Inhibition of PDGF- and TGF-beta 1-induced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster and are more sensitive to transforming growth factor-beta 1 (TGF-beta 1) than those of normotensive Wistar-Kyoto rats. We studied the in vitro effects of tranilast, an anti-allergic drug, on the proliferation, migration and extracellular matrix synthesis in the SHR-VSMC. There were many inhibitory effects of tranilast (30-300 microM) on SHR-VSMC. One is the effect on the proliferation stimulated with fetal bovine serum (FBS), TGF-beta 1 and platelet-derived growth factor-BB (PDGF-BB). Another is the effect on the PDGF-BB-induced migration. Lastly, tranilast exhibited inhibitory effects on spontaneous collagen synthesis and TGF-beta 1-induced collagen and glycosaminoglycan synthesis. On the other hand, collagen induced the VSMC migration concentration-dependently. These results suggest that tranilast may prevent restenosis after percutaneous transluminal coronary angioplasty.

Tranilast antagonizes angiotensin II and inhibits its biological effects in vascular smooth muscle cells.

Recent studies have been reported indicating that angiotensin II may potentiate neointimal formation. In the present study, we examined the antagonistic effect of tranilast on angiotensin II. Losartan was used as the reference compound. First, tranilast inhibited the angiotensin II-induced contraction of rabbit aortic strips in a noncompetitive manner (pD'(2) = 3.7), whereas it had little effect on the contraction induced by noradrenaline or endothelin-l. Second, tranilast inhibited the binding of (125)I-labeled angiotensin II to angiotensin AT1 receptors in rat liver membranes with an IC(50) value of 289 mu M. Finally, functional antagonism of tranilast (100 and 300 mu M) was demonstrated by its blockade of angiotensin II (10(-8)M)-induced (45)Ca(2+) -efflux from human vascular smooth muscle cells (VSMC). However, tranilast (30-300 mu M) exerted no influence on PDGF-induced formation of inositol triphosphates which cause an increase in [Ca(2+)]i in human VSMC. The antagonistic activity of tranilast towards angiotensin II may be involved in part in preventing restenosis after percutaneous transluminal coronary angioplasty (PTCA).

Antiproliferative and c-myc mRNA suppressive effect of tranilast on newborn human vascular smooth muscle cells in culture.

1. Newborn human vascular smooth muscle cells (VSMCs) proliferated faster and were more sensitive to platelet-derived growth factor-BB (PDGF-BB) than those from adults. In this study, we investigated mechanism of the inhibitory effect of tranilast on PDGF-BB-induced proliferation of VSMCs from newborns. 2. Tranilast (30-300 microM) concentration-dependently inhibited the VSMC proliferation in randomly growing cultures stimulated with PDGF-BB. 3. Tranilast (30-300 microM) concentration-dependently inhibited the [3H]-thymidine incorporation into DNA in VSMCs that had been synchronized by 48 h serum depletion and then stimulated by addition of PDGF-BB. However, tranilast had little influence on unscheduled DNA synthesis in quiescent cells or on RNA and protein synthesis, unlike aphidicolin, actimomycin D, and cycloheximide. 4. In synchronized VSMC cultures, tranilast still inhibited the PDGF-BB-induced DNA synthesis even when added 18 h after stimulation of the quiescent cells. The mode of the antiproliferative action of tranilast was different from that of NiCl2, genistein, or staurosporin. In addition, flow cytometry of synchronized VSMCs treated with tranilast revealed a blockade of PDGF-inducible cell-cycle progression at the G1/S checkpoint. 5. Northern blotting showed that tranilast (30-300 microM) concentration-dependently suppressed constitutive c-myc mRNA expression even when added 18 h after PDGF-BB-stimulation of quiescent VSMCs. Tranilast still had an inhibitory effect on the induction of c-myc mRNA when de novo protein synthesis was inhibited by cycloheximide and did not shorten the degradation of c-myc mRNA at the post-transcriptional level, demonstrating that tranilast directly inhibited c-myc mRNA expression at the transcriptional level. 6. These results suggest that the inhibitory effect of tranilast on PDGF-BB-induced proliferation is due to S-phase blockade and may be, at least in part, involved in the direct suppression of c-myc gene expression. Tranilast did not cause cell toxicity and may therefore hold promising potential for the prevention of vascular proliferative diseases.

Effects of thromboxane synthetase inhibitors on aggregation of rabbit platelets.

Thromboxane (TX) synthetase activity was selectively inhibited by (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046) and sodium (E)-3-[4-(3-pyridylmethyl)phenyl]-2-methyl-propenoate (OKY-1581) (OKYs). Their IC50 for the rabbit platelet enzyme were found to be 11nM and 3nM respectively. Arachidonic acid (AA) or collagen induced platelet aggregation, and generated TXA 2 and prostaglandins (PGs) in rabbit platelets. OKYs inhibited platelet aggregation and TXA2 generation without affecting PGs generation, while aspirin inhibited platelet aggregation, and TXA2 and PGs generation. There was a parallel relation between the degree of inhibition of platelet aggregation and TXA2 generation by OKYs, but the inhibitory effects of aspirin on platelet aggregation was related to that on both TXA2 and PGs generation. However, OKYs and aspirin did not inhibit ADP-induced platelet aggregation which did not involve the generation of TXA2 and PGs. These results suggested that TXA2 generation is related to platelet aggregation induced by AA or collagen, and that the inhibitory effect of OKYs on platelet aggregation is due to the inhibition of TX synthetase.

Effect of KMD-3213, an alpha 1a-adrenoceptor-selective antagonist, on the contractions of rabbit prostate and rabbit and rat aorta.

KMD-3213, (-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy) phenoxy]ethyl]amino]propyl]indoline-7-carboxamide, a newly synthesized alpha 1-adrenoceptor antagonist, has been shown to have potent action toward, and to be selective for human cloned and native alpha 1-adrenoceptors. In the present study, we characterized the inhibitory effect of KMD-3213 on the phenylephrine (alpha 1-adrenoceptor-selective agonist)-induced contraction of rabbit prostate, rabbit thoracic aorta and rat thoracic aorta to functionally confirm the tissue selectivity of KMD-3213. The mean pA2 value for KMD-3213 for the inhibition of the rabbit prostatic contraction was 10.05, whereas the values for the rabbit and rat aortic contractions were 9.36 and 8.13, respectively. The order of mean pA2 values for the inhibition of the rabbit prostatic contraction was KMD-3213 > or = tamsulosin >> prazosin, whereas that for the rabbit and rat aortic contractions was tamsulosin > KMD-3213 > prazosin and tamsulosin > or = prazosin >> KMD-3213, respectively. KMD-3213 produced a sigmoidal inhibition curve for single-dose phenylephrine-induced contractions of rabbit prostate, whereas it produced a non-sigmoidal curve for that of rabbit aorta. KMD-3213 had no effect on isoproterenol-induced chronotropic action in guinea-pig atria, and 5-hydroxytryptamine-, histamine- and acetylcholine-mediated contractions of rabbit aorta. These results indicate that the potency of the inhibitory activity of KMD-3213 depends on the tissue subtype expression and that KMD-3213 preferentially antagonizes prostatic contraction.

[Research and development of ozagrel, a highly selective inhibitor of TXA2 synthase].

Highly selective inhibitors of thromboxane (TX) A2 synthase were noted as a therapeutic agent for ischemic heart diseases, thromboembolic disorders, cerebral circulatory disorders, and asthma. The 1-substituted imidazoles and beta-substituted pyridines showed high inhibitory potency on TXA2 synthase. The structure-activity relationships of the imidazole and pyridine derivatives as inhibitors of TXA2 synthase were investigated. Introduction of various substituents into the carboxy-bearing side chain of 1-(7-carboxyheptyl) imidazole and beta-(7-carboxyheptyl) pyridine was found to increase the inhibitory potency. The length of the side chains with the phenylene group was optimum in the region of 8.5 to 10 A for the inhibitory potency on TXA2 synthase. Among the tested imidazole and pyridine derivatives, (E)-4-(1-imidazolylmethyl)cinnamic acid (44) and (E)-3-[4-(3-pyridylmethyl)phenyl]-2-methylacrylic acid (56) showed the highest potency (IC50 = 1.1 x 10(-8) and 3 x 10(-9) M). The inhibition by these derivatives was highly selective for TXA2 synthase, since other enzymes which are involved in the arachidonic acid cascade, such as fatty acid cyclooxygenase, 5-lipoxygenase, prostacyclin (PGI2) synthase, and PGE2 isomerase were not affected. On the basis of the results obtained from the pharmacological, physicochemical and toxicological studies on the two compounds (44 and 56), (E)-4-(1-imidazolylmethyl) cinnamic acid (44; OKY-046, ozagrel) was selected as the best compound of highly selective inhibitors of TXA2 synthase. The pharmacological properties of ozagrel are as follows. The inhibition of TXA2 synthase by ozagrel was more effective on human and rabbit enzymes than those of other species. Ozagrel increased 6-keto-PGF1 alpha, one of stable metabolites of PGI2, in various isolated cells and tissues perhaps via accumulated PG endoperoxides resulted by the inhibition of TXA2 synthase. Such an increase in PGI2 production by ozagrel was also observed in various experimental animals. We obtained the suggestion that, by the reduction of TXA2 production and increment of PGI2 production, ozagrel inhibits the spasms of basilar artery and the decreases in regional cerebral blood flow in dogs which received autologous blood into cisterna magna, and inhibits the decreases in motor function and regional cerebral blood flow, and the formation of infarcted area in the animals of cerebral ischemic treatment. It was also suggested that ozagrel inhibits leukotriene-, platelet-activating factor-, and antigen-induced bronchoconstriction in guinea-pigs and inhibits the induction of airway hyperresponsiveness by various stimuli in several species of animals by both mechanisms. The summarized results of ADME, toxicological, and clinical studies were also described.

[Clinical studies on tobramycin using intravenous drip infusion (author's transl)].

Clinical studies and serum levels on tobramycin (TOB) using intravenous drip infusion were carried out and the following results were obtained. 1. Clinical results Nineteen patients with various infections including 11 respiratory tract infections, 7 urinary tract infections and a wound infection were treated with TOB 20-60 mg b.i.d. and t.i.d. by intravenous drip infusion and for 1 hour during 4 to 16 days. Clinical effects were excellent in 10 cases, good in 4 cases, fair in 2 cases and failure in 3 cases. It was 73.7% of the efficacy rate of the drug. Side effects were observed in 2 cases of oliguria and increase in BUN, S-creatinine and S-potassium. 2. Serum levels of TOB Serum levels of TOB administrated 30 or 60 mg intravenous drip infusion for 1 hour were measured in first and final administration of 8 patients. The peak serum levels were 3.1-5.0 micrograms/ml at the end of infusion in first 60 mg administration of the drug, and 3.8-5.3 micrograms/ml in final 60 mg administration of the drug.

[Double-blind comparative clinical evaluation of cefamandole and cefmetazole in the treatment of respiratory tract infections].

Cefamandole sodium (CMD), a new cephalosporin-derivative, was synthesized in the Laboratory of Eli-Lilly Co. Ltd. U.S.A. in 1972. CMD, which is several times more active than cefmetazole (CMZ, a cephamycin antibiotic) against Gram-positive cocci, is only as active as the latter antibiotic against Gram-negative bacilli. Against Haemophilus influenzae, CMD exhibits an antimicrobial activity which is as strong as that of ampicillin sodium. Our previous comparative tests on efficacy and safety of CMD versus cefazolin (CEZ) demonstrated that CMD was as effective and safe as CEZ in the treatment of respiratory tract infections. In the present clinical trial, the efficacy and safety of CMD are evaluated by a comparative double blind method using CMZ, a more recently synthesized cephamycin antibiotic, as a reference drug. For this purpose, a comparative double blind study was carried out in 50 institutions and clinics in Tohoku and Hokkaido districts in Japan. A total of 272 inpatients, who was aged over 16 years and was diagnosed as having pneumonia, lung abscess or acute infectious exacerbation of chronic obstructive pulmonary diseases, was included in this trial. They received 2 g of CMD or CMZ twice a day by intravenous drip infusions, as a rule, for 14 days. Of these patients, 264 (133 received CMD and 131 CMZ) were available for the evaluation of safety and usefulness. Two hundred and thirty-eight patients (122 received CMD and 116 CMZ) were adopted for the evaluation of efficacy. Prior to the treatment, there was no significant difference with respect to age, sex, severity of infection and underlying diseases between subjects in 2 treatment groups. An excellent or good clinical response was obtained in 82% of the patients treated with CMD, and in 81% of those treated with CMZ. Thus, there was no significant difference in cure rate between 2 treatment groups. However, an excellent clinical response was found in 12.3% of the patients treated with CMD, whereas only in 4.3% of those treated with CMZ. This difference in percentage of excellent clinical response between 2 treatment groups was statistically significant (P less than 0.05). Of the 87 patients with moderate to severe infection who were treated with CMD, 13 showed an excellent response. Only 4 of 90 patients treated with CMZ showed an excellent response. Statistically the difference in the rate of excellent response between these 2 groups was significant (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

[Clinical study on dosage schedule of tobramycin: controlled comparison between 180 mg b.i.d. and t.i.d].

The efficacy and safety of tobramycin (TOB) administration at 90 mg twice a day (90 mg group) to patients with respiratory tract infections (RTI) were objectively investigated by means of a well-controlled comparative study. The results in this 90 mg group were compared with the case of administering 3 times a day of 60 mg each (60 mg group) as the control. In both of these patient groups, TOB was administered by intramuscular injection for 10 consecutive days. This study included 85 patients (90 mg group: 43 patients; 60 mg group: 42 patients). The Committee, however, excluded 19 patients consisting of 13 cases of infection other than RTI and 6 cases of out of protocol drug administration. Thus, 66 patients (90 mg group: 32 patients; 60 mg group: 34 patients) were used for the efficacy analysis. All 85 patients were used, however, for the evaluation of the safety of TOB and for the usefulness of TOB as judged by the physicians in charge. A brief summary of the results is presented below. The cases employed by the Committee consisted of 8 and 17 cases in the 90 mg and 60 mg groups, respectively, with bacterial pneumonia and lung abscess, and 24 and 17 cases in the 90 mg and 60 mg groups, respectively, with chronic RTI. There was a tendency for the distribution of disease cases in the 2 drug groups to be significant. Within the chronic RTI disease classification, the number of patients with chronic bronchitis was almost equal in the 2 drug groups; 16 cases in the 90 mg group and 15 cases in the 60 mg group. However, secondary infections were diagnosed in 8 cases in the 90 mg group and 2 cases in the 60 mg group. The clinical efficacy judged for all cases investigated by the Committee showed efficacy ratios of 56.3% for the 90 mg group and 70.6% for the 60 mg group. The difference between the 2 drug groups was not statistically significant. In the treatment of bacterial pneumonia and lung abscess, the efficacy rate in the 90 mg group was 87.5%, while it was 88.2% in the 60 mg group; these high values were practically identical. Against chronic RTI, the efficacy rates were 45.8% in the 90 mg group and 52.9% in the 60 mg group; within that disease classification, the efficacy rates against the chronic bronchitis cases were nearly the same at 43.8% and 46.7%, respectively. These data showed no statistically significant differences between the 2 drug administration groups. (ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization.

A selective and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of glycyl-L-histidyl-L-lysine (GHK), a liver-cell growth factor isolated from human plasma, and its metabolite, L-histidyl-L-lysine (HK), in rat plasma. Both high selectivity and sensitivity were achieved by the use of solid-phase extraction with a Bond-Elut Certify cartridge, ion-pair chromatography with 1-pentanesulfonate on a 5-microm Capcell Pak C18 UG120 column (250x4.6 mm I.D.) with a guard column, and by post-column derivatization with o-phthalaldehyde (OPA). GHK and HK were extracted from 0.1 ml of rat plasma after addition of o-phenanthroline to protect against degradation. The limit of detection for GHK and HK were 50 and 15 ng/ml, respectively, and the calibration curves were linear in the range 0.1-5.0 microg/ml. The developed method was applied to the pharmacokinetic study of GHK after a single dose was administered intravenously to rats. GHK was rapidly degraded to HK, which was eliminated rapidly.

Low-molecular-weight heparin (dalteparin) demonstrated a weaker effect on rat bone metabolism compared with heparin.

We studied the pharmacological effects of dalteparin (low-molecular-weight heparin) and heparin on bone metabolism in rats. After their 28 days of consecutive intravenous injections, significant loss of bone weight and mineral contents was observed in the heparin-treated rats, whereas dalteparin slightly reduced bone mass. By the end of the experiment, the femora of 7 out of 8 rats fractured in the heparin (10,000 U/kg/day)-treated group, but none had broken in the control and dalteparin-treated groups. Serum osteocalcin levels were significantly decreased in the former group. The growth plate width of the tibia was increased in a dose-dependent manner, especially in the heparin-treated group. Histomorphometric assessment of tibia showed that the osteoid surface and mineral apposition rates were significantly reduced in the heparin-treated group, whereas the eroded surface was significantly increased in the heparin-treated group. The above results suggest that heparin not only augments bone resorption but also suppressed bone formation and that dalteparin has a weaker suppressive effect on bone formation compared with heparin.