Analysis of body constitution of fifty-two patients with Stevens-Johnson Syndrome (SJS) using Kampo Medical Questionnaires: prediction of SJS based on body constitution using decision tree.

UNLABELLED: Reports have indicated a relationship between adverse drug reaction (ADR) and Human Leukocyte Antigen (HLA) polymorphism and a relationship between Body Constitution (BC) and HLA polymorphism. Thus, a relationship between ADR and BC is suggested. We therefore created a questionnaire (hereinafter "Questionnaire") to survey the typical BC of Stevens-Johnson Syndrome (SJS) patients to determine how they differ from healthy persons, and studied the relationship between the development of SJS and BC. The Questionnaire had 30 typical items selected from those relevant to the BC necessary for the diagnosis and therapy of Sho-syndrome in Kampo Medicine. In the comparison of the prevalence of BCs between SJS patients and control persons, the prevalence of three BCs in the SJS group was significantly higher than that in the control group: 1) Does your throat ever feel closed up? ANSWER: Yes, 2) Do you easily feel hot flashes or burning cheeks even though your hands and feet feel cold? ANSWER: Yes, and 3) Do your lips or gums look dull red? ANSWER: Yes. In the analysis using the decision tree, the concentrated group of SJS patients (eighty-fold) was extracted using two decision trees consisting of 3 index variables. Persons with BCs from any of 1) to 3) are suggested to be at high risk of developing SJS.

Penetration of oseltamivir and its active metabolite into the brain after lipopolysaccharide-induced inflammation in mice.

OBJECTIVES: Oseltamivir phosphate is used for the treatment of influenza virus infections. Recently, oral intake has been associated with abnormal behaviour. The present study examined the brain penetration of oseltamivir phosphate and oseltamivir carboxylate, its active metabolite, during inflammation. METHODS: Male C57BL/6 mice were given three i.p. injections of lipopolysaccharide (LPS) or saline. We studied the concentration of Evans blue (a marker of blood-brain barrier function) and oseltamivir phosphate and its active metabolite in the brain and plasma. KEY FINDINGS: The brain-to-plasma ratio of Evans blue compared with saline-treated control mice increased significantly with LPS dose. LPS-induced inflammation increased the permeation of drugs through the blood-brain barrier. The concentration of oseltamivir phosphate in both brain and plasma was 2-fold higher in mice treated with LPS than in control mice. Although the plasma concentration of the active carboxylate was not significantly altered by inflammation, the brain concentration was increased 2.7-fold in mice treated with LPS compared with control mice. CONCLUSIONS: Administration of oseltamivir phosphate in the presence of inflammation increased the brain concentration of both parent drug and active metabolite, which may explain the central nervous system side-effects observed with this agent.

[Questionnaire to examine the prevalence of allergy symptoms and consumer measures by prefecture].

To ascertain the prevalence of allergy symptoms and consumer measures for eye, nose, skin, food, and asthma by prefecture. The self-evaluation questionnaire was mailed to 100,000 subjects throughout Japan by the OTC distribution network of Fujiyakuhin Co., Ltd. The prevalence of allergy symptoms for eye, nose, skin, food, and asthma was 17.3, 26.7, 14.5, 4.5, and 5.4%, respectively. These values were comparable with those of previous studies. The ratio of consultation behavior in respondents reporting allergy symptoms was estimated to be 26.4-82.1%. The usage rate of OTC drugs in respondents reporting non-consultation and the rate of combined use of prescription and OTC drugs were 2.9-51.1% and 7.0-44.1%, respectively. The positive likelihood ratio for subjective allergy in persons having an allergy history of "both parent and child" was 13.9, and this ratio for subjective child allergy in persons having an allergy history of "either parents" and "both parents" was 4.39 and 15.7. The relationship between consumer measures and the allergy history of subjects and families was observed in various combinations. In this study, the allergy prevalence of eye and food not reported in a previous study was estimated. Furthermore, it appears that there are points which make one think anew about the allergy information offered to consumers and patients by both OTC manufacturers/distributors and pharmacists.

Effects of anticancer agents and scavengers on CMV-promoter-driven exogenous gene expression in genetically modified cells.

OBJECTIVES: This study aimed to investigate whether the levels of rsGFP mRNA and the fluorescence levels of cytomegalovirus (CMV)-promoter-driven rsGFP (red-shifted green fluorescent protein) could be changed by using anticancer agents and also to examine the effects of co-treatment with anticancer agents and scavengers. METHODS: The pQBI25 vector, which encodes the CMV promoter and the cDNA for rsGFP, was transfected into FR cells (rat skin fibroblast cell line). FR-pQBI25 cells were then exposed to doxorubicin, 5-fluorouracil, methotrexate or paraquat with or without scavengers such as N-acetyl cysteine (NAC) and edaravone for 48 h. KEY FINDINGS: The levels of rsGFP mRNA were found to be significantly higher following doxorubicin, 5-fluorouracil and paraquat treatment but were not changed by methotrexate. These levels of rsGFP mRNA were found to be significantly lower after paraquat/edaravone co-treatment compared with paraquat alone. The fluorescence levels of rsGFP were found to be significantly higher following doxorubicin and paraquat treatment but were not changed by 5-fluorouracil and methotrexate. The levels were also found to be significantly lower after paraquat/edaravone co-treatment compared with paraquat alone and also after doxorubicin/NAC co-treatment compared with doxorubicin alone. CONCLUSIONS: These findings suggest that CMV-promoter-driven exogenous gene expression may be partly regulated by reactive oxygen species.

Regulation of CMV promoter-driven exogenous gene expression with doxorubicin in genetically modified cells.

The regulation of gene expression after the introduction of an exogenous gene is a problematic aspect of gene therapy. The purpose of this study was to use doxorubicin to regulate exogenous gene expression in a vector containing the cytomegalovirus (CMV) promoter. The pQBI25 vector, which encodes the CMV promoter and the cDNA for red-shifted green fluorescent protein (rsGFP), was transfected into a rat skin fibroblast cell line (FR cells). The pEGFP vector, encoding the CMV promoter and enhanced green fluorescent protein (EGFP) cDNA, was transfected into human hepatoma HepG2 cells. FR-pQBI25 cells were then continuously exposed to doxorubicin and methotrexate for 96 and 48 h, respectively; HepG2-pEGFP cells were continuously exposed to doxorubicin for 48 h. The levels of c-fos, c-jun and rsGFP mRNA, as well as the levels of rsGFP protein, in the FR-pQBI25 cells were found to be significantly higher following exposure to doxorubicin. However, the level of rsGFP protein was not changed by exposure to methotrexate. The level of EGFP protein in the HepG2-pEGFP cells was also significantly higher following exposure to doxorubicin. To examine the effect of cessation of doxorubicin exposure, FR-pQBI25 cells that had been exposed to doxorubicin for 48 h were re-plated in fresh medium without doxorubicin for a further 48 h. The increased levels of c-fos, c-jun and rsGFP mRNA and rsGFP protein seen after treatment with doxorubicin had reduced by 48 h after the cessation of exposure to doxorubicin. These findings suggest that CMV-driven exogenous gene expression may be regulated by doxorubicin.

Regulation of exogenous gene expression by superoxide.

PURPOSE: Regulation of gene expression after gene introduction is a problematic aspect of gene therapy. Transcription regulates gene-specific transcriptional factors, which bind to regulatory regions in the promoter. The cytomegalovirus long terminal repeat (CMV-LTR) has a TPA response element (TRE) as a binding site for activator protein 1 (AP-1), which is induced by oxidative stress. The purpose of this study was to regulate exogenous gene expression in a vector with CMV-LTR using oxygen radicals. METHODS: We used two plasmids (1) pQBI25 encoding CMV-LTR and red-shift green fluorescent protein (rsGFP) cDNA and (2) pRc/CMV-SOD encoding CMV-LTR and human Cu, Zn-superoxide dismutase (SOD) cDNA. FR cells were transfected with pQBI25 (FR-pQBI25 cells), and L2 cells were transfected with pRc/CMV-SOD (L2-pRc/CMV-SOD cells). Each type of cell was exposed to oxygen radicals using paraquat for 24 h. Levels of c-fos, c-jun and rsGFP mRNAs were determined using reverse transcription polymerase chain reaction (RT-PCR). Levels of rsGFP protein were measured by fluorometry. Total SOD activity was measured using the nitrite method. RESULTS: Levels of c-fos, c-jun (AP-1 composition protein) and rsGFP mRNA were induced significantly by oxygen radical exposure in FR-pQBI25 cells. A positive correlation was observed between levels of c-fos mRNA and rsGFP mRNA and also between levels of c-jun mRNA and rsGFP mRNA. Levels of rsGFP protein were also induced significantly. Total SOD activity was induced significantly by oxygen radical exposure in L2-pRc/CMV-SOD cells. CONCLUSIONS: This study suggests that gene expression driven by the CMV- LTR promoter may be regulated by oxygen radicals.

Effect of mixing method on the mixing degree during the preparation of triturations.

By using lactose colored with erythrocin, we investigated the effects of mixing methods on mixing degree during the preparation of trituration with a mortar and pestle. The extent of powder dilution was set to 4 to 64 fold in the experiments. We compared the results obtained by using two methods: (1) one-step mixing of powders after addition of diluents and (2) gradual mixing of powders after addition of diluents. As diluents, we used crystallized lactose and powdered lactose for the preparation of trituration. In the preparation of 64-fold trituration, an excellent degree of mixing was obtained, with CV values of less than 6.08%, for both preparation methods and for the two kinds of diluents. The mixing of two kinds of powders whose distributions of particle sizes were similar resulted in much better degree of mixing, with CV values of less than 3.0%. However, the concentration of principal agents in 64-fold trituration was reduced by 20% due to the adsorption of dye to the apparatus. Under conditions in which a much higher dilution rate and/or much better degree of dilution was required, it must be necessary to dilute powders with considering their physicality and to determine the concentrations of principal agents after the mixing.

Induction of CYP3As in HepG2 cells by several drugs. Association between induction of CYP3A4 and expression of glucocorticoid receptor.

The cytochrome P-450 3A (CYP3A) enzyme family is responsible for most of the drug metabolism in the human liver. In this study, we demonstrated the inductive effects of phenobarbital, rifampicin, carbamazepine, phenytoin, prednisolone, ciclosporin and clotrimazole on CYP3A4, CYP3A5 and CYP3A7 mRNA expression, and established the relationship between the expression of human glucocorticoid receptor alpha (hGR) mRNA and the induction of CYP3A4 mRNA in cultured HepG2 cells by reverse transcription polymerase chain reaction (RT-PCR). Treatment with prednisolone, rifampicin and carbamazepine rapidly induced the level of CYP3A4 mRNA expression by 3- to 6-fold. However, phenytoin and phenobarbital gradually induced CYP3A4 mRNA level by 3 to 4-fold. The induction of CYP3A4 mRNA expression by clotrimazole and ciclosporin was negligible. Treatment with phenytoin, rifampicin, carbamazepine and ciclosporin induced approximately 2-fold increases in the expression of CYP3A5 mRNA, although prednisolone, phenytoin and clotrimazole had no effect. Treatment with rifampicin, phenytoin, clotrimazole and ciclosporin resulted in approximately a 2-fold induction of the CYP3A7 mRNA level. Treatment with rifampicin and ciclosporin induced the expression of hGRalpha mRNA significantly in comparison with controls, although the induction of hGRalpha mRNA following treatment with other drugs was negligible. In cluster analysis, the induced level of CYP3A4, CYP3A5, CYP3A7 and hGRalpha mRNA by these drugs could be classified into four major clusters. This suggested that each cluster might be associated with different mechanism(s) of induction by these drugs. Furthermore, we studied the associations between the expression of hGRalpha mRNA and the induced level of CYP3A4 mRNA by prednisolone and ciclosporin. Treatment with both prednisolone and ciclosporin showed synergistic effects on induction of CYP3A4 mRNA and, following treatment with both drugs, the expression level of CYP3A4 mRNA was 2-fold greater compared with prednisolone alone after the fifth day. Positive correlations were observed between the levels of hGRalpha mRNA expression and those of CYP3A4 mRNA. This observation shows that the regulation of CYP3A4 gene expression was hGRalpha-dependent and that ciclosporin may function as a regulator of expression via hGRalpha.