The absorption, distribution and excretion of a benzoylphenylurea derivative, HO-221 were studied in rats after single oral administration of ¹⁴C(Pc)-HO-221 or ¹⁴C(Pn)-HO-221.
1. The radioactivity level in the blood reached a maximum at 12 hr after administration of ¹⁴C(Pc)-HO-221 to male and female rats at a dose of 25 mg/kg. The absorption ratio was about 9 % as caluculated from the mass balance study. The Cm and AUC were not dose-related at the dose range of 8.3 to 25 mg/kg. The absorption from the digestive tracts tended to decrease by fasting. The C_max and AUC derived from ¹⁴C(Pn)-HO-221 were both 2 times higher than those derived from ¹⁴C(Pc)-HO-221.
2. The radioactivity levels in the fat, liver, brown fat, adrenal gland and Harderian gland were high after administration of ¹⁴C(Pc)-HO-221 to male and female rats. The radioactivity in the tissues was eliminated rapidly, except the kidney and liver.
3. The excretion of radioactivity in the urine and feces was about 2 and 95% of the dose, respectively, within 120 hr after administration of ¹⁴C (Pc)-HO-221 to male and female rats. The excretion of radioactivity in the bile was about 5% of the dose. About 30% of the biliary excreta (1% of the dose) was re-absorbed. The excretion of radioactivity in the urine was 3 % of the dose within 120 hr after administration of ¹⁴C(Pn)-HO 221.
4. The protein binding ratios in vitro and vivo were more than 99% but the binding was reversible.

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The absorption, distribution and excretion of a benzoylphenylurea derivative, HO-221 were studied after repeated administration of ¹⁴C(Pc)-HO-221 once a day for a maximum of 28 days to male rats at a dose of 25 mg/kg. The radioactivity levels in the blood at 24 hr after daily dosing reached a steady state after the 20th dosing, being 2 times higher than after the 7th dosing. The radioactivity levels in all the tissues after the 28th dosing were less than 2 times of those after single dosing. The radioactivity was still detected at 240 hr after administration but only in the kidney and liver. The excretion of radioactivity in the urine and feces 24 hr after daily dosing did not change after the 4th dosing, and after the final repeated dosing was similar to that after single dosing.
The metabolism was investigated after administration of ¹⁴C(Pc)-HO-221 or ¹⁴C(Pn)-HO-221 to male rats. The parent compound accounted for about 70% of the radioactivity in the plasma at 6 hr after administration. M5, M6, M7 and ο-nitrobenzamide were also detected in the plasma. The parent compound and M5 were principally observed in the liver, kid ney and fat. The conjugate of M6 was major metabolite in the urine, accounting for about 46% of the radioactivity in the urine. Unknown metabolites were major components in the bile. The parent compound and M5 were major in the feces. No difference was noted in urinary and fecal metabolites between the single and repeated dosing groups.
UDPG transferase activity increased significantly by repeated dosing of HO-221.

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The transfer into fetus, milk and tumor of a benzoylphenylurea derivative, HO-221 was studied after oral administration of ¹⁴C(Pc) HO-221 to rats at a dose of 25 mg/kg.
The radioactivity levels in the fetuses on the 12th and 18th days of gestation were 35 and 67% of those in the maternal plasma, respectively. The radioactivity level in the fetus on the 12th day of gestation was below the detection limit at 48 hr after administration. The radioactivity levels in the fetal tissues, except the intestinal contents, on the 18th day of gestation were less than 14% of the maximum.
The radioactivity level in the milk was 3-4 times higher than that in the plasma up to 12 hr after administration. The elimination of radioactivity from the milk was more rapid than from the plasma.
The radioactivity level in the tumor was similar to that in the plasma. No difference was noted in the distribution of radioactivity into the tissues and the excretion of radioactivity in the urine and feces between tumor bearing and normal male rats.

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The absorption, metabolism and excretion of benzoylphenylurea derivative, HO-221 in male dogs were studied after oral administration of ¹⁴C(Pc)-HO-221 at a dose of 25 mg/kg.
1. The radioactivity levels in the plasma reached a maximum at 24 hr after administration and declined with a half-life of 220 hr up to 120 hr.
2. The excretion of radioactivity in the urine and feces was 0.2 and 98.2% of the dose, respectively, within 120 hr after administration. The excretion of radioactivity in the bile was 0.5% of the dose within 48 hr after administration.
3. The protein binding ratios in vitro and vivo were more than 99% both in the case of dog or human plasma, but binding was reversible.
4. The parent compound was a major component in the plasma. M5 and M6 were also observed. Polar metabolites in origin accounted for about 80% of the radioactivity in the urine. The parent compound was a major component in the feces and M5 was detected as a minor metabolite. Polar metabolites in origin were major in the bile, part of which were conjugates.

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Plasma levels, distribution, excretion and metabolism of acetaminophen (APAP) in rats in relation to age (3, 7 and 49 days old) and dose (10, 50 and 100 mg/kg) were studied.
AUC and C_max obtained from plasma level in immature rats (3 and 7days old) were higher than those in matured rats (49 days old), regardless of the dose.
T_max was delayed relatively with increase of the doses, and there was a marked tendency for the immature rats.
Changes of distribution patterns in immature and matured rats, except the gastro-intestinal tract, were similar to that in plasma levels respectively, and the elevation of tissue levels in immature rats were delayed in comparison with that of the matured rats. These findings were consistent with the results of the whole body autoradiography.
In immature and matured rats, the recovery of APAP and conjugates in urine show no marked differences. However, the glucuronide/sulfate ratio of immature rats was higher than that of matured rats.
Percentage of plasma protein binding showed on increase with the age of the rats.

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The absorption, excretion, tissue and dermal distribution of amorolfine were studied following topical application of 0.5 mg of ¹⁴C or ³H labelled amorolfine hydrochloride to rat skin.
The results were as follows:
1. Blood levels of radioactivity reached the maximum of 6.3 or 9.6 ng eq./ml at 24 hr after application to intact skin, and 91.7 or 19.3 ng eq./ml after application to stripped skin in the form of solution or cream, respectively.
2. Total recovery in urine and feces within 7 days was 5.7 or 5.6% after application to intact skin, and 53.9 or 12.3% after application to stripped skin as the solution or cream, respectively.
3. High levels of radioactivity were observed in the liver, spleen and pancreas at 24 hr after application of the cream to the intact skin, and were retained in the applied dermal site over an extended period.
4. In the microautoradiography of the intact skin at 24 hr after application of cream, the grains distributed uniformly in whole epidermis from stratum corneum to stratum basale, and partially distributed to dermis and hypodermis.
5. The level of radioactivity in sequential skin layers cut in parallel to surface decreased by two semilogarithmic fashions with increasing depth from the surface at 24 hr after application of the cream.

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Metabolic fate of a high molecular weight ¹⁴C-labeled sodium hyaluronate (¹⁴C-SL-1010), a highly purified preparation from the culture medium of Streptococcus zooepidemicus, was studied in rabbits after intra-articular injection into the shoulder.
1. Radioactivity in the synovial fluid was eliminated biphasically with an apparent half-life of 57 hr during the first phase, when most radioactivity was eliminated. The fraction of ¹⁴C-labeled sodium hyaluronate to total radioactivity recovered from the synovial fluid was above 95%, indicating no metabolism in the synovial fluid.
2. In the joint region, the highest radioactivity was observed in the mesotendon, followed by the synovial tissue and tendon. In the whole body, the extremely high radioactivity was detected in the axillary lymph node, and relatively high radioactivity was observed in the liver and spleen than in other tissues. Approximately 90% of the radioactivity in the synovial tissue was derived from interstitial synovial fluid of synovial tissues. Analysis of distribution of radioactivity revealed that the main route of elimination of ¹⁴C-SL-1010 from the cavity was via the lymphatic system, since the level of radioactivity in the axillary lymph node was much higher than that in the liver.
3. Analysis of metabolism of ¹⁴C-SL-1010 in the synovial fluid and tissue, axillary lymph node, plasma and urine was done by gel filtration. Gel filtration patterns showed that ¹⁴C-SL-1010 was not metabolized in the synovial fluid. However, slight metabolism occurred in the synovial tissue and rapid metabolism occurred in the axillary lymph node. The radioactivity in the plasma 168 hr after injection was derived from plasma protein and that in the urine was mainly detected in the fraction of Vt of gel filtration except for a very small fraction such as oligosaccharide.
4. Excretion of radioactivity at 76 hr was 39, 1.6 and 1.4% in expired air, urine and feces, respectively.
5. The intra-articular injection of ¹⁴C-SL-1010 into the shoulder produced results similar to those after injection to the knee joint except for the site of lymphatic elimination; the elimination occurred mainly via the axillary lymph node after shoulder injection versus via the lumbar-iliac node after knee injection.

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The absorption, distribution, metabolism, excretion and the effect on the drug-metabolizing enzyme activities of 1-[bis (4-fluorophenyl) methyl]-4 (2, 3, 4-trimethoxybenzyl) piperazine dihydrochloride (KB-2796) were studied in male rats during and after multiple oral administration at a dose of 2 mg/kg/day.
1. The blood radioactivities at 24 hr after daily administration increased progressively with an increase in the number of dosing, but remained practically constant after the 15th administration.
2. The blood radioactivity reached C_max of 188.8 ng/ml at 8 hr after the 21st administration, and decreased gradually with the half-life of 67.3 hr. The C_max and AUC after the 21st administration were 4.4 and 5.7 times greater than those after the 1st dosing, respectively.
3. The extents of cumulative excretion in the urine and feces were almost constant after the 15th administration, and those until 120 hr after the 21st administration were about 9 and 86%, respectively, which were similar with those after the 1st administration.
4. The radioactivity in tissues at 24 hr after daily administration increased with an increase in the number of dosing. After the final administration, a high level of radioactivity was observed in the fat, lung, liver, skin, adrenal and kidney. The elimination of radioactivity from tissues was slow, especially from the aorta.
5. The radioactivity in the brain, liver, lung, spleen, thymus and aorta were present predominantly in the unconjugated fraction. The main metabolites in the tissues were bis (4-fluorophenyl) methylpiperazine (M6) and 4, 4'-difluorobenzophenone (M10), and the unchanged drug was confirmed to be present in all their tissues.
6. The drug-metabolizing enzyme activities were increased after 7 day consecutive oral administration of KB-2796.

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The placental transfer and mammary excretion of 1-[bis(4-fluorophenyl)-methyl]-4-(2, 3, 4-trimethoxybenzyl)piperazine dihydrochloride (KB-2796) were studied in rats after oral administration of 14C-labeled KB-2796.
1. The whole-body autoradiograms after oral administration to rats on the 13th day of pregnancy showed that the radioactivity in the fetus was higher than that in the placenta.
2. The maximum levels of radioactivity in the fetus were reached at 1 hr after administration to rats on the 18th day of pregnancy. The elimination of radioactivity in the fetus was slower than that in maternal plasma. The levels of radioactivity in the fetus at 48 hr was about 4 times higher than that in the mater nal plasma. In the fetus, radioactivity in the liver and lung was relatively higher than in other tissues, and the radioactivity in the lung was increasing up to 96 hr after the administration.
3. The maximum levels of radioactivity in the milk reached at 6 hr after the administration to lactating rats. The concentration is 37 times higher than that in the plasma. Thereafter, the concentration in the milk declined with half-life of 12.3 hr by 48 hr after dosing. The elimination was parallel to the plasma level.
4. The main metabolites of KB-2796 in the maternal and fetal lung were bis(4-fluorophenyl)methylpiperazine (M6) and 4, 4'-difluorobenzophenone (M10).
5. The unchanged KB-2796 accounted for about 60% of total radioactivity in the milk. 1-[Bis (4-fluorophenyl) methyl]-4-(3, 4-dimethoxy-2-hydroxybenzyl) piperazine (M2) and M10 were detected as the main metabolites in the milk.

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A nobel N-sulfoconjugate of alicyclic amine was identified in the metabolic study of tiaramide. Highly polar N-and O-sulfoconjugates were analyzed by secondary ion mass spectrometry (SIMS) and negative SIMS was an effective mode to detect the molecular ion peak of the conjugates. Sulfoconjugation of alicyclic, alkyl-and aryl-amines was detected in the hepatic soluble fractions in the presence of PAPS, a biologically active sulfate. Subcellular distribution, coenzyme requirement and tissue distribution of amine sulfoconjugations were similar to those in phenol and hydroxysteroid (alcohol) sulfoconjugations. Some gender-, age and species-related differences of N and O-sulfoconjugations were observed in animal species, but the differences were substrate-dependent. Analyses using purified enzyme proteins and genes of sulfotransferases will contribute to the understanding of multiplicity in N-and O-sulfoconjugations.

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The present review article describes our recent studies to clarify the renal tubular secretion mechanisms of drugs. Renal tubular secretion is a transcellular transport process composed of two kinds of transmembrane transports across the basolateral and luminal membranes of tubular epithelial cells. We attempted to develop a framework to analyze each transmembrane transport process in morphologically intact kidneys by means of the pharmacokinetic as well as molecular biological approach.
A novel method has been established to assess the basolateral and luminal membrane transports in in vivo kidney or the isolated perfused kidney. The method is based upon a noncompartmental moment analysis of the plasma concentration and urinary excretion rate curves following renal artery injection of the compounds. A new parameter, the mean residence time in renal epithelial cells was defined to evaluate the time for drug molecules to pass across the epithelial cells. The basolateral membrane transport from blood into cells was demonstrated to be a step determining the amount of urinary secretion of p-aminohippurate, a typical organic anion. In contrast, in the case of tetraethylammonium, a typical organic cation, the luminal transport was thought to be a rate-limiting step. Renal failure affected primarily on these active transport processes. The tubular secretion mechanism of digoxin was first demonstrated to be mediated by Pglycoprotein which is expressed on the luminal membranes of proximal tubular cells. Clinically important drug interactions, such as digoxin-quinidine and digoxin-verapamil, are caused by the inhibition of P-glycoprotein.
These works are expected to open new fields to investigate the molecular mechanism of renal excretion of drugs, and to correlate the activity of functional transporter protein with whole body pharmacokinetic features.

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