Studies on the isolation and molecular mechanisms of phytochemicals with anti-tumor or anti-inflammatory properties are important to developing new drugs for cancer and neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In the course of a study to screen bioactive isoflavones from Erythrina poeppigiana (Leguminosae), we isolated an isoflavone with potent apoptosis-inducing activity against human leukemia HL-60 cells. It was designated erypoegin K. The studies demonstrated an enantiomer, (S)-erypoegin K, displayed selective cytotoxic activity, was a novel inhibitor of topoisomerase II, and possessed anti-tumor activity both in vitro and in vivo. We identified other apoptosis-inducing isoflavones with the ability to inhibit glyoxalase I. Dimeric acridone alkaloids, carbazole alkaloids, and coumarin and quinoline derivatives—all obtained mainly from plants in the family Rutaceae—induced apoptosis of HL-60 cells via the production of reactive oxygen species and mitochondrial dysfunction. We also identified terpenoid coumarins, carbazole quinones, rotenoid derivatives, and quinolone alkaloids with anti-inflammatory activities. These compounds reduced nitric oxide (NO) production from RAW264.7 macrophage cells stimulated with lipopolysaccharides and interferon-γ. Some of the compounds displayed neuroprotective activity by reducing NO production. This review primarily describes our recent studies on erypoegin K, and other compounds with apoptosis-inducing and anti-inflammatory activities.

This review summarizes one portion of the research for which the author received the Pharmaceutical Society of Japan Award. The complete title of the awarded research is “Pharmacological Studies on Metabolism and Functions of Biomembrane Lipids”. Because the awarded research is a very broad study, this review describes the discovery, physiological functions, and congenital defects of α-tocopherol transfer protein (α-TTP), a critical factor in determining the transport of vitamin E in the body, which has been the focus of the author's work throughout his research career.

Naldemedine (Nal) is widely used as a therapeutic drug against opioid-induced constipation. However, patients in phase III trials are limited to those with good performance status (PS). Cancer patients may have inferior PS owing to progression of symptoms and adverse events from chemotherapy. Therefore, it is important to survey the efficacy of Nal in patients with poor PS. This study aimed to evaluate Nal efficacy in patients with poor PS. We retrospectively investigated patients from July 2017 to June 2019 and compared Nal efficacy between patients with good and poor PS. The efficacy of Nal was evaluated using changes in the number of spontaneous bowel movements 7 days before and after the introduction of Nal with reference to previous reports. Multivariate analysis was performed to reveal whether poor PS affects Nal efficacy. In total, 141 patients at the Hokkaido University Hospital were analyzed. The effective rate of Nal from day 1 to day 7 of administration was 71.7% and 71.4% in the patients with good and poor PS, respectively, that from day 1 to day 2 of administration was 61.1% and 57.1%, respectively, and that from day 3 to day 7 of administration was 60.2% and 71.4%, respectively, suggesting an absence of significant differences. Furthermore, results of multivariate analysis showed that “best supportive care” and “body weight (55 kg and above)” reduced Nal efficacy. In conclusion, Nal showed similar effectiveness in patients with poor PS as that in those with good PS.

Natural medicines, including crude drugs and Kampo prescriptions, have a long history of clinical uses. An important characteristic of natural medicines is that they are multicomponent medicines. Kampo prescriptions, particularly, usually consist of several crude drugs that contain a large number of constituents. The therapeutic effects of a Kampo prescription result from the total effects of its constituents and not from the effect of a representative constituent with a strong pharmacological effect. In fact, only a few of these constituents with strong biological activity have been listed in the Japanese Pharmacopoeia as therapeutic agents. During my research career, I have observed various synergistic effects and interactions among the constituents of natural medicines. This article reviewed our researches on the biologically active constituents of natural medicines, including the analysis of the anti-inflammatory constituents of orengedokuto (a Kampo prescription). Understanding the role of each constituent with therapeutic effects in Kampo prescriptions forms a scientific basis of Kampo medicine. This will enable the better use and quality control of Kampo prescriptions. Moreover, this will aid the future development of a multicomponent mimic for a specific effect of a Kampo prescription using the appropriate mixture of active constituents in amounts found in the prescription. The above is my dream as a natural product chemist, and I believe that the mixture can do more than we anticipate!

Discovery of natural products that possess novel chemical structures and pharmaceutical activities increases opportunities of drug development. Filamentous fungi have been recognized as an attractive source for pharmaceutically beneficial natural products. Genome sequencing innovation represented by Next-generation sequencer opened fungal genomes one after another, suggesting that one fungal strain has far more biosynthetic gene clusters than that are estimated from the number of previously isolated natural products. In addition, bioinformatics analyses have indicated that most biosynthetic gene clusters are silent under laboratory culture conditions and there are a huge number of natural products hidden in the fungal genome. Therefore, we focused on those silent biosynthetic gene clusters as a potential source for novel natural products and developed methods to activate silent biosynthetic gene clusters by using low molecular weight molecules. In this review, we describe on discovery of novel natural products through activating fungal silent biosynthesis by addition of epigenetic modifiers and plant hormones.

The use of flame retardants, namely bis(2,3-dibromopropyl) phosphate (BDBPP) and tris(2,3-dibromopropyl) phosphate (TDBPP), in textile products such as curtains, carpets and sleeping clothes is banned in Japan under the ‘Act on the Control of Household Products Containing Harmful Substances’. Herein, we developed a GC-MS based method to quantify these compounds with greater accuracy and safety than the current official method. For accurate and sensitive quantification, deuterated compounds, BDBPP-d₁₀ and TDBPP-d₁₅, were used as surrogate standards. In consideration of the safety of the analyst, certain solvents and reagents used for the pretreatment that are carcinogenic or have a risk of explosion were replaced. For the extraction step, benzene was replaced by ethyl acetate, and for the methyl derivatization step, the reagent was changed from a self-prepared solution of diazomethane in ether to a solution of trimethylsilyl diazomethane in hexane, a safe and easy-to-use commercially available reagent. The calibration curves were liner in the range of 0.5-8.0 μg/mL for both methylated BDBPP (BDBPP-Me) and TDBPP. The detection limit was 0.05 μg/g for BDBPP-Me and 0.3 μg/g for TDBPP, which is sufficiently low compared to the current detection limits of 10 μg/g for BDBPP-Me and 8 μg/g for TDBPP. The recoveries in various curtain material were 66-108% and relative standard deviations were 1.2-10.2% when 5 μg BDBPP and TDBPP were added to 0.5 g of samples. Thus, the developed method is applicable to textile products of various materials.

Fullerene (C₆₀) and fullerene derivatives are attractive novel compounds not only for carbon materials of nanotechnology but also for medical fields because of its unique chemical and physical properties. We intend to develop fullerene derivatives as novel lead compounds for drug discovery. At first, we synthesized many types of water-soluble fullerene derivatives to investigate their biological activities because of their poor solubility in water. We found that anionic fullerene derivatives possess anti-oxidant activities, whereas di-cationic fullerene derivatives exhibited antiproliferative activities against various cancer cell lines including drug-resistant cells. Proline-type fullerene derivatives showed inhibitory activities against human immunodeficiency virus (HIV) reverse transcriptase, HIV protease, hepatitis C virus (HCV) NS5B RNA polymerase, and HCV NS3/4A protease. These activities may strongly inhibit virus replication via a synergistic effect and fullerene derivatives may be used as novel multi-target drugs for the treatment of AIDS and hepatitis C in the future.

The crystallization of active pharmaceutical ingredients (APIs) in matrix-type transdermal patches has implications for the rate of drug absorption through the skin and patch adhesion strength. Therefore, the presence or absence and the degree of API crystallinity must be controlled to guarantee the quality of patches. In this study, the utility of laboratory-level X-ray diffractometers for the detection and analysis of crystalline APIs in transdermal patches was investigated using medical patches of tulobuterol and isosorbide dinitrate. Several matrix-type patches employ a controlled drug delivery system containing intentionally crystallized API. Both benchtop and high-resolution laboratory X-ray diffractometers can detect several characteristic peaks of the APIs in these patches even if the patches are wrapped in an outer bag, although a benchtop model provides peak heights one-seventh to one-fifth that of a high-resolution instrument. An isosorbide dinitrate patch containing an unintentionally crystallized spot was wrapped in an outer bag, followed by measurements using both X-ray diffractometers. For both instruments, several isosorbide dinitrate-derived peaks were detected only at the crystallized spot, although the signal-to-noise ratio was poorer for the benchtop model. These results show that a high-resolution X-ray diffractometer is advantageous for high-detection sensitivity and offers a high degree of freedom of the measurement position on the sample. It was concluded that a laboratory-level high-resolution X-ray diffractometer can be used to examine the crystalline state of APIs in patches inside an unopened outer bag.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and β/δ) with distinct physiological functions and ligand recognition. PPARs regulate energy metabolism and therefore become therapeutic targets for various metabolic diseases. While PPARα agonists are used as anti-dyslipidemia drugs and PPARγ agonists as anti-type 2 diabetes drugs, PPAR dual/pan agonists (that acts on two or three subtypes) are expected to treat non-alcoholic steatohepatitis (NASH), pulmonary fibrosis, etc. Structural analyses of PPAR-ligand-binding domain (LBD)-ligand co-crystals using X-ray crystallography have been done mainly on PPARγ, in which ligand-free apocrystals were prepared; however, the information on PPARα-LBD and PPARδ-LBD is limited. Recently, we succeeded to obtain 34 novel co-crystal structures of PPARα-LBD and various PPARα ligands (including fibrates) using various co-crystallization techniques. This procedure is applicable to preparation of PPARδ-LBD co-crystals, and contributes to molecular design of new PPAR targeted drugs based on all three PPAR-LBD structures.

In order to understand the actual state of residual solvents contained in commercial supplements, we performed a simultaneous analysis of residual solvents by headspace (HS)-GC-MS with reference to the Japanese Pharmacopoeia's “Residual Solvents”, for 29 products selected from among commercial supplements (e.g., revitalizers, weight loss pills) that are deeply colored or contain coating agents and extract powder. As a result, benzene (class 1) was detected in eight black-colored supplements, and hexane (class 2B) was also detected in one of those products. On the other hand, methanol (class 2A) was detected in four products containing coating agents and extract powders, such as citrus peel extract. None of these residual solvents exceeded the concentration limits set by the Japanese Pharmacopoeia. Benzene was detected at 1.7 μg/g, which was near the concentration limit, in some products. As raw materials used for the manufacture of the black-colored supplements from which benzene was detected commonly included activated carbon, we analyzed the residual solvents contained in activated carbon commercially available for use as food additive and in food production and medicine. As a result, benzene was detected at high concentrations in activated carbon made from hemp (approximately 29 μg/g) and bamboo (approximately 140 μg/g).

Occupational exposure of pharmacists to drugs during powder drug preparation in dispensing pharmacies was investigated. First, we determined frequently prescribed tipepidine hibenzate and ambroxol hydrochloride suspended in the air of the dispensing room. The median concentration of the drugs in the air was 0.01 μg/m³ and 0.02 μg/m³, respectively; these values indicate that the air in the dispensing room was contaminated with powder drug. To estimate drug exposure during powder drug preparation, drug dust was collected near the nose of workers. Analysis of the active ingredients of the drugs used in the preparation revealed that eight drugs, including bethanechol, l-carbocisteine, and zonisamide, were detected in the range of 1.5-1220 μg/m³. Assuming that the respiratory volume of an adult was 0.008 m³/min, it was estimated that 0.4-36 μg of the ingredients were exposed per prescription by multiplying concentration, respiratory volume and sampling time (3-5 min). Furthermore, the effect of wearing a medical mask on the drug powder exposure was evaluated using a self-made apparatus. When the amount of drug powder collected on filters that is either covered with or without a medical mask was compared, the covered filter exhibited reduced drug powder accumulation to less than 10% the amount collected on the uncovered filter. The present data suggested that a medical mask might decrease the drug dust allergies in dispensing pharmacists.

On the occasion of receiving the Pharmaceutical Society of Japan Award 2020, I explained our research activities on the total syntheses of polycyclic alkaloids as an invited review. The structure of lundurine B, which has an unstable cyclopropane fused indoline skeleton, was proved firstly by the total synthesis. I also describe the total syntheses of optically active lundurine B and rapidilectine B utilizing asymmetric desymmetrization of the spiro intermediate. We developed an intramolecular bond formation reaction between the 2-position of the furan ring to the iminium cation (furane-iminium cation cyclization) to synthesize manzamine alkaloids. The reaction was applied to the total synthesis of the core skeleton of nakadomarin A and ircinal A. A ring-closing metathesis reaction effectively applied to the synthesis of medium and large heterocyclic rings containing the cis double bond found in the structures of nakadomarin A and ircinal A. The total synthesis of schizocommunin, a metabolite of Schizophyllum commune isolated from a patient with human allergenic bronchopulmonary mycosis, was accomplished. We could correct the error in the proposed structure by total synthesis of the natural product. A part of the mechanism of cytotoxicity expression was clarified using newly synthesized shizocommunin.

Drug-drug interactions (DDIs) are responsible for an increase in the incidence of adverse drug reactions. Although CYP is known to be involved in metabolic processes, the DDIs among three or more drugs that involve the same CYP molecular species have not been fully investigated. In this study, we retrospectively examined the relationship between the number of drugs and potential DDIs in metabolic processes involving CYPs in patients who picked up their prescribed drugs at 11 pharmacies in the Kojima Branch of the Okayama Pharmaceutical Association. We found that 66.5% of the 924 patients had potential DDIs; more than half of the patients who took six or more drugs had potential DDIs among three or more drugs. The mean number of CYP3A4-related drugs involved in potential DDIs was 3.52±1.56 in patients who took seven drugs, suggesting the need for careful monitoring of specific symptoms and blood test results for the early detection of adverse drug reactions caused by DDIs among three or more drugs.

Heavy metals, both toxic and essential, have long been an important research focus in life science. To investigate the intracellular actions of heavy metals at the molecular level, I have been exploring protein factors involved in induction of metallothionein (MT) genes by heavy metals that specifically bind to a metal responsive element (MRE) in the region upstream of the human MT-IIA gene. Purification of a zinc-dependent MRE-binding factor, and cloning of its cDNA identified a sequence identical to that of metal-responsive transcription factor-1 (MTF-1). MTF-1, which is characterized by six tandem repeats of the C₂H₂ type zinc finger motif, is indispensable for induction of MT gene expression by multiple types of heavy metal, but zinc is the only metal that can directly activate MTF-1 binding to the MRE, indicating that other heavy metal signals act through zinc as a second messenger. Functional analysis of various MTF-1 point mutants revealed several cysteine (Cys) residues critical for DNA binding and/or transactivation activity. Interestingly, six finger motifs seem to mediate several MTF-1 functions other than DNA binding. Immunohistochemical analyses of various mouse tissues revealed selective expression of MTF-1 in spermatocytes among the testicular cells, suggesting roles relevant to spermatogenesis. The zinc regulon, under the control of MTF-1, will likely provide good clues to aid in unraveling novel functions of intracellular zinc ions.

The aims of the present study were to investigate the effects of fenofibrate and bezafibrate on the risk of development of diabetic retinopathy (DR) in patients with type 2 diabetes and dyslipidemia. Japanese working age patients with type 2 diabetes and dyslipidemia were extracted from the Nihon University School of Medicine Clinical Data Warehouse. These patients were divided into three groups: control group (n=2549), fenofibrate group (n=40), and bezafibrate group (n=135). Multivariate logistic regression analysis was performed to assess the association between fibrates and the development of DR. After adjustment for covariates, fenofibrate showed no association with the risk of DR [adjusted odds ratio (OR), 0.160; 95% CI, 0.021-1.209; p=0.0758]. Bezafibrate also showed no association with the risk of DR (adjusted OR, 0.731; 95% CI, 0.411-1.299; p=0.2855). However, poor control of hemoglobin A1c (HbA1c ≥8.0%; adjusted OR, 3.623; 95% CI, 2.649-4.956; p<0.0001) and high low-density lipoprotein cholesterol (LDL-C ≥140 mg/dL; adjusted OR, 1.399; 95% CI, 1.013-1.932; p=0.0415) within the follow-up period of type 2 diabetes and dyslipidemia increased the risk of DR. Our results suggested that to prevent development of DR in patients with type 2 diabetes and dyslipidemia, controlling LDL-C levels as well as HbA1c levels under coexistence type 2 diabetes and dyslipidemia is more important than the selection of fibrate.

The blood-brain barrier (BBB) consists of brain capillary endothelial cells linked by tight junctions and serves to regulate the transfer of endogenous compounds and xenobiotics between the circulating blood and brain interstitial fluid. We have developed a methodology to characterize brain-to-blood efflux transport in vivo, using the Brain Efflux Index and an in vitro culture model of the BBB, i.e., a conditionally immortalized cell line of the neurovascular unit. Employing these methods, we showed that the BBB plays an important role in protecting the brain by transporting neurotransmitters, neuromodulators, metabolites, uremic toxins, and xenobiotics together with atrial natriuretic peptide from the brain interstitial fluid to the circulating blood. We also developed a highly selective, sensitive LC-MS/MS method for simultaneous protein quantification. We found significant species differences in the expression amounts of various BBB transporter proteins among mice, rats, marmosets, cynomolgus monkeys, and humans. Among transporter proteins at the BBB, multidrug resistance protein 1 (Mdr1/Abcb1) is known to generate a concentration gradient of unbound substrate drugs between the blood and brain. Based on measurements of the intrinsic efflux transport rate of Mdr1 and the protein expression amounts of Mdr1 in mouse brain capillaries and Mdr1-expressing cell lines, we predicted the unbound drug concentration gradients of 7 drugs in the mouse brain in vivo. This was the first successful prediction of in vivo drug transport activity from in vitro experimental data and transporter protein concentration in tissues. This methodology and findings should greatly advance central nervous system barrier research.

Pharmacists began preparing drug solutions intraoperatively for postoperative analgesia in the Department of Surgery at Chiba University Hospital from May 2014. To verify the usefulness of pharmacists preparing these drug solutions, we conducted a questionnaire survey among 51 anesthesiologists and received 44 responses (recovery rate 86.3%). Burden on the anesthesiologists was significantly reduced both temporally and mentally when the pharmacists prepared the drug solutions compared with when the anesthesiologists did (p<0.01). The anesthesiologists' degree of anxiety about sometimes having to prepare drug solutions alone without any confirmation was also significantly reduced when pharmacists prepared them (p<0.01), which implies the need for a double-check system. In addition, 88.6% of anesthesiologists said that they were reassured with preparations done by the pharmacists under a sterile environment using a clean bench. Overall, 88.6% of anesthesiologists responded that they were satisfied with the preparation of drug solutions by pharmacists. Based on the results of this survey, pharmacists' preparation of drug solutions for postoperative analgesia is considered to be useful in ensuring the quality and safety of medical care because it reduced anesthesiologists' work to prepare the drug solutions, allowing them to concentrate on anesthesia and related work, it established a double-check system between the two staff teams, and it was done under a sterile environment.

Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.