Augmentation of tumor immunity in mice by intralesional injection of vitamin A.

We investigated the antitumor effect of vitamin A(VA) using the double grafted tumor technique to examine whether VA administered into a primary tumor (intralesionally or i.l.) accelerates antitumor immune reactions so that growth of the secondary tumor may be more effectively inhibited than by other systemic administration routes. In the double grafted tumor system, where BALB/c mice were inoculated with MethA fibrosarcoma cells into the right inguinal region (1 x 10[6] cells) on day 0 and later into the left (3 x 10[6] cells) on day 10, the injection of VA at a dose of 1000 IU/mouse i.l., s.c., i.p., and i.v. on days 3 through 7 inhibited the growth of the secondary tumor to the same extent, while VA at the i.l. dose of 100 IU/mouse into the primary tumor inhibited more effectively than by any other administration route. VA did not inhibit the secondary MethA growth in BALB/c (nu/nu) mice. The spleen cells taken from VA-treated tumor-bearing mice prevented the growth of MethA tumors in naive BALB/c mice when given as a mixture with the MethA inoculum (the Winn assay). The delayed-type hypersensitivity (DTH) response to methylated bovine serum albumin (MBSA) antigen was augmented when VA (1000 IU) was injected at the site of the antigen injection. These results suggest that the direct interaction of VA with the tumor cells may be necessary for the tumor immunity-potentiating effect of VA, and that T-lymphocyte-mediated tumor immunity is involved in the anti-tumor effect of VA. The antitumor mechanism of VA seems to involve retinoid receptors, because the benzoic acid derivative Am80, which has been reported to exert retinoidal activity by binding to specific retinoid receptors, also showed activity.

Study on the mechanism of immunopotentiating antitumor effect of 6-MPG, a water-soluble derivative of 6-mercaptopurine.

We investigated possible mechanisms of the antitumor action of gamma-(9H-purine-6-yl) thiomethyl L-glutamate (6-MPG), a water-soluble derivative of 6-MP. In the double grafted tumor system, BALB/c mice were inoculated intradermally with 10(6) cells of MethA fibrosarcoma at the right inguinal region on day 0 (the primary tumor) and later with 3 x 10(6) cells at the left on day 10 (the secondary tumor). Intraperitoneal administration of 6-MPG at a dose of 100 mg/kg/day from day 3 through 7 completely prevented growth of the secondary tumor. 6-MPG showed no effect on growth of colon 26 adenocarcinoma cells inoculated in place of the secondary MethA cells (antigen specificity). 6-MPG did not inhibit the secondary MethA growth in the BALB/c (nu/nu) mouse. The inhibitory effect of 6-MPG on the secondary tumor growth was diminished by prior treatment of the primed animals with cyclosporin A and anti-Thy antibody. Spleen cells from the tumor-bearing mice treated with 6-MPG showed a tumor-neutralizing activity (Winn assay). Treatment of the spleen cells with anti-CD8 antibody plus complement diminished the tumor-neutralizing effect but that with anti-CD4 antibody plus complement did not, indicating that CD8-positive cells are responsible for potentiation of the tumor immunity. These results suggest that the antitumor effect of 6-MPG against the secondary tumor is elicited by augmenting tumor specific T-cell production.

[Effect of betotastine besilate (TAU-284), a novel anti-allergic agent, on experimental allergic rhinitis].

We investigated the effect of betotastine besilate (betotastine) on the experimental allergic rhinitis. The oral administration of betotastine (1, 3 and 10 mg/kg) inhibited the increase in dye leakage during and after the nasal perfusion of antigen in actively sensitized rats. It also prevented the increase in intranasal pressure induced by topically applied histamine in non-sensitized guinea pigs. Cetirizine and terfenadine dose-dependently inhibited the increase in a similar manner. Ketotifen (0.01-0.3 mg/kg, p.o.) inhibited the increase more than 50% at 0.01 mg/kg. The ID50s of ketotifen, cetirizine, betotastine and terfenadine for this model were more than 0.01 mg/kg, 0.01 mg/kg, 0.03 mg/kg and 0.5 mg/kg, respectively. Furthermore, in actively sensitized guinea pigs, nasal airway resistance showed a biphasic increase after the topical antigen challenge to the nasal cavity; the first peak at 0.5 hr and a second peak at 4 hr. Both the responses of first and second peaks were significantly inhibited by orally administered betotastine besilate, and its inhibitory effect on the second peak was the strongest among drugs tested. Since betotastine showed significantly inhibitory effects in experimental allergic rhinitis models, it was suggested to show a good efficacy for the treatment of allergic rhinitis clinically.

[Anti-allergic activity of betotastine besilate (TAU-284), a new anti-allergic drug].

The anti-allergic activity of betotastine besilate (betotastine), a new anti-allergic drug, was investigated in several allergy models of rats in comparison with other anti-allergic drugs.1) Orally administered betotastine (0.1-30 mg/kg) inhibited homologous passive cutaneous anaphylaxis (PCA) in rats in a dose-dependent manner (ID30-value: 0.38 mg/kg). The inhibitory activity of betotastine was significant at 1 mg/kg and was more potent than that of ketotifen, terfenadine, cetirizine and epinastine. The PCA-inhibitory activity of betotastine lasted more than 8 hr after administration, and the repeated administration of betotastine lasted more than not induce drug-tolerance. 2) Orally administered betotastine inhibited the histamine-induced skin reaction in rats in a dose-dependent manner (ID30:0.10 mg/kg), and the inhibitory activity lasted more than 4 hr after the administration. Its inhibitory activity was significant at 0.1 and 1 mg/kg and was more potent than those of ketotifen, terfenadine, cetirizine and epinastine. 3) Betotastine suppressed histamine release from rat peritoneal mast cells at a high concentration (10(3)(10(-3)M). These results suggest that betotastine has a potent and long acting anti-allergic activity, and these effects are mainly due to histamine antagonistic activity.

Involvement of leukotrienes in allergic pleurisy in actively sensitized rats: inhibition by the lipoxygenase inhibitor T-0757 of the increase in vascular permeability and leukotriene E4 production.

The relative contributions of inflammatory mediators to the increase in vascular permeability in antigen-induced pleurisy were examined in rats actively sensitized with ovalbumin. The effects of various inhibitors were assessed on the exudate volume and plasma exudation rate in the pleural cavity. Two peaks were observed in plasma exudation rate at 0.5 and 3 h after antigen challenge. At 0.5 h, there was a marked decrease in the histamine content of the pleural cells and also a sharp increase in the LTE4 level in the exudate, which was inhibited dose-dependently by the lipoxygenase inhibitor T-0757. Indomethacin and cyproheptadine both depressed exudate volume and exudation rate, whereas T-0757 only reduced the exudation rate. At 3 h, a substantial LTE4 concentration was still detected in the exudate, and the exudation rate was depressed by T-0757 and indomethacin, but not by cyproheptadine. These results suggest that histamine is involved mainly in the early phase, and leukotrienes predominantly contribute to the later phase of exudation. Prostaglandins appear to be involved in both phases. Allergic pleurisy of rats, therefore, may be a suitable model to examine the roles of these inflammatory mediators.

Augmentation of tumor immunity by 6-mercaptopurine (6-MP) and its analogs in the double grafted tumor system in mice.

We investigated the antitumor effect of 6-mercaptopurine (6-MP) and its analogs using the double grafted tumor technique. BALB/c mice were inoculated intradermally with MethA fibrosarcoma cells at the right inguinal region on day 0 (the primary tumor) and at the left on day 10 (the secondary tumor). Intraperitoneal or intra-lesional administration of 6-MP, 6-mercaptopurine riboside (6-MP-r) and 6-mercaptopurine riboside triacetate (6-MPRTA) from day 3 to 7 dose-dependently inhibited growth of the secondary tumor. Without the primary inoculation, 6-MP showed no effect on growth of the tumor inoculated on day 10, indicating that the antitumor effect of 6-MP could not be attributable to its direct antimetabolic or tumoricidal action only, and that the primary tumor inoculation is necessary for these compounds to inhibits growth of the challenging tumor. 6-MP did not inhibit the secondary MethA growth in the BALB/c (nu/nu) mouse. Both CD4+ and CD8+ T cells increased in the spleen of mice treated with 6-MP. Meanwhile, delayed-type hypersensitivity (DTH) reaction to the methylated bovine serum albumin (MBSA) antigen at the footpad was not augmented but inhibited by 6-MP-r and 6-MPRTA in both normal and tumor-bearing mice. Thus, the immunomodulatory activity of 6-MP could be observed in two opposite directions, augmentation of tumor immunity and inhibition of DTH to MBSA. This indicates that the immune mechanism and/or the type of effector cells induced in these two cell-mediated immune systems are different from each other.

Difference among angiotensin-converting enzyme inhibitors in potentiating effects on bradykinin-induced microvascular leakage in guinea pig airways.

We investigated the effect of imidapril, a novel angiotensin-converting enzyme (ACE) inhibitor, on augmentation of airway microvascular leakage induced by bradykinin (BK) and substance P (SP) in guinea pigs and compared it with those of enalapril and captopril. The three ACE inhibitors significantly potentiated BK- and SP-induced airway microvascular leakage in a dose-dependent manner. In spite of the compatible or higher ACE inhibitory activity of imidapril, its potentiating activity in BK-induced leakage was lower than those of enalapril and captopril both by single administration (0.3-30 mg/kg, p.o.) and repeated administration for eight days (0.1-10 mg/kg/day, p.o.). The potentiating activities of the three ACE inhibitors were suppressed by pretreatment with a BK2-receptor antagonist, but not by neurokinin 1 and neurokinin 2 antagonists, suggesting that neurokinins may not be involved in BK-induced leakage under the conditions used. On the other hand, the potentiating effect of imidapril in SP-induced leakage was weaker than those of enalapril and captopril only after single high doses. The present study shows that the ACE inhibitors have different activity in potentiation of the airway microvascular leakage induced by BK, which may be ascribable to the difference in their inhibition of BK hydrolysis. This evidence may partly explain the smaller incidence of dry cough induced by imidapril compared with other ACE inhibitors when clinically used as antihypertensive drugs.

Involvement of leukotriene B4 in zymosan-induced rat pleurisy: inhibition of leukocyte infiltration by the 5-lipoxygenase inhibitor T-0757.

The role of leukotriene B4 (LTB4) in leukocyte infiltration in zymosan-induced rat pleurisy was investigated by studying the effects of 5-lipoxygenase inhibitors, T-0757 and AA-861, and a cyclooxygenase inhibitor, indomethacin, on leukocyte infiltration and LTB4 levels in the inflammatory exudate of rat pleurisy induced by intrapleural injection of zymosan (20 mg/rat). T-0757 and AA-861 inhibited the infiltration of leukocytes, mainly neutrophils, 3 h after injection of zymosan at a dose of 100 mg/kg, p.o., but indomethacin did not do so at a dose of 5 mg/kg, p.o. LTB4 was detected in the exudate 1 h after zymosan injection, and its level peaked at 3 h (45.4 +/- 8.6 ng/rat) and decreased thereafter. These LTB4 levels were depressed by T-0757 and AA-861 in a dose-dependent manner. T-0757 completely prevented LTB4 production at a dose of 100 mg/kg. These observations suggest that the inhibition of leukocyte infiltration is mediated by the inhibition of LTB4 production, and that LTB4 is one of the main chemical mediators of leukocyte infiltration in zymosan-induced rat pleurisy.

Inhibitory effect of 4-acylaminophenol derivatives (T-0799 and T-0757), novel lipoxygenase inhibitors, on arachidonic acid-induced infiltration of polymorphonuclear leukocytes in rat skin.

Effects of 4-acylaminophenol derivatives, novel 5-lipoxygenase inhibitors, on the neutrophil infiltration in arachidonic acid (AA) (10 mg/site)-induced skin inflammation in rats were examined. Myeloperoxidase (MPO) activity in the skin lesion, used as an indicator of neutrophil infiltration, was significantly increased after intradermal injection of AA. Dual inhibitors of cyclooxygenase and 5-lipoxygenase, phenidone (100 mg/kg x 2, i.p. and p.o.) and BW-755C (50 mg/kg x 2, p.o.), and 5-lipoxygenase inhibitors, AA-861 (100 mg/kg x 2, i.p.) and the 4-acylaminophenol derivatives T-0799 and T-0757 (10-100 mg/kg x 2, p.o.), inhibited the increase in MPO activity 5 hr after AA-injection, but the cyclooxygenase inhibitor indomethacin (5 mg/kg, i.p.) showed no effect. These results suggest that products of lipoxygenase, but not of cyclooxygenase, are involved in the MPO activity increase (i.e., neutrophil infiltration), and that this model is useful for in vivo evaluation of 5-lipoxygenase inhibitors. It is suggested that the 4-acylaminophenol derivatives may be useful as orally active drugs for treatment of some leukotriene-mediated diseases.

4-acylaminophenol derivatives as novel lipoxygenase inhibitors: synthesis and inhibitory effect on 5-lipoxygenase and leukotriene B4 production.

Structure-activity relationships in the inhibitory effects of 4-acylaminophenol derivatives on the 5-lipoxygenase (5-LOX) from RBL-1 cells and leukotriene B4 (LTB4) production by guinea pig neutrophils were studied. When the N-acyl group was n-octanoyl or 2-thiophenecarbonyl and the size of the two ortho substituents of phenol was varied, the substituents bulkier than isopropyl, i.e., 2,6-di-tert-butyl and 2,6-dicyclohexyl, substantially weakened the inhibitory activity in both enzymatic and cellular systems. Among the 2,6-dimethyl derivatives with an acyl group of various carbon-chain lengths (C1-13), those with a n-alkyl chain of C5 to C12 showed similarly potent inhibitory activities toward 5-LOX with an IC50 ranging from 0.27 to 0.66 microM; in contrast, maximal inhibitory activities toward LTB4 production were observed in a narrower range of the serial compounds: i.e., those with a n-hexyl, n-heptyl, or n-octyl chain on the carbonyl carbon formed by far the most inhibitory group of the series and the inhibitory activity sharply decreased on either side of the chain length. Nearly all the active compounds also inhibited cyclooxygenase (COX), but the IC50 values for COX inhibition were more than ten times higher than the corresponding IC50 values for 5-LOX inhibition in most cases, indicating that the acylaminophenols are relatively selective 5-LOX inhibitors.

Studies on a new antiallergic pyridinecarboxamide TA-5707F and its sodium salt (TA-5707). II. Mechanism of antiallergic action of TA-5707.

Mechanism of the antiallergic action of 6-methyl-N-(1H-tetrazol-5-yl)-2-pyridinecarboxamide (TA-5707F) was studied using the water-soluble sodium salt (TA-5707). 1) TA-5707 administered p.o. at the dose ca. 3 times the ID50 for the PCA reaction did not inhibit capillary dye leakage induced on the rat skin by intracutaneous injection of histamine, serotonin, bradykinin and leukotriene D4 (LTD4). 2) TA-5707, at concentrations above 10(-7) M, inhibited antigen-induced histamine release and dye leakage in the rat peritoneal cavity (passive peritoneal anaphylaxis). 3) TA-5707 inhibited both anaphylactic and compound 48/80-induced histamine release from the rat peritoneal mast cells, the IC50 being ca. 10(-5) M in both cases. It was concluded that TA-5707F exerts its antiallergic action by inhibiting the release of chemical mediators from sensitized cells.

Studies on a new antiallergic pyridinecarboxamide TA-5707F and its sodium salt (TA-5707). I. Inhibition of IgE-induced passive cutaneous anaphylaxis (PCA) in rats.

Effects of TA-5707F [6-methyl-N-(1H-tetrazol-5-yl)-2-pyridinecarboxamide] and its sodium salt (TA-5707) on IgE-induced homologous PCA in rats were investigated. 1. Both TA-5707 and TA-5707F were found to be orally effective inhibitors of rat PCA. Maximum activity by oral administration was obtained when they were administered 5 min before the challenge. Their ID50's under these conditions were both approximately 1 mg/kg. Administration 5 min after the challenge was no longer effective. 2. TA-5707 was also effective by intravenous administration, and its ID50, ca. 0.1 mg/kg, was less than that of disodium cromoglycate (DSCG). 3. The PCA-inhibitory activity of TA-5707 was not affected by adrenalectomy and adrenomedullectomy. 4. Daily administration of TA-5707 or TA-5707F for 8 days did not induce drug tolerance. 5. Tachyphylaxis and cross-tachyphylaxis were observed when the PCA-inhibitory activities of TA-5707 and DSCG were tested after intravenous pretreatment with a large dose (ca. 30 times the ID50) of either drug, but not after oral pretreatment with a therapeutic dose of TA-5707 or TA-5707F.

Antiallergic agents. 3. N-(1H-tetrazol-5-yl)-2-pyridinecarboxamides.

A series of N-tetrazolylpyridinecarboxamides was prepared and evaluated for antiallergic activity by the passive cutaneous anaphylaxis (PCA) assay. From the structure-activity relationships (SAR) of this class of compounds, it was revealed that the N-tetrazolylcarbamoyl group as an acidic functionality is required to be at the 2-position of the pyridine nucleus and that the phenyl group as a subtituent is not necessarily required for activity. 6-Methyl-N-(1H-tetrazol-5-yl)-2-pyridinecarboxamide (36) showed good oral activity and low toxicity.

Antiallergic agents. 2. N-(1H-tetrazol-5-yl)-6-phenyl-2-pyridinecarboxamides.

A new series of N-(1H-tetrazol-5-yl)-6-phenyl-2-pyridinecarboxamides was prepared to determine the effects of substituents on the benzene and pyridine rings on antiallergic activity in the rat passive cutaneous anaphylaxis (PCA) assay after oral administration. One member of this series, N-(1H-tetrazol-5-yl)-4-methyl-6-[4-(methylamino)-phenyl]-2- pyridinecarboxamide (231), has an ED50 value of 0.8 mg/kg po and is 85 times more potent than disodium cromoglycate (DSCG) on intravenous administration. Further evaluation of 231 as a clinically useful antiallergic agent is in progress.

Quantitative determination of trimetoquinol in plasma as its mixed pentafluoropropionyl-trimethylsilyl derivative by gas chromatography mass spectrometry.

A method is described for the determination of the bronchodilator trimetoquinol in plasma, based on selected ion monitoring gas chromatography mass spectrometry employing a deuterated trimetoquinol as internal standard. N-methyl-(trimethylsilyl)pentafluoropropionamide and N-methyl-bis-pentafluoropropionamide were prepared for derivatization of trimetoquinol into a mixed O-TMS-N-PFP derivative, which was found to be the most suitable form in terms of sensitivity, specificity and efficiency. The calibration curve was linear in the range of 2-50 ng ml-1, and the lower limit of detection was 0.1 ng ml-1 of rat plasma.