Identification of Factors Necessary for Gatekeeper of Overdose.

Overdose has become a global social problem. The Japanese government requires gatekeeper training to detect and prevent indicators of overdose and suicide. However, knowledge of necessary factors for the gatekeeper of overdose (patient intervention) is limited. This study aimed to investigate the characteristics of individuals who experienced intervening persons expected to overdose, and to identify the factors required of gatekeepers. A Google form was used to survey 298 pharmacists and registered sellers in Japan. We searched for factors by logistic analysis. Knowledge of prescription drugs used for overdose was higher among pharmacists than among registered sellers. Conversely, pharmacists and registered sellers had similar knowledge about OTC drugs. Overall multivariate logistic regression analysis revealed countermeasures against overdose at their workplace (odds ratio (OR): 4.01, 95% confidence interval (CI): 2.25-7.15, p < 0.01) and knowledge that overdose is on the rise (OR: 1.93, 95% CI: 1.04-3.69, p < 0.05) to be significantly associated with intervention experience as a gatekeeper. Countermeasures against overdose at their workplace (OR: 2.40, 95% CI: 1.10-5.25, p < 0.05) in pharmacists and years of work experience (OR: 1.13, 95% CI: 1.04-1.24, p < 0.05), countermeasure against overdose at their workplace (OR: 3.43, 95% CI: 1.18-10.0, p < 0.05), and willingness to participate in study sessions and workshops on overdose (OR: 3.50, 95% CI: 1.51-8.10, p < 0.05) in registered seller were significantly associated with intervention experience as a gatekeeper. These results are useful evidences for countermeasures and gatekeeper training for overdose at pharmacies and drugstores in the community.

A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells.

BACKGROUND: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1). METHODS: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1's promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown -(siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E. RESULTS: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by -siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27. CONCLUSIONS: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

Pharmacokinetic Analysis of Intraocular Penetration of Latanoprost Solutions with Different Preservatives in Human Eyes.

PURPOSE: To investigate the effect of excipients on latanoprost penetration into the aqueous humor with clinically available 6 products with different solutions mainly in the types and concentrations of preservatives. METHODS: In 363 patients with cataracts, we instilled 1 latanoprost drop in 1 eye before surgery. The drop was randomly selected by brand name product (A) and 5 generic products (B-F) composed with different excipients. B contains similar excipients to A. C and D contain lower concentrations of benzalkonium chloride than A. E includes sodium benzoate, and F contains no preservatives. At 0.5-1, 3, and 6 h after instillation, samples of aqueous humor were collected to determine the latanoprost free acid by mass spectrometry. The time course of intraocular concentration and the areas under the aqueous humor latanoprost free acid concentration-time curves (AUCs) were calculated. RESULTS: At 0.5-1 h, the aqueous humor concentration of latanoprost free acid was 8.5 ± 1.0 ng/mL for C, which was significantly higher (P < 0.01) than that of A (3.4 ± 0.5 ng/mL). At 3 and 6 h, however, no significant difference was noted in the concentrations of latanoprost free acid between the brand name and generic products. For each of the generic products, the peak free acid concentration was above the known threshold concentration for biological activity. At 6 h postdose, however, the levels of latanoprost free acid were below the threshold for Products C, E, and F. Comparisons of AUC0-6h and AUC0-24h values showed that these parameters were the greatest with A, and E was significantly inferior to A (P < 0.05). CONCLUSIONS: Currently available latanoprost solutions with different preservatives showed sufficient intraocular concentration to activate the FP receptor, but different pharmacokinetic profiles of absorption or elimination.

Urat1-Uox double knockout mice are experimental animal models of renal hypouricemia and exercise-induced acute kidney injury.

Renal hypouricemia (RHUC) is a hereditary disease characterized by a low level of plasma urate but with normal urinary urate excretion. RHUC type 1 is caused by mutations of the urate transporter URAT1 gene (SLC22A12). However, the plasma urate levels of URAT1 knockout mice are no different from those of wild-type mice. In the present study, a double knockout mouse, in which the URAT1 and uricase (Uox) genes were deleted (Urat1-Uox-DKO), were used as an experimental animal model of RHUC type 1 to investigate RHUC and excise-induced acute kidney injury (EIAKI). Mice were given a variable content of allopurinol for one week followed by HPLC measurement of urate and creatinine concentrations in spot urine and blood from the tail. The urinary excretion of urate in Urat1-Uox-DKO mice was approximately 25 times higher than those of humans. With allopurinol, the plasma urate levels of Urat1-Uox-DKO mice were lower than those of Uox-KO mice. There were no differences in the urinary urate excretions between Urat1-Uox-DKO and Uox-KO mice administered with 9 mg allopurinol /100 g feed. In the absence of allopurinol, plasma creatinine levels of some Urat1-Uox-DKO mice were higher than those of Uox-KO mice. Consequently, hypouricemia and normouricosuria may indicate that the Urat1-Uox-DKO mouse administered with allopurinol may represent a suitable animal model of RHUC type 1. Urat1-Uox-DKO mice without allopurinol exhibited acute kidney injury, thus providing additional benefit as a potential animal model for EIAKI. Finally, our data indicate that allopurinol appears to provide prophylactic effects for EIAKI.

Cytotoxicity Induced by a Redox-silent Analog of Tocotrienol in Human Mesothelioma H2452 Cell Line via Suppression of Cap-dependent Protein Translation.

De novo synthesis of proteins is regulated by cap-dependent protein translation. Aberrant activation of the translation is a hallmark of many cancer types including malignant mesothelioma (MM). We previously reported that a redox-silent analog of α-tocotrienol, 6-O-carboxypropyl-α-tocotrienol (T3E) induces potent cytotoxicity against human MM cells. However, the detailed mechanism of cytotoxicity of T3E remains unclear. In this study, we investigated if T3E induced potent cytotoxicity aganist MM cells. T3E reduced the formation of the cap-dependent translation complex and induced inactivation of oncogene from rat sarcoma virus (RAS). These events were associated with T3E cytotoxicity in MM cells. Furthermore, atorvastatin, an inhibitor of RAS function, had similar effects on MM cells. Moreover, 4EGI-1, a specific inhibitor of the cap-dependent translation complex, induced severe cytotoxicity in MM cells. Overall, T3E had a cytotoxic effect on MM cells via disruption of the activated cap-dependent translation complex through inactivation of RAS.

Pharmaceutical studies of levothyroxine sodium hydrate suppository provided as a hospital preparation.

The levothyroxine sodium hydrate suppository (L-T4-suppository) is provided as a hospital preparation for the treatment of hypothyroid patients with dysphagia in Japan because only oral preparations of levothyroxine sodium (L-T4) are approved for the treatment of hypothyroidism. However, it has been found that serum thyroxine and triiodothyronine levels do not increase as expected with the hospital preparation, requiring a higher dosage of L-T4 in the L-T4-suppository than in the oral preparations. In this study, to determine an effective thyroid gland hormone-replacement therapy for patients with dysphagia, the pharmaceutical properties of the L-T4-suppository were investigated. Suppositories containing 300 µg L-T4 in a base of Witepsol H-15 and Witepsol E-75 (ratio of 1 : 1) were prepared according to Chiba University Hospital's protocol. Content uniformity, stability, and suppository release were tested. The L-T4-suppository had uniform weight and content. The content and release property were stable over 90 d when the L-T4-suppository was stored at 4 °C and protected from light. The release rate of L-T4 increased as pH increased. However, no L-T4 was released below pH 7.2. The release rate of L-T4 decreased as temperature decreased. These findings suggest that the low level of release of L-T4 in the rectum under physiological conditions may be the cause of the low serum thyroxine and triiodothyronine levels following L-T4-suppository administration.

Elacridar enhances the cytotoxic effects of sunitinib and prevents multidrug resistance in renal carcinoma cells.

Intrinsic drug resistance occurs in many renal carcinomas and is associated with increased expression of multidrug resistant proteins, which inhibits intracellular drug accumulation. Multidrug resistant protein 1, also known as P-glycoprotein, is a membrane drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. ABC Sub-family B Member 2 (ABCG2) is widely distributed and is involved in the multidrug resistant phenotype. Sunitinib is a tyrosine kinase inhibitor used to treat kidney cancer that disrupts signaling pathways responsible for abnormal cancer cell proliferation and tumor angiogenesis. Multiple drug resistance is important in tyrosine kinase inhibitor-induced resistance. We hypothesized that inhibition of multidrug resistant transporters by elacridar (dual inhibitor of P-glycoprotein and ABCG 2) might overcome sunitinib resistance in experimental renal cell carcinoma. Human renal carcinoma cell lines 786-O, ACHN, and Caki-1 were treated with sunitinib or elacridar alone, or in combination. We showed that elacridar significantly enhanced sunitinib cytotoxicity in 786-O cells. P-glycoprotein activity, confirmed by P-glycoprotein function assay, was found to be inhibited by elacridar. ABCG2 expression was low in all renal carcinoma cell lines, and was suppressed only by combination treatment in 786-O cells. ABCG2 function was inhibited by sunitinib alone or combination with elacridar but not elacridar alone. These findings suggest that sunitinib resistance involves multidrug resistance transporters, and in combination with elacridar, can be reversed in renal carcinoma cells by P-glycoprotein inhibition.

Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology.

Application of a novel multiplex polymerase chain reaction system for 12 X-chromosomal short tandem repeats to a Japanese population study.

The analysis of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several researches, mainly due to its applicability in the investigation of complex kinship cases. A new 12 X-STR multiplex system (GATA172D05, DXS7423, DSX6809, DXS10134, DXS7132, DXS9902, DXS6789, DXS10074, DXS8378, DXS9898, DXS10147, and GATA31E08) was developed and applied to a Japanese population study. DNA samples from 290 males and 160 females were successfully analyzed using the 12 X-STR multiplex system. No mutation was detected in the kinship cases involving 34 family trios. The combined powers of discrimination of the 12 X-STR loci in males and females were 0.999997 and 0.9999999996, respectively. We conclude that the combined analysis of 12 X-STR loci using this single multiplex polymerase chain reaction system is a powerful tool in forensic DNA testing.

[Development of advanced pharmacy practice experience in national universities].

The six-year pharmacy program started in April 2006 in Japan. In the new program, students in the fifth year of the pharmacy course undergo a long-term practice experience (pharmacy clerkship) in community pharmacy and hospital pharmacy settings as compulsory 20 course credits. The new pharmacy practice experience started in May 2010. A start of the new system was a chance as for beginning movement, thus we conducted the questionnaire survey for the following steps. The finding obtained from our questionnaires indicated that many universities had already planned to execute new approaches, such as an advanced practice at outpatient units, an with medical students, and so on.

A demethylating agent enhances chemosensitivity to vinblastine in a xenograft model of renal cell carcinoma.

Renal cell carcinoma (RCC) is resistant to chemo-therapy partly due to the overexpression of the P-glycoprotein. Several tumor suppressor genes have been reported to be silenced by hypermethylation of the promoter region in RCC. We recently reported that the in vitro cytotoxicity of vinblastine (VBL) was enhanced by pre-treatment with the demethylating agent, 5-aza-2'-deoxycytidine (Aza), in the RCC cell line, Caki-1. In this study, we investigated the combined effect of Aza and VBL in a Caki-1 xenograft model and in other RCC cell lines in vitro. In the xenograft model, tumor volume and weight were significantly suppressed in the co-treatment group, compared to the control, and the expressions of P-glycoprotein, Bcl-2 and cyclin B1 were reduced. Thus, this combined effect could be mediated by the accumulation of intracellular VBL and the enhancement of apoptosis and cell cycle arrest. More-over, the cytotoxicity of VBL was enhanced in vitro in three RCC cell lines by Aza treatment. These findings suggest that the combination treatment with Aza and VBL is effective against RCC.

Divergent activities of interferon-alpha subtypes against intracellular hepatitis C virus replication.

BACKGROUNDS: Interferon (IFN)-alpha is represented by several structurally related subtypes that show different antiviral and anti-tumor effects. Here, we analyzed differential effects of IFN-alpha subtypes on intracellular hepatitis C virus (HCV) replication using HCV subgenomic replicon system as a model. METHODS: Huh7 and HeLa cells supporting expression of HCV replicon were treated with various concentrations of five recombinant human IFN-alpha subtypes 1, 2, 5, 8, and 10, and with IFN-alpha con1. The effects of IFNs on various cell-signaling pathways were assayed by using ISRE-, GAS-, AP1-, NF-kappa B-, CRE-, and SRE-luciferase reporter plasmids. RESULTS: Each IFN-alpha subtype suppressed HCV replication in a dose-dependent manner. Among them, IFN-alpha8 was the most effective, while IFN-alpha1 was the least effective with 50% inhibitory concentrations of 0.123IU/ml versus 0.375IU/ml, respectively. These differential effects against HCV replication did not correlate with levels of the IFN-responsive ISRE or GAS reporter activities, nor they did activate the other reporters, AP1, NF-kappa B, CRE and SRE. CONCLUSION: There were divergent effects of IFN-alpha subtypes against HCV replication that may be through JAK-STAT-independent pathways. Exploring further mechanisms of action may elucidate IFN-mediated cellular antiviral mechanisms.

IRF-1 mediates upregulation of LMP7 by IFN-gamma and concerted expression of immunosubunits of the proteasome.

An immunoproteasome subunit low molecular weight protein 7 (LMP7) plays critical roles in major histocompatibility complex class I antigen processing; however, the mechanism for its expression has remained unclear. We demonstrate that interferon (IFN) regulatory factor-1 (IRF-1) has a pivotal role in IFN-gamma-dependent LMP7 expression, as was shown for the other two immunosubunits. A tetracycline-inducible system for IRF-1 revealed its function in the LMP7 expression, and a genomic region functionally interacting with IRF-1 was also determined. Furthermore, the role of IRF-1 in IFN-gamma-inducible LMP7 transcription was confirmed by employing small interfering RNA experiments and IRF-1-/- mice. These results suggest that IRF-1 acts as a master regulator for the concerted expression of immunoproteasome components.

Development and evaluation of pharmaceutical services in the ICU/CCU by medical staffs.

Questionnaires were sent out to the staffs (13 physicians, 52 nurses and 5 medical engineers) of the ICU/CCU at the University of Tokyo Hospital, to evaluate pharmaceutical services by analyzing problems in the services offered. Four components of pharmaceutical services were evaluated: inventory control of drugs, check of drug usage and doses, mixing of injections, and offering drug information. Almost all responses from medical staffs evaluated pharmaceutical services overall as "good". The high response rate (96%) from the nursing staff was attributed to the fact that they were familiar with the pharmacist's role with drug inventory, and mixing injections, when nursing was not available for these tasks. Although 50% of physicians rated the pharmaceutical services of providing drug information as "good", this value was lower than responses on other items of the questionnaires, which suggests some dissatisfaction. The occurrences of drug information obtained by passive offering (121 subjects) was 4 times as common as drug information obtained by active offering (30 subjects). From this finding, and comments on the questionnaires from physicians, it suggests that physicians require more drug information for dosage regimens, and prefer the drug information to be provided more actively. Further, an important comment from physicians and nurses was that the services of pharmacists are not available on all shifts/all days of the week to provide consultation for drug information and mixing of injections. Although having a pharmacist available daily around the clock is desirable and ideal to the medical team, the number of pharmacists under the present system cannot support this. As a solution, we think that it is crucial that pharmacists educate medical staff when they are present to in order to optimize therapy and patient care over time.

[A case study of shifting a patient with percutaneous endoscopic gastrostomy (PEG) to home care].

We studied the primary factor preventing a patient with PEG from being transferred to home care focusing on the dietic situation. The study has revealed differences in not the age of the patient, the days of hospitalization or hematological and biochemical test data but in the condition of use of home care services or family makeup. In other words, insufficient use of social resources is considered to prevent shifting to home care or continuation of home care of patients with PEG. It is necessary to understand not only dietic situation but social backgrounds of patients and supply information.

[A case study of shifting a patient with percutaneous endoscopic gastrostomy (PEG) to home care].

We studied the primary factor preventing a patient with PEG from being transferred to home care focusing on the dietetic situation. The study has revealed differences in not the age of the patient, the days of hospitalization or hematological and biochemical test data but in the condition of use of home care services or family makeup. In other words, insufficient use of social resources is considered to prevent shifting to home care or continuation of home care of patients with PEG. It is necessary to understand not only dietetic situation but social backgrounds of patients and supply information.