The absorption, distribution and excretion of ethyl 2-[4, 5-bis (4-methoxyphenyl) thiazol-2-yl]pyrrol-1-ylacetate (KBT-3022) were studied after administration of ¹⁴C-KBT-3022 to mice.
Maximum plasma levels (Cax) appeared at 0.5 hr after oral administration of ¹⁴C-KBT-3022 to fasted male mice and ¹⁴C-KBT-3022 was absorbed from small intestine, especially from jejunum. The radioactivity decreased with half-life of 5-6 hr. The C_max and the area under the plasma concentration-time curve (AUC) were higher in unfasted mice than in fasted mice.
The percentages of radioactivity excreted in urine and feces, whithin 96 hr after intravenous or oral administration of ¹⁴C-KBT-3022 to male mice, were below 1% and above 96% of dose, respectively. The percentages of radioactivity excreted in urine and feces after oral administration of ¹⁴C-KBT-3022 were similar both in male and female mice. The ¹⁴C-KBT-3022 probably is excreted mainly in bile. The radioactivities in the intestine and liver were higher than in plasma, however, radioactivities were rapidly eliminated from tissues so that no significant remaining was observed in the tissue. After oral administration of ¹⁴C-KBT-3022 to fasted mice, the radioactivites in the platelet and aorta were 2.4-15.7 times higher and about 0.5 times lower than that in plasma, respectively. The elimination half-life of radioactivity in the platelet was close to that in plasma. This result shows KBT-3022 is reversibly distributed in platelet.

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Absorption, distribution, excretion, enzyme induction and effect to platelet were investigated in male mice after oral administration of an antiplatelet drug, KBT-3022.
1. Radioactivity levels in the plasma 24 hr after daily oral administration of ¹⁴C-KBT-3022 reached a steady state by the 4th dosing. The radioactivity levels after the 21st dosing showed a broad peak with about half of the Cmax after single dosing but had a similar AUC_{0-24 h}.
2. Radioactivity levels in tissues 24 hr after daily oral administration of ¹⁴CKBT-3022 increased with the number of doses and reached a steady state by the 21st dosing except the spleen. The radioactivity levels in the lung, heart and testis after the 21st dosing were 5-6 times higher than those after single dosing, but those in the other tissues were lower than 4 times. Elimination of radioactivity from tissues after the 21st dosing was slower than that after single dosing. At 72 hr after the 21st dosing, the radioactivity level in the aorta was 50% of the maximum and those in the skin, testis, lung, fat and spleen were 24-35% of the maximum. High level of radioactivity was noted in the deferent duct as revealed by whole body autoradiography.
3. No marked difference was observed in plasma concentration of 6-keto-PGF_1α and serum concentration of TXB2 between the single and repeated dosing groups, although the accumulation of radioactivity was noted in the aorta after multiple doses of ¹⁴C-KBT-3022. From these results, metabolites in the aorta would not inhibit the production of PGI₂.
4. Excretion of radioactivity in the urine and feces did not change with the number of doses and was 1.3 and 92.7% of the cumulative dose, respectively, within 120 hr after the 21st dosing.
5. No significant effect on hepatic drug-metabolizing enzyme system was observed after repeated dosing of KBT-3022.

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Transfer to fetus in mice and milk in rats were investigated after oral administration of an antiplatelet drug, ¹⁴C-KBT-3022 at a dose of 2 mg/kg.
1. The level of radioactivity in the fetus as revealed by whole body autoradiography, was lower than in the maternal blood and decreased to a trace levels at 24 hr after administration to fasting mice on the 13th day of gestation.
2. The radioactivity level in the fetus was less than 36% of that in the maternal plasma up to 8 hr after administration to fasting mice on the 18th day of gestation. And the radioactivity level at 24 hr decreased to 55% of that found at 8 hr. Fetal intestine showed a high level of radioactivity but the other fetal tissues showed low levels as shown by whole body autoradiograms.
3. The radioactivity levels in the milk reached a maximum of 105 ng/ml at 8 hr after administration to non-fasting lactating rats and were below the detection limit at 48 hr. Compared with the radioactivity levels in the plasma, the radioactivity levels in the milk were 2 and 3 times higher at 8 and 24 hr after administration, respectively.

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The absorption, distribution and excretion of N-[p-[2-(Dimethylamino)-ethoxy]benzyl]veratramide hydrochloride (HSR-803) were investigated in rats and dogs following single administration of ¹⁴C-labelled compound.
1. Blood and serum radioactivity reached the C_max at 0.5 to 1 hr after single oral administration of ¹⁴C-HSR-803 to rats and dogs, and then declined with half-lives of 3.42, 10.30 hr for whole blood and 3.78, 10.12 hr for serum, respectively. The site of absorption in rats was the entire intestine, particularly the duodenum.
2. Radioactivity level was high in tissues such as the small intestine, stomach, kidney and liver of rats after oral administration of ¹⁴C-HSR-803. In the central nervous system, the concentration was low. Elimination from each tissue was rapid. Radioactivity in the stomach wall in rats was high after intravenous administration of ¹⁴C-HSR-803.
3. When ¹⁴C-HSR-803 was administered orally, urinary and fecal excretions were 63.6 and 35.7% of the dose for rats, and 46.7 and 50.9% of the dose for dogs, as determined within 168 hrs. In rats and dogs, 68.1 and 79.3% of the dose were excreted into bile after oral dosing, respectively. In rats, enterohepatic circulation was observed.
4. In vitro protein binding to human, rat and dog serum was 95.5, 79.6 and 57.5% at the concentration range of 0.2-10.0 μg/ml, respectively.

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The absorption, distribution and excretion of N-[p-[2-(Dimethylamino)ethoxy]benzyl]veratramide hydrochloride (HSR-803) in male rats after repeated oral dosing were studied using ¹⁴C-labelled HSR-803. The transfer of radioactivity into the fetus and milk after single oral administration of ¹⁴C-HSR-803 to pregnant rats or lactating rats were examined.
1. When ¹⁴C-HSR-803 was administered repeatedly to male rats, the Cmax of blood radioactivity for each dosing was nearly constant, and the radioactivity in tissues almost reached a plateau after the 7th administration.
In final administration, the time courses of radioactivity in the blood and most tissues were essentially the same as those after single dosing. The urinary and fecal excretion of radioactivity in rats were nearly constant during repeated administrations of ¹⁴C-HSR-803, and similar to those after single administration. In conclusion, there was no accumulation of radioactivity after repeated administration of ¹⁴C-HSR-803.
2. The tissue distribution of radioactivity in the fetus was apparent after oral administrations of ¹⁴C-HSR-803 to pregnant rats on days 13 and 19. Radioactivity in the fetal liver was twice of that in the maternal serum, and that in other fetal tissues was basically the same as in maternal serum.
3. Radioactivity was detected in the milk after oral administrations of ¹⁴C-HSR-803 to lactating rats. Radioactivity in the milk was 1.1 (0.5 hr) and 3.9 times (12 hr) higher than that in maternal serum. The distribution of radioactivity in rat fetus was less than 0.18% of the dose at 6 hr after dosing.

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The metabolism of N-[p-[2-(Dimethylamino)ethoxy]benzyl]veratramide hydrochloride (HSR-803) was investigated using both ¹⁴C-labelled and unlabelled HSR-803.
1. Unchanged HSR-803, the oxidative demethylated product at the position of the dimethylamino-group (N-demethyl metabolite, M1), the additionally oxidized product after leaving the methylamino-group from M1 (Carboxylic acid metabolite, M3) and oxygen added product at the position of dimethylaminogroup (N-oxide metabolite, M2) were identified in the urine after oral administrations of HSR-803 to rats.
2. M2 appeared in the serum and reached C_max 1 hr after single intravenous administrations of ¹⁴C-HSR-803. Serum level of M2 was subsequently almost the same as that of unchanged HSR-803. The level of unchanged HSR-803 was higher than those of other metabolites in all tissues after single intravenous or oral administration of ¹⁴C-HSR-803.
3. Unchanged HSR-803, M1, M2 and M3 were excreted in the urine and bile in rats and dogs after dosing, however, M2 was predominantly excreted. Urinary and biliary excretion of unchanged HSR-803 in rats exceeded those in dogs.

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The imaging plate technique (radioluminography, RLG) is a fascinating new tool in whole-body autoradiography (WBAR). While being a standard technique for the qualitative evaluation of the distribution of new drugs for many years, WBAR has some major drawbacks preventing its broader use for quantitative studies (time consuming, difficult to standardize, very limited linearity, low sensitivity). Most of these problems are now solved by RLG, thus offering the possibility to perform also quantitative distribution studies with autoradiographic methods (QWBAR). However, before using RLG during drug development, a careful validation of the method has to be done. Crucial points are linearity for concentration and exposure time, reproducibility, as well as stability of information and resolution. Influences of exposure temperature, shielding, and imaging plate handling have to be standardized. By the use of internal standards and correction for selfabsorption in different tissues, calibration was performed and compared to a tissue dissection study. It is concluded that the RLG is a real break through from the scientific point of view. It clearly will replace WBAR with X-ray film for carbon-14. For its quantitative use, RLG may combine the advantages of WBAR (high resolution, completeness of the distribution pattern) and the quantitative distribution studies (concentrations, accumulation factors, time course of elimination from organs and tissues). However, scientific and economic aspects have to be carefully evaluated before replacing the quantitative distribution studies as a whole by QWBAR. To date only a selective use of the RLG for the evaluation of accumulation in substructures, placental transfer studies, and urgent issues during drug development can be proposed.

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A method alternative to liquid scintillation counting (LSC) for detecting β emitters was developed. The new method involves putting an aqueous ¹⁴C sample onto the flatt-bottomed wells of a polystyrene microplate, drying the sample and determining the radioactivity using radioluminography (RLG). It provides a simple, inexpensive and reliable technique for determining the radioactivity of a few hundred samples simultaneously. ³H can be determined by using a microplate having wells interconnected with shallow vent channels and exposing in helium atmosphere. ³²P can be determined by placing the sample planchettes in shielding adapter made of brass, which prevents each sample from cross-talking. This method was also applied to an off-line counting of ¹⁴C in HPLC eluate. A succession of aliquots of the eluate were collected directly in the wells of microplates and evaporated to dryness. The detection limit was 0.35 Bq per injection. The RLG off-line counting method was utilized to determine [¹⁴C] EPA metabolites formed by rat hepatic microsomes.
Radioluminography, microplate, liquid scintillation counting, β emitters, cross-talk, off-line counting of HPLC eluate, EPA
A substitute for liquid scintillation counting, a simple, inexpensive, sensitive and reliable technique for determining β emitters, applicable to off-line counting of HPLC eluate.

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To test and evaluate possible application of radioluminography (RLG) for pharmacokinetic and drug metabolism studies, the validation studies were investigated with research laboratories from 21 of industrial companies. Special attention were made on evaluation of applicability of the methods to quantify regional radioactive intensity on whole body autoradiographic sections. For this purpose, comparison were made on radioactive quantification of tissue distribution by both conventional tissue dissection method quantified with liquid scintillation and quantitative whole body autoradiography (WBA) with the use of RLG. ¹⁴C labelled indomethacin and chloramphenicol were used and an experiment with one compound for one quantification method were carried out in a laboratory. After summerized the results obtained, it was indicated that quantification of radioactivity with RLG were proved to be sensitive and accurate for determining radioactive concentration in biological materials after drug administration, and to be useful tool for quantifying tissue distribution by WBA, suggesting that WBA-RLG methodncould be an equivalent, and replaceable in some cases, to the conventional method as a common method for all facilities.
On the course of the study, several technical problems to be considered were pointed out as follows. (1) Calibration of the relative intensity of RLG should be needed to convert to the tissue concentration of the radioactivity (nCi/g) based on data obtained from liquid scintillation counting of tissues corresponding to the tissue remaining in the same sectioned body. (2) In WBA-RLG, over estimation of tissue concentration due to the presence of blood in the section will be considered, if required to show true intra-tissue concentration. (3) In some tissues like pulmonary tissue having low specific gravity intensity, essential difference of the quantification between the conventional and WBA-RLG method could give different data due to essential difference of two methods, i.e. the former is based on the direct tissue weighing and the latter on the determination of the surface intensity of the radioactivity.
As the results, it was demonstrated that WBA-RLG method could offer the accurate tissue distribution data, and be used for new drug registration, if above mentioned three items were satisfied.
The present study also demonstrated that RLG is excellent tool for determining concentration of metabolites in several biological fluids after separation with thin layer chromatography.

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