A remarkable feature of the disposition of polypeptide hormones and their analogues is the contribution of specific binding sites (receptors) to the polypeptides distribution and clearance in the body. The concept of"clearance receptor"is now well established, and receptor-mediated endocytosis (RME) is known as a general mechanism in the uptake of biologically important polypeptides. This review article focuses on the kinetic analysis of the RME of epidermal growth factor (EGF) based mainly on our observations of EGF handling by the liver and kidney. Pharmacokinetic analysis of the tissue distribution of EGF in rats performed in vivo clarified that the uptake of EGF by the liver and kidney exhibited clear saturation with Km values of 7 nM and 0.4 nM, respectively, and that both tissues could account for more than 80% of the total plasma clearance of EGF in the distribution phase. The hepatic and renal handlings of EGF by isolated perfused organs were also analyzed to obtain the kinetic parameters with respect to RME. In the overall RME process in the liver, the EGF-receptor association and dissociation processes are rapid (mean time <1 min), the degradation process is much slower (mean time 4-5h) and the internalization process is intermediate (mean time 4-5 min). The mean time required for the recovery of receptors after being down-regulated in the liver is approximately 30 min, and was much shorter than those in other tissues. Such rapid recovery from the receptor down regulation in the liver reflects the process of recycling of internalized EGF receptors to the cell surface. The kinetic parameters obtained from the perfused livers and from isolated hepatocytes were compared, and a significant difference was observed only for the association rate constant (kon). A hypothesis accounting for this difference is proposed, in which the association rate of EGF to the cell surface receptor is considered to be limited by the diffusion in the unstirred water layer in the interstital space of the intact liver such as the perfused liver. The comparison of EGF handling between the filtering and nonfiltering perfused kidneys demonstrated that the bulk of receptor-mediated EGF uptake takes place via the antiluminal plasma membrane with very high affinity (Kd=0.1nM).
The drug metabolism studies in which we have been engaging for about 40 years since 1952 are briefly reviewed in this paper. Our main efforts were initially made to elucidate the metabolic fates of various abused drugs including barbiturates, carbamates, opioids, amphetamines and cannabinoids in mammals from pharmacological and toxicological points of view. Among the interesting findings obtained from these studies, the most remarkable one was that morphine-6-glucuronide, a minor metabolite of morphine, has much stronger analgesic activity than morphine. Recently we have also been interested in clarifying the enzyme system involved in the metabolic pathways of the above drugs. Several cytochrome P-450 isozymes were thus purified from the liver microsomes of mammals and their role in oxygenation of amphetamines and cannabinoids were elucidated. The finding that MALDO (microsomal aldehyde oxygenase), a purified P-450 isozyme, could catalyze an oxidation of lipid-soluble aldehydes to the corresponding carboxylic acids was most noticeable. Metabolic and toxicologic studies on furylfuramide (AF-2) and polychlorinated biphenyl (PCB) have also been performed using rats and other animal species, and some interesting results were obtained.
A new sesquiterpenic ketoacid, named chloranthalic acid (1), was isolated from the roots of Chloranthus japonicus and characterized as selin-4α-hydroxy-7 (11)-en-8-oxo-12-oic acid. Two new sesquiterpenic dimers were also obtained. One of them was purified as an acetate, named methyl chloranthadimeric acid acetate (2) and the structure determined by means of the spectroscopic and X-ray crystal analyses. The other dimer (3) was elucidated as an artifact from chloranthalactone A (5) under irradiation of sun light. Chloranthalactone B (4) was elucidated to be derived from chloranthalactone A (5) through peroxyradical reaction.
Effects of tissue cultured ginseng on the function of the stomach and small intestine were compared with those of cultivated ginseng. Fifty percent ethanol extracts of the tissue cultured and cultivated ginseng stimulated gastrointestinal propulsion in mice. The tissue cultured ginseng also inhibited ulcer formation induced by water immersion and restraint stress, and ligature of the pylorus. In contrast, the cultivated ginseng showed no such inhibitory action.
Effects of the tissue cultured and cultivated ginseng on gastric secretion and pepsin activity were investigated. Fifty percent ethanol extracts of both cultured and cultivated ginsengs reduced gastric secretion and acid output in pylorus-ligated rats. They did not affect pepsin activity. The tissue cultured ginseng inhibited histamine and pentagastrin-induced acid secretion in rats, whereas the cultivated ginseng showed no such effect. They also suppressed acid secretion induced by 2-deoxy-D-glucose and baclofen [β-(p-chlorophenyl)-γ-aminobutyric acid], which are known to stimulate gastric acid secretion via the central nervous system. However, they had no effect on acid secretion induced by vagal stimulation. These results suggest that both tissue cultured and cultivated ginsengs may have an inhibitory effect on gastric secretion. The effect seems to be due to the inhibition of acid secretion via the central nervous system.
A 3α-reducing activity of 5α-dihydrotestosterone (5α-DHT) was found in pig adrenal cytosol. The enzyme (3α-hydroxysteroid dehydrogenase : 3α-HSD) has been purified to homogeneity from pig adrenal cytosol by ammonium sulfate precipitation followed by DEAE-cellulose, 2', 5'-adenosine diphosphate-Sepharose and Sephadex G-100 column chromatographies. The molecular weight was estimated to be 33000 and 39000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric point was estimated to be 8.5 by isoelectric focusing. The Km and Vmax values for 5α-DHT in the reduction were 10.2μM and 10.6 nmol/min/mg. The enzyme utilized reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotine amide adenine dinucleotide (NADH) in the reduction as a cofactor, but it preferencially required NADPH rather than NADH. Furthermore, the purified enzyme catalyzed not only 3α-reduction of 5α-DHT (9.65nmol/min/mg), but also catalized 20α-reduction of 17α-hydroxyprogesterone (0.58nmol/min/mg). The enzyme activity of 3α-HSD was strongly inhibited by Hg2+, but it was not inhibited by medroxyprogesterone acetate and some anti-infulammatory agents. No remarkable differences was demonstrated between 3α-HSD and 20α-HSD activity under the influence of heat treatment, divalent cation, anti-infulammatory agents and some inhibitory steroids. These results strongly suggest that 3α-HSD purified from pig adrenal cytosol is a bi-functional enzyme catalizing 3α- and 20α-HSD activities.
A comment aid system for therapeutic drug monitoring (TDM), which produces the primary comment on the dosage regimen of valproate, was developed. This system produces consulting comments with a fuzzy theory, by using the dosage regimen of valproate, pharmacokinetic parameters estimated by the Bayesian method, and therapeutic effect and toxicity of anticonvulsants evaluated by the physician. For nearly 90% in 42 cases, comments of the system were agreeable comparing with the notes of TDM technologists. There are a few disagreements between this system and experts, mainly because the system does not use enough information about combination therapy with other anticonvulsants. Developed comment aid system shows some clinical and educational utility, and suggests significance and possibility of more useful and allround TDM expert system. Then this system might be available as a prototype of the TDM expert system.
The quantitative analysis of (-)-epigallocatechin gallate (EGCG) in tea (Camellia sinensis L.) was performed by high-performance liquid chromatography (HPLC) with a C-18 reversed-phase column. EGCG was then eluted within 20 min by using methanol-water-acetic acid (20 : 75 : 5 (v/v/v)) as an eluent. As an internal standard, tryptophan was used. The content of EGCG in five kinds of green tea (sencha, gyokuro, bancha, matsucha and oolong tea) and in a cup of those was determined by both the extraction method with 50% (v/v) methanol and the infusion method with water. The largest amount of EGCG was obtained from matsucha by the extraction method, or from sencha by the infusion method. Furthermore, EGCG contents in various parts of the tea plant were examined. The first leaf had the highest concentration of EGCG, and the concentration of EGCG decreased with the aging of the leaf.
The relationship between the oral absorption and gastrointestinal transit time of nitrofurantoin was investigated in dogs by the double-marker method using acetaminophen and salicylazosulfapyridine as markers. The extent of bioavailability of nitrofurantoin gave correlations with both gastric emptying time and small intestinal transit time. The results indicate that the slower the passage from the stomach into the small intestine and/or the longer the residence time in the small intestine, the oral absorption efficiency of nitrofurantoin increases. These observations were consistent with the reported findings from in situ absorption study in rats and intestinal intubation study in humans, respectively. Moreover, it was clarified that small intestinal transit time is the most important determinant of absorption of nitrofurantoin, since the good correlation was observed between the small intestinal transit time and the extent of bioavailability. The double-marker method appears consequently to be useful tool for the determination of the gastrointestinal transit time in dogs.