Masaaki Kurata

Flush induced by fluoroquinolones in canine skin

The flush induced by two fluoroquinolone antibacterial agents, balofloxacin and ofloxacin, was studied in beagle dogs. Intradermal injection of the fluoroquinolones at concentrations above 10−5M produced a localized flushed area. The flush responses to fluoroquinolones were inhibited by co-administration with H2-antagonist(s) (ranitidine or cimetidine), but not with H1-antagonist(s) (mepyramine or chlorpheniramine). Similar inhibitory effects of these H2-antagonists were observed for the response to histamine. The flush responses to fluoroquinolones were inhibited by a local pretreatment with compound 48/80 administered to deplete the local stores of mast cell-bound histamine. When the fluoroquinolones were orally administered at a dose of 400 mg/kg, the concentration of histamine in plasma was increased, being accompanied by systemic erythema. These results indicate that the flush induced by fluoroquinolones is mediated by histamine release from canine cutaneous mast cells and H2-receptor stimulation.

A comparative study of whole-blood platelet aggregation in laboratory animals: its species differences and comparison with turbidimetric method.

We undertook a systematic comparison of whole-blood platelet aggregation concerning species difference of commonly used laboratory animals (rabbits, guinea pigs, rats and dogs), agonist difference (collagen, ADP and platelet activating factor [PAF] and technical difference against a traditional method of turbidimetry. Collagen-induced aggregation with whole-blood aggregometry was less variable among species tested and was similar to the results obtained by turbidimetry, which measures platelet aggregation in citrated plasma. In contrast, whole-blood aggregometry in responses to ADP and PAF showed marked species difference, being different from turbidimetry. Our results provide species difference among laboratory animals and suggest that the modulations of platelet aggregation by other blood elements probably differ in each species and with stimulating agonists.

Postprandial change in canine blood viscosity

1. Postprandial variation in blood viscosity was studied in beagle dogs. 2. Blood viscosity increased following feeding. This change was caused by haematocrit elevation, which resulted mainly from splenic contraction. 3. Haemoconcentration, plasma viscosity and erythrocyte deformability did not contribute to the postprandial increment in blood viscosity.

Different roles of endothelium-derived substances on inhibiting platelet aggregation in whole blood.

1. The inhibitory effects of adenosine, nitroprusside (a nitric oxide donor) and prostacyclin on collagen-induced rabbit platelet aggregation were studied under two different conditions: in whole blood with an impedance method and in platelet-rich plasma (PRP) with a turbidimetric method. 2. All substances tested were less potent in whole blood than in PRP, and the differences in IC50 value between whole blood and PRP were not of the same order of magnitude; adenosine (669-fold), nitroprusside (54-fold) and prostacyclin (2-fold). 3. These results imply that (a) some other, as yet unknown, factors in blood modulate the platelet aggregation; (b) adenosine and nitric oxide act close to the endothelium, and (c) prostacyclin acts as a relatively long lasting circulating hormone.

Establishment of an in silico phospholipidosis prediction method using descriptors related to molecular interactions causing phospholipid-compound complex formation.

Although phospholipidosis (PLD) often affects drug development, there is no convenient in vitro or in vivo test system for PLD detection. In this study, we developed an in silico PLD prediction method based on the PLD-inducing mechanism. We focused on phospholipid (PL)-compound complex formation, which inhibits PL degradation by phospholipase. Thus, we used some molecular interactions, such as electrostatic interactions, hydrophobic interactions, and intermolecular forces, between PL and compounds as descriptors. First, we performed descriptor screening for intermolecular force and then developed a new in silico PLD prediction using descriptors related to molecular interactions. Based on the screening, we identified molecular refraction (MR) as a descriptor of intermolecular force. It is known that ClogP and most-basic pKa can be used for PLD prediction. Thereby, we developed an in silico prediction method using ClogP, most-basic pKa, and MR, which were related to hydrophobic interactions, electrostatic interactions, and intermolecular forces. In addition, a resampling method was used to determine the cut-off values for each descriptor. We obtained good results for 77 compounds as follows: sensitivity = 95.8%, specificity = 75.9%, and concordance = 88.3%. Although there is a concern regarding false-negative compounds for pKa calculations, this predictive ability will be adequate for PLD screening. In conclusion, the mechanism-based in silico PLD prediction method provided good prediction ability, and this method will be useful for evaluating the potential of drugs to cause PLD, particularly in the early stage of drug development, because this method only requires knowledge of the chemical structure.

Substrates for glutathione regeneration in mammalian erythrocytes

The efficacy of hexoses, their intermediates and analogues, as substrates for glutathione (GSH) regeneration, were tested in rabbit and guinea-pig erythrocytes. Glucose was found to be the most effective substrate in both species. Compared to glucose, the efficacy of other substrates were: fructose (85%), mannose (68%), galactose (47%), 2-deoxyglucose (43%), glucose-1-phosphate (29%), 2-deoxyglucose-6-phosphate (21%), fructose-6-phosphate (12%) and glucose-6-phosphate (11%) in rabbit erythrocytes, and mannose (93%), fructose (52%) and 2-deoxyglucose-6-phosphate (11%) in guinea-pig erythrocytes. Our results provide evidence that in addition to enzyme activities, the availability of the substrate is also an important factor in determining GSH regeneration in mammalian erythrocytes.

Characteristics of corneal phospholipidosis induced by topical ocular application of chloroquine and amiodarone in rabbits

Several cationic-amphiphilic drugs such as chloroquine and amiodarone are known to induce phospholipidosis in the cornea by systemic administration. However, the characteristics of ophthalmological and pathological changes when phospholipidosis-inducing drugs are topically applied have not been well studied. This study was conducted to investigate the characteristics of corneal changes caused by topical application of chloroquine and amiodarone to Japanese white rabbits. The changes were evaluated by ophthalmological, histopathological, and ultrastructural examinations. An in vivo confocal microscopy was also applied to the chloroquine-treated corneas. In both chloroquine- and amiodarone-treated corneas, diffuse cloudiness was observed by slit-lamp biomicroscopy, and its transparency increased with duration of dosing. Confocal microscopy showed punctate dots in the corneal epithelium. Histopathologically, cytoplasmic vacuolation was found in the corneal epithelium and keratocytes in both chloroquine- and amiodarone-treated eyes. Furthermore, foamy cytoplasm of the corneal endothelium was observed in the chloroquine-treated eyes. Ultrastructural examination showed multi-lamellar inclusion bodies or membrane-like debris in the lysosome-like vacuoles in the cytoplasm of corneal cells, which is a characteristic of the lesions of phospholipidosis. These changes disappeared after a withdrawal period. Continuous dosing of chloroquine resulted in corneal erosion and focal corneal opacity as shown by gross observation and slit-lamp biomicroscopy. Confocal microscopy could detect the corneal changes prior to the appearance of these ophthalmological changes. The present study showed that phospholipidosis caused by ocular administration of chloroquine and amiodarone first induces reversible diffuse corneal cloudiness. Confocal microscopy is a useful method for monitoring induction of corneal phospholipidosis.

Comparison of heavy metals in inhibiting glutathione regeneration in mammalian erythrocytes

The effect of heavy metals (copper (Cu2+), iron (Fe2+), lead (Pb2+) and cobalt (Co2+)) on glutathione (GSH) regeneration were examined in erythrocytes from rabbits and guinea pigs. Cu2+ and Fe2+ oxidised GSH, and decreased GSH regeneration rate. Pb2+ did not oxidise GSH, but decreased GSH regeneration rate. Co2+ did not show any effects. In an in vitro study using haemolysate, Cu2+ inhibited the activities of hexokinase and 6-phosphogluconate dehydrogenase (6-PGD); Pb2+ also inhibited 6-PGD; Fe2+ inhibited 6-PGD, glucose-6-phosphate dehydrogenase (G-6-PD) and glutathione reductase (GR). There were no striking differences between the two species used. The present results suggest that the decline of GSH generation by heavy metals involves oxidation of GSH and inhibition of its associated enzymes, and that the inhibitory mechanism is different for different metals.

ATP levels and glutathione regeneration in canine erythrocytes

The effect of ATP levels on GSH regeneration was examined in canine erythrocytes. The main findings were: (1) The GSH regeneration was dependent on glucose and ATP; (2) cytochalasin B and polymyxin B, both glucose transport inhibitors, reduced ATP synthesis and GSH regeneration; (3) inosine, a substrate of the salvage pathway, was not effective for ATP synthesis and GSH regeneration. These results indicate that glucose transport and its metabolism play an important role in oxidant defence systems in general and GSH regeneration in particular in canine erythrocytes.