Validation of procedures for quantitative whole-body autoradiography using digital imaging.

The objective of this study was to demonstrate that tissue concentrations of radioactivity derived by digital analysis of autoradiograms were comparable to values derived from direct sampling and analysis of tissues. In addition, we describe the preparation and calibration of standards for use in quantitative whole-body autoradiography. For this study, three male Long-Evans hooded rats were administered 14C radioactivity intravenously. The animals were sectioned for whole-body autoradiography, with concomitant sampling of blood and 16 selected tissues. After 3 weeks of film exposure, the optical densities of the resulting autoradiograms were analyzed with a RAS3000 digital imaging system to estimate tissue concentrations of radioactivity. These concentrations were then compared with those obtained by direct analysis of the tissue samples. The concentrations derived from digital analysis of the autoradiograms were very highly correlated with those determined from direct tissue analysis (r = 0.956). Linear regression analysis yielded a straight line with a slope of 0.97 and a goodness of fit (r2) of 0.913. This analysis suggested that there is an approximate 1:1 correlation between concentration values determined by the two methods. Marked differences between the values derived via the two techniques were observed for only three tissues. However, this subset of the data accounted for only 6% of the total data, and the differences were probably due to contamination from adjacent tissues during excision. Overall, the concentrations of radioactivity derived from digital analysis of the autoradiograms were comparable to those derived from direct analysis of tissue samples. The results indicated that the digital analysis procedure for film can serve as a valuable adjunct to conventional tissue analysis for radioactivity.

Automated-extraction, high-performance liquid chromatographic method and pharmacokinetics in rats of a highly A2-selective adenosine agonist, CGS 21680.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine, I) is a highly A2-selective (A2/A1 = 140), high-affinity adenosine agonist. A method has been devised to extract the compound from biological matrices with automated solid-phase extraction using C18 bonded silica columns. This is followed by reversed-phase, paired-ion chromatography on a Supelco LC-18-S column with fluorescence detection. The limit of quantitation is 5 ng/ml, but 1 ng/ml (five times the signal-to-noise ratio) can readily be detected. Tritium-labeled compound was used to study the pharmacokinetics in rats. After an intravenous dose of 0.3 mg/kg, biphasic elimination kinetics were observed for parent I, characterized by half-lives of 1.8 min (distribution) and 15 min (elimination). The volume of distribution in the terminal phase (V beta) was low (0.27 l/kg) and plasma clearance was moderate (0.83 l/kg/h). Although the compound was rapidly absorbed (mean Tmax = 13 min), low concentrations (mean Cmax = 94 ng/ml) were observed after an oral dose of 3.0 mg/kg, and bioavailability was only approximately 1.4%. Radioactivity persisted in plasma longer than parent compound after either dose, but levels were too low for isolation and structure identification of drug-derived compounds.

Physicochemical factors associated with binding and retention of compounds in ocular melanin of rats: correlations using data from whole-body autoradiography and molecular modeling for multiple linear regression analyses.

The relationship between the physicochemical characteristics of 27 new drug candidates and their distribution into the melanin-containing structure of the rat eye, the uveal tract, was examined. Tissue distribution data were obtained from whole-body autoradiograms of pigmented Long-Evans rats sacrificed at 5 min and 96 hr after dosing. The physicochemical parameters considered include molecular weight, pKa, degree of ionization, octanol/water partition coefficient (log Po/w), drug-melanin binding energy, and acid/base status of the functional groups within the molecule. Multiple linear regression analysis was used to describe the best model correlating physicochemical and/or biological characteristics of these compounds to their initial distribution at 5 min and to the retention of residual radioactivity in ocular melanin at 96 hr post-injection. The early distribution was a function primarily of acid/base status, pKa, binding energy, and log P(o/w), whereas uveal tract retention in rats was a function of volume of distribution (V1), log P(o/w), pKa, and binding energy. Further, there was a relationship between the initial distribution of a compound into the uveal tract and its retention 96 hr later. More specifically, the structures most likely to be distributed and ultimately retained at high concentrations were those containing strongly basic functionalities, such as piperidine or piperazine moieties and other amines. Further, the more lipophilic and, hence, widely distributed the basic compound, the greater the likelihood that it interacts with ocular melanin. In summary, the use of multiple linear regression analysis was useful in distinguishing which physicochemical characteristics of a compound or group of compounds contributed to melanin binding in pigmented rats in vivo.

Calculation of drug-melanin binding energy using molecular modeling.

Conformational analysis and molecular graphics are used to model a representative melanin structure to estimate a chemical's in vitro affinity for melanin. The modelling data fit to a simple linear equation relative to a logarithmic transformation of the experimentally-derived binding data (r = 0.901). The goodness of fit, as evidenced by the F-statistic, F (1,14) = 60.09 (p = 0.000002), for the regression indicates that this technique gives an accurate representation of the interaction of these chemicals with melanin in vitro.