Breast cancer resistance protein (BCRP) is a drug efflux transporter expressed on the epithelial cells of the small intestine and on the lateral membrane of the bile duct in the liver; and is involved in the efflux of substrate drugs into the gastrointestinal lumen and secretion into bile. Recently, the area under the plasma concentration–time curve (AUC) of rosuvastatin (ROS), a BCRP substrate drug, has been reported to be increased by BCRP inhibitors, and BCRP-mediated drug–drug interaction (DDI) has attracted attention. In this study, we performed a ROS uptake study using human colon cancer-derived Caco-2 cells and confirmed that BCRP inhibitors significantly increased the intracellular accumulation of ROS. The correlation between the cell to medium (C/M) ratio of ROS obtained by the in vitro study and the absorption rate constant (ka) ratio obtained by clinical analysis was examined, and a significant positive correlation was observed. Therefore, it is suggested that the in vitro study using Caco-2 cells could be used to quantitatively estimate BCRP-mediated DDI with ROS in the gastrointestinal tract.

Enoxaparin and daikenchuto are commonly administered to prevent venous thromboembolism and intestinal obstruction after gynecological malignancy surgery. However, the effects of their combined use on hepatic function are not well studied. This study aimed to clarify the effects of the coadministration of enoxaparin and daikenchuto on hepatic function. First, Japanese Adverse Drug Event Report (JADER) data were analyzed to identify signals of hepatic disorders. Second, a retrospective observational study of patients who underwent surgery for gynecological malignancies was conducted. This study defined hepatic disorders as an increase in aspartate aminotransferase (AST) or alanine aminotransaminase (ALT) levels above the reference values, using 1-h postoperative values as the baseline. The analysis of JADER data revealed an increased risk for hepatic disorders with the coadministration of enoxaparin and daikenchuto. An observational study also showed higher odds ratios (95% confidence intervals) for the occurrence of hepatic disorders in the coadministration group (4.27; 2.11–8.64) and enoxaparin alone group (2.48; 1.31–4.69) than in the daikenchuto alone group. The median increase in the ALT level was also higher in the coadministration group (34; 15–59) than in the enoxaparin alone (19; 6–38) and daikenchuto alone groups (8; 3–33). In conclusion, our study suggests that compared with the use of enoxaparin or daikenchuto alone, enoxaparin and daikenchuto coadministration increases the risk of hepatic disorders, with more significant increases in AST and ALT levels. Healthcare workers need to be aware of these potential side effects when combining these drugs after surgery for gynecological malignancies.

L-Lactate transport via monocarboxylate transporters (MCTs) in the central nervous system, represented by the astrocyte-neuron lactate shuttle (ANLS), is crucial for the maintenance of brain functions, including memory formation. Previously, we have reported that MCT1 contributes to L-lactate transport in normal human astrocytes. Therefore, in this study, we aimed to identify transporters that contribute to L-lactate transport in human neurons. SH-SY5Y cells, which are used as a model for human neurons, were differentiated using all-trans-retinoic acid. L-Lactate uptake was measured using radiolabeled L-lactate, and the expression of MCT proteins was confirmed Western blotting. L-Lactate transport was pH-dependent and saturated at high concentrations. Kinetic analysis suggested that L-lactate uptake was biphasic. Furthermore, MCT1, 2 selective inhibitors inhibited L-lactate transport. In addition, the expression of MCT1 and 2 proteins, but not MCT4, was confirmed. In this study, we demonstrated that MCT1 and 2 are major contributors to L-lactate transport in differentiated human neuroblastoma SH-SY5Y cells from the viewpoint of kinetic analysis. These results lead to a better understanding of ANLS in humans, and further exploration of the factors that can promote MCT1 and 2 functions is required.

No progress has been made in using remote communication tools for less urgent but equally important health support services, such as preventive medicine and health education. In this study, we developed a remote health support program by pharmacists for community residents and conducted a randomized controlled study on its effectiveness in proper self-medication through pharmacists. People over the age of 20 years who lived in the vicinity of Gifu City, Japan were eligible to participate in this study. Participants were recruited using posters and brochures. This program comprised a lecture, based on the health belief model and behavioral economics, and access to remote health support. The participants were randomly assigned to two groups: the medicine/health class only (control) and the medicine/health class along with the program (intervention) groups. The participants were administered questionnaire surveys immediately before (the first survey) and 2 months after (the second survey) the medicine/health class, which allowed us to compare the changes in the two groups’ behavior regarding performing proper self-medication through pharmacists. The percentage of individuals who started consulting pharmacists about self-medication in the intervention group (63.9%, 23/36) was significantly higher than that in the control group (15.2%, 5/33; p < 0.001). The percentage of individuals who started recording information about their self-medication in their medication notebooks in the intervention group (16.7%, 6/36) was significantly higher than that in the control group (0%, 0/33; p = 0.026). We clarified the effectiveness of this program for behavioral changes toward proper self-medication using support from pharmacists.

Midazolam (MDZ) is clinically used for its sedative and anticonvulsant properties. However, its prolonged or potentiated effects are sometimes concerning. The main binding protein of MDZ is albumin, and reduced serum albumin levels could lead to MDZ accumulation, thereby potentiating or prolonging its effects. Previous investigations have not thoroughly examined these phenomena from a behavioral pharmacology standpoint. Consequently, this study aimed to evaluate both the prolonged and potentiated effects of MDZ, as well as the effects of serum albumin levels on the action of MDZ in low-albumin rats. Male Wistar rats were classified into control (20% protein diet), low-protein (5% protein), and non-protein groups (0% protein diet) and were fed the protein-controlled diets for 30 d to obtain low-albumin rats. The locomotor activity and muscle relaxant effects of MDZ were evaluated using the rotarod, grip strength, and open-field tests conducted 10, 60, and 120 min after MDZ administration. Serum albumin levels decreased significantly in the low-protein and non-protein diet groups compared with those in the control group. Compared with the control rats, low-albumin rats demonstrated a significantly shorter time to fall, decreased muscle strength, and a significant decrease in the distance traveled after MDZ administration in the rotarod, grip strength, and open-field tests, respectively. Decreased serum albumin levels potentiated and prolonged the effects of MDZ. Hence, serum albumin level is a critical parameter associated with MDZ administration, which should be monitored, and any side effects related to decreased albumin levels should be investigated.

The number of patients with type 2 diabetes is increasing worldwide. The mechanisms leading to type 2 diabetes and its complications is being researched; however, the pathological mechanisms of diabetes in the small intestine remain unclear. Therefore, we examined these pathological mechanisms in the small intestine using a mouse model of type 2 diabetes (KK-Ay/TaJcl) aged 10 and 50 weeks. The results showed that diabetes worsened with age in the mice with type 2 diabetes. In these mice, advanced glycation end products (AGEs) in the small intestine and mast cell expression increased, whereas diamine oxidase (DAO) decreased; increased tumor necrosis factor (TNF)-α and histamine levels in the plasma and small intestine were also detected. Additionally, the expression of zonula occludens (ZO)-1 and Claudin1 and cell adhesion molecules in the small intestine reduced. These results exacerbated with age. These findings indicate that type 2 diabetes causes AGE/mast cell/histamine and TNF-α signal transmission in the small intestine and decreases small intestinal wall cell adhesion molecules cause TNF-α and histamine to flow into the body, worsening the diabetic condition. In addition, this sequence of events is suggested to be strengthened in aged mice with type 2 diabetes, thus exacerbating the disease. These findings of this study may facilitate the elucidation of the pathological mechanisms of type 2 diabetes and its associated complications.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Isoliquiritigenin formation is a key reaction during deoxyflavonoid biosynthesis, which is catalyzed by two enzymes, chalcone synthase (CHS) and reductase (CHR). The substrates for CHS are established. However, the substrate for CHR is unknown. In this study, an in vitro reaction was performed to confirm whether naringenin chalcone can be a substrate. Naringenin chalcone was used as a substrate during the CHR reaction. Analyzing the product revealed that isoliquiritigenin was produced from naringenin chalcone, indicating that naringenin chalcone is a substrate. This study is the first to identify a substrate for CHR, reveals that deoxyflavonoid biosynthesis diverges from naringenin chalcone, endorses the term “chalcone reductase,” and answers the long-standing questions about doubly-labeled acetic acid uptake pattern in deoxyflavonoid biosynthesis.

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT₁ receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

Polypharmacy exacerbates lower urinary tract symptoms (LUTS). Japan exhibits a higher prevalence of concomitant medication use in drug therapy than other countries. Previous age- and sex-specific reports exist; however, none include patients of all ages. Therefore, this retrospective study determined the impact of polypharmacy and its associated risk factors on LUTS exacerbation in outpatients with urological conditions. We included patients receiving medication who visited the Department of Urology at the Gifu Municipal Hospital (Gifu, Japan) between January, 2018 and December, 2018. The association between LUTS and polypharmacy and the risk factors for LUTS exacerbation were investigated. Patients were categorized into two groups according to their polypharmacy status. We performed propensity score matching and compared the International Prostate Symptom Score (IPSS) between the groups using the unpaired t-test. Multiple logistic regression analysis was performed to examine the risk factors, including “polypharmacy” and “taking multiple anticholinergic medications” for LUTS exacerbation. When comparing the IPSS between the groups, the polypharmacy group was found to have significantly higher scores than the non-polypharmacy group in six items, including “total score” and “storage score.” Multiple logistic regression analysis results showed high significance in three items, including “polypharmacy” (odds ratio (OR) = 1.67, 95% confidence interval (CI): 1.03–2.71) and “taking multiple anticholinergic medications” (OR = 8.68, 95% CI: 1.05–71.7). In conclusion, this study revealed that “polypharmacy” and “taking multiple anticholinergic medications” were risk factors for LUTS. Particularly, “polypharmacy” is associated with storage symptom exacerbation. Therefore, eliminating “polypharmacy” and “taking multiple anticholinergic medications” is expected to improve LUTS.

Parkinson’s disease (PD) is a common neurodegenerative disease with progressive loss of dopaminergic neurons in substantia nigra and the presence of α-synuclein-immunoreactive inclusions. Gaucher’s disease is caused by homozygous mutations in β-glucocerebrosidase gene (GBA). GBA mutation carriers have an increased risk of PD. Coptis chinensis (C. chinensis) rhizome extract is a major herb widely used to treat human diseases. This study examined the association of GBA L444P mutation with Taiwanese PD in 1016 cases and 539 controls. In addition, the protective effects of C. chinensis rhizome extract and its active constituents (berberine, coptisine, and palmatine) against PD were assayed using GBA reporter cells, LC3 reporter cells, and cells expressing mutated (A53T) α-synuclein. Case-control study revealed that GBA L444P carriers had a 3.93-fold increased risk of PD (95% confidence interval (CI): 1.37–11.24, p = 0.006) compared to normal controls. Both C. chinensis rhizome extract and its constituents exhibited chemical chaperone activity to reduce α-synuclein aggregation. Promoter reporter and endogenous GBA protein analyses revealed that C. chinensis rhizome extract and its constituents upregulated GBA expression in 293 cells. In addition, C. chinensis rhizome extract and its constituents induced autophagy in DsRed-LC3-expressing 293 cells. In SH-SY5Y cells expressing A53T α-synuclein, C. chinensis rhizome extract and its constituents reduced α-synuclein aggregation and associated neurotoxicity by upregulating GBA expression and activating autophagy. The results of reducing α-synuclein aggregation, enhancing GBA expression and autophagy, and protecting against α-synuclein neurotoxicity open up the therapeutic potentials of C. chinensis rhizome extract and constituents for PD.

Trastuzumab, an anti-HER2 monoclonal antibody, is the mainstay treatment for of HER2-positive breast cancer. However, trastuzumab resistance is often observed during treatment. Therefore, new therapeutic strategies are needed to enhance the clinical benefits of trastuzumab. Maitake β-glucan MD-Fraction, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. In this study, we examined the effect of MD-Fraction on trastuzumab treatment of HER2-positive breast cancer. MD-Fraction did not directly inhibit the survival of HER2-positive breast cancer cells, alone or in the presence of trastuzumab in vitro. In HER2-positive xenograft models, the combination of MD-Fraction and trastuzumab was more effective than trastuzumab alone. Peripheral blood lymphocytes and splenic natural killer cells isolated from BALB/c nu/nu mice treated with MD-Fraction showed enhanced trastuzumab-induced antibody-dependent cellular cytotoxicity (ADCC) ex vivo. MD-Fraction-treated macrophages and neutrophils did not show enhanced trastuzumab cytotoxicity in the presence of heat-inactivated serum, but they showed enhanced cytotoxicity in the presence of native serum. These results suggest that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced complement-dependent cellular cytotoxicity (CDCC). Treatment of HER2-positive breast cancer cells with MD-Fraction in the presence of trastuzumab and native serum increased C3a release and tumor cell lysis in a dose-dependent manner, indicating that MD-Fraction enhanced trastuzumab-induced complement-dependent cytotoxicity (CDC) by activating the complement system. This study demonstrates that the combination of trastuzumab and MD-Fraction exerts a greater antitumor effect than trastuzumab alone by enhancing ADCC, CDCC, and CDC in HER2-positive breast cancer.

In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.

The C3 carbon of glucose molecules becomes the C1 carbon of pyruvate molecules during glycolysis, and the C1 and C2 carbons of glucose molecules are metabolized in the tricarboxylic acid (TCA) cycle. Utilizing this position-dependent metabolism of C atoms in glucose molecules, [1-¹³C], [2-¹³C], and [3-¹³C]glucose breath tests are used to evaluate glucose metabolism. However, the effects of chronic ethanol consumption remain incompletely understood. Therefore, we evaluated glucose metabolism in ethanol-fed rats using [1-¹³C], [2-¹³C], and [3-¹³C]glucose breath tests. Ethanol-fed (ERs) and control rats (CRs) (n = 8 each) were used in this study, and ERs were prepared by replacing drinking water with a 16% ethanol solution. We administered 100 mg/kg of [1-¹³C], [2-¹³C], or [3-¹³C]glucose to rats and collected expired air (at 10-min intervals for 180 min). We compared the ¹³CO₂ levels (Δ¹³CO₂, ‰) of breath measured by IR isotope ratio spectrometry and area under the curve (AUC) values of the ¹³CO₂ levels-time curve between ERs and CRs. ¹³CO₂ levels and AUCs after administration of [1-¹³C]glucose and [2-¹³C]glucose were lower in ERs than in CRs. Conversely, the AUC for the [3-¹³C]glucose breath test showed no significant differences between ERs and CRs, although ¹³CO₂ levels during the 110–120 min interval were significantly high in ERs. These findings indicate that chronic ethanol consumption diminishes glucose oxidation without concomitantly reducing glycolysis. Our study demonstrates the utility of ¹³C-labeled glucose breath tests as noninvasive and repeatable methods for evaluating glucose metabolism in various subjects, including those with alcoholism or diabetes.

Taguchi et al. reported that postmenstrual age (PMA) is a promising factor in describing and understanding the developmental change of caffeine (CAF) clearance. The aim of the present study was to quantify how developmental changes occur and to determine the effect of the length of the gestational period on CAF clearance. We performed a nonlinear mixed effect model (NONMEM) analysis and evaluated the fit of six models. A total of 115 samples were obtained from 52 patients with a mean age of 34.3 ± 18.2 d. The median values of gestational age (GA) and postnatal age (PNA) were 196 and 31 d, respectively. Serum CAF levels corrected for dose per body surface area (BSA) (C/D ratio_BSA) were dependent on PMA rather than PNA, which supports the findings of a previous study. NONMEM analysis provided the following final model of oral clearance: CL/F = 0.00603∙WT∙∙0.877^{GA ≤ 196} L/h. This model takes into account developmental changes during prenatal and postnatal periods separately. The model successfully described the variation in clearance of CAF. Our findings suggest that the dosage of CAF in preterm infants should be determined based not only on body weight (WT) but also on both PNA and GA.

Restoration of blood flow in skeletal muscle after a prolonged period of ischemia induces muscular ischemia–reperfusion injury, leading to local injury/dysfunction in muscles followed by systemic inflammatory responses. However, preventive/curative agents for skeletal muscle ischemia injury are unavailable in clinics to date. Increasing evidence has validated that carbon monoxide (CO) prevents the progression of ischemia–reperfusion injury in various organs owing to its versatile bioactivity. Previously, we developed a bioinspired CO donor, CO-bound red blood cells (CO-RBC), which mimics the dynamics of RBC-associated CO in the body. In the present study, we have tested the therapeutic potential of CO-RBC in muscular injury/dysfunction and secondary systemic inflammation induced by skeletal muscle ischemia–reperfusion. The results indicate that CO-RBC rather than RBC alone suppressed elevation of plasma creatine phosphokinase, a marker of muscular injury, in rats subjected to both hind limbs ischemia–reperfusion. In addition, the results of the treadmill walking test revealed a significantly decreased muscular motor function in RBC-treated rats subjected to both hind limbs ischemia–reperfusion than that in healthy rats, however, CO-RBC treatment facilitated sustained muscular motor functions after hind limbs ischemia–reperfusion. Furthermore, CO-RBC rather than RBC suppressed the production of tumour necrosis factor (TNF)-α and interleukin (IL)-6, which were upregulated by muscular ischemia–reperfusion. Interestingly, CO-RBC treatment induced higher levels of IL-10 compared to saline or RBC treatments. Based on these findings, we suggest that CO-RBC exhibits a suppressive effect against skeletal muscle injury/dysfunction and systemic inflammatory responses after skeletal muscle ischemia–reperfusion.

The formalin test is one approach to studying acute pain in rodents. Similar to formalin, injection with glutamate and veratrine can also produce a nociceptive response. This study investigated whether opioid-related compounds could suppress glutamate- and veratrine-induced nociceptive responses in mice at the same dose. The administration of morphine (3 mg/kg), hydromorphone (0.4 mg/kg), or fentanyl (0.03 mg/kg) suppressed glutamate-induced nociceptive response, but not veratrine-induced nociceptive response at the same doses. However, high doses of morphine (10 mg/kg), hydromorphone (2 mg/kg), or fentanyl (0.1 mg/kg) produced a significant reduction in the veratrine-induced nociceptive response. These results indicate that high doses are required when using morphine, hydromorphone, or fentanyl for sodium channel-related neuropathic pain, such as ectopic activity. As a result, concerns have arisen about overdose and abuse if the dose of opioids is steadily increased to relieve pain. In contrast, trimebutine (100 mg/kg) and fentanyl analog isobutyrylfentanyl (iBF; 0.1 mg/kg) suppressed both glutamate- and veratrine-induced nociceptive response. Furthermore, nor-isobutyrylfentanyl (nor-iBF; 1 mg/kg), which is a metabolite of iBF, suppressed veratrine-induced nociceptive response. Besides, the optimal antinociceptive dose of iBF, unlike fentanyl, only slightly increased locomotor activity and did not slow gastrointestinal transit. Cancer pain is a complex condition driven by inflammatory, neuropathic, and cancer-specific mechanisms. Thus, iBF may have the potential to be a superior analgesic than fentanyl.