Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. Hepatoma Prevention Study Group.

BACKGROUND: In patients with hepatocellular carcinoma (hepatoma), the rate of recurrent and second primary hepatomas is high despite surgical resection and percutaneous ethanol-injection therapy. We developed an acyclic retinoid, polyprenoic acid, that inhibits hepatocarcinogenesis in the laboratory and induces differentiation and apoptosis in cell lines derived from human hepatoma. In a randomized, controlled study, we tested whether the compound reduced the incidence of recurrent and second primary hepatomas after curative treatment. METHODS: We prospectively studied 89 patients who were free of disease after surgical resection of a primary hepatoma or the percutaneous injection of ethanol. We randomly assigned the patients to receive either polyprenoic acid (600 mg daily) or placebo for 12 months. We studied the remnant liver by ultrasonography every three months after randomization. The primary end point of the study was the appearance of a histologically confirmed recurrent or new hepatoma. RESULTS: Treatment with polyprenoic acid significantly reduced the incidence of recurrent or new hepatomas. After a median follow-up of 38 months, 12 patients in the polyprenoic acid group (27 percent) had recurrent or new hepatomas as compared with 22 patients in the placebo group (49 percent, P = 0.04). The most striking difference was in the groups that had second primary hepatomas--7 in the group receiving polyprenoic acid as compared with 20 in the placebo group (P = 0.04 by the log-rank test). Cox proportional-hazards analysis demonstrated that as an independent factor, polyprenoic acid reduced the occurrence of second primary hepatomas (adjusted relative risk, 0.31; 95 percent confidence interval, 0.12 to 0.78). CONCLUSIONS: Oral polyprenoic acid prevents second primary hepatomas after surgical resection of the original tumor or the percutaneous injection of ethanol.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils.

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2-200 microM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 trisphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

Heterogeneity of circulating and exudated polymorphonuclear leukocytes in superoxide-generating response to cyclic AMP and cyclic AMP-elevating agents. Investigation of the underlying mechanism.

It has been found that cyclic AMP and cyclic AMP-elevating agents inhibit formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide production from polymorphonuclear leukocytes (PMNs). The quantitative differences of this inhibitory effect on human and rabbit blood versus human salivary and rabbit peritoneal (tissue) PMNs were investigated. PMNs from all sources showed the same pattern of fMLP-stimulated superoxide generation, although it was slightly higher in tissue PMNs. However, treatment with salbutamol differentially blunted fMLP-stimulated superoxide production from blood PMNs compared with tissue PMNs in both human and rabbit. While it could inhibit production from blood PMNs by 30-60%, it had only a negligible effect on generation from tissue PMNs. Similarly, forskolin, phosphodiesterase IV inhibitor Ro-201724, and dibutryl cyclic AMP showed significantly higher inhibitory effects on superoxide generation from blood PMNs than tissue PMNs in both species. beta-Adrenergic receptors, cyclic AMP accumulation, and protein kinase A activity were investigated in blood versus tissue PMNs to clarify the mechanism underlying the above-mentioned differences. At the beta-adrenergic receptor level, no significant changes were detected in the number or the binding affinity of the receptors in tissue versus blood PMNs of human and rabbit. On the other hand, cyclic AMP accumulation was significantly higher in response to salbutamol and Ro-201724 in fMLP-stimulated blood versus tissue PMNs in human and rabbit. At the same time, blood PMNs showed significantly higher cyclic AMP-dependent protein kinase A activity than tissue PMNs in human and rabbit. We concluded that tissue PMNs are less responsive to the effect of cyclic AMP-elevating agents in terms of fMLP-stimulated superoxide inhibition. This is due to differences, at least, at two levels. The first is lower accumulation of cyclic AMP and the second is lower protein kinase A activity in tissue versus blood PMNs.

A proposal for purification of salivary polymorphonuclear leukocytes by combination of nylon mesh filtration and density-gradient method: a validation by superoxide- and cyclic AMP-generating responses.

Isolation and purification of salivary polymorphonuclear leukocytes (SPMNs) from accompanying epithelial cells was presented by using a density-gradient method with Ficoll. SPMNs samples prepared by already established methods (nylon mesh filtration) was compared with SPMNs samples after further purification by Ficoll (d = 1.083). Microscopically, SPMNs samples after nylon mesh filtration contain higher percentage of epithelial cells than SPMNs samples after Ficoll centrifugation. In response to stimulation of superoxide generation, both samples showed the same pattern of response. However, in response to forskolin and prostaglandin E1, cyclic AMP levels in samples after nylon mesh purification were significantly higher than in samples after Ficoll purification because of the presence of contaminating epithelial cells. We can conclude that, although nylon mesh filtration is satisfactory when we need to examine superoxide generation but further purification is necessary when we want to measure factors like intracellular cyclic AMP formation.

Glucagon modulates superoxide generation in human polymorphonuclear leucocytes.

It has been found that leucocytes possess receptor sites for glucagon and glucagon was shown to increase during bacterial infection. To verify the interconnection between glucagon, leucocytes and bacterial infection we studied the effect of glucagon on superoxide generation and second messenger transduction in PMNs. We found that glucagon could not stimulate chemiluminescence by itself but it could enhance FMLP- but not PMA-induced chemiluminescence in a concentration (50-800 pg/ml) dependent manner. However, after incubation of PMNs with 10 microM of ST-638 (a tyrosine kinase inhibitor) the enhancement effect converted into inhibitory effect. We also found that glucagon treatment of PMNs increased both IP3 and cyclic AMP levels as second messengers. ST-638 greatly attenuated the IP3 increment in the glucagon-treated FMLP-stimulated PMNs. From these results we can conclude that glucagon could enhance superoxide generation from FMLP-stimulated PMNs by elevating IP3. Inhibition of IP3 increment by tyrosine kinase blockade uncover the inhibitory effect of the increasing cyclic AMP on superoxide production.

Platelet prostaglandin E1 hyposensitivity in schizophrenia: decrease in cyclic AMP formation and in inhibitory effects on aggregation.

Cyclic adenosine monophosphate (cAMP) formation via prostaglandin E1 (PGE1) or forskolin stimulation was determined in washed platelets from 35 schizophrenic patients and 34 normal controls. The inhibitory effects of PGE1 and forskolin on platelet aggregation response (PAR) elicited by adenosine diphosphate (ADP) were also examined simultaneously. Both PGE1- and forskolin-stimulated cAMP formation decreased in platelets from schizophrenic patients, as compared with those from normal controls. PGE1 inhibition of PAR was significantly lowered in schizophrenic patients compared to normal controls, but forskolin inhibition was not. No correlations between PGE1- or forskolin-stimulated cAMP formation and inhibitory effects of PGE1 or forskolin on PAR, respectively, were explained by the complex factors involved in PAR. In conclusion, platelet hyposensitivity to PGE1 in schizophrenic patients is partially based on aberrant adenylate cyclase (AC) activity and includes other variables related to PAR.

Platelet prostaglandin E1 hyposensitivity in schizophrenia: reduction of prostaglandin E1- or forskolin-stimulated cyclic AMP response in platelets.

Cyclic 3',5'-adenosine monophosphate (cAMP) formation via prostaglandin E1 (PGE1)-or forskolin-stimulation were determined in washed intact platelets from 32 schizophrenic patients and 30 normal controls. Regarding basal cAMP levels in the platelets, there were no differences between schizophrenic patients and normal controls. Both PGE1-and forskolin-stimulated cAMP response reduced in platelets from schizophrenics compared with normal controls. These results suggested that platelets in schizophrenics were impaired not only in the adenylate cyclase unit per se but also extensively in the cAMP generating system coupled to a PGE1 receptor.

[Characterization of adrenal medullary opioid receptors. I. Binding of opioids to adrenal medullary opioid receptors].

We studied the binding of [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) and [3H] diprenorphine to crude plasma membrane fraction obtained from the bovine adrenal medulla (bovine adrenal medullary membranes) in order to characterize adrenal medullary opioid receptors. The [3H] diprenorphine binding was the highest in crude plasma membrane-mitochondrial fraction among all subcellular fractions studied. The amount of [3H] diprenorphine bound to bovine adrenal medullary membranes was proportional to the protein concentration. Association kinetics of the [3H] diprenorphine binding to bovine adrenal medullary membranes showed that the maximal binding was achieved following 8 min incubation and that the binding conformed the second-order kinetics. [3H] DADLE and [3H] diprenorphine bound to bovine adrenal medullary membranes with high affinities. The Kd and Bmax for the [3H] DADLE binding were found to be 2.9 nM and 57.5 fmole/mg protein, respectively, while those for the [3H] diprenorphine binding were 0.31 nM and 250 fmole/mg protein, respectively. Displacement studies showed that the [3H] diprenorphine binding was inhibited dose-dependently by levorphanol, dynorphin (1-13), beta-endorphin and DADLE. Levorphanol was at least 1000-fold more potent to inhibit the [3H] diprenorphine binding than dextrorphan, indicating stereospecificity of the [3H] diprenorphine binding. Na+, Li+ and K+ (100 mM) diminished the [3H] DADLE binding and enhanced [3H] diprenorphine binding. Na+ (100 mM) increased the Kd value for the [3H] DADLE binding from 2.9 nM to 14.1 nM. Mn++, Ca++ and Mg++ diminished the [3H] diprenorphine binding. Mn++ (1 mM) increased the Bmax value for the [3H] DADLE binding from 95 fmole/mg protein to 450 fmole/mg protein. These effects of Na+ and Mn++ on the [3H] diprenorphine binding were found to be dose-dependent. [3H] Diprenorphine binding to the digitonin-solubilized opioid receptor was also inhibited dose-dependently by Mn++. These results suggest that bovine adrenal medullary membranes contain high affinity and stereospecific opioid receptors and that the binding of opioids to the bovine adrenal medullary opioid receptors is influenced by cations. Binding study also revealed the presence of opioid receptors in human malignant pheochromocytoma. The Kd and Bmax of the [3H] diprenorphine binding to crude membrane fraction obtained from malignant pheochromocytoma were found to be 0.14 nM and 10.4 fmole/mg protein, respectively.

Interaction of BW755C, a potent inhibitor of lipoxygenase and cyclo-oxygenase, with mitochondrial cytochrome oxidase.

The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytochrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes.

[Ossification arising in the atypical epithelium in primary stomach cancer--report of a case].

We report a case of primary stomach carcinoma arising in the atypical epithelium, which was accompanied with ossification. The patient was a 73-year-old man, who was operated because of the possibility of advanced Type IIa carcinoma. Grossly, a large papillary polypoid lesion was recognized in the anterior wall of the antrum near the pylorus ring. Histological study revealed well differentiated tubular adenocarcinoma arising in the atypical epithelium; ossification was found in the stroma without necrosis, calcification or chondrification. Ossification arising in primary stomach carcinoma is very rare. Only five cases of ossification in primary stomach carcinoma have been reported in the world literature; two of these were Japanese patients. Probably ours is the first reported case with both stomach carcinoma and ossification arising in the atypical epithelium.

[Anti-inflammatory, analgesic and antipyretic activities of alpha-(p-thenoylphenyl)-propionic acid (TN-762) (author's transl)].

We reported in our previous paper that TN-762, a potent inhibitor of prostaglandin biosynthesis, has marked inhibitory activity on acute experimental inflammation. In this paper, the anti-inflammatory, analgesic, and antipyretic activities of TN-762 were assessed in animal models, and compared with those of indomethacin, ketoprofen and ibuprofen. TN-762 inhibited the sustained paw edema induced by mustard in rats during administration for 3 days, but after final administration, the inhibitory activity was decreased rapidly and was less then that of ketoprofen and indomethacin. TN-762 also inhibited the proliferation of granuloma induced by means of cotton pellet and granuloma pouch methods, and the adjuvant arthritis in rats. The inhibitory activity of the compound on inflammatory proliferation was more potent than that of ibuprofen, but slightly less than that of ketoprofen and less than about 1/10 times that of indomethacin. Indomethacin markedly inhibited the body weight gain at a high dose, while TN-762 did not affect it. Therefore, TN-762 was proven to have an inhibitory effect on subacute and chronic inflammation at low doses without toxic effects, but the compound appeared to have a less of an inhibitory effect on secondary or late stages of inflammation than on primary stage inflammation. TN-762 inhibited the acute paw edema induced by nystatin, and the inhibitory activity was the same as that of ketoprofen and indomethacin. The pathogenesis of nystatin edema has been considered to be due to lysosomal labilization. This result suggests that TN-762 has a potent membrane stabilizing action which is considered to be one of the necessary mechanisms in anti-inflammatory action. On the other hand, TN-762 showed the same potent analgesic effect as ketoprofen and indomethacin as observed by the acetic acid writhing and modified Haffner's methods in mice and by the Randall-Selitto's method in rats. However the antipyretic effect of TN-762 was significantly less than that of ketoprofen and indomethacin.

[Clinical studies on cefoxitin in the field of gastro-surgery (author's transl)].

Cefoxitin (CFX) was administered to the total of 21 hospitalized patients at a daily dose of 1 to 6 g for the duration of 6 to 23 days and the following results were obtained. 1) Clinical results of the 10 patients with surgical infections were excellent in 3 patients, good in 5, fair in 1 and poor in 1, with the efficacy rate of 90%. 2) CFX was also administered to 11 cases for prophylaxis of postoperative infections and the clinical efficacy rate was 100%. 3) Susceptibility tests showed all clinical isolates such as E. coli, Klebsiella and Gram-negative anaerobes were highly susceptibility to CFX except for Pseudomonas. 4) There were no subjective nor objective side effects related to CFX. The above results indicate that CFX is exceedingly useful for the treatment of infections in the field of gastro-surgery.

Urinary excretion of acid glycosaminoglycans and hydroxyproline in a patient with oculo-cerebro-renal syndrome.

The metabolism of ground substance in connective tissue of an 18-year-old boy with oculo-cerebro-renal syndrome was studied. He had characteristic clinical and laboratory findings described by Lowe et al. such as growth retardation, mental deficiency, glaucoma, cataracta, decreased muscle tone, metabolic acidosis, aminoaciduria and osteomalacia. The urinary excretion of acid glycosaminoglycans and of total hydroxyproline were 27 mg/day (as glucuronic acid) and 280 mg/day respectively on admission. Both values decreased to the upper limits of normal level transiently during treatment with alkali and vitamin D2. At that time, an improvement in bone abnormalities, a decrease of serum alkaline phosphatase, and an elevation of serum inorganic phosphate were observed. The therapy prevented him from progressive osteomalacia and cured him of it, but mucopolysacchariduria and hydroxyprolinuria did not disappear. Analytical electrophoresis on cellulose acetate sheets showed that urinary acid glycosaminoglycans were composed of undersulfated chondroitin 4-/6-sulfate and heparan sulfate with a ratio of 6:4, on admission. After oral administration of alkali, the excretion of heparan sulfate decreased and undersulfated chondroitin 4-/6-sulfate was determined as a main component of urinary acid glycosaminoglycans. The clinical and laboratory data in this case suggested that the increased excretion of acid glycosaminoglycans and total hydroxyproline was caused by abnormal metabolism in connective tissues, especially by the bone abnormalities, in this syndrome.

[Interactions between 6-chloro-5-cyclohexyl-1-indancarboxylic acid (TAI-284) and biopolymers (author's transl)].

TAI-284, a new non-steroidal acidic analgesic and anti-inflammatory agent was investigated and the interactions with biopolymers were compared with those of indomethacin (IMC) and ibuprofen (IP). TAI-284 inhibited the heat denaturation of bovine serum albumin at pH 5.3, similar to that seen with IMC and weaker than that seen with phenylbutazone. TAI-284 prevented the rat erythrocyte from heat-induced hemolysis and was twice as potent as IMC. TAI-284 produced alterations in platelet function as characterized by loss of secondary aggregation in response to ADP and inhibition of aggregation by collagen. Both these effects were one fifth as potent as those seen with IMC. In rats, platelet aggregation induced by collagen and secondary ADP aggregation was reduced in a dose-dependent manner by a single oral administration of TAI-284. The inhibitory activity was approximately one fourth that of IMC or twice that of IP in the former and in the latter one fifth that of IMC or similar as that of IP. These results suggest that the essential feature of TAI-284 is its potent membrane stabilizing action which is considered to be an necessary mechanism in the action of anti-inflammatory drugs. It is considered that TAI-284 may be less active than IMC in inhibiting prostaglandin biosynthesis in platelets.