The aim of this study was to investigate the time-to-onset of drug-induced interstitial lung disease (DILD) following the administration of small molecule molecularly-targeted drugs via the use of the spontaneous adverse reaction reporting system of the Japanese Adverse Drug Event Report database. DILD datasets for afatinib, alectinib, bortezomib, crizotinib, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, osimertinib, sorafenib, sunitinib, temsirolimus, and tofacitinib were used to calculate the median onset times of DILD and the Weibull distribution parameters, and to perform the hierarchical cluster analysis. The median onset times of DILD for afatinib, bortezomib, crizotinib, erlotinib, gefitinib, and nilotinib were within one month. The median onset times of DILD for dasatinib, everolimus, lapatinib, osimertinib, and temsirolimus ranged from 1 to 2 months. The median onset times of the DILD for alectinib, imatinib, and tofacitinib ranged from 2 to 3 months. The median onset times of the DILD for sunitinib and sorafenib ranged from 8 to 9 months. Weibull distributions for these drugs when using the cluster analysis showed that there were 4 clusters. Cluster 1 described a subgroup with early to later onset DILD and early failure type profiles or a random failure type profile. Cluster 2 exhibited early failure type profiles or a random failure type profile with early onset DILD. Cluster 3 exhibited a random failure type profile or wear out failure type profiles with later onset DILD. Cluster 4 exhibited an early failure type profile or a random failure type profile with the latest onset DILD.

The aim of this study has been to investigate the time-to-onset and onset-pattern of drug-induced interstitial lung disease (DILD) after the administration of monoclonal antibodies through the use of the spontaneous adverse reaction reporting system of the Japanese Adverse Drug Event Report database. DILD datasets for adalimumab, bevacizumab, cetuximab, denosumab, golimumab, infliximab, nivolumab, panitumumab, pembrolizumab, tocilizumab, and trastuzumab were used to calculate the median time-to-onset of DILD, as well as the Weibull distribution parameters. The median time-to-onset of DILD for pembrolizumab and infliximab was within 1 month. The median time-to-onset of DILD for cetuximab, nivolumab, panitumumab, bevacizumab, golimumab, trastuzumab, and tocilizumab ranged from 1 to 2 months. The median time-to-onset of DILD for denosumab and adalimumab was more than 2 months. Infliximab, trastuzumab and tocilizumab, and denosumab were estimated to fit the early failure type profile of the Weibull distribution parameters. Cetuximab, nivolumab, panitumumab, bevacizumab, golimumab, and adalimumab were estimated to fit the random failure type profile. Pembrolizumab was estimated to fit the wear out failure type profile. Cluster analysis was performed to classify the time-to-onset patterns of DILD. Hierarchical cluster analysis showed 3 clusters. The findings of this study established both the most likely time period and onset-pattern of DILD that can occur in patients after the administration of monoclonal antibody agents.

ABSTRACTObjective: The objective of this study was to quantitatively evaluate anticancer drug exposure of non-health care professionals who administer drugsthrough a tube employing a method devised by us.Methods: The subjects were 30 general volunteers aged 22-84 years. They wore gloves and administered Indian ink, simulating an anticancer drug, toa multipurpose adult human-type patient care simulator through a tube using 5 types of syringe, and the area stained with Indian ink was measured.Results: When comparing the number of pixels among the syringes regardless of age, Syringe B showed the lowest number (11.8±3.1 cm2), and therewas a significant difference between Syringes B and E. Furthermore, we compared the total number of pixels in each age group regardless of the typeof syringe. In the 20-year-old group, it was the lowest (10.9±2.3 cm2) showing significant differences in comparison with the other groups. WhenSyringe B was used, the number of pixels was markedly lower than on adopting the other syringes.Conclusion: It was clarified that the level of exposure to anticancer drugs markedly varies depending on the type of syringe and age. It was alsoclarified that the method to evaluate exposure to anticancer drugs using Indian ink devised by us is simple and useful.Keywords: Oral anticancer drugs, Simple suspension method, Drug-administering persons’ exposure.

Objective: Pharmacogenomics(PGx)is the study of the relationship between the efficacy and / or safety of drugs and the genetic polymorphism. Since PGx information can be used to personalize medical treatments, there has been a recent increase in the development of drug and companion diagnostic devices based on genome-wide analyses. Therefore, we surveyed the contents of PGx information in package inserts and interview forms (IF) of Japanese pharmaceuticals, and investigated potential problems with the PGx information supplied by Japanese pharmaceuticals. Methods: PGx information content in package inserts and the IF used by Japanese pharmaceuticals was compared with that listed in the U.S.pharmaceuticals“Table of Pharmacogenomic Biomarkers in Drug Labeling.” Results: There were 166 PGx information content listings for 137 drugs described in the “Table of Pharmacogenomic Biomarkers in Drug Labeling.” However,there were 31 PGx information content listings for 20 biomarkers of 24 drugs that were described in the U.S.but not the Japanese package inserts. In addition,there was no PGx information for 17 biomarkers of 20 drugs in both the Japanese package inserts and the IF. We additionally found that 57.7% of the biomarkers with PGx information listed in the package inserts were for drugs that are normally covered by in vitro diagnostic medical insurance. These biomarkers were mainly the gene mutations and expression of the target molecules. Conclusions: The Japanese PGx information associated with gene mutations and expression of the target molecules was similar to the U.S.PGx information. However,the contents of the PGx information for drug-metabolizing enzymes differed widely among each of the drugs. In order to more effectively use PGx information, a more careful inspection of the information regarding efficacies and side effects will need to be undertaken to ensure better evaluations of patient therapies.

To receive a screening test for hepatitis virus B (HBV) and C (HCV) is one of the top priorities for liver cancer prevention in Japan. The aim of this study is to clarify the factors, which affect the decision of acceptance or refusal the HBV (cid:2) HCV screening test, and we conducted a survey in the form of a questionnaire. Of the 447 subjects who received the annual health check-up, 373 subjects accepted the additional HBV (cid:2) HCV screening test according to the information of mortality rate of liver cancer and benefit of anti-virus therapy whereas 74 subjects refused according to no intent to take anti-virus therapy for economical reasons and saw themselves as outside of HBV (cid:2) HCV infection. Results indicated that meaning information about natural history of viral hepatitis and liver cancer prevention could contribute to the improvement of consultation rate of screening test for HBV & HCV.

The simple suspension method has now gained much popularity. Saiseikai Matsuyama Hospital introduced this method in August 2005. Five years after its introduction, we aimed to investigate its application and user opinion of the method by carrying out a questionnaire-based survey. The questionnaires were distributed among 114 ward nurses, responses were collected, and data were collated. We obtained 57 valid and justified responses (overall response rate, 50%). On the basis of the collected responses, it was found that 47.4% of the ward nurses had experienced feeding tube obstruction. We therefore analyzed the data of the questionnaire-based survey focusing on factors related to tube obstruction. Data analyses were performed by applying a decision tree algorithm, which is used as a data mining technique.Analyses revealed the possibility that the feeding tube obstruction in the simple suspension method was caused by the use of drugs that could not be suspended in hot water. In addition, another contributory factor that was proposed was adherence of the suspension to fingers or other part of the hand during administration.

We have proposed a method for high cell culture throughput by means of a micro 3D scaffold and density with high surface-to-volume ratio, and have successfully demonstrated that the throughput and density of cell culture significantly increased under a finite regime of scaffold size and figuration. We have succeeded in obtaining high density cell culture and also found that the size and figuration of capillaries in the micro 3D scaffolds significantly affect cell culture and surface adhesion.

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.

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.

1,8-di-O-alkylaloe-emodin derivatives (namely, methyl-, propyl-, hexyl-, dodecyl-, and octadecyl) were synthesized from naturally occurring aloe-emodin. Further, derivatives having various substituents such as diethylamino, pyrrolidinyl, piperidinyl, methylpiperazinyl, imidazolyl, thiocyano and selenocyano groups at the 15 position of chrysophanol and 1,8-di-O-hexylchrysophanol from aloe-emodin were synthesized. The cytotoxic effects of these derivatives on less P-glycoprotein (P-gp)-expressing HCT 116 cells and stably P-gp-expressing Hep G2 cells were evaluated by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among these products, several of them exhibited markedly higher potent cytotoxic effects not only on HCT116 cells but also Hep G2 cancer cells as compared to aloe-emodin.

Seven hydroxyanthraquinone derivatives, 1–7, were isolated from the root of Rheum palmatum (Polygonaceae). Two propionated anthraquinone derivatives, 8 and 9, were synthesized. Four hydroxynaphthoquinone derivatives, 13, 14, 16 and 21, were isolated from the root of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) and also three naphthoquinone derivatives, 19, 22 and 23, were isolated from the root of Macrotomia euchroma (Royle) Pauls. (Boraginaceae). The cytotoxicity of the anthraquinone and naphthoquinone derivatives on P-gp-underexpressing HCT 116 cells and P-gp-overexpressing Hep G2 cells was examined by MTT assay. Among the anthraquinone derivatives, compounds 3–5 which had OH, CH2OH and COOH substituent groups on the anthraquinone skeletons, respectively, showed potent growth inhibitory activities against both types of cancer cells (IC50 values: 5.7 ± 0.9 to 13.0 ± 0.7 μM in the case of HCT 116 cells and 5.2 ± 0.7 to 12.3 ± 0.9 μM in the case of Hep G2 cells). All hydroxynaphthoquinone derivatives isolated in this study exhibited extremely potent growth inhibitory activities against both types of cancer cells (IC50 values: 0.3 ± 0.09 to 0.46 ± 1.0 μM in the case of HCT 116 cells and 0.22 ± 0.03 to 0.59 ± 0.06 μM in the case of Hep G2 cells) as well as shikonin 10 (IC50 values: 0.32 ± 0.02 μM in the case of HCT 116 cells and 0.24 ± 0.03 μM in the case of Hep G2 cells).

Purpose. The purpose of this study is to prepare and evaluate gene-transfected cultured skin to establish a dermal patch consisting of cultured skin as a new and novel delivery system for severely burned skin.Materials and Methods. Plasmid DNA encoding the green fluorescent protein (GFP) gene was used as a model gene and transfected to rat and human cultured dermis models (CDMs) using the hemagglutinating virus of Japan envelope vector (HVJ-E) to prepare gene-transfected CDM and evaluate GFP expression in the CDM. Two kinds of transfection methods were evaluated. In pre-transfection, the gene was first transfected into fibroblasts and then CDM was prepared using these gene-transfected cells. In post-transfection, the gene was transfected directly into CDM.Results. GFP expression was observed in both the pre- and post-transfected CDMs. The post-transfection method showed higher GFP expression in the CDM than pre- transfection, although no statistically significant difference was observed. The cell viability of these transfected CDMs was also examined with MTT assay. Slight decrease in viability was observed in these transfected CDMs. These methods could be useful in preparing gene-transfected cultured skins with low cell damage.Conclusion. Gene transfection to cultured skin may produce several dermal patches that release potent endogenous bioactive peptides.

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.

26-Iodopseudodiosgenin (8) and 26-iodopseudodiosgenone (9) were reacted with various nucleophiles (KSCN, KOCN, NaCN, NaN(3) and various amines) to give pseudodiosgenin derivatives (4, 12, 16-20, 26) and pseudodiosgenone derivatives (5, 13, 21-25, 27), respectively. The reactions of 8 and 9 with KOCN gave the elimination products (10) and (11), respectively. The reaction of 9 with NaCN gave 5alpha,26- (14) and 5beta,26-dicyanocholestan-3-one (15). The reaction of 8 with NaN3 gave triazepine derivative (30), while that of 9 gave 26-azidopseudodiosgenone (31). Compound 31 was converted into triazepine derivative (32) by heating at 120 degrees C. The cytotoxicity of the pseudodiosgenins and pseudodiosgenones on P-gp-underexpressing HCT 116 cells and P-gp-overexpressing Hep G2 cells was examined by MTT assay. Pseudodiosgenins 2, 4, 12 and 30 showed strong cytotoxic activity (IC50 values: 2.6+/-0.3-6.7+/-1.4 microM), as did pseudodiosgenones 3, 5, 11, 13, 21-25 and 27 (IC50 values: 1.3+/-0.3-6.4+/-0.3 microM) toward HCT 116 cells. Pseudodiosgenins 12, 16 and 30 (IC50 values: 1.2+/-0.7-2.2+/-0.6 microM) and pseudodiosgenones 22, 23, 25 and 27 (IC50 values: 0.6+/-0.1-2.5+/-0.3 microM) were highly cytotoxic to Hep G2 cells. Compounds 3 and 27 showed efficient antibacterial activity (MIC: 15.6, 10.4 microg/ml) and (MIC: 7.8, 15.6 microg/ml) against Bacillus subtilis and Staphylococcus aureus, respectively.

Vitamin D analogs 12 and 13 having a spiro ring in the side chain, various spirostanols 18-21, 26, 27, 29 and 37, and furostanols 34-36 having SCN and SeCN groups at the 26 position were prepared from diosgenin 1 via (20S,22R,25R)-spirost-1alpha,2alpha-epoxy-4,6-dien-3-one 19 as a key intermediate. The cytotoxic activities of these derivatives as well as 1 on scarcely P-gp-expressed HCT 116 cells and P-gp-overexpressed Hep G2 cells were examined by MTT assay. Furostanols 34 (IC(50) value: 4.9+/-0.3 microM) and 36 (IC(50) value: 1.3+/-0.2 microM) exhibited marked cytotoxic effects on HCT 116 cells, and spirostanol 29 (IC(50) value: 2.4+/-0.8 microM) and furostanol 36 (IC(50) value: 2.8+/-0.4 microM) on Hep G2 cells. Furthermore, the effects of vitamin D analog 12, spirostanol 26 and furostanol 36 on apoptosis-signaling pathways were investigated. Compounds 12 and 26 overexpressed p53 and Bax mRNAs, while compound 36 overexpressed only Bax mRNA.

PURPOSE:For the individual optimization of drug therapy with sulfasalazine (SASP), we studied the influence of the N-acetyltransferase 2 (NAT2) genotype on the pharmacokinetics, efficacy, and incidence of adverse reactions of SASP in patients.METHODS:Ninety-six rheumatoid arthritis (RA) patients were treated or had been treated with 0.5 and/or 1.0 g/day of SASP. The wild-type allele (NAT2*4) and three variant alleles (NAT2*5B, *6A, and *7B) of NAT2 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. Plasma concentrations of SASP and its two metabolites, sulfapyridine (SP) and N-acetylsulfapyridine (AcSP), were estimated by HPLC. Therapeutic efficacy and incidence of adverse reactions were also monitored as recommended by the American College of Rheumatology.RESULTS:Patients were classified into three groups by NAT2 genotyping: Rapid Type (homozygote for NAT2*4), Intermediate Type (heterozygote for NAT2*4 and variant alleles), and Slow Type (homozygote for variant alleles). There was no clear difference in the genotype frequencies between RA patients and healthy subjects. NAT2 genotypes significantly affected both the plasma concentration ratios of SP to AcSP (SP/AcSP) and the efficacy of SASP (p < 0.05). Adverse reactions to SASP were found in 26 (27.1%) out of 96 patients, and there was no difference among the three genotype groups.CONCLUSIONS:NAT2 gene polymorphism is related to the plasma SP/AcSP ratio and the efficacy of SASP.

The cytotoxic effects on HCT 116, Hep G2 and HCT 116/VCR 100-1-1 cell lines of synthetic 4′-O-alkylaloenins (2–17), 4′-O-benzylaloenin (18) and 4′-O-allylaloenin (19) were examined by MTT assay, and compared with that of aloenin (1) isolated from Aloe arborescens Mill. Var. natalensis Berger which showed no marked effect (IC50 value: >100μM). The cytotoxic effects of 4′-O-alkylaloenin sulfates (21–29) were also examined on the same cell lines. The introduction of a longer alkyl group at the O-4′ position of 1 resulted in a higher cytotoxic action on HCT 116 and Hep G2 cells. Among 4′-O-alkylaloenins 2–17, 4′-O-tetradecylaloenin 14 was the most cytotoxic to both on HCT 116 cells (IC50 value: 5.3±2.3μM) and Hep G2 cells (IC50 value: 4.0±0.6μM). Also among 4′-O-alkylaloenin sulfates 21–29, 4′-O-dodecylaloenin sulfate 29 was the most cytotoxic to both on HCT 116 (IC50 value: 4.8±0.2μM) and Hep G2 cells (IC50 value: 4.0±0.5μM). 4′-O-Alkylaloenins 7–14 and 4′-O-alkylaloenin sulfates 24–29 were also cytotoxic to Hep G2 and HCT 116/VCR 100-1-1 cell lines, which overexpress P-glycoprotein, as well as HCT 116 cell lines which scarcely express it.