The effects of pH, dietary proteins and trypsin inhibitors on the hydrolytic rate of recombinant human granulocyte colony-stimulating factor (G-CSF) by digestive enzymes were studied by in vitro incubation experiments. The bile obtained by cannulation into the rat common bile duct and the eluates obtained by infusing distilled water into the gastrointestinal tract were used as sources of digestive enzymes. Some proteins including dietary proteins such as casein and ovalbumin, and two kinds of trypsin inhibitors, chicken egg white and soybean ones, were used to examine the protective action on the hydrolysis of G-CSF by digestive enzymes. With an experiment using the digestive enzyme fluids obtained after centrifugation of bile by ultra-filters with a molecular weight (M_r) cut off of 30000, 10000 and 5000, the proteolytic activity to G-CSF decreased as the cut off M_r decreased. The enzyme solution obtained with a membrane with a M_r cut off of 5000 still had an enzyme activity against G-CSF. The protease activity was mainly ascribed to the pancreatic fluid, but the hepatic bile still had an enzyme activity. The hydrolytic rate of G-CSF was dependent on the pH of the enzyme fluid, and the intestinal fluid. The hydrolytic rate of G-CSF was studied at pHs of 1.68, 4.01, 6.86 and 9.18. Especially, as the pH decreased to 1.68, the hydrolytic rate of G-CSF considerably decreased. Some proteins including dietary proteins also affected the hydrolytic rate of G-CSF. The strength of the inhibitory effect of the proteins is the following order ; ovalbumin > casein > mucin > keratin. Trypsin inhibitors considerably decreased the hydrolytic rate of G-CSF by the digestive enzymes. The inhibitory effect of trypsin inhibitors on the hydrolytic rate of G-CSF is predominantly stronger than that of the dietary proteins.

View full abstract

We studied the effect of dietary konjac mannan on serum and liver cholesterol levels and biliary bile acid composition in hamsters. In hamsters fed a control diet supplemented with konjac mannan for 6 weeks, serum and liver cholesterol levels were decreased significantly compared to hamsters fed the control diet. Addition of konjac mannan to the cholesterol-enriched diet reduced the elevation of serum cholesterol caused by feeding cholesterol. However, the absorption of cholesterol from the intestine was not changed between the four groups. The increase in the proportion of chenodeoxycholic acid in biliary bile acids was significantly enhanced in the hamsters fed both cholesterol and konjac mannan compared to bamsters fed the control diet. The change of the bile acid composition may suggest that konjac mannan inhibits the intestinal absorption of bile acids.

View full abstract

Upon incubation of 23-O-benzyl-5-mycaminosyl-tylonolide (TMC-101) with liver microsomes in the presence of an nicotinamide adenine dinucleotide phosphate-generating system, at least four metabolites were formed : two of them were also formed by an enzyme (s) in rat serum. One of the metabolites formed by liver microsomes possessed antibacterial activity comparable to TMC-101 as examined by bioautography using Micrococcus luteus ATCC 9341 as a tester strain. Incubation of TMC-101 with rat serum degraded most of the parent compound and did not form the active metabolite. The capacity of liver microsomes to produce the active metabolite was increased by pretreatment of rats with 3-methylcholanthrene, phenobarbital and polychlorinated biphenyl. The metabolite with the antibacterial activity was estimated not to be formed by the N-demethylation of TMC-101, and was chemically unstable.

View full abstract

The effects of a series of fatty acids on the rectal absorption of propranolol (PL) were examined in vitro, using macrogol 1500 base and rat rectal tissue. Lauric acid, at a fatty acid : PL molar ratio of 1 : 1, produced the largest increase in permeation rate (J₃), penetration coefficient and partition coefficient of PL. PL flux was increased 2.5-fold in the presence of lauric acid compared to that without the fatty acid. However, the J₃ value of PL was decreased at increased molar ratios (e.g., 3 : 1) of lauric acid. The permeation rate of lauric acid across the rectal membrane was much larger than that of PL. Furthermore, the apparent partition coefficient of PL in an n-octanol/buffer system was significantly increased at a 1 : 1 molar ratio to lauric acid compared with that of PL alone. These results suggest that a complex-mediated mechanism facilitates PL transport, thereby partially contributing to the enhancement of PL rectal absorption. A similar mechanism is applicable to percutaneous drug absorption, as reported previously. Thus, a portion of PL, after first forming a complex with fatty acids, may rapidly permeate across rectal membranes.

View full abstract

The effects of neomycin on substance P (SP)-induced inositol phospholipid hydrolysis and muscle contraction were studied in longitudinal smooth muscle of guinea pig ileum. Various tachykinin peptides including SP stimulated inositol phospholipid hydrolysis in guinea pig ileum. Neomycin (2.5mM) reduced both the contractions and the accumulation of [³H] inositol phosphates induced by SP. These results suggest that the initial transient contractions induced by SP are coupled to inositol phospholipid hydrolysis.

View full abstract

An experimental cerebral embolic model was prepared by an injection of [¹²⁵I] fibrin clot particles (20-100μm) into the left internal carotid artery in rats, and the changes in radioactivity of the brain were continuously monitored by a γ-ray detector. The autoradiograms of the caput transections showed the existence of emboli in small vessels of the left hemicerebrum. After the injection of [¹²⁵I] fibrin clots, the radioactivity spontaneously decreased to a half of the initial radioactivity at 90 min. The decrease in radioactivity which represented the embolus dissolution was markedly suppressed by an antiplasmin agent, trans-4-aminomethyl cyclohexane carboxylic acid, indicating that the endogenous fibrinolysis through the activation of fibrin clots caused a summation of the radioactivity and decreased the rate of dissolution at every embolus preparation. The thrombolytic agents were infused via the left internal carotid artery for 30 min after the second successive injection of fibrin clots. Although the spontaneous dissolution of emboli was observed during the infusion of saline, tissue plasminogen activator (t-PA) as well as urokinase plasminogen activator (u-PA) produced a further dissolution. Approximately half of the emboli disappeared 60 min after the infusion of t-PA at a dose of 75μg/kg and at a dose of 10000 IU/kg respectively.

View full abstract

Effect of intracranial injection of spermine on feeding and drinking behavior was studied in rats. A significant and long-term suppression of feeding and drinking behavior appeared when 30 nmol/hemisphere of spermine was bilaterally injected into the lateral hypothalamic area, substantia nigra or ventral noradrenergic bundle. Micro-injected spermine into the medial amygdaloid nucleus produced a weak and short-time but significant suppression of ingestive behavior. On the other hand, a significant increase in feeding and drinking behavior was observed when spermine was injected into the ventromedial hypothalamic nucleus. These results suggest that spermine inhibits neuronal activity of all the brain regions studied here.

View full abstract

Hepatic estradiol-17β dehydrogenase from chickens catalyzed the reduction of the 3-keto group of androgens such as 5α-dihydrotestosterone and 5α-androstane-3, 17-dione as well as the 17-ketoreduction of 4-androstene-3, 17-dione and dehydroepiandrosterone. The reaction products from 17-ketosteroid and 3-ketosteroid substrates were identified as 17β-hydroxysteroids and 3β-hydroxysteroids, respectively, by thin layer chromatography, high performance liquid chromatography and gas chromatography. Barbital inhibited both 17β-estradiol dehydrogenase and 5α-androstane-3, 17-dione reductase activity noncompetitively giving the same kinetic constant, K_i=50 μM. 5α-Androstane-3, 17-dione competitively inhibited 17β-estradiol dehydrogenase activity. These results indicate that chicken liver estradiol-17β dehydrogenase is in fact a 3β-and 17β-hydroxysteroid dehydrogenase and that both 3- and 17-ketosteroids bind to the same catalytic site.

View full abstract

Cannabidiol hydroxy-quinone (CBDHQ) was identified as an air oxidation product of cannabidiol (CBD). The in vitro incubation of mouse hepatic microsomes with CBDHQ resulted in a decrease of cytochrome P-450 content. CBDHQ inhibited the hepatic microsomal drug-metabolizing enzymes of mice. This inhibitory effect was stronger than that of CBD. CBDHQ (150μM) inhibited aniline hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase in the microsomes by 70, 52 and 77%, respectively, whereas the same concentration of CBD caused the inhibition by 39, 30 and 26%, respectively. CBDHQ (91.5μM) significantly decreased total heme content by 21% and free SH groups by 11% in the microsomes. The results indicate that CBDHQ, which is an oxidation product of CBD, inhibits the hepatic microsomal drug-metabolizing enzymes through the decrease of cytochrome P-450 content.

View full abstract