Shin-Ichi Hirano

Synthesis of NF-κB activation inhibitors derived from epoxyquinomicin C

In order to develop new inhibitors of NF-κB activation, we designed and synthesized dehydroxymethyl derivatives of epoxyquinomicin C, namely, DHM2EQ and its regioisomer DHM3EQ. These derivatives were synthesized from 2,5-dimethoxyaniline in 5 steps. Since DHM2EQ was more active and less toxic than DHM3EQ, its stereochemical configuration was determined by X-ray crystallographic analysis. Each enantiomer of the protected DHM2EQ was separated by a chiral column and deprotected. DHM2EQ inhibited TNF-α-induced activation of NF-κB in human T cell leukemia cells, and also inhibited collagen-induced arthritis in a rheumatoid model in mice.

Effects of Dried Bonito (Katsuobushi) and Captopril, an Angiotensin I-Converting Enzyme Inhibitor, on Rat Isolated Aorta: A Possible Mechanism of Antihypertensive Action

In order to elucidate the mechanism of the antihypertensive action of dried bonito (katsuobushi), we compared the effects of dried bonito extracts with those of captopril, an angiotensin I-converting enzyme (ACE) inhibitor, on aorta preparations isolated from rats. Dried bonito extracts (3×10−4 to 3×10−3 g/ml) more potently relaxed contractions induced by norepinephrine (10−7 M) than contractions induced by KCl (55.9 mM). Dried bonito extracts (3×10−3 g/ml) slightly inhibited 10−7 M angiotensin I-induced contractions. In contrast, captopril (10−8 to 10−7 M) did not affect 10−7 M norepinephrine- or 55.9 mM KCl-induced contractions, but a higher concentration of captopril (10−6 M) very slightly relaxed it. Captopril (10−8 to 10−6 M) markedly inhibited 10−7 M angiotensin I-induced contractions in a concentration-dependent manner. These results suggest that antihypertensive mechanism of action induced by dried bonito involves direct action on vascular smooth muscle in addition to ACE-inhibitory activity.

Prevention of Pathogenic Escherichia coli Infection in Mice and Stimulation of Macrophage Activation in Rats by an Oral Administration of Probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-κB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-α production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.

Effects of a Casein Hydrolysate Prepared from Aspergillus oryzae Protease on Adjuvant Arthritis in Rats

We evaluated the effects of a casein hydrolysate (CH) prepared from Aspergillus oryzae protease on rat adjuvant arthritis, a model of human rheumatoid arthritis. CH was administered orally once a day to the animals for 22 d after the adjuvant injection. CH suppressed swelling in the adjuvant-uninjected hind paws, and a higher dose of CH suppressed the increase in arthritic score and swelling of the adjuvant-injected hind paws. A histopathological examination revealed evidence that the higher dose of CH suppressed the articular changes in the rats. In addition, CH suppressed the production of nitric oxide and prostaglandin E2 in the plasma of the rats. These results suggest that CH had a suppressive effect on adjuvant arthritis by inhibiting the acute and chronic inflammatory reactions.

Pirarubicin-induced Endothelium-dependent Relaxation in Rat Isolated Aorta

Abstract— The mechanism of relaxation produced by pirarubicin [(2″R)‐4′‐O‐tetrahydropyranyladriamycin, THP] has been studied in rat isolated aorta. THP (1·5 × 10−6–4·5 × 10−5 m) markedly relaxed contractions induced by noradrenaline (10−7 m) in the aorta with endothelium, but not in that without endothelium. The relaxation induced by 1·5 × 10−5 m THP was inhibited by methylene blue (5 × 10−6 m), hydroquinone (10−4 m), phenidone (5 × 10−5 m), haemoglobin (10−6 m) and p‐bromophenacyl bromide (5×10−5 m), but not by indomethacin (2·5 ×10−5 m). The relaxation induced by THP (1·5 × 10−7 − 4·5 × 10−5 m) was inhibited by NG‐nitro‐l‐arginine (10−5 m), but enhanced by superoxide dismutase (10 units mL−1) or by L‐arginine (10−2 m). However, the THP‐induced relaxation was not inhibited by various receptor antagonists such as atropine (10−6 m), cimetidine (10−5 m), diphenhydramine (3 × 10−6 m) and [D‐Pro4, D‐Trp7,9,10]‐substance P(4–11) (1·5 × 10−6 m). In fifteen anthracycline analogues, THP and 13‐dihydropirarubicin (both with a tetrahydropyranyl group) produced endothelium‐dependent relaxations. These results suggest that the THP‐induced relaxation which is probably mediated by endothelium‐derived relaxing factor (EDRF) was not produced by an activation of muscarine, histamine H1 or H2, or substance P receptor, and further that the tetrahydropyranyl group must play an important role in the THP‐induced relaxation.

Effects of pirarubicin, an antitumor antibiotic, on the cardiovascular system

SummaryIn the present study we examined the effects of pirarubicin [(2″R)-4′-0-tetrahydropyranyladriamycin, THP] on a cardiovascular system. An injection of THP (0.39–3.13 mg/kg, i. v.) reduced the mean blood pressure and caused an increase in the respiratory air rate in anesthetized rats. At 1.5×10−6–1.5×10−5m, THP markedly relaxed a contraction induced by 10−7m norepinephrine in rat aorta with endothelium but not in that without endothelium. At a dose of 0.02–0.5 mg, THP produced an increase in the contractile force and the perfusion flow of isolated perfused guinea pig hearts. At a higher concentration (4.5×10−5–1.5×10−4m), it produced a slight increase in the contractile force of the left atria in guinea pigs. This positive inotropic action of THP was inhibited by diphenhydramine (10−6–5×10−5m), chlorpheniramine (3×10−7–3×10−5m), and tripelennamine (3×10−7–3×10−5m) but not by propranolol (10−6m), cimetidine (10−5m), diltiazem (10−6m), or ryanodine (10−8m). THP given i. v. at 2.5 mg/kg elevated the plasma histamine level in anesthetized dogs. From these data, we conclude that THP mainly relaxed the rat aorta in the presence of endothelium and that at higher concentrations, it increased the contractile force in the cardiac muscle, probably mediated through the release of histamine.

Effects of pirarubicin in comparison with epirubicin and doxorubicin on the contractile function in rat isolated cardiac muscles.

1. We have examined the effects of pirarubicin (THP), compared with epirubicin (EPI) and doxorubicin (DXR), on the contractile function in papillary muscles isolated from rats. 2. In in vivo experiments, in which the rat was treated once a week for 4 weeks with DXR (total dose 10 mg/kg) and thereafter once a week for 4 weeks with THP, EPI or DXR (total dose 10 mg/kg), a positive instead of negative force-frequency relationship was observed in the muscles treated with EPI and DXR, but not with THP, and an increase in contractile response to extracellular Ca2+ was observed more markedly in the muscles treated with DXR than in those treated with EPI and THP. 3. In in vitro experiments, in which the muscle preparations were incubated with the drugs at 100 or 200 microM for 2 hr, EPI and DXR caused a negative inotropic effect and a prolongation of tension duration, while THP caused a slight positive inotropic effect and a slight prolongation of tension duration. 4. Furthermore, a decrease in the potentiated postrest contraction was observed more markedly in the muscles incubated with EPI and DXR at 200 microM than in those with THP. 5. These results suggest that both EPI and DXR show a cardiotoxicity by impairing the function of cardiac sarcoplasmic reticulum, and that the switching of the treatment from DXR to THP produces less impairing effects.

A possible mechanism of endothelium-dependent relaxation induced by pirarubicin and carbachol in rat isolated aorta

Abstract— The mechanism of endothelium‐dependent relaxation induced by pirarubicin, (2″R)‐4′‐O‐tetrahydropyranyladriamycin, THP, or carbachol was investigated in the rat isolated aorta. The relaxant effect of THP (1·5 × 10−6‐4·5 × 10−5 m) or carbachol (10−8‐10−4 m) on the aorta with endothelium was decreased by lowering Ca2+ in the medium. The relaxation induced by THP was not inhibited by pretreatment with verapamil (10−6–10−5 m), and that induced by carbachol was only partially inhibited. However, on replacement of all but 20 Mm Na+ with either Li+ or choline, the THP‐ or carbachol‐induced relaxation was inhibited. Furthermore, the relaxing effect of THP or carbachol was inhibited by pretreatment with amiloride (10−4‐3 × 10−4 m), with ouabain (10−4‐10−3 m), or with K+‐depletion. These results suggest that the THP‐ or carbachol‐induced relaxation depending on endothelium was affected by modifying the calcium ion concentration, and that a Na+–Ca2+ exchange process is involved.

Inhibitory Effects of Tiamulin on Contractile and Electrical Responses in Isolated Thoracic Aorta and Cardiac Muscle of Guinea‐pigs

Abstract— The inhibitory effect of tiamulin, an antibiotic produced by Pleurotus mutilis, on contractile and electrical responses in isolated thoracic aorta and cardiac muscle of guinea‐pigs was studied. In the thoracic aorta, tiamulin with an IC50 of 9·7 × 10−6 m inhibited sustained contractions induced by isosmotically added 60 Mm KCl. The inhibitory effect of tiamulin on a Ca2+‐induced contraction in a depolarized muscle was competitively antagonized by raising external Ca2+ concentration. Bay K 8644 (10−7 m) antagonized tiamulins inhibition of the Ca2+ ‐induced contraction. Tiamulin (2×10−5 m) decreased the elevated cytoplasmic Ca2+ level measured by the fura 2 AM method in the depolarized muscle. In high K+‐isoprenaline‐treated left atria, tiamulin (2 × 10−5–2 × 10−4 m) produced negative inotropic effects. On the other hand in the membrane action potential of papillary muscles, tiamulin (2 × 10−6–2 × 10−4 m) produced decreases in action potential and durations and 2 × 10−4 m tiamulin depressed the slow response action potential in depolarized muscles. Tiamulin produced prolongations of the PR interval in ECG, negative chrono‐ and inotropic effects, and an increase in perfusion flow in guinea‐pig isolated and perfused hearts. These effects of tiamulin on the aorta or cardiac muscle were similar to those of verapamil and nifedipine. These results suggest that both the inhibitory action of tiamulin on the high K+‐induced contraction in the aorta and the negative inotropic effect of tiamulin on the cardiac muscle are due to an inhibition of Ca2+ entry through the voltage‐dependent Ca2+ channels of cells of both these muscles.