Development of a novel moisture-suppression bag for the preservation of hygroscopic medications.

BACKGROUND: One-dose packaging is frequently used in Japan for elderly patients who are prescribed several medications. It is useful for easy administration and the prevention of misuse or missed medications. Hygroscopic medications are not suitable for one-dose packaging because moisture absorption may alter their properties. Plastic bags with desiccating agents are sometimes used to store hygroscopic medicines in one-dose packaging. However, the relationship between the quantity of desiccating agents and their safety in the storage of hygroscopic medications is poorly understood. Furthermore, older adults might accidentally consume desiccating agents used in food preservation. In this study, we developed a bag that suppresses the moisture absorption of hygroscopic medications without the use of desiccating agents. METHODS: The bag was manufactured using polyethylene terephthalate, polyethylene, and aluminum film on the outside, and unified with a desiccating film on the inside. RESULTS: The relative humidity (RH) in the bag was maintained at approximately 30-40% when the bag was stored at 75% RH and 35 °C. The manufactured bag's moisture suppressing effect was better than that of plastic bags with desiccating agents when the hygroscopic medications, potassium aspartate and sodium valproate tablets, were stored at 75% RH and 35 °C for 4 weeks. CONCLUSIONS: The moisture-suppression bag effectively stored and preserved hygroscopic medications and was more effective in inhibiting moisture absorption than plastic bags with desiccating agents under high temperature and humidity conditions. The moisture-suppression bags are expected to be useful for elderly patients who are prescribed several medications in one-dose packaging.

Olanzapine enhances adipogenesis and suppresses lipolysis in 3T3­L1 adipocytes under low­glucose and weak differentiation/maturation conditions.

Olanzapine, a second-generation antipsychotic used in the treatment of schizophrenia, is classified as a multi-acting receptor-targeted antipsychotic. Abnormal weight gain is one of the most common side effects of this drug, along with an increased appetite and food intake. However, weight gain has also been reported in patients taking olanzapine without an increase in appetite. Olanzapine has been reported to be directly associated with enhanced adipogenesis; however, whether olanzapine increases lipid content in adipocytes under weak stimulus conditions, such as low glucose concentrations and weak differentiation and/or maturation conditions, is poorly understood. The present study examined the stimulatory effect of olanzapine during the differentiation and maturation of 3T3-L1 pre-adipocytes under low-glucose and weak stimulation conditions by evaluating the expression levels of PPARγ by western blotting and oil red O staining. Western blotting revealed that olanzapine suppressed perilipin phosphorylation, which is an important lipolysis step in adipocytes. The findings of the present study provide novel insights to explain weight gain in patients taking olanzapine but not presenting with increased food intake.

Effects of yogurt as a deglutition aid on disintegration and dissolution of oral tablets.

Patients with dysphagia have difficulty swallowing oral medications. Swallowing aid foods, such as deglutition aid jellies and food thickeners, are often used to help such patients take oral medications. Yogurt is occasionally used to help swallow medications. It is also advantageous as it is nutritious and easy to swallow. However, the influence of yogurt on the pharmacokinetics of oral medications is poorly understood. In this study, we aimed to evaluate yogurt as a potential swallowing aid for the intake of oral tablets, by comparing the physical properties and effects of yogurt on disintegration and dissolution profiles of various oral tablets with deglutition aid jelly and xanthan gum-based food thickener. Yogurt and the food thickener were found to extend the disintegration time of several tablets; however, this increase was unremarkable. Although dissolution of magnesium oxide tablets decreased by 6%, 14%, and 25% after immersion in deglutition aid jelly, food thickener, and yogurt, respectively, at 15 min, this impact on dissolution reduced over time (dissolution rates of all samples at 120 min were over 90%). Rheological measurements showed that yogurt and food thickeners have a weak gel structure and therefore have better fluidity than deglutition aid jelly. The adhesiveness and dynamic viscosity of yogurt were higher than those of the food thickener, which delayed tablet disintegration and reduced the dissolution rate. However, these effects were not substantial. We can thus conclude that yogurt may be a useful swallowing aid for patients with deglutition disorders who take oral medications.

Comparison of the effects on tablet disintegration of solvents used to dissolve food thickeners.

Food thickening agents help patients with dysphagia to eat, drink, and take medications. Taking medications with food thickeners has been reported to cause problems such as reduction of pharmacological effects through the delayed disintegration or non-disintegration of tablets. We previously reported that long immersion periods in food thickeners causes delayed disintegration and non-disintegration, while an immersion time of 1 min prevents these problems. However, in many studies including ours, water was used as the solvent, and patients with dysphagia use various drinks as food thickener solvents. Therefore, in this study, we examined the effects on tablet disintegration of food thickeners dissolved in 12 solvents. The line spread test (LST) and pH of the food thickeners differed among solvents, whereas the disintegration times of tablets immersed in food thickeners for 1 min were similar. Magnesium oxide tablets immersed in food thickeners for 30 min experienced delayed disintegration or non-disintegration in all solvents. These results suggested that the effects of solvents on the disintegration of medications hardly differ. Therefore, patients taking medications with food thickeners may refer to reports in which water was used as the solvent, regardless of their drink of choice.

Relative humidity and qualities of hygroscopic medications stored in different one-dose packaging papers.

The one-dose package is useful for patients who are prescribed multiple medications. However, the one-dose packaging of hygroscopic medications is difficult because the quality of the medication is reduced by moisture absorption. Cellophane polyethylene laminating paper at 20 µm or 30 µm thickness and glassine papers are widely used for one-dose packaging. The basic characteristics, such as water permeability, of these packaging papers have been demonstrated by companies; however, the quality changes of hygroscopic medications stored in these packaging papers are poorly understood. In this study, we compared the relative humidity in packaging papers and the qualities of the stored hygroscopic medications among 20 µm and 30 µm thick cellophane polyethylene laminating paper and glassine paper. Glucobay® 50 mg, Magmitt® 330 mg, and Phosblock® 250 mg tablets were used as hygroscopic medications to be packaged and the relative humidity, weight change, and hardness of tablets were measured. The relative humidity decreased in the order of glassine paper, 20 µm thick cellophane polyethylene laminating paper, and 30 µm thick cellophane polyethylene laminating paper. Additionally, tablets inside the 30 µm thick cellophane polyethylene laminating paper gained the least weight. Therefore, tablets in a 30 µm thick polyethylene laminating paper absorb less moisture than those in other papers. However, the effect was less pronounced at high temperature, even if the relative humidity remained the same. We expect that the results will be used by hospitals and clinical pharmacies to understand the characteristics of packaging papers and ensure appropriate usage.

Appropriate usage of food thickening agents to prevent non-disintegration of magnesium oxide tablets.

Food thickening agents are used to aid the administration of medicine to elderly patients with dysphagia. Magnesium oxide tablets are sometimes administered with food thickening agents. Non-disintegration and disintegration delay of these tablets in the body are problems associated with food thickening agent use. However, the appropriate usage of food thickening agents for administering tablets is not established. Here, the reasons for the non-disintegration of magnesium oxide tablets administered with food thickeners and appropriate usage of food thickeners were examined using a disintegration test of newly opened and moisture-absorbed magnesium oxide tablets. Immersion of magnesium oxide tablets for 10 and 30 min in xanthan and guar gum-based food thickening agents caused disintegration delay and non-disintegration in the first fluid (pH 1.2). However, tablets immersed for 1 min quickly disintegrated. The disintegration of xanthan gum-based food thickening agents was faster than guar gum-based food thickening agents. Moisture absorption by magnesium oxide tablets caused a significant delay in their disintegration in water. The tablets that absorbed moisture disintegrated within 1 min in the first fluid, even when immersed in food thickening agents for a short time. Overall, a short immersion of magnesium oxide tablets in food thickening agents can avoid non-disintegration.

2-Deoxy-D-glucose enhances the anti-cancer effects of idarubicin on idarubicin-resistant P388 leukemia cells.

Cancer cells switch from mitochondrial oxidative phosphorylation to glycolysis, even in the presence of normal oxygen concentrations. Inhibition of the glycolytic pathway is therefore a critical strategy in cancer therapy. A non-metabolic glucose analog, 2-deoxy-D-glucose (2-DG), has been the focus of research on glycolytic inhibitors for use in cancer treatment. The current study examined the anti-cancer effects of 2-DG on idarubicin (IDA)-resistant P388 (P388/IDA) leukemia cells. P388/IDA cells were established following continuous exposure of IDA to P388 cells. Characterization of P388/IDA cells revealed increased lactate production and glucose consumption compared with P388 parent cells. The results of a cell viability assay determined that 2-DG induces higher toxicity in P388/IDA cells compared with P388 cells. Although 2-DG also exhibits endoplasmic reticulum (ER) stress-inducing activity, the cytotoxic effect of the ER stress inducer, tunicamycin, on P388/IDA cells was lower than that of P388 cells. A combination of 2-DG and IDA enhanced P388/IDA cell death compared with each agent alone. The results indicated that P388 cells activated glycolysis after acquiring IDA resistance and therefore, inhibition of the glycolytic pathway via 2-DG might be a useful strategy for cancer therapy against IDA- resistant leukemia cells.

The GALNT6­LGALS3BP axis promotes breast cancer cell growth.

Polypeptide N­acetylgalactosaminyltransferase 6 (GALNT6), which is involved in the initiation of O­glycosylation, has been reported to play crucial roles in mammary carcinogenesis through binding to several substrates; however, its biological roles in mediating growth­promoting effects remain unknown. The present study demonstrated a crucial pathophysiological role of GALNT6 through its O­glycosylation of lectin galactoside­binding soluble 3 binding protein (LGALS3BP), a secreted growth­promoting glycoprotein, in breast cancer growth. The Cancer Genome Atlas data analysis revealed that high expression levels of GALNT6 were significantly associated with poor prognosis of breast cancer. GALNT6 O­glycosylated LGALS3BP in breast cancer cells, whereas knockdown of GALNT6 by siRNA led to the inhibition of both the O­glycosylation and secretion of LGALS3BP, resulting in the suppression of breast cancer cell growth. Notably, LGALS3BP is potentially O­glycosylated at three sites (T556, T571 and S582) by GALNT6, thereby promoting autocrine cell growth, whereas the expression of LGALS3BP with three Ala substitutions (T556A, T571A and S582A) in cells drastically reduced GALNT6­dependent LGALS3BP O­glycosylation and secretion, resulting in suppression of autocrine growth­promoting effect. The findings of the present study suggest that the GALNT6­LGALS3BP axis is crucial for breast cancer cell proliferation and may be a therapeutic target and biomarker for mammary tumors.

Effects of thickened drinks on the disintegration of various oral tablets.

Food thickeners are widely used to aid the oral administration of medications to patients with dysphagia. Powder-type food thickeners are used to modulate the viscosity of therapeutic solutions depending on the swallowing capacity of patients. Food thickeners inhibit or delay the disintegration of some medications, resulting in reduced pharmaceutical effects of the medications and/or their excretion in the stool. A short immersion time (within 1 min) is important to overcome these problems. Although thickened drinks are commercially available, their use as vehicles for medications is not well understood. In this study, we evaluated the effects of thickened drinks on the disintegration time of therapeutic tablets. Furthermore, we compared the thickened drinks with powder-type xanthan gum-based food thickeners. Forty tablets were used, including naked tablets, film-coated tablets, orally disintegrating tablets, enteric-coated tablets, and sugar-coated tablets. For the disintegration test, the tablets were immersed in thickened drinks or food thickeners for 1 min. The changes in the disintegration time of the 40 tablets immersed in the thickened drinks were comparable with those in food thickeners. The disintegration time of several tablets was shorter or unchanged after immersion in the thickened drinks. The disintegration time of rapidly disintegrating tablets tended to increase when immersed in thickened drinks, but it was less than 2 min for the majority of the tablets. These results demonstrate that thickened drinks, similar to food thickeners, could help administer medications to patients. Overall, our study provides valuable information for pharmacists and clinicians to decide the most suitable way to deliver medications to patients with dysphagia.

In Vitro Anticancer Activities of B6 Vitamers: A Mini-review.

Vitamin B6 compounds, including pyridoxine, pyridoxal, pyridoxamine, and their phosphorylated forms, have been investigated with regard to their cancer preventive and therapeutic effects through epidemiological, in vivo, and in vitro studies. In particular, in vitro studies in cancer cells have evaluated the effects of several B6 vitamers such as pyridoxine, pyridoxal, pyridoxamine, and pyridoxal-5'-phosphate, which is a bioactive form of vitamin B6 However, the anticancer activity and concentration required to influence cancer cells vary among B6 vitamers. In this review, the various in vitro effects of vitamin B6 compounds on cancer cells are presented and discussed.

Lactic Acid Promotes Cell Survival by Blocking Autophagy of B16F10 Mouse Melanoma Cells under Glucose Deprivation and Hypoxic Conditions.

In solid tumors, cancer cells are exposed to microenvironment stress, including hypoxia and insufficient nutrients. An acidic microenvironment in tumors is facilitated by the increase in synthesis of lactic acid; this is known as Warburg effect. We previously showed that B16F10 melanoma cells were induced autophagic cell death by glucose-deprivation stress, and lactic acid suppressed the cell death through the inhibition of autophagy. In this study, effects of lactic acid on cell death of B16F10 cells under hypoxic and glucose-depleted double stress conditions were investigated. The double stress promoted autophagic cell death earlier than glucose-depleted stress alone. Lactic acid repressed the double stress-induced cell death by inhibiting autophagy. These results suggest that lactic acid serves for cell survival under microenvironmental stress conditions in B16F10 melanoma cells.

The effects of vitamin B6 compounds on cell proliferation and melanogenesis in B16F10 melanoma cells.

B16F10 murine melanoma cells are frequently used for the study of cancer and melanogenesis. The cells are usually cultured in Dulbecco's Modified Eagle Medium, with the addition of 20 µM pyridoxal (PL) or pyridoxine (PN) for vitamin B6. The difference between these vitamin B6 compounds is thought not to affect cell proliferation, whereas their influence on other physiological effects is poorly understood. In the present study, the effects of PL and PN on cell proliferation and melanogenesis in B16F10 cells were compared. At 500 µM PL significantly suppressed cell growth but the growth inhibitory effect of PN was weak. Although neither of the vitamin B6 compounds affected cell growth at 20 µM, melanogenesis was suppressed by 20 µM PL compared with the effect of PN. In addition, the expression levels of tyrosinase, which is the rate-limiting enzyme, correlated with the melanin content. The results of the present study indicate that PL may be more useful for melanoma therapy and suppression of skin pigmentation than PN. The results also signify the importance of medium selection for cell culture.

Extracellular acidification by lactic acid suppresses glucose deprivation-induced cell death and autophagy in B16 melanoma cells.

In solid tumors, cancer cells survive and proliferate under conditions of microenvironment stress such as poor nutrients and hypoxia due to inadequate vascularization. These stress conditions in turn activate autophagy, which is important for cancer cell survival. However, autophagy has a contrary effect of inducing cell death in cancer cells cultured in vitro under conditions of glucose deprivation. In this study, we hypothesized that supplementation of lactic acid serves as a means of cell survival under glucose-deprived conditions. At neutral pH, cell death of B16 murine melanoma cells by autophagy under glucose-deprived conditions was observed. However, supplementation of lactic acid suppressed cell death and autophagy in B16 melanoma cells when cultured in glucose-deprived conditions. Sodium lactate, which does not change extracellular pH, did not inhibit cell death, while HCl-adjusted acidic pH suppressed cell death under glucose-deprived conditions. These results suggested that an acidic pH is crucial for cell survival under glucose-deprived conditions.

Immunoblotting with Peptide Antibodies: Differential Immunoreactivities Caused by Certain Amino Acid Substitutions in a Short Peptide and Possible Effects of Differential Refolding of the Peptide on a Nitrocellulose or PVDF Membrane.

Immunodetection using antibodies, e.g., Western blotting, is generally utilized to measure the amount of a certain protein in a protein mixture. For valid interpretation of results observed by immunodetection, strict attention must be paid to the factors affecting the immunoreactivities of the antibodies. We here describe the step-by-step procedures to demonstrate that substitution of certain amino acids in a peptide can cause remarkable differences in its immunoreactivity with antibodies against epitope tags in the immobilized peptide. Refolding of the peptide on the membrane in a way that masks the epitope to different degrees was the possible reason for their distinct immunoreactivities with the antibodies. The results in this chapter suggest that we need to interpret carefully the experimental results involving immunodetection.

BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells.

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.

Early growth response 4 is involved in cell proliferation of small cell lung cancer through transcriptional activation of its downstream genes.

Small cell lung cancer (SCLC) is aggressive, with rapid growth and frequent bone metastasis; however, its detailed molecular mechanism remains poorly understood. Here, we report the critical role of early growth factor 4 (EGR4), a DNA-binding, zinc-finger transcription factor, in cell proliferation of SCLC. EGR4 overexpression in HEK293T cells conferred significant upregulation of specific splice variants of the parathyroid hormone-related protein (PTHrP) gene, resulting in enhancement of the secretion of PTHrP protein, a known mediator of osteolytic bone metastasis. More importantly, depletion of EGR4 expression by siRNA significantly suppressed growth of the SCLC cell lines, SBC-5, SBC-3 and NCI-H1048. On the other hand, introduction of EGR4 into NIH3T3 cells significantly enhanced cell growth. We identified four EGR4 target genes, SAMD5, RAB15, SYNPO and DLX5, which were the most significantly downregulated genes upon depletion of EGR4 expression in all of the SCLC cells examined, and demonstrated the direct recruitment of EGR4 to their promoters by ChIP and luciferase reporter analysis. Notably, knockdown of the expression of these genes by siRNA remarkably suppressed the growth of all the SCLC cells. Taken together, our findings suggest that EGR4 likely regulates the bone metastasis and proliferation of SCLC cells via transcriptional regulation of several target genes, and may therefore be a promising target for the development of anticancer drugs for SCLC patients.

Involvement of B3GALNT2 overexpression in the cell growth of breast cancer.

A number of glycosyltransferases have been identified and biologically characterized in cancer cells, yet their exact pathophysiological functions are largely unknown. Here, we report the critical role of ß1,3-N-acetylgalactosaminyltransferase II (B3GALNT2), which transfers N-acetylgalactosamine (GalNAc) in a ß1,3 linkage to N-acetylglucosamine, in the growth of breast cancer cells. Comprehensive transcriptomics, quantitative PCR and northern blot analyses indicated this molecule to be exclusively upregulated in the majority of breast cancers. Knockdown of B3GALNT2 expression by small interfering RNA attenuated cell growth and induced apoptosis in breast cancer cells. Overexpression of B3GALNT2 in HEK293T cells prompted secretion of the gene product into the culture medium, suggesting that B3GALNT2 is potentially a secreted protein. Furthermore, we demonstrated that B3GALNT2 is N-glycosylated on both Asn-116 and Asn-174 and that this modification is necessary for its secretion in breast cancer cells. Our findings suggest that this molecule represents a promising candidate for the development of a novel therapeutic targeting drug and a potential diagnostic tumor marker for patients with breast cancer, especially TNBC.

Targeting BIG3-PHB2 interaction to overcome tamoxifen resistance in breast cancer cells.

The acquisition of endocrine resistance is a common obstacle in endocrine therapy of patients with oestrogen receptor-α (ERα)-positive breast tumours. We previously demonstrated that the BIG3-PHB2 complex has a crucial role in the modulation of oestrogen/ERα signalling in breast cancer cells. Here we report a cell-permeable peptide inhibitor, called ERAP, that regulates multiple ERα-signalling pathways associated with tamoxifen resistance in breast cancer cells by inhibiting the interaction between BIG3 and PHB2. Intrinsic PHB2 released from BIG3 by ERAP directly binds to both nuclear- and membrane-associated ERα, which leads to the inhibition of multiple ERα-signalling pathways, including genomic and non-genomic ERα activation and ERα phosphorylation, and the growth of ERα-positive breast cancer cells both in vitro and in vivo. More importantly, ERAP treatment suppresses tamoxifen resistance and enhances tamoxifen responsiveness in ERα-positive breast cancer cells. These findings suggest inhibiting the interaction between BIG3 and PHB2 may be a new therapeutic strategy for the treatment of luminal-type breast cancer.

Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis.

Triple negative breast cancer (TNBC) has a poor outcome due to the lack of beneficial therapeutic targets. To clarify the molecular mechanisms involved in the carcinogenesis of TNBC and to identify target molecules for novel anticancer drugs, we analyzed the gene expression profiles of 30 TNBCs as well as 13 normal epithelial ductal cells that were purified by laser-microbeam microdissection. We identified 301 and 321 transcripts that were significantly upregulated and downregulated in TNBC, respectively. In particular, gene expression profile analyses of normal human vital organs allowed us to identify 104 cancer-specific genes, including those involved in breast carcinogenesis such as NEK2, PBK and MELK. Moreover, gene annotation enrichment analysis revealed prominent gene subsets involved in the cell cycle, especially mitosis. Therefore, we focused on cell cycle regulators, asp (abnormal spindle) homolog, microcephaly-associated (Drosophila) (ASPM) and centromere protein K (CENPK) as novel therapeutic targets for TNBC. Small-interfering RNA-mediated knockdown of their expression significantly attenuated TNBC cell viability due to G1 and G2/M cell cycle arrest. Our data will provide a better understanding of the carcinogenesis of TNBC and could contribute to the development of molecular targets as a treatment for TNBC patients.