The linear no-threshold model (LNT) was recommended in 1956, with abandonment of the traditional threshold dose-response for genetic risk assessment. Adoption of LNT by the International Commission on Radiological Protection (ICRP) became the standard for radiation regulation worldwide. The ICRP recommends a dose limit of 1 mSv/year for the public, which is too low and which terrorizes innocent people. Indeed, LNT arose mainly from the lifespan survivor study (LSS) of atomic bomb survivors. The LSS, which asserts linear dose-response and no threshold, is challenged mainly on three points. 1) Radiation doses were underestimated by half because of disregard for major residual radiation, resulting in cancer risk overestimation. 2) The dose and dose-rate effectiveness factor (DDREF) of 2 is used, but the actual DDREF is estimated as 16, resulting in cancer risk overestimation by several times. 3) Adaptive response (hormesis) is observed in leukemia and solid cancer cases, consistently contradicting the linearity of LNT. Drastic reduction of cancer risk moves the dose-response curve close to the control line, allowing the setting of a threshold. Living organisms have been evolving for 3.8 billion years under radiation exposure, naturally acquiring various defense mechanisms such as DNA repair mechanisms, apoptosis, and immune response. The failure of LNT lies in the neglect of carcinogenesis and these biological mechanisms. Obstinate application of LNT continues to cause tremendous human, social, and economic losses. The 60-year-old LNT must be rejected to establish a new scientific knowledge-based system.
The author has studied nerve growth factor (NGF) and its family of neurotrophic factors (neurotrophins) for over 40 years. During the first 20 years, my laboratory established a highly sensitive enzyme immunoassay for NGF and analyzed the regulatory mechanism of NGF synthesis in cultured primary cells. Fibroblast cells cultured from peripheral organs such as the heart and astrocytes from the brain produced a substantial amount of NGF in a growth-dependent manner. Furthermore, synthesis of NGF in these cells could be upregulated by catechol compounds including catecholamines. This observation might explain a physiological relation between the level of NGF mRNA and the density of innervation in the peripheral sympathetic nervous systems. Over the subsequent 20 years, my laboratory investigated the physiological functions of neurotrophic factors, including neurotrophins, during development or post-injury and found that brain-derived neurotrophic factor (BDNF) plays a role in the formation of the laminar structure of the cerebral cortex. In addition, my laboratory discovered that endogenous glial cell line-derived neurotrophic factor (GDNF) contributes to the amelioration of motor activity after spinal cord injury. Therefore we aimed to develop low-molecular weight compounds that generate neurotrophic factor-like intracellular signals to protect or ameliorate neurological/psychiatric diseases. 2-Decenoic acid derivatives and other similar molecules could protect or ameliorate in animal models of mood disorders such as depression and enhance recovery from spinal cord injury-induced motor paralysis. Compounds that can generate neurotrophin-like signals in neurons are expected to be developed as therapeutic drugs for certain neurological or psychiatric disorders.
Although exercise and drug therapy are important to prevent progression of arteriosclerotic disease, exercise leads to an increase in muscular disorder induced by HMG-CoA reductase inhibitors (statins). Elucidation of this mechanism is needed to prevent the occurrence of muscular disorders. Since exercise induces expression of monocarboxylate transporter (MCT) 4, we focused on the association between MCT4 function and statin-induced muscle injury. First, we examined the transport of L-lactate via MCT4 using MCT4 cRNA-injected Xenopus laevis oocytes. L-lactate uptake by MCT4-expressing oocytes was markedly reduced by alkalizing the buffer pH and saturated at higher L-lactate concentrations. On the other hand, AMP-activated protein kinase (AMPK) and protein kinase C (PKC) are activated by exercise. We next examined whether AMPK and PKC activation affects the expression and function of MCT4 in rat skeletal muscle and RD cells as an in vitro skeletal muscle model. AMPK and PKC activation increased MCT4 expression level and lactate efflux by MCT4. Finally, we examined the association between MCT4 function and statin-induced cytotoxicity. Statins inhibited transport of L-lactate via MCT4 in a concentration-dependent manner. Statin-induced cytotoxicity was associated with intracellular acidification and caspase-3/7 activation. On the other hand, bicarbonate suppressed statin-induced pH alteration, caspase activation, and morphological change. The results suggest that statin-induced muscle injury exacerbated by exercise is associated with intracellular acidification and that the effects of statins on L-lactate transport are mediated by MCT4.
Esterases hydrolyze compounds containing ester, amide, and thioester bonds, causing prodrug activation or detoxification. Among esterases, carboxylesterases have been studied in depth due to their ability to hydrolyze a variety of drugs. However, there are several drugs for which the involved esterase(s) is unknown. We found that flutamide, phenacetin, rifamycins (rifampicin, rifabutin, and rifapentine), and indiplon are hydrolyzed by arylacetamide deacetylase (AADAC), which is highly expressed in human liver and gastrointestinal tissues. Flutamide hydrolysis is considered associated with hepatotoxicity. Phenacetin, a prodrug of acetaminophen, was withdrawn due to side effects such as methemoglobinemia and renal failure. It was demonstrated in vitro and in vivo using mice that AADAC is responsible for phenacetin hydrolysis, which leads to methemoglobinemia. In addition, it was shown that AADAC-mediated hydrolysis attenuates the cytotoxicity of rifamycins. Thus AADAC plays critical roles in drug-induced toxicity. Another orphan esterase, α/β hydrolase domain containing 10 (ABHD10), was found responsible for deglucuronidation of acyl-glucuronides including mycophenolic acid acyl-glucuronide and probenecid acyl-glucuronide. Because acyl-glucuronides appear associated with toxicity, ABHD10 would function as a detoxification enzyme. The roles of orphan esterases are becoming increasingly understood. Further studies will facilitate our knowledge of the pharmacologic and toxicological significance of orphan esterases in drug therapy.
Multi-substituted β-lactam compounds have not only attracted considerable interest as core structures of pharmaceutical compounds such as antibiotics but also have been used as building blocks for the construction of β-amino acids. Electrophilic β-lactams can be used to enhance essential biological activities. Furthermore, the ring-opening reactions of electrophilic β-lactams can be used to provide facile access to β-amino acids. The introduction of an electronegative fluorine atom to a β-lactam ring to give the corresponding fluoro-β-lactam can be used as an effective strategy for the preparation of electrophilic β-lactams. In this review, we provide a summary of our recent research towards the direct functionalization of fluoro-β-lactams. This review has been divided into four topics, including: 1) the alkylation and hydroxyalkylation of α-bromo-α-fluoro-β-lactams (1); 2) the nickel-catalyzed cross coupling reaction of 1; 3) the asymmetric synthesis of fluoro-β-lactams using chiral ligands; and 4) the utilization of fluoro-β-lactams as highly electrophilic building blocks.
Arynes, such as benzyne, are highly strained and kinetically unstable intermediates that have been widely employed in organic synthesis. In particular, since the initial report regarding the in situ generation of arynes under neutral conditions from ortho-(trimethylsilyl)aryl triflates and fluoride ions in 1983, numerous nucleophilic additions to arynes have been utilized in the synthesis of carbocycles, heterocycles, and multi-substituted arenes. Recently, we reported the insertion of arynes into the π-bond in formamides, as well as related cascade reactions. First, we synthesized salicylaldehydes and aminophenols via the insertion of arynes into the C=O bond of sterically less hindered N,N-dimethylformamide (DMF). Notably, computational studies revealed the generation of unstable benzoxetene and ortho-quinone methide as reactive intermediates. We also studied multi-component coupling reactions leading to 2H-chromenes, coumarins, and xanthen-1-ones under mild conditions through the stepwise release of the strain energy in arynes. Furthermore, we studied the synthesis of O-heterocycles such as dihydrobenzofuran, benzofuran, and 4H-chromene as an application of our method. In this review, we will describe these studies in detail.
The endogenous circadian clock drives robust oscillations in physiology and behavior, such as hormone secretions and sleep/wake cycles, with a period of about 24 h. We are rarely aware of this internal clock system because it is usually synchronized with environmental light-dark cycles. However, travelling rapidly across multiple time zones in a jet airplane suddenly makes us aware of the desynchrony between the body clock and external time, causing sleep disturbances and gastrointestinal problems. Although jet lag is recognized as a chronobiological problem, its specific molecular and neural mechanisms are poorly understood. To address this issue, we identified genes highly expressed in the suprachiasmatic nucleus of the anterior hypothalamus (SCN), the mammalian master clock that controls rhythmic behavior, then analyzed the behavior of knock-out mice for these genes under jet lag condition. We found that the circadian rhythms of locomotor activity and clock gene expression rapidly re-entrained to phase-shifted light-dark cycles in mice genetically deficient in V1a and V1b receptors. Real-time imaging of cellular rhythms in the SCN suggested that interneuronal communication through V1a and V1b confers on the SCN an intrinsic resistance to external perturbation, enhancing the robustness of the SCN clockwork. Pharmacological blockade of V1a and V1b in the SCN of wild-type mice accelerated their recovery from jet lag symptoms, suggesting vasopressin signaling as a potential pharmaceutical intervention for the management of circadian rhythm misalignment.
Drug-induced hepatotoxicity is of concern in drug discovery and development. Reactive metabolites generated by drug metabolizing enzymes in the liver contribute to the induction of hepatotoxicity. Therefore, drug-induced hepatotoxicity, drug metabolism, and pharmacokinetics were evaluated in vitro and in vivo in this pre-clinical study. First, hepatotoxicity was tested in vitro using three-dimensional hepatocyte cultures. Hepatocyte spheroids formed in the three-dimensional culture systems maintain various liver functions such as the expression of drug metabolizing enzymes. High dose exposure to acetaminophen (APAP) induces hepatotoxicity because of the formation of reactive metabolites by CYP. Using fluorescence imaging, we observed that cell viability and glutathione levels were reduced in hepatocyte spheroids exposed to APAP mediated by the metabolic activation of CYP. On the other hand, there are species differences in the expression of drug metabolizing enzymes and metabolite profiles between animals and humans. Therefore, chimeric mice transfected with human hepatocytes were used for the in vivo assessment of metabolic profiles in humans. We found that drug metabolism and pharmacokinetics mediated by CYP and non-CYP enzymes, such as UDP-glucuronosyltransferase and aldehyde oxidase, in chimeric mice with humanized liver were similar to those in humans. The combination of in vitro and in vivo assessments using spheroids and chimeric mice with humanized liver, respectively, during the screening of drug candidates may help to reveal hepatotoxicity induced by the formation of metabolites.
The recently discovered high mannose (HM)-binding lectin family in lower organisms such as bacteria, cyanobacteria, and marine algae represents a novel class of anti-viral or anti-tumor compounds. This lectin family shows unique carbohydrate binding properties with exclusive high specificity for HM glycans with core trisaccharide comprising Manα(1-3)Manα(1-6)Man at the D2 arm. At low nanomolar levels, these lectins exhibit potent antiviral activity against HIV and influenza viruses through the recognition of HM glycans on virus spike glycoproteins. In addition, some of these lectins, such as bacterial PFL, show cytotoxicity for various cancer cells at low micromolar levels. Cell surface molecules to which PFL bound were identified as integrin alpha 2 and epidermal growth factor receptor (EGFR) by peptide mass finger printing with MALDI-TOF MS. Upon PFL binding, these molecules were rapidly internalized to cytoplasm. EGFR was time dependently degraded in the presence of PFL, and this process was largely responsible for autophagy. Furthermore, PFL sensitizes cancer cells to the EGFR kinase inhibitor, gefitinib. In vivo experiments showed that intratumoral injection of PFL significantly inhibited the growth of tumors in nude mice. PFL-mediated down regulation of integrin/EGFR ultimately contributed to the inhibition of tumor growth both in vitro and in vivo. Thus, the novel anti-cancer mechanism of PFL suggests that this lectin is potentially useful as an anti-cancer drug or as an adjuvant for other drugs. This class of proteins will likely have beneficial impact as a tool for biochemical and biomedical research because of its unique carbohydrate specificity and various biological activities.
Drug delivery techniques to tumor cells have attracted considerable attention. For instance, folic acid (FA) as a tumor-targeting ligand is widely used because of overexpression of folate receptor-α (FR-α) in various kinds of epithelial tumor cells. On the other hand, methyl-β-cyclodextrin (M-β-CyD) is acknowledged to induce cell death through the extraction of cholesterol from lipid rafts. It was recently reported that intraperitoneal administration of M-β-CyD exerted antitumor activity in human tumor xenografted athymic nude mice. However, the cytotoxic activity of M-β-CyD is known to lack tumor cell selectivity. Therefore in the present study, in an attempt to confer tumor cell selectivity to M-β-CyD, we newly synthesized folate-appended M-β-CyD (FA-M-β-CyD) and evaluated its potential as a novel antitumor agent. FA-M-β-CyD showed potent antitumor activity in various FR-α-positive cells such as KB cells, Ihara cells, and M213 cells but not in FR-α-negative cells, A549 cells. FA-M-β-CyD induced the formation of autophagic vacuoles in KB cells. In addition, the antitumor activity of FA-M-β-CyD, but not M-β-CyD, was inhibited by addition of the autophagy inhibitors chloroquine and bafilomycin A1 in KB cells. A single intravenous injection of FA-M-β-CyD drastically inhibited tumor growth and significantly improved survival rate in Colon-26 cells-allografted or M213 cells-xenografted mice. In conclusion, FA-M-β-CyD has potential as a novel tumor-selective anticancer agent due to FR-α-mediated cellular uptake. The present results provide useful information for the design and development of novel antitumor drug carriers and antitumor drugs based on CyDs.
Metallo-β-lactamases (MBLs) are di-Zn(II) metalloenzymes that efficiently hydrolyze most β-lactam antibiotics used in clinical settings. Bacteria producing MBLs have been isolated from clinical settings and from natural environments such as rivers and soils, and are now recognized as a new potential threat to human health. No effective inhibitors are available for clinical use, making the treatment of infectious diseases caused by bacteria producing MBLs more difficult. IMP-1 is encoded on a plasmid which can be horizontally transferred between bacterial strains. Our studies on MBLs, and especially on IMP-1, focus on understanding the role of Zn(II) ion(s) in the hydrolysis of β-lactam antibiotics and on the detailed structure of the IMP-1 active site in order to develop efficient inhibitors. We investigated the role of the two Zn(II) ions in IMP-1 by kinetic, spectroscopic and thermodynamic analyses. The results revealed that the first Zn(II) ion is necessary for the hydrolysis of β-lactam antibiotics while the second Zn(II) ion enhances enzyme activity and structural stability, thus helping the enzyme achieve maximum activity. The detailed structures of the IMP-1 active site were examined by X-ray crystallography. Thiol compounds for irreversibly inhibiting IMP-1 were developed and the binding mode of these inhibitors was investigated in detail. These findings will aid the design of inhibitors that target MBLs.
The dose of a transdermal fentanyl patch is proportional to its application site area. Therefore, the absorption of fentanyl may decrease if the patch detaches, leading to insufficient analgesia. Sixteen healthy volunteers were enrolled in a study to investigate the appropriate application sites and clinical utility of three transdermal fentanyl patches available in Japan. Three placebos, Fentanyl 1-day (Fentos; Fen), Fentanyl 3-day (Durotep; Dur), and Generic Fentanyl 3-day (HMT) were administered using a crossover study design. The placebos were applied to 11 different sites, including both sides of the upper arm, abdomen, back, thigh, chest, and the middle of the chest. We determined the patch detachment area and incidence of patch-induced itching every 24 h and evaluated differences between each application site using the Wilcoxon signed-rank test. Significant patch detachment was observed on the abdomen and upper arms with Fen, on the abdomen and chest with Dur, and on the chest with HMT compared with that at other sites (p<0.005). Although no significant difference in itching was observed between regions when administering Fen, itching significantly increased on the chest and back when using Dur and on the abdomen when using HMT as compared with that at other sites (p<0.05). Our results indicate that the three transdermal Fen patches exhibit different adhesive properties and local adverse events, indicating that the application site should be cautiously selected for each patch type.
In recent years, the necessity of a bioanalytical method validation has been discussed and guidance/guidelines have been released from regulatory agencies. However, none of these provides any details for partial validation (PV) in case of a partial change in the validated analytical method. Therefore eleven scientists have launched a discussion group (DG) with the approval of Japan Bioanalysis Forum (JBF), and have been discussing PV for chromatographic methods based on survey results of Japanese bioanalysts. This document reports the results of discussion on PV for a change of analytical instruments such as: 1) full system (limited to same manufacturer and model); 2) pump; 3) autosampler; and 4) mass spectrometer. The DG members agreed on an outline that validation items required for PV are as follows: calibration curve and reproducibility in case 1); calibration curve, reproducibility, and selectivity in case 2); calibration curve, reproducibility, and carryover in case 3); and nearly full validation items without recovery, dilution integrity, and stability in case 4), in consideration of instrument specification and characteristics of each analytical method. Note that this report does not represent a consensus of all the members of JBF, but is a recommendation from the DG members at this stage. Thus further thought is recommended for future discussions.