Switching subtype-selectivity: Fragment replacement strategy affords novel class of peroxisome proliferator-activated receptor α/δ (PPARα/δ) dual agonists

Peroxisome proliferator-activated receptors (PPARs) are important drug targets for treatment of dyslipidemia, type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and great efforts have been made to develop novel PPAR ligands. However, most existing PPAR ligands contain a carboxylic acid (CA) or thiazolidinedione (TZD) structure (acidic head group) that is essential for activity. We recently discovered non-CA/TZD class PPARα/δ partial agonists, which contain an acetamide moiety and adjacent methyl group, linked to a 1,2,4-oxadiazole ring (“fragment a”). We hypothesized that the acetamide structure might interact with the CA/TZD-binding pocket. To test this idea, we firstly replaced fragment a in one of our compounds with the α-alkoxy-CA structure often found in PPAR agonists. Secondly, we replaced the α-alkoxy-CA head group of several reported PPAR agonists with our acetamide-based fragment a. The agonistic activities of the synthesized hybrid compounds toward PPARs (PPARα, PPARγ and PPARδ) were evaluated by means of cell-based reporter gene assays. All the hybrid molecules showed PPAR-agonistic activities, but replacement of the α-alkoxy-CA head group altered the maximum efficacy and the subtype-specificity. The acetamide-based hybrid molecules showed partial agonism toward PPARα and PPARδ, whereas the α-alkoxy-CA-based molecules were generally selective for PPARα and PPARγ, with relatively high activation efficacies. Thus, the fragment replacement strategy appears promising for the development of novel acetamide-based PPARα/δ dual agonists.     

Maximal lactate steady-state prediction through quadratic modeling of selected stages of the lactate minimum test

In this study, we compared the maximal lactate steady state (MLSS) with lactate minimal (LM) intensities determined visually and through a quadratic polynomial function of selected stages of LM test. Eleven male recreational cyclists (27.7 +/- 4.5 years, 175.7 +/- 5.6 cm, 69.5 +/- 10.8 kg, and 12.0 +/- 5.5% body fat) performed one LM test under previous induction of hyperlactaemia with an initial intensity of 75 W with 30-W increments every 3 minutes with blood lactate concentration (HLa) and rating of perceived exertion (RPE) measurements. The LM intensity was determined visually (VLM) and by modeling the lactate response through polynomial function by using: 1) all stages (LMP); 2) the first stage, the stage corresponding to RPE-13 and the last stage/exhaustion (LMP3max); 3) the three lowest lactate concentration stages (LMP3adj); and 4) the initial, RPE-13, and RPE-16 stages (LMP3sub). The MLSS was determined as the highest intensity at a variation not greater than 0.05 mmol.l.min of HLa during the last 20 minutes of a 30-minute exercise session. The MLSS (204.0 +/- 16.0 W), VLM (198.6 +/- 15.2 W), LMP3adj (190.4 +/- 12.9 W), and LMP3sub (192.1 +/- 27.2 W) were not different, well correlated, and in agreement to each other. In conclusion, the polynomial modeling of HLa response to three submaximal stages produced exercise intensities that did not differ from MLSS.     

Discovery of potent CCR4 antagonists: Synthesis and structure-activity relationship study of 2,4-diaminoquinazolines

A new series of quinazolines that function as CCR4 antagonists were discovered during the screening of our corporate compound libraries. Subsequent compound optimization elucidated the structure-activity relationships and led the identification of 2-(1,4'-bipiperidine-1'-yl)-N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine 14a, which showed potent inhibition in the [(35)S]GTPgammaS-binding assay (IC(50)=18nM). This compound also inhibited the chemotaxis of human and mouse CCR4-expressing cells (IC(50)=140nM, 39nM).     

Cinnamon extract inhibits the postprandial overproduction of apolipoprotein B48-containing lipoproteins in fructose-fed animals

We have reported previously that a cinnamon extract (CE), high in type A polyphenols, prevents fructose feeding-induced decreases in insulin sensitivity and suggested that improvements of insulin sensitivity by CE were attributable, in part, to enhanced insulin signaling. In this study, we examined the effects of CE on postprandial apolipoprotein (apo) B-48 increase in fructose-fed rats, and the secretion of apoB48 in freshly isolated intestinal enterocytes of fructose-fed hamsters. In an olive oil loading study, a water-soluble CE (Cinnulin PF, 50 mg/kg body weight, orally) decreased serum triglyceride (TG) levels and the over production of total- and TG-rich lipoprotein-apoB48. In ex vivo ³⁵S labeling study, significant decreases were also observed in apoB48 secretion into the media in enterocytes isolated from fructose-fed hamsters. We also investigated the molecular mechanisms of the effects of CE on the expression of genes of the insulin signaling pathway [insulin receptor (IR), IR substrate (IRS)1, IRS2 and Akt1], and lipoprotein metabolism [microsomal TG transfer protein (MTP), sterol regulatory element-binding protein (SREBP1c) in isolated primary enterocytes of fructose-fed hamsters, using quantitative real-time polymerase chain reaction. The CE reversed the expression of the impaired IR, IRS1, IRS2 and Akt1 mRNA levels and inhibited the overexpression of MTP and SREBP1c mRNA levels of enterocytes. Taken together, our data suggest that the postprandial hypertriglycerides and the overproduction of apoB48 can be acutely inhibited by a CE by a mechanism involving improvements of insulin sensitivity of intestinal enterocytes and regulation of MTP and SREBP1c levels. We present both in vivo and ex vivo evidence that a CE improves the postprandial overproduction of intestinal apoB48-containing lipoproteins by ameliorating intestinal insulin resistance and may be beneficial in the control of lipid metabolism.     

Development of photoaffinity probes for γ-secretase equipped with a nitrobenzenesulfonamide-type cleavable linker

We have developed photoaffinity probes for γ-secretase with a nitrobenzenesulfonamide-type linker that can be cleaved with 2-mercaptoethanol under physiological conditions.     

Oxidative renal tubular injuries induced by aminocarboxylate-type iron (III) coordination compounds as candidate renal carcinogens

Oxidative renal tubular injuries and carcinogenesis induced by Fe^III-nitrilotriacetate (NTA) and Fe^III–ethylenediamine-N,N′-diacetate (EDDA) have been reported in rodent kidneys, but the identity of iron coordination structure essential for renal carcinogenesis, remains to be clarified. We compared renal tubular injuries caused by various low molecular weight aminocarboxylate type chelators with injuries due to NTA and EDDA. We found that Fe^III-iminodiacetate (IDA), a novel iron-chelator, induced acute tubular injuries and lipid peroxidation to the same extent. We also prepared Fe^III-IDA solutions at different pHs, and studied resultant oxidative injuries and physicochemical properties. The use of Fe^III-IDA at pH 5.2, 6.2, and 7.2 resulted in renal tubular necrosis and apoptotic cell death, but neither tubular necrosis nor apoptosis was observed at pH 8.2. Spectrophotometric data suggested that Fe^III-IDA had the same dimer structure from pH 6.2 to 7.2 as Fe^III-NTA; but at a higher pH, iron polymerized and formed clusters. Fe^III-IDA was crystallized, and this was confirmed by X-ray analysis and magnetic susceptibility measurements. These data indicated that Fe^III-IDA possessed a linear μ-oxo bridged dinuclear iron (III) around neutral pH.     

Responses to Apical and Basolateral Application of Glutamate in Mouse Fungiform Taste Cells with Action Potentials

In taste bud cells, glutamate may elicit two types of responses, as an umami tastant and as a neurotransmitter. Glutamate applied to apical membrane of taste cells would elicit taste responses whereas glutamate applied to basolateral membrane may act as a neurotransmitter. Using restricted stimulation to apical or basolateral membrane of taste cells, we examined responses of taste cells to glutamate stimulation, separately. Apical application of monosodium glutamate (MSG, 0.3 M) increased firing frequency in some of mouse fungiform taste cells that evoked action potentials. These cells were tested with other basic taste compounds, NaCl (salty), saccharin (sweet), HCl (sour), and quinine (bitter). MSG-sensitive taste cells could be classified into sweet-best (S-type), MSG-best (M-type), and NaCl or other electrolytes-best (N- or E/H-type) cells. Furthermore, S- and M-type could be classified into two sub-types according to the synergistic effect between MSG and inosine-5′-monophosphate (S1, M1 with synergism; S2, M2 without synergism). Basolateral application of glutamate (100 μM) had almost no effect on the mean spontaneous firing rates in taste cells. However, about 10% of taste cells tested showed transient increases in spontaneous firing rates (>mean + 2 standard deviation) after basolateral application of glutamate. These results suggest the existence of multiple types of umami-sensitive taste cells and the existence of glutamate receptor(s) on the basolateral membrane of a subset of taste cells.     

In Vivo Role of Phosphorylation of Cryptochrome 2 in the Mouse Circadian Clock

The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2::LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.     

High Rate of Awarding Compensation for Claims of Injuries Related to Clinical Trials by Pharmaceutical Companies in Japan: A Questionnaire Survey

Introduction

International norms and ethical standards have suggested that compensation for research-related injury should be provided to injured research volunteers. However, statistical data of incidence of compensation claims and the rate of awarding them have been rarely reported.

Method

Questionnaire surveys were sent to pharmaceutical companies and medical institutions, focusing on industry-initiated clinical trials aiming at new drug applications (NDAs) on patient volunteers in Japan.

Results

With the answers from pharmaceutical companies, the incidence of compensation was 0.8%, including 0.06% of monetary compensation. Of the cases of compensation claims, 99% were awarded. In turn, with the answers from medical institutions, the incidence of compensation was 0.6%, including 0.4% of serious but not death cases, and 0.04% of death cases. Furthermore, most claims for compensation were initiated by medical institutions, rather than by the patients. On the other hand, with the answers from clinical trial volunteers, 3% of respondents received compensations. These compensated cases were 25% of the injuries which cannot be ruled out from the scope of compensation.

Conclusion

Our study results demonstrated that Japanese pharmaceutical companies have provided a high rate of compensation for clinical trial-related injuries despite the possibility of overestimation. In the era of global clinical development, our study indicates the importance of further surveys to find each country''s compensation policy by determining how it is being implemented based on a survey of the actual status of compensation coming from statistical data.