessary to investigate the tissue distribution of [³H]DE in rats was 865 µCi/kg. Results from experiments 2, 3, and 4 illustrated A) the identification of adipose as the tissue compartment responsible for an extended terminal elimination phase, B) sustained penetration of a DE into brain tissues for at least 24 h, and C) tissue distribution differences between two DEs of the same therapeutic class, respectively. These experiments exemplify the value of WBAL as a screening tool to assist with the selection of a drug candidate. WBAL utilization in drug discovery provides insightful data toward designing pharmacology and toxicology experiments. Substituting the use of tritium for carbon-14 is of crucial importance in drug discovery since [³H]DEs are more readily obtainable than [¹⁴C]DEs.

The utilization of WBA in the drug discovery process has been severely limited by time constraints of film exposures. Until the early 1990s, the only practical method to detect radioactivity in whole-body cryosections was with X-ray film. X-ray film detection, although providing superior autoradiographs, is at best only semiquantitative due to the method's limited linearity resulting from saturation of the silver halide grains of the film (Som et al., 1983). Also, X-ray film development may take more than 2 months (or more), simply because low levels of the radiolabeled compound in certain tissues require this amount of time to produce reliable data. Whole-body cryosections usually contain a wide range of radioactive concentrations. Thus, multiple exposure times, typically ranging from 2 weeks to 2 months, are required when using X-ray film to obtain semiquantitative data. Overexposures with X-ray film also require a new set of samples to be evaluated, which necessitate the acquisition of multiple samples from each study animal during the cryosectioning phase. Because of these factors, WBA studies using X-ray film typically take 3 to 9 months, which severely limit their utility in drug discovery. The second limiting factor for utilizing WBA is obtaining a radiolabeled compound early enough in the drug discovery process to provide data at crucial decision-making stages.

The development of the phosphorimager and storage phosphor plates provided an improved method to detect radioactivity in whole-body cryosections (Johnson et al., 1990). Storage phosphor technology (i.e., autoradioluminography) has now replaced X-ray film (i.e., autoradiography) as the technology of choice in conducting whole-body autoradioluminography (WBAL) tissue distribution studies where quantification is mandatory. Quantification of [¹⁴C]radioactivity using this newer technology has been well documented and overcomes the severe limitations of X-ray film (Potchoiba et al., 1995, 1998). Whole-body cryosections containing tissues with a wide range of [¹⁴C]radioactivity concentrations can be readily quantified across a linear dynamic range of 1.6 to 36,000 nCi/g after one 4-day exposure. This significant reduction in exposure time has resulted in a study turnaround duration of 6 to 10 weeks for WBAL studies required for regulatory filing. The application of WBAL in the discovery process has been limited due mainly to the perception that WBAL (i.e., storage phosphor methodology) studies would be as time consuming as WBA (i.e., X-ray film-based) studies. The present experiments were designed to illustrate more rapid turnaround times for discovery-based WBAL studies, to determine quantification limits of the storage phosphor technology employing tritium, and to evaluate the overall utility of WBAL in the drug discovery process.

	Materials and Methods

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Drug Entities and Chemicals. D-[2-³H(N)]Glucose (20-30 Ci/mmol) in ethanol/water (9:1) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Radiochemical purity of this [³H]glucose product was 97% when determined by HPLC and TLC. The synthesis of [³H]DE-1 was outsourced to Chemsyn (Lenexa, KS). The specific activity of [³H]DE-1 was 46.5 Ci/mmol, with an HPLC radiochemical purity that was >98%. The Radiosynthesis Group of Pfizer Global Research and Development (Groton, CT) synthesized [³H]DE-2. The specific activity of [³H]DE-2 was 74.9 Ci/mmol, with a radiochemical purity that was >98.5% (HPLC) and >99% (TLC). The synthesis of [³H]DE-3 (61.9 Ci/mmol) was outsourced to PerkinElmer Life and Analytical Sciences. HPLC and TLC radiochemical purity of [³H]DE-3 was >99%. The synthesis of [³H]DE-4 (41 Ci/mmol) was outsourced to Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Radiochemical purity for [³H]DE-4 (HPLC) was >98%. The caveat of using [³H]labeled compounds for metabolism studies is the exchange of tritium between the [³H]DE and body water. Lyophilization (for WBAL, dehydration) must be used to expel [³H]water from biological fluids or tissues prior to analysis to obtain accurate quantification of [³H]DE-associated radioactivity (Kim et al., 2004). The tritium exchange rate for the five radiochemicals utilized in this study was minimal.

When necessary, nonradiolabeled DEs were combined with their [³H]DE counterparts to achieve the desired milligrams per kilogram and microcuries per kilogram doses. Nonradiolabeled DE compounds were synthesized by Pfizer Central Research chemists and were of the highest chemical purity achievable. All other reagents were of the highest obtainable commercial purity.

Animals. Jugular vein-cannulated Sprague-Dawley (SD) and Long-Evans rats were purchased from Charles River Laboratories (Raleigh, NC). Spontaneously hypertensive (SH) rats with indwelling jugular vein cannula were purchased from Taconic Farms (Germantown, NJ). The care of rats and treatment administration followed corresponded to the Animal Care and Use Procedures established at Pfizer. All rats were allowed to acclimate to environmental surroundings for 2 days prior to experimentation. Dosing solutions were intravenously administered through indwelling jugular vein cannula. Immediately following dose administration, sterile saline was used to flush residual dose from each cannula. Rats were individually housed in stainless steel metabolism cages. Feed and water were provided ad libitum throughout each experiment. All rats were euthanized by CO₂ asphyxiation at times specified below for each experiment.

WBAL. The whole-body cryosectioning technique developed by Ullberg (1977) was used to prepare whole-body cryosections from all experimental rats for autoradioluminography. Autoradioluminographic imaging and quantification methods for [¹⁴C]xenobiotics in whole-body cryosections described by Potchoiba et al. (1995) were employed for evaluating tissue concentrations of tritium with several notable modifications. For carbon-14 studies, a thin piece of Mylar film is typically applied over each sample to prevent contamination of the phosphorimager screens. The use of Mylar film with samples containing tritium must be avoided to prevent total quenching of the radioactive signal generated from the energy released during decay. Likewise, a suitable imaging plate for detecting tritium must also be utilized since the protective overcoat layer of cellulose acetate found on carbon-14 imaging screens is of sufficient thickness to totally prevent the capture of energy release by this weak ß-emitter. Standard curve calibrators (STD) and cryosection quality control samples (CQCS) were freshly prepared with [³H]glucose in rat control whole blood. STD and CQCS were independently prepared across a stepwise range of radioactivity concentrations. STD were necessary to construct the calibration curve for the quantification of [³H]compounds present in tissues of whole-body digital images. CQCS were necessary to determine the limits of quantification for each experiment and to assess the quantification quality of the WBAL assay. For all experiments, STD and CQCS were embedded in the same carboxymethyl cellulose blocks as the specimens. Half of the study animals were embedded along with the STD; the remaining half from the same study were embedded along with the CQCS. Seven cryosections, each from 5 to 7 sectioning levels, were obtained from each rat based on sampling of all major tissues. Four cryosections from each sectioning level were apposed to Fuji imaging plates (BAS-TR2040; 20 x 40 cm) (Fuji Medical Systems, Stamford, CT) for 17-day exposures. Each imaging plate contained two whole-body cryosections with STD and two whole-body cryosections with CQCS. This apposition procedure ensured that each imaging plate had its own calibration curve in duplicate while effectively assessing quantification with appropriate acceptance and rejection controls in the form of CQCS. All imaging plates were placed in a lead- and copper-lined cabinet to decrease background signals from environmental radiation. Imaging plates were scanned either with a 425F or STORM PhosphorImager (Amersham Biosciences). The MicroComputer Imaging Device (Imaging Research, St. Catharines, Ontario, Canada) was used to quantify the concentration of tritium in STD, CQCS, and tissues of whole-body cryosections for all experiments. Calibration curves for each tritium imaging plate were generated from the STD by weighted (1/x) linear regression analysis. The linear regression curve was then utilized to assess the radioactivity concentration in the CQCS and to determine the concentration of unknown radioactivity in the tissues of whole-body cryosection computerized images.

Pharmacokinetics. AUC_{(0-T last)} and t_1/2 values were calculated from mean tissue levels using an in-house pharmacokinetics computer program (PKPARAM). AUC_{(0-T last)} values were calculated using linear trapezoidal approximation. The t_1/2 was calculated as the ln2/elimination rate constant (determined from the regression of the terminal phase tissue-radiolabeled material concentration).

Experiment 1. The objective of this experiment was to determine the dose administration level necessary to investigate the tissue distribution of [³H]DE using WBAL. [³H]Glucose in ethanol/water (9:1) was dried under nitrogen and reconstituted in sterile saline. The concentration of [³H]glucose in this saline dosing solution was 490 µCi/ml as determined by liquid scintillation analysis (Wallac 1409 Liquid Scintillation Counter; Amersham Biosciences). Four male SD rats were administered an intravenous dose containing 49, 147, 368, or 735 µCi of [³H]glucose. Each rat was prepared for WBAL analysis 15 min after dose administration. STD ranged in radioactivity from 5 to 1.9 x 10⁶ nCi/g and were embedded in the specimen blocks with rats administered 49 and 147 µCi of [³H]glucose. CQCS ranging in radioactivity from 7 to 68,400 nCi/g were embedded in the specimen blocks with rats administered 368 and 735 µCi of [³H]glucose.

Experiment 2. The sequestration of DE-1 into an unknown deep compartment from which it slowly equilibrates back into the systemic circulation was revealed by previous pharmacokinetics studies. The primary objective of this experiment was to identify this deep tissue compartment. Definitive quantification of tissue concentrations for [³H]DE-1 was not essential for the qualitative assessment of distribution of this compound, so the incorporation of CQCS was not necessary. [³H]DE-1 was dissolved in an aqueous solution (pH 2.5) to provide a concentration of 3.17 mg/ml (1.72 ml/kg dosing volume). The potency of this radioactive dosing solution was 525 µCi/ml. [³H]DE-1 (998 µCi/kg) was administered to five male SD rats weighing 210 to 250 g. Rats were prepared for WBAL analysis at 2, 12, 48, 96, and 168 h postdose (n = 1 per time point). STD ranged in radioactivity from 31 to 19,800 nCi/g.

Experiment 3. DE-2 was the lead candidate in its therapeutic class. The primary objectives of this experiment were to characterize the tissue distribution of this compound and to assess its distribution into brain tissues. [³H]DE-2 and nonradioactive DE-2 were codissolved in physiological saline to provide a concentration of 5 mg/ml. The radioactive potency of this dose was 1880 µCi/ml. Delivery volumes were adjusted from 0.18 to 0.20 ml in order for each rat to receive 5 mg/kg and 1880 µCi/kg [³H]DE-2. The dosing volume was 1 ml/kg. Each Long-Evans male rat was euthanized by CO₂ asphyxiation at 0.25, 0.5, 1, 6, 12, and 24 h postdose (n = 1 per time point) and prepared for WBAL analysis. STD ranged in radioactivity from 16 to 15,600 nCi/g and were embedded in the specimen blocks with the 0.25-, 0.5-, and 1-h rats. CQCS ranging in radioactivity from 29 to 14,200 nCi/g were embedded in the specimen blocks with the 6-, 12-, and 24-h rats.

Experiment 4. The purpose of this experiment was to compare the tissue distribution, including brain distribution, of two [³H]DEs in the same therapeutic class in two rat models. [³H]DE-3 and nonradioactive DE-3 were codissolved in physiological saline to provide a final concentration of 5.0 mg/ml. The delivery volume (2 ml/kg) of the dosing solution was adjusted for each rat based on body weight. The radioactive potency was 1330 µCi/ml. The dosing solution containing [³H]DE-4 and nonradioactive DE-4 was prepared as described for [³H]DE-3. The final concentration, delivery volume, and radioactive potency of this [³H]DE-4 dosing solution were similar to those for the [³H]DE-3 dosing solution. Two SD and SH male rats were intravenously administered [³H]DE-3 or [³H]DE-4 at a dose of 10 mg/kg (2660 µCi/kg). One rat of each strain for each [³H]DE was euthanized by CO₂ asphyxiation at 0.33 and 2 h postdose. STD ranged in radioactivity from 27 to 34,500 nCi/g and were embedded in the specimen blocks with the SD rats. CQCS ranging in radioactivity from 27 to 28,800 nCi/g were embedded in the specimen blocks with the SH rats.

	Results

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Calibration, Linear Dynamic Range, and Limits of Detection. Precision and accuracy were used as the fundamental criteria to determine the analytical error of the phosphorimager signals generated by the radioactivity concentrations of the STD and CQCS. Measurements of phosphorimager signals were considered acceptable if the radioactivity concentrations were within ±20% of nominal concentrations. The relationship between the radioactivity concentration of each STD and the relative intensity of the phosphorimager signal is shown in Fig. 1. Calibration curves for tritium imaging plates scanned with the 425F PhosphorImager were prepared over the concentration range of 5 to 1.9 x 10⁶ nCi/g. Two of the five STD representing the lowest radioactivity concentrations (5 and 10 nCi/g) did not elicit a phosphorimager signal. Precision and accuracy for the 19, 38, and 68 nCi/g STD were above the 20% acceptable limits and excluded from the standard curves. Precision and accuracy of five additional STD of radioactivity concentrations in the range of 110,000 to 1,900,000 nCi/g were greater than 20% of nominal concentrations and consequently disqualified based upon the failure to meet this acceptance criterion. Curve fitting of these STD obtained from 16 imaging plates after 17-day exposures by weighted (1/x) linear regression resulted in a mean correlation coefficient (r) of 0.998 ± 0.002. Precision for the STD ranged from 11 to 19%. Accuracy for these same STD was calculated to be in the range of 89 to 117% of nominal concentrations. Sixteen CQCS having radioactivity concentrations from 7 to 68,400 nCi/g were used to assess the quality and acceptability of this analytical method (Table 1). At the three lowest concentrations of 7, 9, and 11 nCi/g, digital images of the expected radioactive patterns were not observed on any autoradioluminogram obtained with the 425F PhosphorImager. Precision of the highest radioactivity concentration (68,400 nCi/g) was 12% and met acceptance criteria. However, accuracy of this CQCS did not satisfy acceptance criteria, indicating that the upper limit of quantification was between 37,500 and 68,400 nCi/g. For experiments 1 and 2 involving the 425F PhosphorImager, the linear dynamic range of the STD was 120 to 59,000 nCi/g. The lower limit of quantification based upon CQCS evaluation was 127 nCi/g. These weighted (1/x) linear regression calibration curve and CQCS parameters were then utilized to determine the concentrations of unknown radioactivity in tissues of whole-body cryosection digital images from rats administered [³H]glucose and [³H]DE-1.

View larger version (6K): [in this window] [in a new window]   FIG. 1. A representative whole-body autoradioluminographic tritium standard curve for the Amersham Biosciences PhosphorImagers using Fuji BAS-TR2040 imaging plates. Radioactivity concentration (nanocuries per gram) versus MicroComputer Imaging Device response (MCID; Amersham Biosciences) (counts per micrometer squared minus background counts) of STD prepared by serial dilution of D-[2-3H(N)]glucose with control blood. Duplicate sets of STD were apposed to disposable tritium imaging plates.

Standard curve concentration ranges were variable for experiments 3 and 4 but encompassed values between 16 to 34,500 nCi/g. Curve fitting of these STD obtained from STORM PhosphorImager scans for 84 imaging plates after 17-day exposures by weighted (1/x) linear regression resulted in mean r values of 0.998 ± 0.002 and 0.996 ± 0.005 for experiments 3 and 4, respectively. Precision for the STD ranged from 0.2 to 16%. Accuracy for these same STD ranged from 89 to 107% of nominal concentrations. Three STD representing the lowest radioactivity concentrations of 16, 27, and 30 nCi/g were excluded from the calibration curves, because either the precision or accuracy was ±20% of nominal concentrations. Precision and accuracy for STD of the greatest radioactivity concentrations were well within acceptable limits and consequently included in the calibration curves for experiments 3 and 4. For the two experiments involving the STORM PhosphorImager, the linear dynamic range was 47 to 16,000 nCi/g and 54 to 34,500 nCi/g for experiments 3 and 4, respectively. The lower limit of quantification based upon CQCS evaluation was 54 nCi/g for experiment 3 and 72 nCi/g for experiment 4 (Table 2). These weighted (1/x) linear regression calibration curve and CQCS parameters were then utilized to determine the concentrations of unknown radioactivity in tissues of whole-body cryosection digital images from rats administered [³H]DE-2, [³H]DE-3, and [³H]DE-4.

Experiment 1. Fifteen minutes following a single intravenous bolus injection of [³H]glucose, radioactivity distributed into most tissues regardless of the amount administered (Table 3). Although radioactivity was visibly present in tissues of a rat given [³H]glucose at 275 µCi/kg, the concentration of the [³H]radioactivity was below the 127 nCi/g limit of quantification for adipose, bone marrow, heart, kidney, muscle, pancreas, salivary gland, skin, spleen, and thymus. Only muscle was still devoid of quantifiable concentrations of tritium radioactivity 15 min after receiving 865 µCi/kg [³H]glucose. In rats administered 2360 and 4320 µCi/kg [³H]glucose, radioactivity was above 127 nCi/g for all tissues examined. The eye had the highest concentration of [³H]glucose at all dosages. The uptake of [³H]glucose radioactivity in muscle was the lowest observed for any tissue regardless of dose. The concentration of [³H]glucose radioactivity was lower in the heart, kidney, pancreas, salivary gland, skin, spleen, and thymus than that noted for systemic blood and central nervous system (CNS) tissues. Liver and lung concentrations of [³H]glucose radioactivity appeared to be similar to that of systemic blood and CNS tissues. In addition to the eye, only the Harderian gland and intestinal mucosa had a greater uptake of [³H]glucose radioactivity than systemic blood and CNS tissues, except at the dose of 4320 µCi/kg for the Harderian gland. The uptake of [³H]glucose in most tissues appeared to be proportional to the amount administered up to 2360 µCi/kg. At 4320 µCi/kg, [³H]glucose uptake into tissues continued to increase but was no longer proportional to the increase in dosage. Increasing the dose from 2360 to 4320 µCi/kg (1.8-fold) increased the concentration of radioactivity in tissues only by 1.3-fold. For muscle, this increase in dose resulted in concentrations that were similar to those at 2360 µCi/kg. Concentrations of [³H]glucose were quantifiable in the cerebellum, cerebrum, medulla oblongata, and spinal cord of all four rats at 15 min postdose, regardless of dose. Mean brain to whole-blood ratios remained relatively constant at 0.95 but did fluctuate to 0.84 for the rat administered [³H]glucose at 2360 µCi/kg.

The results of this experiment illustrate that a dose of 275 µCi/kg [³H]glucose was insufficient to quantify levels in approximately half of the tissues examined (Fig. 2). At a dose of 865 µCi/kg, all tissues except muscle were shown to uptake [³H]glucose radioactivity in quantifiable amounts. It appears from our investigation that the minimum amount of tritium necessary to investigate the tissue distribution of [³H]DE in rats using WBAL was a dose of 865 µCi/kg, whereas a dose of 2360 µCi/kg provided excellent qualitative visualization of [³H]radioactivity distribution (Fig. 3).

View larger version (86K): [in this window] [in a new window]   FIG. 2. Distribution of radioactivity 15 min following an intravenous dose of 275 µCi/kg [3H]glucose to a male Sprague-Dawley rat. Note the low concentrations of glucose radioactivity in tissues relative to Fig. 3.

View larger version (61K): [in this window] [in a new window]   FIG. 3. Distribution of radioactivity 15 min following an intravenous dose of 2360 µCi/kg [3H]glucose to a male Sprague-Dawley rat. Note the high concentrations of glucose radioactivity in tissues relative to Fig. 2.

Experiment 2. The uptake of [³H]DE-1 was observed in 17 tissues following a single intravenous bolus injection (Table 4). Thirteen tissues had higher concentrations of [³H]DE-1 than blood at 2 h. The highest concentration of [³H]radioactivity was found in the liver. The concentration of [³H]DE-1 in white adipose was approximately half of that measured in liver. The uptake of [³H]DE-1 in muscle was the lowest observed for any tissue in which quantifiable levels could be measured. The lack of quantifiable [³H]DE-1 in the tissues of the CNS, other than the pituitary and pineal gland, suggested that this DE does not readily distribute into brain tissues. By 12 h postdose, [³H]radioactivity had declined below the WBAL assay quantification limits for all tissues except adipose (brown and white), Harderian gland, kidney (cortex), liver, and lung. At 96 and 168 h postdose, only white adipose still had quantifiable concentrations of [³H]DE-1, demonstrating that [³H]DE-1-related material was slowly eliminated from this tissue compartment. The largest decline in [³H]radioactivity from white adipose occurred from 2 to 12 h, and elimination was slower from 12 to 168 h.

Experiment 3. The uptake of [³H]DE-2 was observed in most tissues at 0.25 h postdose following intravenous administration (Table 5). Exceptions included white adipose, lens, and vitreous body. These three tissues were devoid of [³H]DE-2-associated radioactivity at all sampling times. Twenty tissues had higher concentrations of [³H]radioactivity than blood at 0.25 h. The highest concentrations of [³H]radioactivity were found in CNS tissues, bile, and thyroid. High concentrations of [³H]radioactivity present in the brain at 0.25 h indicated that [³H]DE-2 readily distributed into brain tissues. [³H]DE-2 in blood, muscle, and skin were the lowest observed for any tissue. Depending upon the tissue, maximum mean concentrations of [³H]radioactivity occurred either at 0.25, 0.5, or 1 h following the administered intravenous dose. Tissue [³H]radioactivity appeared to decrease during the course of the study in all tissues except for testis and skin. Testicular concentrations of [³H]radioactivity were relatively unchanged throughout the 0.25- to 24-h time course, suggesting a slow elimination of drug-related material from this organ. Although skin concentrations of [³H]DE-2 declined between 1 and 12 h, the 24-h concentration was similar to 12-h levels.

The t_1/2 of [³H]radioactivity measured for five brain tissues ranged from 2.9 to 6.9 h (mean, 4.8 h). Radioactivity was eliminated from whole blood with a t_1/2 of 3.6 h. Based on AUC_{(0-T last)}, the cerebrum to whole-blood ratio was 20. These WBAL findings provided visual and quantitative confirmation that [³H]DE-2 distributed into brain tissues following intravenous administration (Fig. 4).

View larger version (49K): [in this window] [in a new window]   FIG. 4. Distribution of radioactivity 0.5 h following an intravenous dose of 1880 µCi/kg [3H]DE-2 to a male Long-Evans rat. Note the high concentrations of DE-2 radioactivity in cerebellum, cerebrum, olfactory bulb, spinal cord, kidney, lung, spleen, thyroid, and some segments of the GIT.

[³H]Radioactivity present in bile ducts and renal pelvis indicated that [³H]DE-2 (or its metabolites) was eliminated by the liver and kidneys. The presence of [³H]radioactivity in the gastrointestinal tract (GIT) contents represents biliary excretion and possible intestinal secretion of [³H]DE-2 radioactivity. The urinary bladder of the 0.25- and 6-h rats contained [³H]radioactivity (5180 and 1480 nCi/g, respectively).

Experiment 4. In SD and SH rats, the penetration of [³H]DE-3 and [³H]DE-4 after an intravenous dose occurred rapidly into each region of the brain. It was apparent from the autoradioluminograms and quantitative data that the penetration of [³H]DE-3 into brain tissues from whole blood occurred more freely than the penetration of [³H]DE-4 (Figs. 5 and 6). It was also apparent that the penetration of [³H]DE-3 and [³H]DE-4 into tissues occurred more freely in SD rats than in SH rats.

View larger version (44K): [in this window] [in a new window]   FIG. 5. Distribution of radioactivity 15 min following an intravenous dose of 2660 µCi/kg [3H]DE-3 to a male Sprague-Dawley rat. Note the high concentrations of DE-3 radioactivity in cerebellum, cerebrum, spinal cord, bone marrow, preputial gland, salivary gland, urine, and some segments of the GIT relative to Fig. 6.

View larger version (46K): [in this window] [in a new window]   FIG. 6. Distribution of radioactivity 15 min following an intravenous dose of 2660 µCi/kg [3H]DE-4 to a male Sprague-Dawley rat. Note the lower concentrations of DE-4 radioactivity in the cerebellum, cerebrum, spinal cord, bone marrow, preputial gland, salivary gland, urine, and some segments of the GIT relative to Fig. 5.

In general, regardless of rat strain, substantially higher maximum mean concentrations of [³H]DE-3-associated radioactivity were measured in all regions of the brain than those measured for [³H]DE-4 (Table 6). [³H]DE-3 radioactivity was ca. 3-fold greater than [³H]DE-4 radioactivity in selected brain tissues (i.e., caudate putamen, cerebellum, cerebrum, and olfactory bulb). The pituitary contained the highest concentrations of radioactivity after the administration of either [³H]DE-3 or [³H]DE-4. The penetration of radioactivity into the pituitary was 2.6- and 2.4-fold higher for [³H]DE-3 than for [³H]DE-4 in SD and SH rats, respectively. The penetration of [³H]DE-3 and [³H]DE-4 radioactivity into the medulla oblongata and spinal cord of SD rats occurred in a manner similar to the penetration noted for other brain tissues. In SH rats, the penetration of radioactivity into the medulla oblongata and spinal cord was 2.4- and 2.1-fold higher for [³H]DE-3 than for [³H]DE-4, respectively. By 2 h postdose, [³H]DE-3 and [³H]DE-4 radioactivity in all brain tissues had declined from 0.33-h concentrations in both SD rats and SH rats. Most notably, all brain tissues except the pituitary in the SH rat were devoid of [³H]DE-4 radioactivity at 2 h.

Mean concentrations of radioactivity in glandular tissues were consistently higher in rats administered [³H]DE-3 than for rats administered [³H]DE-4. For SD rats, most glandular tissue [³H]radioactivity concentrations were 1.6- to 2.7-fold higher following [³H]DE-3 administration than those measured after [³H]DE-4 administration, except for the Harderian gland. For SH rats, glandular tissue [³H]radioactivity concentrations (except for the pancreas) were 1.5- to 3.0-fold higher following [³H]DE-3 administration than those measured after [³H]DE-4 administration. Pancreatic concentrations of [³H]DE-3 and [³H]DE-4 radioactivity in SH rats at 0.33 h postdose were similar. Radioactivity concentrations of [³H]DE-3 and [³H]DE-4 in all other tissues where radioactivity was measured followed similar patterns of distribution differences noted in brain and glandular tissues of SD and SH rats. Adipose was devoid of [³H]DE-3 and [³H]DE-4 radioactivity in both rat strains.

	Discussion

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The linearity properties and relatively shorter exposure times of storage phosphor imaging plates make them more ideal than X-ray film for use in tissue distribution studies that require accurate and precise quantification. In any analytical method, the calibration curve needs to be constructed with STD over a concentration range expected in the samples being analyzed. The inclusion of CQCS provides a quantitative measurement of sensitivity and the foundation for determining the error associated with the storage phosphor technology. Calculating the accuracy and precision of quality control samples assesses the quality of any analytical method. In this study, CQCS were used to monitor the quality of the storage phosphor calibration curve. The 425F PhosphorImager calibration curves for experiment 1 were prepared over a dynamic range that spanned 7 orders of magnitude (5 to 1.9 x 10⁶ nCi/g). The linear response of these STD was limited to 3 orders of magnitude encompassing 120 to 59,000 nCi/g. The quality of these curves was evaluated with CQCS that covered a radioactivity range of 7 to 68,400 nCi/g. CQCS response verified the linearity, reproducibility, limit of quantification, and ruggedness of 17 calibration curves. Based upon this CQCS response, the working linear dynamic range of the 425F PhosphorImager calibration curves was constrained from 127 to 37,500 nCi/g. High background levels resulting from cosmic and environmental radiation over the 17-day exposure period influenced the lower limit of quantification. The upper limit of quantification was restricted for the following reason: at high concentrations of radioactivity, the 425F PhosphorImager generates flare. Increases in radioactivity concentration results produce a proportional increase in flare production. Flare is due to stray laser light generated after striking high-intensity signals on the imaging plate. It is characterized on digital images by a halo effect surrounding the target tissue and causes a loss of clarity of well-defined boundaries between tissues. The intensity of flare signals can obliterate the observation of an entire tissue in close proximity to the signal causing the flare event. Consequently, the ability to quantify the radioactivity in tissues covered with flare becomes impossible, and valuable tissue distribution data are lost. The necessity of a 17-day exposure to maximize the lower limit of quantification also allows for the generation of increased flare intensity and a nonlinear response at the upper limits of quantification. The results of this validation of the tritium WBAL assay developed the calibration and CQCS foundation for experiments 2, 3, and 4. In addition, to reduce flare intensity, the STD of high radioactivity was restricted to 19,800 nCi/g for experiment 2. Nominal concentrations of tritium radioactivity for the high STD for experiments 3 and 4 were limited to 14,200 and 28,800 nCi/g, respectively.

The STORM PhosphorImager response to tritium revealed improvements in quantification compared with that observed for the 425F PhosphorImager. The dynamic range was still limited to 3 orders of magnitude, but a lower quantification limit was obtained in experiments 3 (54 nCi/g) and 4 (72 nCi/g). This improvement in response may be attributed to the redesigned mechanical operations of the scanning laser beam and scanner platform resulting in lower background readings and a significant reduction of flare generation for the STORM PhosphorImager. These modifications were responsible for an average 2-fold increased sensitivity as realized by an improved lower limit of quantification over the results obtained with the 425F PhosphorImager. The use of CQCS ensured that any variation in obtaining cryosections of the specimens would also be reflected in the phosphorimager signal produced by the CQCS. Variations in section thickness would produce similar variations in the intensity of the phosphorimager signal. The greater the variation in section thickness, the greater the deviation will be from the expected nominal concentration of radioactivity. Thus, the inclusion of CQCS in the same block as the specimens was an appropriate measure of accuracy and precision for testing the validity and quality of this WBAL analytical method.

The results of these experiments illustrated that a good linear correlation exists between the concentration of tritium radioactivity and the PhosphorImager response. In comparison, the STORM PhosphorImager has a lower limit of quantification of 1.6 nCi/g for carbon-14 and a linear dynamic range that expands across 4 orders of magnitude (i.e., 1.6-39,000 nCi/g). Based upon the lower quantification limits, WBAL analysis employing tritium appears to be at least 35-fold less sensitive than that observed for carbon-14.

The length of the WBAL study was shown to be dependent on the objectives of the study. In experiment 2, for example, which was designed to determine distribution to a deep compartment and for which the use of CQCS was not necessary, exposure was held constant at 17 days, and the study was successfully completed in 32 days. In comparison, experiment 3 was conducted to characterize the distribution of a lead discovery candidate, particularly to assess its ability to distribute into brain tissues. Since the study design primarily required the use of CQCS, 38 days were required to complete this experiment. In contrast, 45 days were required to complete experiment 4, in which the disposition of two discovery compounds in two different rat strains was assessed.

A comparison of study lengths between WBAL and WBA experiments employing tritium demonstrates that the amount of time required to complete a WBAL study was always less than the 2 or 3 months typically required just for exposure of samples to X-ray films in WBA studies. The results of these three experiments clearly illustrated that the application of storage phosphor technology (i.e., WBAL) in the discovery process required significantly less time (greater than 2-fold) than those studies using X-ray film (i.e., WBA) as the medium to capture data. Utilization of WBAL to address specific discovery issues to aid in the selection of a candidate for full development was accomplished in a timely and efficient manner while generating useful quantitative data. To decrease the WBAL study turnaround time even further, the 17-day exposure time can be shortened provided that the lower quantification limit can be sacrificed without jeopardizing the objectives of the experiment.

Shimada et al. (1976) reported the uptake of [U-¹⁴C]glucose in albino mice at 30 min after an intraperitoneal injection was greatest in the Harderian gland and intestinal mucosa based upon the autoradiographic density as measured with the aid of a microdensitometer. Relatively intense autoradiographic densities were also observed for cerebral gray matter, pancreas, and bone marrow, indicative of a high uptake of glucose that was less than the uptake noted for the Harderian gland and intestinal mucosa. The exposure length of 1 week for these mouse cryosections to Softex HS X-ray film allowed for the characterization of [U-¹⁴C]glucose uptake into approximately 17 tissues. This current WBAL study that investigated the quantitative distribution of D-[2-³H(N)]glucose in the nonpigmented rat revealed similarities to the aforementioned mouse study. Both studies illustrated very similar distribution patterns, with some of the same tissues having the greatest glucose uptake. Both studies also clearly revealed a high uptake of glucose into the brain. In this rat WBAL study, the eye was observed to contain the greatest amount of tritium radioactivity. Ocular uptake of [¹⁴C]glucose was not discussed by Shimada et al. (1976). This WBAL investigation in the rat illustrated that storage phosphor quantification of [³H]glucose provided quantification data in addition to generating comparable qualitative results to the qualitative assessment of Shimada et al. (1976) with [¹⁴C]glucose using X-ray film.

The application of WBAL routinely investigates the fate of a radiolabeled DE from the site of administration to its tissue distribution sites, including small anatomical structures, within the intact whole-body. Over a predetermined time course based on the T_max of the parent DE, WBAL can provide pivotal screening data pertaining to tissue pharmacokinetics; tissue accumulation and retention; penetration into brain, tumors, or other target tissues of interest; and routes of elimination. Depending upon specific issues generated during the selection process for the best suitable DE, the utilization of WBAL can successfully provide answers in a timely manner. As illustrated in experiment 2, adipose was identified as the tissue compartment responsible for selective partitioning of [³H]DE-1. The retention and, consequently, the slow elimination of [³H]DE-1 from adipose explained the observed triphasic decline in DE-1 plasma concentrations over time in pharmacokinetic studies in rats. In experiment 3, the utilization of WBAL simply illustrated the penetration of [³H]DE-2 into brain tissues in concentrations that were sustainable for at least 24 h postdose. This experiment also revealed that [³H]DE-2 was retained in the testis, Harderian gland, and lacrimal gland that exhibited little or no deviation in concentration over the time course of the study. This observed retention of [³H]DE-2 raised potential toxicological concerns that could be incorporated into prospective toxicology studies. Experiment 4 compared the distribution of two [³H]DEs in SD and SH rats. WBAL results revealed the ability of [³H]DE-3 to distribute into tissues at higher concentrations than those noted for [³H]DE-4 regardless of rat stain. The distribution of [³H]DE-3 and [³H]DE-4 achieved greater concentrations into tissues of the SD rat relative to the SH rats. This type of informative WBAL data can be instrumental in aiding in the selection of the lead candidate for drug development.

In summary, WBAL can be successfully utilized to characterize the distribution of [³H]DE in drug discovery. The use of tritium as the radiolabel of choice is of key importance, because this material can be synthesized more readily than [¹⁴C]DE. Typically, a tritium synthesis on average can be completed in about 3 weeks while achieving a [³H]DE with a radiochemical purity of >95%. The synthesis of a [¹⁴C]DE requires a minimum of 3 months. Since the preparation of radiolabeled test materials is a major gating factor in study turnaround, the use of tritium is key to the timely completion of experiments in the discovery setting. These WBAL experiments have demonstrated that the use of tritium can play a significant role in the discovery process for new drugs. In programs where the distribution of the drug to target tissues is an issue, resolution is achievable within a 2-month time frame using tritium as opposed to a minimum of 5 months for WBAL studies using carbon-14 when factoring in the time required to synthesize these radiochemicals.

	Footnotes

 
ABBREVIATIONS: WBA, whole-body autoradiography; WBAL, whole-body autoradioluminography; DE, drug entity; HPLC, high-performance liquid chromatography; TLC, thin layer chromatography; SD, Sprague-Dawley; SH, spontaneously hypertensive; STD, standard curve calibrator(s); CQCS, cryosection quality control sample(s); AUC, area under the curve; CNS, central nervous system; GIT, gastrointestinal tract.

Address correspondence to: Michael J. Potchoiba, Pfizer Inc., Global Research and Development, PDM Development, MS4096, Eastern Point Road, Groton, CT 06340. E-mail: michael_j_potchoiba{at}groton.pfizer.com

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