Following the oral administration of [¹⁴C] pranoprofen to mice, 70-80% of radioactivity was excreted in the urine, and 15-25% in the feces within 3 d at 5, 25, 50, 100 and 200 mg/kg. This showed that no significant change in urinary and fecal excretion was observed among these doses. The radioactivity levels in the blood also increased in proportion to the doses, indicating that no repression of the absorption of pranoprofen was found even at increased doses. The major metabolites in mouse urine were acyl glucuronide and the glucoside of pranoprofen. At low doses acyl glucoside was predominantly excreted in urine, whereas acyl glucoside decreased relative to acyl glucuronide at increased doses. Although 43.2% of the acyl glucoside was recovered in the 24 h urine samples after the intravenous administration of acyl glucuronide, no acyl glucuronide was found in the urine after the intravenous administration of acyl glucoside. These results demonstrated the interesting observation that pranoprofen had a preference for glucosidation rather than glucuronidation in mice at low doses in spite of having a higher capacity of glucuronidation.

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The formation of acyl glucuronide and glucoside of 2-(5H-[1] benzopyrano [2, 3-b] pyridin-7-yl) propionic acid (pranoprofen), an anti-inflammatory drug, was studied in homogenates and microsomes of mouse tissues by using S (+)-, R (-)-and RS (±)-pranoprofen. Acyl glucuronidation occurred mainly in the liver, whereas acyl glucosidation was predominant in the kidney. Furthermore, both conjugations occurred by enzymatic transfer of glucuronic acid of uridine diphosphate glucuronic acid (UDPGA) and glucose of uridine diphosphate glucose (UDPG) to pranoprofen, respectively. No conjugation reactions were observed in the lung, plasma or gut. The amount of conjugates in the liver and kidney increased by the prolongation of the incubation times and reached the maximum at 15-30 min for glucuronidation and 45 min for glucosidation. After that, both conjugates decreased with the lapse of time. Both acyl conjugates were the least stable in the liver, then in the kidney and the most stable in the plasma. In the liver, acyl glucoside was converted to acyl glucuronide and pranoprofen in the presence of UDPGA, but only a small amount of acyl glucuronide was changed to acyl glucoside in the kidney in spite of the presence of UDPG. In the kindney, acyl glucoside decreased relative to acyl glucuronide at increasing doses for both S (+)-and R (-)-pranoprofen, but the concentration of acyl glucoside was much higher for S (+)-pranoprofen than R (-)-enantiomer 1 h after the oral administration of S (+)-and R (-)-pranoprofen. No acyl glucoside was detected in the liver and plasma. Although only a small difference in acyl glucuronidation in the liver was observed between S (+)-and R (-)-enantiomers, acyl glucosidation in the kidney occurred more predominantly for S (+)-pranoprofen than for the R (-)-enantiomer.

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Following the oral administration of RS (±)-pranoprofen to mice at a dose of 25 mg/kg, 10.7% of the acyl glucuronide and 46.4% of the acyl glucoside of pranoprofen were excreted in the urine within 24 h. The recovery of acyl glucoside in the urine decreased relative to that of acyl glucuronide at increasing doses (100, 200 mg/kg). Following the oral administration of S (+)-pranoprofen to mice at a dose of 25 mg/kg, 5.0% of the acyl glucuronide and 56.5% of the acyl glucoside were excreted in the urine within 24 h, while 10.8% of the acyl glucuronide and 13.9% of the acyl glucoside were excreted after the oral administration of R (-)-pranoprofen, respectively. The absolute configuration of the aglycone of acyl glucuronide was almost R (-)-enantiomer (92.5-96.1%) in the 0-24 h urine, whereas that of acyl glucoside contained 15.3-24.7% of S (+)-enantiomer after the oral administration of R (-)-pranoprofen. On the other hand, only the S (+)-isomer was found as the aglycone of both acyl glucuronide and glucoside after the oral administration of S (+)-pranoprofen. The present results showed that stereoselective conjugation was observed in glucosidation in mice. Nevertheless, a dose-dependent shift in glucuronidation and glucosidation was found for both the administrations of S (+)-and R (-)-enantiomers as well as RS (±)-pranoprofen. Also a chiral inversion of R (-)-enantiomer to S (+)-antipode may occur slightly but significantly in mice.

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Absorption, metabolism and excretion of 2-(5H-[1] benzopyrano-[2, 3-b] pyridin-7-yl) propionic acid (pranoprofen), an anti-inflammatory drug, were investigated in mice, rats, guinea pigs and rabbits using ¹⁴C-labeled compound ([¹⁴C] pranoprofen) at a dose of 5 mg/kg. After the oral administration of [¹⁴C] pranoprofen the radioactivity was rapidly and almost completely absorbed from the digestive organs of the animals tested. The radioactivity in the blood reached the maximum at 30-60 min after the oral administration of [¹⁴C] pranoprofen in all species tested, and the biological half-lives of the radioactivity were 4.1 h in rats, 2.6 h in guinea pigs, 1.3 h in mice and 0.9 h in rabbits, respectively. When [¹⁴C] pranoprofen was orally administered, urinary and fecal excretions of the radioactivity within 3 d were 81.1% and 18.7% of the dose in mice, 51.5% and 39.4% in rats, 81.8% and 9.0% in guinea pigs, and 93.2% and 3.6% in rabbits, respectively. A major metabolite of pranoprofen was its acyl glucuronide in rats, guinea pigs and rabbits. However, it was shown that acyl glucosidation is also a predominant metabolic pathway of pranoprofen in mice.

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The binding affinity ; incorporation and adsorption, of human tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) for blood clots was investigated in vitro. In order to study the incorporation, the blood clot formation was performed after mixing [¹²⁵I] t-PA or [¹²⁵I] u-PA with the blood obtained from human and several animal species. The radioactivities of [¹²⁵I] t-PA incorporated in blood clots of human, dog, rat and rabbit were higher than those of [¹²⁵I] u-PA. The adsorption study was carried out by immersing the blood clot in saline containing the [¹²⁵I] plasminogen activators (PAs). The adsorptions of [¹²⁵I] t-PA to blood clots of human and animals were higher than those of [¹²⁵I] u-PA. The results suggest that t-PA has a much higher affinity for fibrin in blood clots than u-PA. The blood clot lysis caused by the clot-bound PAs was investigated using the [¹²⁵I] fibrincontaining blood clots pretreated with t-PA or u-PA. In the human blood clot, the clot-bound t-PA showed a dose-dependent clot lysis at the concentrations of t-PA ranging from 0.3 to 3 nM, while the clot-bound u-PA has little clot-lytic activity. The t-PA bound to the human blood clot showed the most effective clot lysis as compared with those bound to the animal blood clots.

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The localization of tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA) on thrombi was investigated in disseminated intravascular coagulation rats (DIC rats) induced by thrombin. One hour after the intravenous infusion of thrombin to rats, the plasma fibrinogen level decreased, while the plasminogen activator activity in the plasma euglobulin fraction increased. The whole body autoradiography was studied after an injection of [¹²⁵I] fibrinogen in DIC rats. The high radioactivity which indicated the presence of microthrombi was observed in the renal cortex, liver, spleen and lung. Futhermore, a large venous thrombus with higher radioactivity was observed in the abdominal vena cava. These results show that the thrombin-treated animal is one of the best DIC models. After the intravenous administration of [¹²⁵I] t-PA, the autoradiograms of DIC rats showed a radioactivity in the blood and much higher radioactivities in the renal cortex, spleen and lung in comparison with the normal rat. However, there was no difference in the distribution of [¹²⁵I] u-PA between normal and DIC rats at all. The strong radioactivity of [¹²⁵I] t-PA but not [¹²⁵I] u-PA was observed on the surface of large thrombus in the vena cava. These results suggest that t-PA localizes more preferentially on microthrombi than u-PA. The ratio of the radioactivity in the tissue to that in the blood was calculated to compare quantitatively the localization of [¹²⁵I] t-PA and [¹²⁵I] u-PA on microthrombi formed in organs. The ratios of the [¹²⁵I] t-PA radioactivities in the kidney, lung and liver showed 1.6 to 2.5 times higher amounts in DIC rats as compared with normal rats. On the other hand, the ratios of the [¹²⁵I] u-PA radioactivities did not show a significant difference between normal and DIC rats. These results further support the preferential localization of t-PA on microthrombi formed in organs.

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The influence of suppository bases and adjuvants on the release rate of drugs and the absorption of non-absorbable drugs such as sulfanilic acid (SA) and sulfaguanidine (SG), was investigated following the rectal administration of suppositories. The suppository bases used were lipophilic bases such as Witepsol W 35, H 15, S 55, E 75 and hydrophilic base such as macrogol. SA was rapidly released from macrogol, W 35, H 15 and S 55, except E 75. On the other hand, SG was rapidly released from macrogol, whereas the release of SG from lipophilic bases was slow. Rectal absorption of SA and SG following administration of each drug alone in suppository form was slight. On the addition of diclofenac sodium (DF) as absorption promoter the blood levels of SA and SG released from all suppositories increased by about two to four fold compared with those suppositories containing only SA or SG, respectively. However, the absorption of SG still did not attain sufficient levels by the administration of DF only. The rectal absorption of SG was markedly increased by the release rate of the drug from the suppository. These results indicate that after administration of these suppositories the bioavailability of non-absorbable drugs was sufficiently improved by enhancing both the release rate from the suppositories and the rectal membrane permeability.

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Angiotensin-converting enzyme (ACE) inhibitors were excised from the molecule of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of baker's yeast by heating at 120°C in 1 M AcOH-20 mM HCl. Three inhibitors were then purified by gel-permeation and reversephase chromatographies. One of the yeast ACE inhibitors, YG-3, was GAPDH peptide 79-89 (Pro-Ala-Asn-Leu-Pro-Trp-Gly-Ser-Ser-Asn-Val, IC₅₀ : 18 μM), and contained the sequence homologous to vertebratc ACE inhibitors (GAPDH peptides 79-86 or 81-88). Other inhibitors, YG-1 (Gly-His-Lys-Ile-Ala-Thr-Phe-Gln-Glu-Arg, IC₅₀ : 0.4 μM) and YG-2 (Gly-Lys-Lys-Ile-Ala-Thr-Tyr-Gln-Glu-Arg, IC₅₀ : 2 μM), corresponded to amino acid residues 68-77 in two different forms of yeast GAPDH, respectively. Their sequences were quite different from those of the venom peptide family. YG-1 was the most potent ACE inhibitor among yeast and vertebrate GAPDH peptides excised by acid-limited proteolysis. Thus, yeast GAPDH seems to be an excellent source of naturally occurring ACE inhibitors.

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Age-related changes in the acquisition and retention of memory based on the step-through active avoidance response were studied in rats and the effects of cholinergic drugs and cerebral metabolic activators on memory impairment in old rats were also tested. Six-and 12-month-old rats showed lower rates of acquisition of the active avoidance response than did 2-month-old rats. In addition, the retention of the active avoidance response in 6-and 12-month-old rats diminished rather rapidly compared with that observed in 2-month-old rats. Intraventricular injection of acetylcholine at doses of 20 and 50 ng caused a significant improvement of memory impairment in old rats. Physostigmine and arecoline also caused a significant ameliorating effect at doses of 0.02 and 0.05 mg/kg i.p. and 0.2 and 0.5 mg/kg i.p., respectively. Hopantenate calcium (100 mg/kg, p.o.), idebenone (20 and 50 mg/kg, i.p.), indeloxazine (50 mg/kg, p.o.) and DM-9384 (30 mg/kg, p.o.) also proved useful to improve memory impairment in old rats.

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