Involvement of de novo ceramide synthesis in radiocontrast-induced renal tubular cell injury.

We reported previously that various radiocontrast media cause apoptosis in porcine proximal tubular (LLC-PK(1)) cells, in which reduction in B-cell lymphoma (Bcl)-2 expression and caspase-3 activation are implicated. In the present study, we investigated a role for ceramide in radiocontrast media-induced apoptosis in renal tubular cells. LLC-PK(1) cells were exposed to radiocontrast media for 30 min, followed by incubation for 24 h in normal medium. Cell viability was assessed by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, while apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling stain. Immunofluorescent stains were performed using antibodies against phosphorylated Akt (pAkt) and cAMP response element binding protein (CREB) (pCREB), and ceramide. The mRNA expression and protein content of Bcl-2 were determined by reverse transcriptase-polymerase chain reaction and enzyme immunoassay, respectively. In vivo model of contrast-induced renal injury was induced in mice with unilateral renal occlusion. The cell injury induced by the nonionic radiocontrast medium ioversol was reversed by inhibiting de novo ceramide synthesis with fumonisin B(1) (FB(1)) and L-cycloserine, but not by suppressing sphingomyelin breakdown with D609. FB(1) reversed ioversol-induced decrease in the immunoreactivities of pAkt and pCREB, reduction in Bcl-2 expression and caspase-3 activation. Like ioversol, C2 ceramide and the Akt inhibitor Src homology-6 induced apoptosis by reducing pAkt and pCREB-like immunoreactivities, lowering Bcl-2 expression and enhancing caspase-3 activity. Indeed, various radiocontrast media, excluding iodixanol which showed the least nephrotoxicity, enhanced ceramide-like immunoreactivity. The role for de novo ceramide synthesis was also shown in the in vivo model of radiocontrast nephropathy. We demonstrated here for the first time that the enhancement of de novo ceramide synthesis contributes to radiocontrast nephropathy.

[Clinical trial of bradykinin-enhancing chemotherapy for a recurrent malignant glioma: a case report].

Patients with malignant glioma undergo a combined treatment with surgical resection, radiotherapy, and chemotherapy. Although those treatments usually show some restraining effects on the tumor, a relapse occurs in most of the patients within a few years. We have investigated the feasibility and safety of intra-arterial chemotherapy for malignant brain tumors by enhancing vascular permeability using intra-arterial bradykinin infusion. In 2001, The Committee of Ethics in Kyushu University approved our clinical trial of the bradykinin-enhancing chemotherapy for recurrent malignant gliomas. We here report the first case of our clinical trial. A 31-year-old man, who had undergone surgical resection followed by chemotherapy and irradiation for malignant progression of the left frontal astrocytoma over a period of 2 years, had a relapse of the tumor in the bilateral frontal lobes. After obtaining informed consent, bradykinin and carboplatin were infused through a microcatheter at the left A1 portion under general anesthesia. By dose escalation of bradykinin, the enhanced lesion in the bilateral frontal lobes diminished on magnetic resonance imaging after 3 trials with 3-week intervals, regardless of new lesions outside of the treated area. No neurological or physiological complication including myelosuppression was noted. Bradykinin-enhancing chemotherapy appeared to be effective and safe for malignant glioma. Because it was able to increase drug delivery to the tumor, it was possible to reduce the size of the dose of chemotherapeutic agent, which resulted in minimum complication.

Protein binding and the metabolism of thiamylal enantiomers in vitro.

UNLABELLED: Thiamylal, a chiral thiobarbiturate, is marketed as a racemic product. We studied the serum protein binding and microsomal metabolism of thiamylal enantiomers in vitro. The unbound fraction of R(+)-thiamylal was greater than that of S(-)-thiamylal. The analysis of binding data revealed that both enantiomers bound to human serum albumin through only one site. In displacement studies with site-specific probes, dansylsarcosine, but not warfarin, significantly decreased the binding of both enantiomers. The bindings of enantiomers were also decreased by octanoate and a large concentration of oleate. These findings suggest that both enantiomers bind to Site II of albumin with higher affinity for S(-)-enantiomer. R(+)-thiamylal was metabolized more rapidly than S(-)-enantiomer by human liver microsomes. An experiment with isoform-selective inhibitors and cytochrome P-450 (CYP) isoforms showed that CYP2C9 had the highest activity for the metabolism of both enantiomers, the activity being 7 to 10 times that of CYP2E1 and CYP3A4. CYP2C9 showed a significantly rapid metabolism of R(+)-enantiomer, suggesting that CYP2C9 is mainly involved in the enantioselective metabolism of thiamylal. IMPLICATIONS: Because clinically marketed thiamylal is a racemic compound, a pharmacokinetic study of each enantiomer may be beneficial. We found that the enantioselectivity of thiamylal existed in protein binding and metabolism. This may be caused by the differences in the affinities of enantiomers for albumin and cytochrome P-450 isoform.

Potentiation of vancomycin-induced histamine release by muscle relaxants and morphine in rats.

The intravenous injection of vancomycin sometimes causes anaphylactoid reactions, in which histamine release may play a major role. These reactions are more frequently manifested when vancomycin is injected into anesthetized patients. We examined the vancomycin-induced histamine release and the interaction of vancomycin with muscle relaxants or opioid in rats. In an in vitro study with rat peritoneal mast cells, treatment with vancomycin at concentrations of greater than 1.25 mM produced significant histamine release. Tubocurarine, vecuronium, pancuronium, succinylcholine, and morphine up to concentrations of 0.25, 1, 5, 30, and 5 mM, respectively, produced no significant histamine release. However, the nonsignificant histamine release induced by 0.5 mM vancomycin was clearly enhanced by combining vancomycin with any of these agents. In the in vivo study, the intravenous injection of vancomycin significantly increased the plasma histamine levels in rats when vancomycin was injected at 200 mg/kg of body weight (63.2 +/- 34.0 ng/ml [mean +/- standard deviation]) but not when it was injected at 100 mg/kg (30.8 +/- 20.2 ng/ml) compared with that in the saline-treated rats (22.5 +/- 11.4 ng/ml). Although the subcutaneous administration of morphine (10 mg/kg) never increased the plasma histamine levels, the intravenous injection of vancomycin (100 mg/kg) 30 min after this morphine treatment markedly increased the plasma histamine levels (56.0 +/- 26.9 ng/ml). These findings provide experimental evidence that the combination of muscle relaxants or an opioid with vancomycin may increase the risk of anaphylactoid reactions by enhancing the release of histamine.

Inhibition of GABA system involved in cyclosporine-induced convulsions.

In this study, we attempted to clarify the mechanisms mediating cyclosporine-evoked convulsions. Cyclosporine (50 mg/kg, i.p.) significantly enhanced the intensity of convulsions induced by bicuculline (GABA receptor antagonist), but not those induced by strychnine (glycine receptor antagonist), N-methyl-D-aspartic acid, quisqualic acid or kainic acid (glutamate receptor agonists). Bicuculline plus cyclosporine-induced convulsions were significantly suppressed by an activation of GABAergic transmission with diazepam, phenobarbital and valproate. The GABA turnover estimated by measuring aminooxyacetic acid-induced GABA accumulation in the mouse brain was significantly inhibited by cyclosporine (50 mg/kg, i.p.). When cultured rat cerebellar granule cells were exposed to 1 microM cyclosporine for 24 hr, the specific [3H]muscimol (10 nM) binding to intact granule cells decreased to 53% of vehicle controls. The present study provides the first evidence suggesting that cyclosporine inhibits GABAergic neural activity and binding properties of the GABAA receptor. These events are closely related to the occurrence of adverse central effects including tremors, convulsions, coma and encephalopathy under cyclosporine therapy.

Improved method of determining thiamylal enantiomers in human serum by high-performance liquid chromatography.

Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a simple and rapid method for measuring the thiamylal enantiomers in human serum. The method involves a liquid-liquid extraction procedure followed by chiral resolution using a 5 microm silica-bonded alpha1-acid glycoprotein column (Chiral-AGP). The thiamylal enantiomers and internal standard were eluted within 15 min and were well-resolved. At concentrations of 1, 5 and 20 microg ml(-1), the relative standard deviations of R(+)- and S(-)-thiamylal were 1.35-2.88% and 1.37-3.01%, respectively, for the intra-day assay, and 2.93-4.46% and 2.46-4.84%, respectively, for the inter-day assay. This method facilitates the routine monitoring and pharmacokinetic studies of thiamylal enantiomers.

[Inflammatory reactions and microorganisms cultured from sputum and blood in association with terminal stage infection of patients with lung cancer].

We reviewed our experience with terminal stage infections in patients with lung cancer over an 11 year period at Kurume University Hospital. In patients with end-stage lung cancer, the infection is common and a mortal disease. We examined the clinical features and significance of pathogenic microbes isolated from sputum and blood in patients with lung cancer during their last month. Bacteriological examinations from blood done frequently in patients with episodes of fever revealed that bacteremia was one of the most important disease in terminal stage infection. In the blood cultures from the 22 patients various species of pathogenic microbes were recovered, and nine of which were fungi; five Candida albicans, three Candida tropicalis and one Candida parapsilosis. The major species of bacteria isolated from sputum were Staphylococcus aureus, including methicillin-resistant strain, and Gram-negative bacilli; P. aeruginasa, A. calcoaceticus, K. pneumoniae and E. cloacae, which are known to be frequently involved in hospital-acquired infections. However, S. pneumoniae and H. influenzae which were well known to be microbes of respiratory infections were rare. We concluded that we had to reveal the feature of terminal stage infection in order to reduce the fee for medical treatment and improve the QOL of patients with terminal stage lung cancer.

Enhancement of serotonergic neural activity contributes to cyclosporine-induced tremors in mice.

A single cyclosporine injection (50 mg/kg, i.p.) significantly enhanced harmine- but not oxotremorine-induced tremors in mice. This potentiation became more apparent when cyclosporine (50 mg/kg, i.p.) was administered once a day for seven days. These findings suggest an involvement of monoaminergic mechanisms in cyclosporine-induced tremors. The effects of cyclosporine were examined on the dynamics of noradrenaline, dopamine and serotonin in the mouse brain. Both single and repeated treatment with cyclosporine significantly facilitated the serotonin turnover as estimated from the probenecid-induced accumulation of 5-hydroxyindoleacetic acid, but either mode of treatment failed to change the contents of monoamines and their metabolites or the turnover of noradrenaline and dopamine. Therefore, the cyclosporine-enhanced activity of serotonin neurons may be interpreted as producing adverse central effects, including tremors.

Micellar electrokinetic capillary chromatography for therapeutic drug monitoring of zonisamide.

The simultaneous determination of zonisamide, a new type of antiepileptic drug, and the typical antiepileptic drugs phenobarbital, phenytoin and carbamazepine in human serum was developed using micellar electrokinetic capillary chromatography (MECC) with a diode array detector. A high correlation was revealed between the zonisamide levels in human serum obtained by MECC and those obtained by high-performance liquid chromatography (r=0.981). The serum levels of phenobarbital, phenytoin and carbamazepine determined by MECC were almost equal to those obtained by fluorescence polarization immunoassay. The reproducibility of separation and quantification with MECC analysis was appropriate for the intra- and inter-day assay coefficients. Therefore, the MECC method established here could provide a simple and efficient therapeutic drug monitoring method for antiepileptic drugs in patients, especially those treated with a combination of zonisamide and other antiepileptic drugs.

Enhancement by morphine of radiographic contrast media-induced histamine release in rat peritoneal mast cells.

In intravascular application of radiographic contrast media causes hypersensitivity reactions, in which histamine release may play a major role. We examined the interaction between contrast medium and morphine. Among the four nonionic contrast media examined, iopamidol showed the most marked histamine release from rat peritoneal mast cells in vitro. Although iopamidol and morphine themselves did not induce histamine release at concentrations up to 65 mgI/ml and 3 mM, respectively, their combination resulted in a significant histamine release. These findings suggest that patients with exposure to medicines that induce histamine release may have a higher incidence and severity of hypersensitivity reactions to radiographic contrast media.

Pharmacokinetics of thiamylal enantiomers in humans.

Thiamylal, a chiral thiobarbiturate, is marketed as the racemate. The pharmacokinetic behavior of thiamylal enantiomers was studied in patients undergoing thiamylal treatment. The percentage of R(+)-thiamylal unbound to serum protein was 1.5 times greater than that of S(-)-enantiomer (17.5 +/- 2.6% and 11.7 +/- 2.0% mean +/- SD, p < 0.001, n = 7). The pharmacokinetic parameters of enantiomers were estimated in 6 patients. S(-)-thiamylal serum concentration was higher than R(+)-enantiomer in all patients at all time points examined. Total clearance of R(+)-thiamylal (0.27 +/- 0.23 1/hr/kg) was 1.8 times greater (p < 0.05) than that of S(-)-thiamylal (0.15 +/- 0.13). The volume of distribution at steady state of R(+)-thiamylal (3.66 +/- 1.99 l/kg) was 1.4 times higher (p < 0.05) than that of S(-)-enantiomer (2.60 +/- 1.35). The differences in these parameters may be due mainly to enantioselective binding to serum protein.

Enantioselective determination of thiamylal in human serum by high-performance liquid chromatography.

Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a novel and simple method for measuring thiamylal enantiomers in human serum using reversed-phase high-performance liquid chromatography. R(+)- and S(-)-Thiamylal were separated using a chiral mobile phase containing beta-cyclodextrin, and detected at the range of 50 ng/ml-25 micrograms/ml in serum. The relative standard deviations of R(+)- and S(-)-thiamylal were 3.4-8.7% and 2.8-8.7% for the intra-day assay, and 2.8-12.0% and 2.8-13.0% for the inter-day assay. This method may be applied to enantioselective pharmacokinetic studies of thiamylal.

Some quantitative evaluation of first-pass metabolism of salicylamide in rabbit and rat.

The first-pass metabolism of salicylamide (SAM) was studied in rabbits and rats paying main attention to the amounts of the glucuronide (SAMG) and sulfate (SAMS) formed. The roles of the intestine and liver for SAMG and SAMS formation during the first-pass were also examined quantitatively. Further, an equation was developed to estimate the hepatic first-pass metabolism based on the experiments of intravenous and oral administration and intestinal perfusion experiment. Significant species differences were found in these two animals. The extent of the first-pass metabolism was larger in rabbits (ca95%) than in rats (ca60%). Of the 95% first-pass metabolism in rabbits, about 30% is due to the intestine whereas the remaining 65% is due to the liver. Of the 60% in rats, about 20 and 40% are estimated to be due to the intestinal and hepatic first-pass metabolism, respectively. In rabbits SAMG and SAMS are formed together in nearly equal amounts in the liver and at the ratio 4 : 1 in the intestine. In rats, on the other hand, SAMG is exclusively formed in the intestine and SAMS in the liver. In this context, the equation described here was applied to the data of Gugler et al. (J. Pharmacol. Exp. Ther., 195, 416-423 (1975)) who carried out a study on the first-pass metabolism of SAM in dogs, and consequently, reported that glucuronidation occurs primarily in the liver whereas sulfation occurs primarily in the intestine in dogs.