Global increase in lifestyle-related diseases, such as type 2 diabetes mellitus and hypertension, and infertility rate due to late marriage has become alarming and problematic. Lifestyle-related diseases and infertility rate are strongly linked to urological diseases and are issues that must be resolved to maintain a high QOL. In this review, we elucidated the pathophysiology of urologic diseases, which authors have been researching, and demonstrated the creation of new therapeutic methods for clinical applications. We have clarified the effects of testosterone on the tissue structure and endothelial function of the corpus cavernosum using castrated rats. Additionally, we reported that the administration of testosterone to model rats with type 2 diabetes suppressed the inflammatory response in the corpus cavernosum and improved erectile function in rats. Furthermore, the effect of anti-cancer agents on erectile function was analyzed using the database obtained from the U.S. Food and Drug Administration (FDA) Adverse Event Reporting System (AERS). Finally, we found that the above-mentioned anti-cancer agents had an affected erectile function in rats. Our study may shed light on possible treatment strategies for improving the QOL of patients with erectile dysfunction, including maintenance of erectile function.
Repetitive behavior, a form of compulsivity, is a component of several neuropsychiatric disorders, including obsessive-compulsive disorder and addiction. Dysfunction of dopaminergic modulation in the striatum is thought to be a key neural mechanism underlying compulsive behavior repetition; however, the mechanistic links between dopaminergic abnormalities and compulsivity remain unclear. This review discusses our recent work demonstrating the contribution of the NOX1 isoform of the superoxide-producing enzyme, nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), to compulsive-like repetitive behavior in mice that received repeated stimulation of D2 receptors. Nox1 deficiency inhibited compulsive-like repetitive behaviors, as assessed by observation of spontaneous behavior patterns and perseveration in the reversal learning test. Repeated stimulation of D2 receptors also upregulated expression of Nox1 in the central striatum (CS), and induced excitatory synaptic potentiation in CS indirect pathway medium spiny neurons. Such synaptic potentiation required recruitment of β-arrestin and was blocked by Nox1 deficiency or acute pharmacological inhibition of NOX1. Furthermore, upregulation of NOX1 in the CS contributed to accumulation of activated Src kinase following stimulation of D2 receptors. Local inhibition of NOX1 or neuron-specific Nox1-knockdown in the CS was sufficient to reduce repetitive behavior. Collectively, these results reveal a novel role for NOX1 in D2 receptor-mediated excitatory synaptic potentiation in the striatum, suggesting the potential of NOX1 inhibition as a treatment for compulsivity.
Magnetic nanoparticle-incorporated liposomes (magnetic liposomes) are considered a promising site-specific drug delivery carrier. Although there are many reports on the development of magnetic liposomes, most of them focus on the characteristics of magnetic nanoparticles, rather than liposomes. Therefore, we first evaluated the effect of the physicochemical properties of magnetic liposomes on their interaction with cells. The highest cellular uptake and retention under a magnetic field was observed using small magnetic cationic liposomes. However, magnetic cationic liposomes exhibited strong cytotoxicity. Based on these results, we constructed complexes of less toxic magnetic anionic liposomes (Mag-AL) and atelocollagen (ATCOL), a biocompatible cationic biomaterial. The cellular associated amount of Mag-AL under a magnetic field was significantly increased when Mag-AL was complexed with ATCOL, and it was comparable to that of magnetic cationic liposomes. Additionally, Mag-AL/ATCOL complexes produced no cytotoxic effect. Moreover, liver accumulation of Mag-AL/ATCOL complexes was significantly increased at a magnetic field-exposed region after intravenous injection in rats. These results indicate that Mag-AL/ATCOL complexes may be a safe and efficient magnetic responsive drug carrier. Next, we applied Mag-AL/ATCOL complexes to prepare magnetized cells for effective cell therapy. Mesenchymal stem cells (MSCs), which have the capacity to suppress tissue inflammation, were efficiently magnetized by incubation with Mag-AL/ATCOL complexes under a magnetic field. Intramuscularly injected magnetized MSCs were significantly retained in mouse skeletal muscle in the presence of a magnetic field and modulated tissue inflammatory responses. These results suggest that magnetized MSCs are useful for muscle regeneration.
Antibody engineering is a powerful method used to generate high-affinity antibodies that enables sensitive immunoassays. It is commonly performed with the following steps: First, antibody fragments (e.g., single-chain Fv fragments; scFvs) with various mutations are displayed on the surface of filamentous bacteriophages to generate a diverse scFv-phages library. Then, rare clones with improved affinities are selected from the library via “panning” against target antigens immobilized on solid phases. However, this process often fails because of biased proliferation of scFv-phage clones and competition with large excesses of clones with weaker affinities. To overcome these limitations, we developed a clonal array profiling (CAP) method in which scFv-phage members in a library are individually examined for their antigen-binding ability. The advantage of CAP over conventional panning was evident in a comparative study that explored a library of anti-cortisol scFvs. CAP isolated five scFv mutants with >30-fold enhanced affinity (Ka) compared with wild-type scFv, enabling >11-fold more sensitive immunoassays. In contrast, no clones showing >5-fold higher Ka were found via panning. Considering the unique features of the primary structures of the improved scFvs found via CAP, we constructed new anti-cortisol scFv libraries where amino acid substitutions or insertions were introduced into the heavy-chain framework region 1 (VH-FR1). As expected, we obtained 21 mutants with >15-fold enhanced affinities. This VH-FR1-targeting mutagenesis also succeeded in generating affinity-matured scFvs against psilocybin, a hallucinogenic compound found in some mushrooms, which could be applied for developing on-site identification systems for hallucinogenic mushrooms, e.g. as immunochromatography devices.
Humans are continually exposed to various chemicals in the environment. Some of these environmental chemicals not only induce malignant transformation but also enhance the malignant potential of the cancer. In this review, the author summarizes the findings on the effects of environmental chemicals on cancer with a focus on inorganic cadmium (Cd) and organic bisphenol A (BPA). Cd, an established human carcinogen, enhances the invasive capacity of rat liver TRL 1215 cells during malignant transformation by downregulating apolipoprotein E (ApoE), a suppressor of cell invasion, via induction of DNA hypermethylation in its promoter region by the oxidative stress/ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-mediated machinery. BPA, which is recognized as an endocrine disruptor, raises the concern that very high concentrations (beyond environmental levels) of BPA are required for activation of estrogen receptors α/β (ERα/β) in vitro. We identified a BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which exhibited more potent estrogenic activity than BPA. Thus, MBP may be a key candidate for explaining the endocrine-disrupting effects of BPA. In addition, the estrogenic action of MBP can be enhanced by repeated exposure of MCF-7 cells to the actualized ERβ subtype because of the downregulation of ERα in human breast cancer MCF-7 cells. MBP downregulates the expression of the tumor suppressor gene, G protein-coupled estrogen receptor 1 (GPER1), via ERβ signaling.
Cytochrome P450 (P450, CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) are major drug-metabolizing enzymes known to catalyze substrate oxidation and glucuronidation, respectively. Both enzymes are located within the endoplasmic reticulum (ER) membrane; however, their membrane topologies differ, with P450 facing cytosol and a major part of UGTs located on the luminal side. Because of the large differences in the reactions that they catalyze and their membrane topologies, it had been believed that P450 and UGT function separately. However, some chemicals are oxidized by P450 and undergo further conjugation by UGT. Therefore, it is important to consider that P450 and UGT may form a complex within the ER membrane and regulate each other's function through this interaction. To prove this hypothesis, we constructed a co-expression system for CYP3A4, P450 reductase, and UGT2B7/1A9 with a baculovirus-insect cell expression system. This system allowed us to compare CYP3A4 activity in the presence and absence of UGT2B7/1A9 co-expression and revealed that the UGTs suppress CYP3A4 activity. The suppressive effect of UGT2B7 was not limited to the enzymatic activity of CYP3A4 but also extended to the entire catalytic cycle, which may be resulted from the inhibition of substrate-binding to the P450. Analysis using UGT mutants indicated that one of the hydrophobic regions within the luminal portion of the UGT interacts with CYP3A4. Furthermore, we suggested that UGT1A suppresses CYP3A activity in vivo by treating rats with dexamethasone. Thus, the functional interactions between P450 and UGT would advance our understanding of the large inter-individual differences in drug-metabolizing capacity.
Disulfide bond A oxidoreductase-like protein (DsbA-L) is a protein (previously named glutathione S-transferase kappa 1) found primarily in peroxisomes and mitochondria. DsbA-L is a key molecule in the multimer formation of adiponectin (APN), which has anti-obesity and anti-inflammatory properties. In humans, DsbA-L mRNA levels in adipocytes were reported to be negatively correlated with the body mass index (BMI). Therefore, we focused on the clinical significance of the DsbA-L gene in obesity and obesity-related diseases in Japanese subjects. First, we showed that the DsbA-L rs1917760 polymorphism is associated with APN multimerization and a high BMI among participants of the health screening program and that this polymorphism is indirectly associated with non-alcoholic fatty liver disease. Second, we showed that the DsbA-L rs1917760 polymorphism was associated with a reduction in the respiratory function and with the elevation of the prevalence of airway obstruction among participants of a health screening program. Finally, we showed in a randomized, double-blind, placebo-controlled clinical trial among healthy volunteers that melinjo seed extract promotes APN multimerization, with a greater association in subjects with the DsbA-L rs1917760 T allele. Through these efforts, we showed that DsbA-L is an important molecule associated with obesity and obesity-related diseases and that it may be a useful target for the treatment or prevention of these diseases.
Vancomycin is the first-choice antimicrobial for the lethal methicillin-resistant Staphylococcus aureus infections. Therefore, the therapeutic performance of vancomycin must be enhanced. The narrow therapeutic range between clinical efficacy and toxicity necessitates therapeutic drug monitoring. Therapeutic targets were previously established by trough concentrations (10-20 μg/mL) but are now commonly determined with the area under the concentration-time curves (AUC, 400-600 μg·h/mL). However, there has not been a strategy for efficiently calculating individual AUC. This review focuses on studies pertaining to activating AUC-guided dosing of vancomycin in clinical settings. First, the author suggested a table for determining empirical dosing of vancomycin by population pharmacokinetic analysis, where weight-normalized dosing corresponded to the estimated glomerular filtration rate, also as in the case during continuous renal replacement therapy (CRRT). The author then demonstrated that Bayesian forecasting was effective for CRRT patients in regards to compliance with a target therapeutic range. The author also developed a web application to enable AUC-guided dosing. Finally, the author demonstrated a reduction in patients developing nephrotoxicity following an intervention against the concomitant use of vancomycin and tazobactam/piperacillin, a known risk factor for acute kidney injury.
The role of β-estradiol (E2) in lipoprotein metabolism in mammary tumors remains unknown. Therefore the effect of E2 on secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells was examined. The E2-treated FM3A cells increased active LPL secretion in a time- and dose-dependent manner. The activity of mitogen-activated protein kinase (MAPK) was elevated in the tumor cells treated with E2, and E2-stimulated secretion of LPL was suppressed by the MAPK kinase 1/2 inhibitor PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor FR180204, p38 MAPK inhibitor SB202190, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In addition, the effect of E2 on active LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but not by the mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed the secretion of LPL by E2. Stimulatory secretion of LPL by E2 from the tumor cells is closely associated with activation of mTORC2 rather than mTORC1, possibly via the MAPK cascade.
ATP-binding cassette (ABC) transporters, which comprise the largest gene-family in humans, are membrane proteins that transport various substrates, depending on ATP hydrolysis. Among these transporters, several include ABCB1 (P-glycoprotein), identified here for the first time in humans, which exports anti-cancer drugs from cancer cells, thus participating in multidrug resistance (MDR). ABC transporters also export drugs, in general, from the human body, therefore affecting overall pharmacokinetics. We have contributed, here, to a better understanding of the role of these exporter proteins in two aspects. First, we have cloned the human ABCC2 gene and identified mutations in hereditary hyperbilirubinemia patients, demonstrating the role of ABCC2 as a xenobiotic export pump. Second, we also found an unexpected role of ABCB1 in cancer, in that it promotes tumor initiation independently of the MDR phenomenon, which was further confirmed by a chemoprevention experiment using verapamil, an ABCB1 inhibitor. In this review, I discuss the role of ABC transporters, both in biodefense against xenobiotics and in cancer development and malignant alterations, based on our results as well as the studies of others.
This review describes the analytical methods for percutaneous absorption and skin permeation of chemical substances and the transdermal drug delivery system (TDDS) that I have been researching for about half a century. This period coincides with that of the dawn and early stage of TDDS through its development to the maturity stage. First, as an introduction, the skin structure, definitions of scientific terms such as percutaneous absorption, skin permeation, skin penetration, types of chemical substances that make contact with the skin, and the skin permeation pathway of chemical substances are outlined. Next, the experimental methods established for percutaneous absorption and skin permeation of chemical substances are described. Then, analytical methods for the percutaneous absorption rate are outlined: i.e., partition to skin barrier and diffusion in the stratum corneum of chemical substances, the meaning of the permeability coefficient, and Fick's 1st and 2nd laws of diffusion. Furthermore, after showing the modeling of the skin barrier as a diffusion layer, the analytical method of skin concentration is also explained. In addition, after explaining the concept of thermodynamic activity of chemical substances for percutaneous absorption, the usefulness of skin-penetration enhancers and physical means to increase skin permeation is reviewed. Finally, I introduce my dream of an installable DDS on or in the skin in future therapeutic modalities.
The globalization of drug trade has led to the increased production of falsified medicines. In addition, poor medication adherence increases the costs of healthcare. The need to manage medication has given rise to marketing of highly functional networked digital medicine. Therefore, a growing need has emerged to ensure the traceability of pharmaceutical products from shipment to patient distribution. Microtaggant technologies that can encode individual numbers on pharmaceutical products are expected to serve achieving this goal. Taggants are a class of materials that can be applied to an object to make it identifiable, like barcodes and holograms. Since the smaller size of microtaggant make it invisible to naked eyes, it is more difficult to reverse-engineer than conventional taggants. The U.S. Food and Drug Administration (FDA) has established guidelines for the use of microtaggants. Many studies have explored the use of various analytical technologies and materials as the microtaggants. However, the advantages and disadvantages of each method have not been established yet. In this review, recent research on the use of microtaggants for anti-counterfeiting is summarized and compared to current anti-counterfeiting technologies with spectrographic methods, distribution management systems with barcodes, and medication management systems with sensor devices. We also discuss the microtaggants implementation costs and security level.
The need for ensuring the traceability of pharmaceutical products from shipment to patient distribution is growing with the spread of falsified medicine and poor medication adherence. The microtaggant technologies that can encode individual numbers on pharmaceutical products has gained much attention to solve these problems. The author well organized the advantages and disadvantages of each encoding method of the microtaggants. This review is useful to understand the emerging traceability system.
During the treatment of cardiogenic shock, various continuous infusion drugs are used simultaneously. However, administration from the same route may result in stability changes due to mixing of drugs. In addition, stability tests after mixing more than three types of drugs have hardly been conducted. In this study, noradrenaline, milrinone, dobutamine hydrochloride, and landiolol hydrochloride were used to evaluate the chemical stability of the mixture. Chemical stability was evaluated by measuring the change in each drug concentration over time and calculating the content. The concentration of each drug was measured using an optimized gradient elution method by HPLC. In a four-drug mixed sample, noradrenaline, milrinone, dobutamine hydrochloride, and landiolol hydrochloride had retention times of 2.1 min, 5.2 min, 9.3 min, and 11.9 min, respectively. The concentration immediately after mixing each drug was almost the same as the theoretical concentration at the time of mixing each drug. Furthermore, noradrenaline, milrinone, and dobutamine hydrochloride concentrations were maintained up to 99% in each drug mixture until 24 h after mixing all the samples. However, the content of landiolol hydrochloride was 90% or less 24 h after mixing, except for two types of mixed solutions with dobutamine hydrochloride. This result suggested that landiolol hydrochloride was being degraded owing to acidic conditions. The results of this study suggest that noradrenaline, milrinone, and dobutamine hydrochloride can be administered from one route, while it is recommended that landiolol hydrochloride be administered from another route.
Neuropsychiatric disorders are among the most represented disorders in the 2013 revision of the Model Core Curriculum for Pharmaceutical Education in Japan. However, it is reported that many pharmacy students have a negative impression of neuropsychiatric disorders because they do not know how to interact with psychiatric patients. To deepen students' understanding of pharmacotherapy for psychiatric disorders, Chiba University Hospital provides a one-day psychiatric clinical practicum in which pharmacy students perform a pharmaceutical intervention. In this practicum, students first had a group discussion about the case, assessed the pharmaceutical problem, and considered pharmacotherapy solutions to the problem on their own. Then, the students interviewed the patients directly to collect pharmaceutical-related information. Finally, the students participated in a conference and presented their own pharmacotherapy solutions to the problem. In this study, we conducted a questionnaire survey and assessed the students' reports in order to investigate their satisfaction with the practicum, changes in their impression of psychiatric disorders or patients with psychiatric disorders, and the level of knowledge they attained. The results showed that almost all students were satisfied with the practicum. The students' impression of psychiatric disorders or patients with psychiatric disorders changed in a positive direction. It seems that students came to realize the role and responsibility of the pharmacist in medical teams. These findings suggest that the practicum led to a positive change in the students' impression of psychiatric patients through conversations with the patients, assessment of pharmacotherapy from a pharmaceutical point of view, and proposing prescriptions.
Experience-based clinical training has been introduced in long-term practical training at pharmacies in Japan. Satisfaction and motivation of pharmacy students differs among pharmacies; however, the underlying causes of the differences are unclear. The purpose of this study was to investigate items for improvement for long-term practical training for raising pharmaceutical problem-solving capability at pharmacies based on students' perspectives. We performed a questionnaire survey to evaluate student satisfaction in clinical education and learning environments. The results were analyzed using customer satisfaction (CS) analysis. After long-term practical training at pharmacies, questionnaires were distributed to 126 pharmacy students at Fukuyama University between November 2021 and March 2022. Ninety-eight students responded. “The practice hours per prescription or pharmacotherapy screening and intervention (improvement factor: 14.954)”, “the discussion hours for optimization of prescribing and rational medication use, or patient education with pharmacist (9.493)”, and “the self-learning place (3.490)” were identified as items requiring improvement. Our findings suggest that the university should work together with pharmacies to improve the learning strategy and environments to increase direct and continuous interaction with pharmacists at pharmacies. Such interventions may result in improvement of students' satisfaction and a wide variety of practical pharmacy skills.