Biological & pharmaceutical bulletin最新文献

The mechanisms by which erythropoietin (EPO) promotes hepatocyte proliferation in primary cultures of adult rat hepatocytes were studied. EPO stimulated cell proliferation in a time- and dose-dependent manner, significantly increasing the number of hepatocyte nuclei and DNA synthesis. EPO-induced hepatocyte proliferation was completely suppressed by specific inhibitors targeting Janus kinase 2 (JAK 2), phospholipase C (PLC), protein kinase C (PKC), intracellular Ca²⁺ mobilization, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), and mammalian target of rapamycin (mTOR). In contrast, inhibition of signal transducer and activator of transcription 3/5 (STAT 3/5) or granule secretion had no effect, indicating that EPO acts through a pathway distinct from the classical JAK2-STAT signaling pathway. Western blot analysis showed rapid phosphorylation of ERK 2, but not ERK 1, following EPO stimulation. In addition, EPO induced phosphorylation of PLC and C-rapidly accelerated fibrosarcoma (C-Raf), with PKC acting downstream of PLC and upstream of C-Raf. In contrast, intracellular Ca²⁺ concentration and activated Ras were transiently increased in hepatocytes after EPO stimulation, and EPO-induced activated Ras was significantly suppressed by the specific PKC inhibitor GF109203X. These results indicate that EPO engages the JAK2/PLC/PKC-Ca²⁺ signaling cascade, leading to the sequential activation of Ras, C-Raf, and ERK2, ultimately promoting hepatocyte proliferation in vitro.

Photoacoustic imaging (PAI) is a hybrid imaging modality that captures ultrasound signals produced by thermoelastic expansion when pulsed laser light is absorbed by optical absorbers, enabling visualization of biological tissues at greater depths than those of conventional fluorescence imaging. While PAI has been conventionally used for biological, structural, and morphological studies, showing promise for imaging calcium dynamics in cardiac tissue, its practical implementation remains undemonstrated. In this study, we implemented a sectional excitation strategy using a thin, sheet-shaped laser beam to confine optical excitation to defined tissue planes. We applied this approach to photoacoustic imaging of a perfused bullfrog heart loaded with liposome-encapsulated calcium-sensitive dye. We successfully achieved real-time visualization of calcium dynamics within the atrial cross-section, while simultaneous electrocardiographic recordings enabled temporal correlation between photoacoustic signal fluctuations and cardiac electrical activity. This method provides a less-invasive approach to assess calcium transients in deep tissue, and broadens the application of PAI from morphology to physiological function. These findings highlight the potential of this optical-acoustic hybrid modality as a powerful tool for calcium imaging in physiological and pharmacological studies involving deep-tissue organs-particularly in applications such as heart imaging, where conventional optical techniques are limited by shallow penetration depth.

Parkinson's disease (PD) is a progressive neurological disorder with an unclear etiology. Nonetheless, abnormal cholesterol metabolism is considered an environmental risk factor. MitoNEET (mNT) is an iron-sulfur cluster protein located in the outer mitochondrial membrane. mNT dysfunction is involved in the pathology of several diseases, including PD. However, the cause of mNT dysfunction remains unclear. Therefore, we hypothesized that increased intracellular cholesterol levels may reduce mNT function and increase reactive oxygen species (ROS) levels, resulting in neuronal cell death and PD progression. In this study, we investigated the effects of cholesterol on cell viability and mNT levels in human neuroblastoma (SH-SY5Y) cells. Cholesterol reduced cell viability and mNT protein levels and increased ROS generation. Pioglitazone, an mNT activator, decreased cholesterol-induced ROS generation but did not rescue cell viability, suggesting that reduced mNT levels may be involved but are not the sole cause of cholesterol-induced cell death. Additionally, the viability of cells treated with rotenone, 6-hydroxydopamine, and 1-methyl-4-phenylpyridinium in the presence of cholesterol was measured. However, no significant enhancement in cell death was observed. Moreover, the toxic compounds did not reduce mNT levels, indicating that mNT is not involved in toxic compound-induced cell death. In summary, these results indicate that cholesterol induces cell death, reduces mNT protein levels without suppressing transcription, and increases ROS generation, which may affect PD development. Further assessment of the mechanisms associated with abnormal intracellular cholesterol metabolism, reduced mNT levels, and cell death may lead to the discovery of novel treatments for PD.

Maintaining medication adherence remains a major clinical challenge, as higher daily dosing frequencies are often associated with decreased adherence. Although once-daily regimens are generally preferred, specific patient preferences regarding dosing frequency are unclear, despite implications for improving patient satisfaction and optimizing pharmacotherapy. Here, we evaluated the frequency at which patients begin to perceive dosing as excessive. A web-based questionnaire survey using the personal health record infrastructure of electronic medication notebooks was administered between July 20 and 26, 2023. Eligible participants were aged ≥20 years and had received oral tablets or capsules within 90 d of the survey. The questionnaire consisted of 6 items, including whether participants felt that the daily medication frequency was excessive. A multivariate logistic regression analysis was performed to identify the frequency beyond which patients perceive dosing as excessive, adjusting for confounders, such as medication formulation, number of medications, and patient characteristics. Of 1478 respondents, 1236 were included in the analysis. In total, 28.9% of participants reported that their medication frequency felt excessive. In the multivariate logistic regression analysis, twice-daily or more frequent dosing was significantly associated with the perception of excessiveness, using once-daily dosing as the reference. Twice-daily dosing is the frequency beyond which patients are significantly more likely to perceive medication as excessive. Our findings emphasize the importance of simplifying dosing regimens.

Graft-versus-host disease (GVHD) is a clinically significant problem with high mortality that is gradually increasing. Ruxolitinib (RUX) is the only drug used for steroid-refractory GVHD treatment and is thereby crucial. Therapeutic drug monitoring of RUX may be effective because of the relationship between the plasma RUX concentration and treatment outcomes. Posaconazole (PCZ) has also been the recent focus of combined treatment with RUX owing to its pharmacokinetics. We established a simultaneous LC-tandem MS (LC-MS/MS) method and performed plasma drug concentration measurements and monitoring using clinical laboratory values for both RUX and PCZ. We also compared our technique to a simple LC-MS/MS method for clinical application. Moreover, the utility of the automated pretreatment LC-MS/MS (auto-LC-MS/MS) method was tested for further applications. The simultaneous quantification LC-MS/MS method satisfied analytical validation criteria under clinical conditions. Our method demonstrated linearity over the range of 0.3-500 ng/mL for RUX and 3-5000 ng/mL for PCZ, with intra- and inter-day precision and accuracy within ±15%. A possible correlation between plasma RUX concentration and kidney injury was observed in 1 of the 6 patients. Notably, plasma PCZ concentrations were decreased by changing the administration route. Moreover, the plasma concentration levels obtained using the auto-LC-MS/MS method were highly concordant with those obtained using the LC-MS/MS method. The validated LC-MS/MS method was found to be useful in clinical applications; thus, further research into its applications in clinical practice is desirable.

Diabetic nephropathy (DN) is among the most serious diabetes-related microvascular complications, a disease with risks leading to end-stage kidney disease (ESKD). However, only limited DN treatment options are currently available. DN development and progression involve different pathological mechanisms, including inflammation and oxidative stress. Stachybotrys microspora is a fungus producing the triphenyl phenol SMTP-44D, which exhibits anti-inflammatory and antioxidant properties in several disease models. In this study, we aimed to evaluate the effects of SMTP-44D in a DN mouse model, which was created by removing the right kidney of 6-week-old db/db mice. We administered SMTP-44D for 10 weeks between weeks 6 and 16 of age to observe blood glucose levels, renal function parameters, inflammatory factors, oxidative stress markers, and histopathological characteristics. SMTP-44D treatment did not reduce blood glucose level but significantly decreased serum creatinine and urinary albumin as renal function parameters, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and nicotinamide adenine dinucleotide phosphate oxidase-1 as inflammation and oxidative stress in the kidney. In addition, histopathological assessment revealed its preventive effect against glomerulosclerosis and local regenerative tubule. Therefore, we discovered that SMTP-44D might protect renal function without affecting blood glucose level in DN possibly via suppression of inflammation and oxidative stress. In conclusion, SMTP-44D could be a potential DN treatment agent, even in patients with poor glycemic control.

Idiosyncratic drug-induced liver injury (iDILI) is an unpredictable and potentially severe adverse drug reaction, in which immune-mediated mechanisms are suspected to play a central role. Although eosinophilia is often considered a marker of hypersensitivity reactions, the role of eosinophils in iDILI, including drug-specific risks, remains poorly understood. We conducted a case-control study using electronic medical records to evaluate drug-specific risks associated with drug-induced liver injury with eosinophilia (DILI-Eos). Among 17129 Japanese adult patients who underwent serial liver function tests and eosinophil counts, we extracted 631 DILI-Eos cases and 16498 non-DILI-Eos controls. Multivariable logistic regression analysis was performed for 38 drugs that were newly prescribed in more than 50 DILI-Eos cases within 60 d prior to liver injury onset. Sulbactam/cefoperazone showed the strongest association with DILI-Eos (adjusted odds ratio (OR) 14.51; 95% confidence interval (CI) 10.09-20.85), followed by meropenem (OR 5.68; 95% CI 4.10-7.82) and tazobactam/piperacillin (OR 3.55; 95% CI 2.63-4.75). Several commonly used drugs, such as mosapride, lansoprazole, furosemide, and ambroxol, were also significantly associated with increased risk. These findings suggest that DILI-Eos can be triggered by a wide range of drugs across various therapeutic classes potentially via immune-mediated pathways. Notably, substantial variability in risk was observed even within the same drug classes, such as β-lactam antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDs), underscoring the importance of drug-specific evaluation. Further studies are needed to clarify the causality and mechanisms underlying eosinophilic responses in DILI.

Fruquintinib is a small-molecule tyrosine kinase inhibitor of vascular endothelial growth factor receptors (VEGFR) used in patients with metastatic colorectal cancer. Fruquintinib reportedly inhibits the phosphorylation of VEGFR-2 by >80% at plasma trough concentrations >176 ng/mL. Therefore, maintaining a stable trough concentration of fruquintinib is clinically necessary. To date, the only reported method for quantifying fruquintinib concentrations in human plasma is LC-MS/MS, which is challenging to use routinely in clinical settings given its high cost and technical demands. Therefore, in this study, we aimed to develop a method for determining fruquintinib levels in human plasma and validate it for therapeutic drug monitoring (TDM). For the analysis, a 50-μL human plasma sample was obtained, and protein precipitation was performed using acetonitrile extraction. Fruquintinib and acetanilide (internal standard) were separated on a reversed-phase column using a mobile phase consisting of 0.5% KH₂PO₄ (pH 2.6) and acetonitrile (69/31, v/v), pumped at a flow rate of 1.0 mL/min. The detection wavelength was set at 237 nm. The calibration curves for fruquintinib were linear (r² = 0.999) in the range of 50-2000 ng/mL. The accuracy and precision in all validation experiments adhered to the guidelines of the U.S. Food and Drug Administration. Using the validated method, we successfully measured fruquintinib concentrations in the plasma of a patient. This article presents a simple, novel, and sensitive HPLC-UV-based method for determining the plasma concentration of fruquintinib and confirms its applicability in the TDM of fruquintinib in the clinical setting.

Hepatocyte growth factor (HGF) exhibits mitogenic, motogenic, and morphogenic activities. Enhancing HGF production could serve as a therapeutic approach for organ regeneration, wound healing, and embryogenesis. Notably, HGF demonstrates therapeutic potential in the treatment of neurodegenerative diseases. In this study, we found that quercetin promotes HGF production in normal human dermal fibroblasts (NHDF) at low concentrations. cAMP response element binding protein (CREB), a transcription factor, is phosphorylated. Additionally, this activity may result from quercetin's interaction with the β2 adrenaline receptor (β2AR). Further pharmacological analysis suggested that HGF production is promoted via PKA pathway. In conclusion, quercetin shows potential as a drug for treating organ-related diseases, including neurodegenerative disorders, by enhancing HGF production.

In the development of pharmaceuticals and other chemical substances, it is important to evaluate their efficacy and safety. There is a growing trend toward reducing reliance on traditional in vivo testing using animals for safety assessments and utilizing new evaluation methods, such as in vitro and in silico testing, to refine human safety assessments. Furthermore, in medical and environmental fields, there is a growing demand for the utilization of vast amounts of information. This has led to the development of data-driven approaches that utilize large-scale medical information and artificial intelligence (AI). Machine learning enables computers to learn from known data, discover new patterns, and predict unknown data. This technology is also useful for in silico prediction of chemical toxicity and adverse reactions in humans. Recently, explainable AI, which presents the basis for forecasts obtained from machine learning models in a user-understandable manner, has attracted attention and is a useful technology for decision-making support. We have developed machine learning models focusing on a quantitative structure-activity relationship approach to predict toxicity and adverse reactions based on the structural information of chemical substances. Furthermore, we have begun to develop a model to predict package insert revisions based on post-marketing adverse reaction information. These efforts will contribute to solving regulatory science issues regarding the appropriate use of chemical substances such as pharmaceuticals.