[Early Prediction of Support Necessity for Pharmacy Clinical Internship Using Deep Learning].

The duration of undergraduate study was extended in 2006 to six years for pharmaceutical education aimed at training highly qualified pharmacists. Clinical internship in current pharmaceutical education is positioned as being important for fostering the qualities required of a pharmacist, and the support of faculty members is essential. Based on the above, we thought that support from faculty members should be provided easily and positively, which would enrich community pharmacy clinical internships. This study aimed to examine the method of predicting the need for support from weekly reports of community pharmacy practice trainees at Showa Pharmaceutical University. It became evident that the level of necessary support could not be predicted by using the support needs listed. However, application of deep learning to the contents of the weekly report for the first to fifth weeks in 2019 enabled the prediction of the level of support needed in 2020 with 97% accuracy. Although this research is currently limited to predicting the level of support required for community pharmacy practical internship at our university, it demonstrates the use of deep learning to predict the level of support needed based on five weeks' worth of weekly reports.

[Orolingual Angioedema after Recombinant Tissue Plasminogen Activator Treatment in Acute Cardiogenic Cerebral Embolism Patient Using Olmesartan: A Case Report].

Recombinant tissue plasminogen activator (rt-PA), which is used to treat acute cerebral infarction, may cause angioedema immediately after administration particularly in patients who are taking angiotensin-converting enzyme (ACE) inhibitors. On the other hand, unlike ACE inhibitors, angiotensin II receptor blockers (ARBs) do not act directly on bradykinin, and are therefore considered an alternative to ACE inhibitors in patients with bradykinin-related side effects. We report a case of orolingual angioedema in an 82-year-old male patient who is taking ARB, which occurred after rt-PA administration for acute cerebral infarction. The patient, who has been on medications for hypertension including ARB (olmesartan 40 mg/day) and for hyperuricemia, was transported to our hospital with the chief complaint of right conjugate deviation of the eyes and left hemiplegia. Head magnetic resonance imaging revealed cerebral infarction in the right mesencephalic artery area including the insular cortex. He was diagnosed with cardiogenic cerebral embolism, and rt-PA administration was started 4 h after onset. The patient developed eyelid edema 2.5 h after the start of administration, and orolingual angioedema and breathing difficulty 15.5 h after. The patient was treated with methylprednisolone, d-chlorpheniramine maleate, and famotidine, and the symptoms improved gradually in 1.5 h. We should pay attention to the occurrence of orolingual angioedema not only at the beginning of rt-PA administration but also for a long time thereafter when it is used in patients taking ARBs.

Bcl-2/Bcl-xL inhibitor navitoclax increases the antitumor effect of Chk1 inhibitor prexasertib by inducing apoptosis in pancreatic cancer cells via inhibition of Bcl-xL but not Bcl-2.

In our previous study, we showed that prexasertib, a checkpoint kinase 1 (Chk1) inhibitor, enhances the effects of standard drugs for pancreatic cancer, including gemcitabine (GEM), S-1, and the combination of GEM and S-1 (GS). The combination of prexasertib and GS has a strong antitumor effect and induces apoptosis in pancreatic cancer cells by downregulating anti-apoptotic protein Bcl-2. In the present study, we investigated the combined effect of GEM, S-1, and prexasertib with a selective Bcl-2 inhibitor (venetoclax) and a non-selective Bcl-2 inhibitor (navitoclax) in SUIT-2 pancreatic cancer cells. An MTT assay revealed that the combination of prexasertib with navitoclax showed a synergistic effect but the combination with venetoclax did not. Investigation of the pancreatic cancer cell lines SUIT-2, MIA PaCa-2, and BxPC-3 revealed that BxPC-3 also showed a high synergistic effect when combined with prexasertib and navitoclax but not venetoclax. Mechanistic analysis of the combined effect showed that apoptosis was induced. Bcl-2 knockdown with siRNA and prexasertib treatment did not induce apoptosis, whereas Bcl-xL knockdown with siRNA and prexasertib treatment resulted in strong induction of apoptosis. In addition, among the three cell lines, the combined effect of prexasertib and navitoclax resulted in increased apoptotic cell death because the protein expression levels of Bcl-xL and Chk1 were higher. Our results demonstrate that the combination of prexasertib and navitoclax has a strong antitumor effect and induces apoptosis in pancreatic cancer cells by downregulating Bcl-xL. Simultaneous inhibition of Chk1 and Bcl-xL could be a new strategy for treating pancreatic cancer.

Prexasertib increases the sensitivity of pancreatic cancer cells to gemcitabine and S­1.

Our previous study demonstrated that gemcitabine (GEM), S­1, and a combination of GEM and S­1 (GS) induced S­phase arrest and increased the phosphorylation of checkpoint kinase 1 (Chk1), which is a critical mediator of cell survival under impaired DNA replication, in pancreatic cancer cell lines. The aim of the present study was to investigate the combined effect of the Chk1 inhibitor prexasertib and antitumor drugs (GEM and S­1) on pancreatic cancer cell line SUIT­2. Furthermore, we conducted mechanistic analysis of the combined effect. The MTT assay revealed that a combination of prexasertib and GS showed a strong effect. Mechanistic analysis of the combined effect showed effective induction of apoptosis. Furthermore, a combination of prexasertib and GS downregulated the expression of antiapoptotic protein Bcl­2. Chk1 knockdown with small interfering RNA and GS treatment resulted in strong induction of apoptosis. Our results provide evidence to show that the combination of prexasertib and GS has a strong antitumor effect and effectively induces apoptosis in pancreatic cancer cells through downregulation of the antiapoptotic protein Bcl­2. Prexasertib could possibly enhance the effects of standard drugs, including GEM, S­1, and GS, against pancreatic cancer.

Effects of proton pump inhibitors on severe haematotoxicity induced after first course of pemetrexed/carboplatin combination chemotherapy.

WHAT IS KNOWN AND OBJECTIVE: Pemetrexed/carboplatin combination chemotherapy has shown efficacy as a first-line treatment for advanced non-small-cell lung cancer. However, severe haematotoxicity is often observed during this combination chemotherapy. Some studies have suggested that concomitant drugs may be the risk factors for severe adverse events. However, those studies identified the predictive risk factors without paying attention to the relative dose intensities (RDIs) of the anticancer drugs. The objective of this study was to clarify the effects of concomitant drugs on the severe haematotoxicity induced by pemetrexed/carboplatin combination chemotherapy using multiple logistic regression analysis incorporating RDIs of the anticancer drugs. METHODS: We retrospectively reviewed the records of 61 patients who had received first-line treatment with this combination chemotherapy at Yamato Municipal Hospital between April 2011 and May 2017. Severe haematotoxicity was defined as grade 3 or 4 according to the Common Terminology Criteria for Adverse Events, version 4.0. To clarify the influence of concomitant drugs on haematotoxicity, we performed multiple logistic regression analysis. RESULTS: Among the 61 patients, 18 (29.5%) developed grade 3 or 4 haematotoxicity. Multiple logistic regression analysis showed that body weight <54.5 kg [odds ratio: 5.21, 95% confidence interval (CI): 1.17-23.08, P = 0.030], haemoglobin <12.0 g/dL [odds ratio: 7.13, 95% CI: 1.54-33.11, P = 0.012], and coadministration of proton pump inhibitors (PPIs) [odds ratio: 5.34, 95% CI: 1.06-26.94, P = 0.042] were significantly associated with severe haematotoxicity in patients receiving pemetrexed/carboplatin combination chemotherapy after adjustment using non-steroidal anti-inflammatory drugs and RDIs of the anticancer drugs. WHAT IS NEW AND CONCLUSION: Multiple logistic regression analysis incorporating RDIs of the anticancer drugs revealed that low baseline body weight, low baseline haemoglobin level, and coadministration of PPIs were the independent risk factors for predicting severe haematotoxicity induced by pemetrexed/carboplatin combination chemotherapy.

[Visualization and analysis of adverse reactions of molecularly targeted anticancer agents using the self-organizing map (SOM)].

Molecularly targeted anticancer agents cause a variety of adverse reactions compared with conventional anticancer agents because of their unique mechanisms of action. Sources of drug information such as package inserts (PIs) provide primarily document-based and numerical information. Therefore it is not easy to obtain a complete picture of drugs with similar effects, or to understand differences among drugs. In this study we used the self-organizing map (SOM) technique to visualize the adverse reactions indicated on PIs of 23 molecularly targeted anticancer agents as of March 2013. In both the presence/absence version and the frequency version, SOM was divided into domains according to mechanism of action, antibody drug or low-molecular weight drug, and molecular target. The component planes of the 753 adverse reaction items in the frequency version enabled us to grasp all available information and differences among the drugs. In some component planes in the presence/absence version, an adverse reaction that had not been reported for a drug but had already been reported for its proximally positioned drug(s) as of March 2013, was found to be reported thereafter by the Drug Safety Update (DSU) or the Adverse Event Report Search System "CzeekV," which is based on FDA Adverse Event Reporting System (FAERS). Our results suggest that visualization of the adverse reactions of molecularly targeted anticancer agents by the SOM technique is useful not only to acquire all available information and differences among drugs, but also to predict the appearance of adverse reactions.

Nanoparticles electrostatically coated with folic acid for effective gene therapy.

We developed a novel vector, electrostatically coated poly(ethylenimine) (PEI)/pDNA complexes with folic acid (FA). Without covalent binding, the FA molecules could coat the PEI/pDNA complexes, and stable anionic nanoparticles were formed at a charge ratio greater than 60. The addition of FA markedly decreased the cytotoxicity of the cationic PEI/pDNA complexes to the melanoma cell line, B16-F10 cells, which regularly expressed FA-specific receptor (FR). Furthermore, the anionic FA60/PEI/pDNA complexes showed high transgene efficiency via the FR-mediated pathway in B16-F10 cells. The FA60/PEI/pDNA complexes did not show agglutination with erythrocytes. After the intravenous injection of FA60/PEI/pDNA complexes into mice, a higher transgene efficiency than PEI/pDNA complexes was observed in the liver, kidney, spleen, and lung with FR. The gene expressions of FA60/PEI/pDNA complexes were significantly inhibited by preadministration of FA. Thus, the FA60/PEI/pDNA complexes were useful for effective gene therapy.

Gamma-polyglutamic acid-coated vectors for effective and safe gene therapy.

In the present study, we developed some novel gene delivery vectors, coated cationic complexes with gamma-polyglutamic acid (gamma-PGA) for effective and safe gene therapy. Cationic complexes were constructed with pDNA and cationic vectors, such as poly-L-arginine hydrochloride (PLA), poly-L-lysine hydrobromide (PLL), N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol (Chol) liposomes, and DOTMA-dioleylphosphatidylethanolamine (DOPE) liposomes. The cationic complexes showed high gene expression with strong cytotoxicity in melanoma B16-F10 cells. The cationic complexes were also strongly toxic to erythrocytes. On the other hand, the gamma-PGA was able to coat all cationic complexes and form stable nano-sized particles with negative charges. These gamma-PGA-coated complexes had high gene expression without cytotoxicity and toxicities to the erythrocytes. In in vivo transfection experiments, polyplexes showed high transfection efficiency over 10(5) RLU/g in the lung tissue after intravenous injection, although gamma-PGA-coated polyplexes showed a high value in the spleen. High transfection efficiency in lipoplexes and gamma-PGA-coated lipoplexes was observed in the spleen and lung. Thus, gamma-PGA-coated vectors are useful for clinical gene therapy.

Pulmonary gene delivery of hybrid vector, lipopolyplex containing N-lauroylsarcosine, via the systemic route.

We newly prepared lipopolyplexes containing N-lauroylsarcosine (LS) as a hybrid vector for pulmonary gene delivery via the systemic route. Lipopolyplexes were composed of polyethylenimine (PEI), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA), LS, and plasmid DNA (pDNA). The particle size of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was not largely changed by the addition of LS, although the addition of LS decreased the high zeta potential. Lipopolyplexes containing LS with low zeta potential showed little aggregation with erythrocytes and low cytotoxicity. In HepG2 cells, lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D. After intravenous injection of the complexes into mice, lipopolyplexes containing LS showed high transgene efficiency, comparable to lipopolyplex 2P-2D. In particular, lipopolyplexes containing LS showed extremely high transgene efficiency in the lung. As a result of the analysis to identify optimum formulations, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index. These results suggest that lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity.

Hybrid vector including polyethylenimine and cationic lipid, DOTMA, for gene delivery.

We developed polyethylenimine (PEI) lipopolyplexes with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA) and pDNA to investigate their usefulness for in vitro and in vivo gene delivery. The charge ratio of the complex to pDNA was calculated with molar values of nitrogen of PEI, and nitrogen of DOTMA to phosphate of pDNA. The polyplexes were prepared at charge ratio 2 (polyplex 2P) and 8 (polyplex 8P). DOTMA solution was added to polyplex 2P to prepare lipopolyplexes at charge ratio 1 (lipopolyplex 2P-1D), 2 (lipopolyplex 2P-2D), and 4 (lipopolyplex 2P-4D). The particle size of the complex was significantly reduced by the addition of DOTMA and settled to 74-114nm, indicating pDNA compaction. The addition of DOTMA to polyplex 2P decreased pDNA dissociation from the complex and degradation in serum. The addition of DOTMA to polyplex 2P remarkably increased gene expression in HepG2 cells in the absence or presence of FBS. These lipopolyplexes showed little cytotoxicity in the presence of FBS. After intravenous injection of the lipopolyplexes into mice, high-gene expression in the liver, spleen, and lung was observed with lipopolyplex 2P-2D, lipopolyplex 2P-4D, and polyplex 8P. In particular, lipopolyplex 2P-4D showed the highest gene expression.

p53 status-dependent sensitization of human tumour cells to hyperthermia by plant flavonol.

PURPOSE: Quercetin (QCT), an important flavonol, is known to sensitize tumour cells to hyperthermia by suppressing heat shock protein 72 (Hsp72) induction, and is also reported to inhibit p53 accumulation. This study was conducted to examine the effects of QCT on the heat sensitivities of human tumour cell lines with different p53 statuses. MATERIAL AND METHODS: Cell lines derived from human cancers and p53-inducible cells were used. After heat treatment at 43 degrees C for 2 h with or without QCT, cell survival was determined in a clonogenic assay. The cellular and nuclear content of Hsp72 as well as that of p53 was determined by Western blotting analysis. RESULTS: Treatment of cells with 150 microM QCT, which completely abolished Hsp72 induction, potentiated the lethal effects of hyperthermia in all tumour cell lines. Particularly, remarkable enhancement of cell death was observed in tumour cell lines having little or no p53 proteins. Although nuclear translocation of Hsp72 is induced by hyperthermia, it was significantly compromised in p53-deficient cells. CONCLUSIONS: These results indicate that p53 is a component for nuclear accumulation of Hsp72; therefore, p53 status is an important determinant of the sensitization of human tumour cells to hyperthermia by QCT.

Exploitation of De Novo helper-lipids for effective gene delivery.

PURPOSE: In gene delivery, a fusogenic lipid such as dioleyl phosphatidylethanolamine (DOPE) which is a component of cationic liposomal vector is important factor for effective transfection efficiency. We investigated the effect of penetration enhancers as alternative helper-lipids to DOPE. METHODS: Transdermal penetraion enhancers such as N-lauroylsarcosine (LS), (R)-(+)-limonene (LM), vitamin E (VE), and phosphatidyl choline from eggs (EggPC) were used in this experiments as helper-lipids with N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethlylammonium chloride (DOTMA) and cholesterol (CHOL). We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of plasmid DNA/cationic liposomes complexes. RESULTS: In transfection experiments in vitro, the cationic lipoplexes containing LS had highest transfection efficiency among the other lipoplexes independently of FBS. Furthermore, the lipoplexes containing LS had lowest cell toxicity among the other lipoplexes in the presence of FBS. As the results of erythrocytes interaction experiment, DOTMA/LS/CHOL, DOTMA/VE/CHOL, and DOTMA/EggPC/CHOL lipoplexes showed extremely lower hematotoxicity. On the basis of these results, the in vivo transfection efficiencies of the lipoplexes were examined. The lipoplexes containing LS had the highest transfection activity among the other lipoplexes. CONCLUSION: In conclusion, several transdermal penetration enhancers are available for alternative helper-lipids to DOPE in cationic liposomal vectors. Among them, DOTMA/LS/CHOL lipoplexes showed superior characteristics in in vitro transfection efficiency, cell toxicity, hematotoxicity, and in vivo transfection efficiency.

Differences in effects of oncogenes on sensitivity to anticancer drugs.

Methods to predict the responsiveness of a particular tumor to a particular anticancer drug are desirable not only for chemotherapy but also for chemoradiotherapy. Here, we examined the effects of viral or activated oncogenes on sensitivity to anticancer drugs by using SHOK (Syrian hamster Osaka-Kanazawa) cells and their transfectants. The IC50 of each transfectant was compared with that of the pSV2Neo transfected control. Cells transfected with the c-myc, v-mos, or v-fgr gene increased their sensitivity to bleomycin, while those transfected with the H-ras gene developed resistance. Resistance to cisplatin was conferred by the introduction of the H-ras or c-cot gene. In the case of adriamycin, the c-myc or c-cot transfectant increased sensitivity and the H-ras transfectant decreased it. Mitomycin C resistance was observed by the introduction of the K-ras gene. Thus, the H-ras gene was found to be involved in the development of resistance to three of the four anticancer drugs. In addition, we have for the first time shown that mos and cot have an effect on sensitivity to three and all of the four anticancer drugs, respectively. These results suggest that the expression of each oncogene would differently affect sensitivity to the four anticancer drugs used in this study, and this property could be a possible marker to predict chemosensitivity.