Role of nitric oxide in the convulsions following the coadministration of enoxacin with fenbufen in mice.

Effects of nitric oxide (NO) synthase inhibitors on the enoxacin-induced convulsions were examined in mice pretreated with fenbufen. 7-nitroindazole markedly suppressed the incidence of convulsions, whereas L-arginine did not modify the convulsions at all. The suppression of the convulsions by 7-nitroindazole was not reversed by the pretreatment of L-arginine. Brain NO synthase activity was significantly raised at 30 min after enoxacin when combined with fenbufen. The increased NO synthase activity was found to be suppressed by the pretreatment of 7-nitroindazole. These findings suggest that endogenous NO may be involved as a proconvulsant substance in the development of enoxacin-induced convulsions in mice pretreated with fenbufen.

Circadian variation in enoxacin-induced convulsions in mice coadministered with fenbufen.

Susceptibility to enoxacin (80 mg/kg, p.o.)-induced convulsions was examined in mice coadministered with fenbufen (100 mg/kg, p.o.) over 24 hr at 3-hr intervals (light 7:00-19:00 hr). There was a marked circadian variation in the incidence of clonic and tonic convulsions and mortality. The susceptibility to enoxacin was higher around 15:00-18:00 hr and lower around 3:00-9:00 hr; the 50% clonic convulsive dose (CD50) at 9:00 and 15:00 hr was 95.0 and 56.5 mg/kg, respectively, its ratio being 1.64. Under these conditions, brain enoxacin level at 15:00 hr was increased 2.43-fold over that at 9:00 hr 30 min after enoxacin administration. Thus, the change of brain enoxacin may contribute to one of the causes of the above circadian variation.

Sustained release of flufenamic acid from a drug-triacetyl-beta-cyclodextrin complex.

Triacetyl-beta-cyclodextrin (TA-beta-CyD), a hydrophobic cyclodextrin derivative that is insoluble in water, was used to form a complex with flufenamic acid (FA). Complexes of FA with TA-beta-CyD (FA-TA-beta-CyD) at various molar ratios (1:1, 1:2, 1:3) were prepared by a kneading method, using ethanol as a solvent. FA-TA-beta-CyD complex formation was demonstrated by differential scanning calorimetry and powder X-ray diffractometry. The release rate of FA from the FA-TA-beta-CyD complexes was measured in both the Japanese Pharmacopoeia XII 1st fluid pH 1.2 and isotonic phosphate buffer pH 6.8. The release rate of FA from the FA-TA-beta-CyD complexes in the isotonic phosphate buffer pH 6.8 was significantly retarded compared to the release rate of FA from the FA-glucose mixture. After 1 h, 100% of the drug was released from the FA-glucose mixture and 10-25% was released from the complexes. When either the powder of the FA-glucose mixture or the FA-TA-beta-CyD mixture was administered directly into the intraduodenal lumen in rats, the plasma concentration of FA reached a maximum level within 40 min after administration. On the other hand, when the FA-TA-beta-CyD complexes were administered into the intraduodenal lumen, the plasma concentration of FA did not show a sharp peak, but remained at a plateau level (10-18 microg/ml) for 6-8 h. An increased mean residence time of FA following FA-TA-beta-CyD complexes administration was observed; however, the AUC(0-10) for the FA-TA-beta-CyD complexes showed no significant difference from that for the FA-TA-beta-CyD mixture. These results indicate that TA-beta-CyD may serve as a hydrophobic carrier in sustained-release preparations of FA. The drug-TA-beta-CyD complexes may therefore be useful in oral administration to achieve prolonged action and reduced side effects.

Protection against alloxan-induced diabetes by various dialkyldithiocarbamates.

Dialkyldithiocarbamates injected into mice 0.5 h prior to alloxan protected dose-dependently against the diabetogenic action of alloxan, and increased blood glucose levels at the time of alloxan injection. Furthermore, they exhibited anti-oxidative properties in vitro such as inhibition of lipid peroxidation, removal of hydrogen peroxide and reduction of the stable free radical, 1, 1-diphenyl-2-picrylhydrazyl (DPPH). These results suggest that dialkyldithiocarbamates protect against the development of alloxan-induced diabetes by the indirect mechanism of producing hyperglycemia at the time of alloxan injection and possibly by their anti-oxidative effects as well.

Improvement of the rectal bioavailability of latamoxef sodium by adjuvants following administration of a suppository.

The absorption of an antibiotic, latamoxef sodium (LMOX), following the rectal administration of a suppository containing adjuvants was investigated. A lipophilic base (Witepsol H15) was used. The rectal absorption of LMOX following the administration of a suppository without adjuvants was very low. Diclofenac sodium (DF) was used as an absorption promoter; it enhances rectal membrane permeability. The blood level of LMOX following the addition of DF(10 mg) to the base was increased only about 1.3-fold compared with that achieved with LMOX alone (difference not significant); even with a higher dose of DF, the absorption of LMOX was not sufficient. The release rate of LMOX from the base was slow. When Tween 80, a non-ionic surfactant, was added to improve the release rate of LMOX, the rate was sufficiently increased. The rectal absorption of LMOX on the addition of both Tween 80 and DF was markedly increased compared to that achieved with LMOX alone or with DF. These results indicate that the rectal absorption of LMOX after administration by a suppository was sufficiently improved by enhancing both the release rate from the base and the membrane permeability of the rectum. Lymphatic uptake and blood levels of LMOX were also investigated after the rectal administration of the LMOX preparation containing both Tween 80 and DF; the lymphatic uptake of LMOX was significantly enhanced compared with the LMOX preparation in which only DF was used as an adjuvant. The mechanism whereby adjuvants lead to the absorption of a non-absorbable drug, and the subsequent drug transportation routes through the membrane are discussed.

Involvement of blood glucose in the dimethylthiourea-induced protection against alloxan-induced diabetes.

Dimethylthiourea (DMTU, 4.0 mmol/kg) injected into mice 30 min prior to alloxan injection markedly protected mice against the diabetogenic actions of 75 mg/kg alloxan. At 30 min after the above dose of DMTU alone (no alloxan), there was a marked rise in blood glucose. Mannoheptulose, an antagonist of glucose action at pancreatic beta-cells, when given 24 min after DMTU and 6 min before alloxan, eliminated the DMTU-induced protection. The protection was also removed in the fasted mice in which DMTU did not cause hyperglycemia. These results indicate that DMTU protected mice from alloxan-induced diabetes by the indirect mechanism of producing hyperglycemia at the time of alloxan injection.

Protection against alloxan-induced diabetes by diethyldithiocarbamate and disulfiram in mice.

Diethyldithiocarbamate (DEDC, 0.25-2.00 mmol/kg) injected into mice at 0.5 hr prior to alloxan administration dose-dependently protected the mice against the diabetogenic actions of 75 mg/kg alloxan. Disulfiram (DS, 0.50-2.00 mmol/kg), a corresponding disulfide form, also exhibited similar protection. The maximum effect of DEDC was found by dosing at 0.5 hr prior to alloxan, and the effect afforded by DEDC pretreatment persisted up to 3 hr, whereas the effect of DS was exhibited when the compound was given 0.5 hr prior to alloxan. Of the metabolites of DEDC, diethylamine and carbon disulfide had no effect. At 0.5 hr after injection, DEDC alone had a potent increasing ability on blood glucose in a dose-dependent manner, but DS was less potent. Mannoheptulose, an antagonist of glucose action at pancreatic beta-cells, when given 24 min after DEDC and 6 min before alloxan, eliminated the DEDC-induced protection. Fasted mice did not exhibit hyperglycemia at 0.5 hr after DEDC injection, and alloxan given at that time produced diabetes. These findings indicate that DEDC itself protected mice from alloxan-induced diabetes by the indirect mechanism of producing hyperglycemia at the time of alloxan administration. The anti-diabetogenic action of low doses of DS and DEDC, in animals lacking hyperglycemia at the time of alloxan injection, is likely based on a mechanism other than one involving hyperglycemia.

Protection by disulfiram and diethyldithiocarbamate against hypoxia-induced lethality in mice.

The effects of disulfiram (DS) and its major metabolite, diethyldithiocarbamate (DEDC), on the survival time under normobaric and hypobaric hypoxia were examined in mice. At an ambient temperature of 24 degrees C, DS at 0.5-3.0 mmol/kg (i.p.) caused a marked dose-dependent prolongation of the survival time in mice subjected to both types of hypoxia. DEDC also prolonged the survival time, but the effect was less at its higher doses with decreased brain superoxide dismutase. The maximum effects of DS and DEDC were found at 3 hr and 1 hr after injection, respectively. Of the metabolites of DEDC, the copper complex with DEDC caused a significant effect, whereas neither diethylamine nor carbon disulfide did. Furthermore, DS, DEDC and copper complex caused marked hypothermia, and the time course changes of hypothermia by DS and DEDC closely paralleled those of the degree of anti-hypoxic effects, respectively. At an ambient temperature of 36 degrees C, in which the body temperature was maintained near the normal level, both DS and DEDC still exhibited a weak anti-hypoxic effect. These results suggest that DEDC itself, formed as a metabolite of DS, and partly the copper complex produced the anti-hypoxic effect, which could not be explained by concomitant hypothermia alone.

Circadian rhythm in the cerebral resistance to hypoxia in mice.

The cerebral resistance to hypoxia in mice was investigated by measuring the survival time under both hypobaric and normobaric hypoxic conditions. In the ad libitum fed mice, there was a circadian variation in the survival time that was longer during the light period than during the dark period under hypobaric hypoxic conditions. The survival time under normobaric hypoxic conditions also exhibited a similar circadian variation in the ad libitum fed mice, whereas the rhythm of the survival time was completely reversed by the restriction of food presentation (9:00-15:00). These findings suggest that there is a circadian rhythm in the cerebral resistance of mice to hypoxia, which can be shifted by the time of food presentation. Furthermore, regression analyses revealed a negative correlation between the survival time of mice exposed to hypoxia and body temperature, and blood glucose levels. These indicate that the cerebral resistance to hypoxia was intimately associated with body temperature and blood glucose that both show a circadian rhythm in mice.

Circadian periodicity in the duration of decapitation-induced gasping in mice.

The circadian variation of decapitation-induced gasping was investigated by measuring the gasping duration of isolated mouse head after decapitation under both normal and restricted feeding conditions. In the normally fed mice, there was a circadian periodicity in the gasping duration: it was longer during the light period than during the dark period. The circadian periodicity was completely reversed by the restriction of food. The circadian periodicity of the gasping duration were conversely parallel to those of body temperature in both normal and feeding restricted mice, and regression analysis revealed a negative correlation between the gasping duration and body temperature. Furthermore, pentobarbital and ethanol, agents that caused hypothermia, markedly prolonged the gasping duration. These findings suggest that there is a circadian periodicity in the brain reactivity after complete ischemia, which may be associated with the changes of body temperature.

Analysis of false negative cytology by a chromosome study.

Forty-two cases of pre-invasive and invasive cancer of the cervix diagnosed by histology were studied cytologically and cytogenetically. Preinvasive lesions showed a good correlation between cytologic impression and the histologic diagnosis. However, among 17 patients with invasive cancer, there were 2 completely negative cytologic impressions. These were in cases having modal chromosome counts of 47 and 48, respectively. This study demonstrates that some invasive cancers of the cervix are resistant to cytologic diagnosis because of their peridiploid chromosome counts and lack of anisokaryosis.

Brain glutathione and the anti-hypoxic effect of glutathione depletors in mice.

The anti-hypoxic effect (assessed by a standard hypoxia survival test) 3 hr after 2-cyclohexene-1-one (CHX) was completely abolished by injections of L-cysteine after CHX. Under these conditions, brain GSH was depleted to about 50% of the control level in CHX-treated mice and recovered to about 80% of control level following L-cysteine post-treatment. However, D-cysteine could not cause such effects. Intracerebroventricular injection of CHX, which caused a selective depletion of brain GSH, produced an anti-hypoxic effect. Thus, the anti-hypoxic effect may be related to the decrease of brain GSH levels.

2-cyclohexene-1-one-induced hyperglycemia in the mice.

2-Cyclohexene-1-one (CHX) dose-dependently caused the elevation of blood glucose levels in both fed and fasted mice. Adrenalectomy considerably prevented the elevated blood glucose with CHX, and plasma adrenaline assays revealed about a three- to fifteen-fold rise after CHX treatment. These findings indicate that the CHX-induced hyperglycemia may be largely mediated by adrenaline released from the adrenal medulla.

Methods for depleting brain glutathione.

To search for a technique to deplete reduced glutathione (GSH) in brain, the influence of various types of compounds on brain GSH levels was investigated in mice. Of the compounds tested, cyclohexene-1-one, cycloheptene-1-one and diethyl maleate were shown to be potent GSH depletors in brain as well as in liver. The depletion of cerebral GSH ranged about 40-60% of control levels at 1 and 3 hr after intraperitoneal injection. Cyclohexene, cycloheptene, phorone, acetaminophen, and benzyl chloride caused mild depletion of cerebral GSH, but buthionine sulfoximine did not alter cerebral GSH levels. Further, intracerebroventricular injection of cyclohexene-1-one and cycloheptene-1-one caused depletion of brain GSH to about 60-80% of control levels at 1 hr after injection, and the effects persisted for at least 6 hr. Under these conditions, hepatic GSH was not altered. These results demonstrated that cyclohexene-1-one and cycloheptene-1-one can cause not only a marked depletion of brain GSH by systemic administration, but also depletion of cerebral GSH by intracerebroventricular injection by virtue of being water-soluble compounds. Thus, methods for depleting brain GSH employing both compounds are available for exploring possible functions of cerebral GSH in in vivo systems.

Anti-hypoxic effect of glutathione depletors.

The effect of various reduced glutathione (GSH) depletors on the survival time under normobaric and hypobaric hypoxia was examined in mice. The survival time was markedly prolonged in mice treated with glutathione S-transferase substrate, 2-cyclohexene-1-one (50-100 mg/kg, ip) and phorone (100-250 mg/kg, ip). The anti-hypoxic effect lasted for at least 3 hr and the maximum effect was found 0.5 hr after injection. Further, both compounds significantly elevated blood glucose levels 0.5-1 hr after treatment. The extent of the elevated blood glucose was nearly comparable to that of the mice treated with glucose (1-2 g/kg, ip), which was found to possess an anti-hypoxic effect. However, a GSH synthesis inhibitor, buthionine sulfoximine, could cause neither a prolongation of survival time of hypoxic mice nor an elevation of blood glucose. Moreover, unlike the depletion of hepatic GSH, brain GSH was markedly decreased by 2-cyclohexene-1-one and phorone, but not by buthionine sulfoximine. These findings suggest that the elevated blood glucose may involve in one of the mechanisms of the anti-hypoxic effect of 2-cyclohexene-1-one and phorone. A relationship between the anti-hypoxic effect and the depletion of brain GSH was also discussed.

Possible regulation mechanism of microsomal glutathione S-transferase activity in rat liver.

After rats were injected with the reduced glutathione (GSH) depletor phorone (diisopropylidene acetone, 250 mg/kg, i.p.), there was a significant increase in microsomal glutathione S-transferase activity in the liver. The maximum activity was observed 24 hr after injection and was about 2-fold that of the control activity. Diethylmaleate (500 mg/kg, i.p.) had the same effect. Twenty-four hours after phorone injection (250 mg/kg, i.p.), the concentrations of GSH and oxidized glutathione (GSSG) in the liver were increased about 2-fold. Under the same conditions, the level of mixed disulfides with microsomal proteins (GSS-protein) was also increased. Further, the activity of microsomal glutathione S-transferases was increased by the in vitro addition of disulfide compounds such as GSSG, cystine and homocystine, and the activity increased by GSSG was reduced to control levels by incubating with the corresponding sulfhydryl compounds such as GSH, cysteine and homocysteine respectively. Thus, microsomal glutathione S-transferase activity appears to be regulated by the formation and/or cleavage of a mixed disulfide bond between the sulfhydryl group present in the enzyme and GSSG. Therefore, the increase of microsomal glutathione S-transferase activity after phorone injection may be due to the formation of a mixed disulfide bond between the sulfhydryl group in the enzyme and GSSG.

In vivo corneal and conjunctival epithelial nuclear stain.

Epithelium plays a very important structural and functional role in the cornea and conjunctiva. Evaluation of the epithelium is the first step in diagnosing many pathologic states. We have developed a new technique for the in vivo staining of nuclei of corneal and conjunctival epithelium in rabbits and guinea pigs. Several drops of 0.01%, 0.1%, 0.25%, 0.5%, or 1.0% toluidine blue or 1.0% methylene blue were applied to the conjunctival sac to stain epithelial cells. The cells picked up the vital dye within 5 minutes and could be photographed at 30X with the Keeler-Konan wide-field specular microscope. Cells and nuclei were clearly observable. Photographs could be further enlarged to enhance details. Wash out time was rapid and no toxic effects were observed. This technique adds a new dimension to the study of epithelium in normal and pathologic states in experimental animals. This technique may also be applicable to human eyes for discerning such diseases as carcinoma, herpes simplex, or superior limbic keratoconjunctivitis.

Tissue-specific induction of intestinal glutathione S-transferases by alpha beta-unsaturated carbonyl compounds.

Glutathione S-transferase activity in rat intestinal mucosa was increased by the injection of alpha beta-unsaturated carbonyl compounds such as phorone and diethylmaleate, but that in the liver and kidney was not. Since the administration of cycloheximide completely blocked the increase of the enzyme activity by phorone, the increase of the activity may be due to de novo synthesis rather than enzyme activation.