As part of a program to identify potent GPR40 agonists with drug-like properties suitable for clinical development, the incorporation of polar substituents was explored with the intention of ...decreasing the lipophilicity of our recently disclosed phenylpropanoic acid derivative 1. This incorporation would allow us to mitigate the cytotoxicity issues observed with compound 1 and enable us to move away from the multifunctional free fatty acid-like structure. Substitutions on the 2',6'-dimethylbiphenyl ring were initially undertaken, which revealed the feasibility of introducing polar functionalities at the biphenyl 4'-position. Further optimization of this position and the linker led to the discovery of several 4'-alkoxybiphenyl derivatives, which showed potent GPR40 agonist activities with the best balance in terms of improved cytotoxicity profiles and favorable pharmacokinetic properties. Among them, 3-{2-fluoro-4-({4'-(4-hydroxy-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy-2',6'-dimethylbiphenyl-3-yl}methyl)aminophenyl}propanoic acid (35) exhibited a robust plasma glucose-lowering effect and insulinotropic action during an oral glucose tolerance test in rats with impaired glucose tolerance.
We determined if a single amino acid substitution in a self protein causes autoantibody responses. Mouse lysozyme (ML) was used as a model self protein, and a mutant ML (F57L ML) was prepared by ...replacing
57Phe of ML to Leu, an approach which resulted in introducing into ML the immunogenic sequence of peptide 50–61 of hen egg lysozyme (HEL) restricted to I-A
k MHC class II molecule. We found that F57L ML but not native ML primed HEL specific T cells and triggered ML specific autoantibody responses in B10.A and C3H mice (I-A
k, I-E
k). Peptide regions, ML 14–69 and ML 98–130, were major epitopes of autoantibodies in both strains of mice. These findings indicate that a single amino acid substitution in self proteins can cause an autoantibody response when the mutated region is presented by MHC class II molecules and recognized by T cells.
Fasiglifam (TAK‐875) is a free fatty acid receptor 1 (FFAR1)/G‐protein–coupled receptor 40 (GPR40) agonist that improves glycemic control in type 2 diabetes with minimum risk of hypoglycemia. ...Fasiglifam potentiates glucose‐stimulated insulin secretion (GSIS) from pancreatic β‐cells glucose dependently, although the precise mechanism underlying the glucose dependency still remains unknown. Here, we investigated key cross‐talk between the GSIS pathway and FFAR1 signaling, and Ca2+ dynamics using mouse insulinoma MIN6 cells. We demonstrated that the glucose‐dependent insulinotropic effect of fasiglifam required membrane depolarization and that fasiglifam induced a glucose‐dependent increase in intracellular Ca2+ level and amplification of Ca2+ oscillations. This differed from the sulfonylurea glimepiride that induced changes in Ca2+ dynamics glucose independently. Stimulation with cell‐permeable analogs of IP3 or diacylglycerol (DAG), downstream second messengers of Gαq‐FFAR1, augmented GSIS similar to fasiglifam, indicating their individual roles in the potentiation of GSIS pathway. Intriguingly, the IP3 analog triggered similar Ca2+ dynamics to fasiglifam, whereas the DAG analog had no effect. Despite the lack of an effect on Ca2+ dynamics, the DAG analog elicited synergistic effects on insulin secretion with Ca2+ influx evoked by an L‐type voltage‐dependent calcium channel opener that mimics glucose‐dependent Ca2+ dynamics. These results indicate that the Gαq signaling activated by fasiglifam enhances GSIS pathway via dual potentiating mechanisms in which IP3 amplifies glucose‐induced Ca2+ oscillations and DAG/protein kinase C (PKC) augments downstream secretory mechanisms independent of Ca2+ oscillations.
Mitochondria-targeted H
S donors are thought to protect against acute ischemia-reperfusion (IR) injury by releasing H
S that decreases oxidative damage. However, the rate of H
S release by current ...donors is too slow to be effective upon administration following reperfusion. To overcome this limitation here we develop a mitochondria-targeted agent, MitoPerSulf that very rapidly releases H
S within mitochondria. MitoPerSulf is quickly taken up by mitochondria, where it reacts with endogenous thiols to generate a persulfide intermediate that releases H
S. MitoPerSulf is acutely protective against cardiac IR injury in mice, due to the acute generation of H
S that inhibits respiration at cytochrome c oxidase thereby preventing mitochondrial superoxide production by lowering the membrane potential. Mitochondria-targeted agents that rapidly generate H
S are a new class of therapy for the acute treatment of IR injury.
Immunotolerogenic activity of monomethoxypolyethylene glycol- (mPEG) conjugated proteins is a beneficial property in protein pharmaceutics. However, procedures for the preparation of tolerogenic mPEG ...proteins have not yet been defined. We prepared mPEG proteins with different mPEG contents using three proteins, hen egg lysozyme, ovalbumin and bovine gamma globulin, and their tolerogenicities to antigen-specific T and B cell responses were examined. We found the most appropriate ratio of tolerance induction to be 1.5–2.0, which is the molecular weight ratio of conjugated total mPEGs to protein. This value may assist in the preparation of tolerogenic mPEG proteins.
Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by ...activating FFAR1 expressed in pancreatic beta cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca2+ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand gamma -linolenic acid ( gamma -LA). Augmentation of glucose-induced insulin secretion by fasiglifam, gamma -LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca2+ influx activities of fasiglifam and gamma -LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.
Summary
We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant ...T‐cell epitope of hen egg lysozyme (HEL), HEL 107–116. As T cells specific for HEL 107–116 were primed in these mice, the anti‐ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107–116. Serum IgG responses against ML were dominantly focused on the ML 14–69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14–69 and examined their antigen specificities in detail, to characterize the nature of the remaining B‐cell repertoire specific for ML. mAbs specific for ML 14–69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six‐ to 80‐fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B‐cell repertoire in normal mice.
Self proteins including foreign T cell epitope induce autoantibodies. We evaluated the relationship between the size of foreign Ag introduced into self protein and the magnitude of autoantibody ...production. Mouse lysozyme (ML) was used as a model self protein, and we prepared three different ML derivatives carrying T cell epitope of hen egg white lysozyme (HEL) 107-116, i.e, heterodimer of ML and HEL (ML-HEL), chimeric lysozyme that has residue 1-82 of ML and residue 83-130 of HEL in its sequence (chiMH), and mutant ML that has triple mutations rendering the most potent T cell epitope of HEL (sequence 107-116). Immunization of BALB/c mice with these three ML derivatives induced anti-ML autoantibody responses, whereas native ML induced no detectable response. In particular, mutML generated a 10(4) times higher autoantibody titer than did ML-HEL. Anti-HEL107-116 T cell-priming activities were almost similar among the ML derivatives. The heterodimerization of mutant ML and HEL led to significant reduction of the autoantibody response, whereas the mixture did not. These results show that size of the nonself region in modified self Ag has an important role in determining the magnitude of the autoantibody response, and that decrease in the foreign region in a modified self protein may cause high-titered autoantibody response.
Objective
To analyze the role of adjuvant chemotherapy in lymph node‐positive patients with upper tract urothelial carcinoma undergoing radical nephroureterectomy, and identified the prognostic ...adjuvant chemotherapy parameters.
Methods
The clinicopathological records of 74 lymph node‐positive upper tract urothelial carcinoma patients who underwent radical nephroureterectomy at multiple institutions were retrospectively reviewed. A total of 45 patients (60.8%) received adjuvant chemotherapy, and 29 (39.2%) underwent radical nephroureterectomy only. Kaplan–Meier analyses and Cox proportional hazard modeling were used to study the association between adjuvant chemotherapy status and both recurrence‐free survival and cancer‐specific survival.
Results
Estimated 5‐year recurrence‐free survival was 33.6% in patients undergoing radical nephroureterectomy plus adjuvant chemotherapy compared with 13.5% in patients undergoing radical nephroureterectomy only (hazard ratio 0.52; P = 0.014, log–rank test). Estimated 5‐year cancer‐specific survival was 42.5% in patients undergoing radical nephroureterectomy plus adjuvant chemotherapy, compared with 12.0% in patients undergoing radical nephroureterectomy only (hazard ratio 0.36; P = 0.0003, log–rank test). Multivariate analysis showed that adjuvant chemotherapy was a significant prognostic factor for cancer‐specific survival (P = 0.001), but not for recurrence‐free survival (P = 0.076). When patients undergoing radical nephroureterectomy plus adjuvant chemotherapy were dichotomized, based on preoperative C‐reactive protein levels above or below the normal value, higher C‐reactive protein levels were significantly associated with poor survival (P = 0.012).
Conclusion
Adjuvant chemotherapy seems to improve cancer‐specific survival in lymph node‐positive patients with upper tract urothelial carcinoma. Preoperative C‐reactive protein levels could carry a prognostic value in this setting, and lymph node‐positive patients with low preoperative CRP values should be considered for adjuvant chemotherapy. Further studies are necessary to validate these observations.
Seven biopsied sural nerves from patients with beriberi were morphometrically evaluated. In teased fiber analysis the mean frequency of myelinated fibers showing axonal degeneration and segmental ...demyelination was 37.5 and 5.3%, respectively. In two cases with frequency of segmental demyelination higher than 5%, segmental demyelination was shown by statistical criteria to have occurred on certain fibers in a clustered fashion. Therefore, the segmental demyelination in beriberi may be secondary to axonal degeneration. Electromyographic findings and slow improvement of muscle weakness were compatible with axonal degeneration of motor fibers. Determinations of fiber densities revealed preferential decrease of the density of large myelinated fibers with the preservation of the density of small myelinated and unmyelinated fibers. The preferential nerve fiber involvement in beriberi was not associated with pain in the lower limbs and this fact is contrary to the expectation of the proponents of the gate control theory.
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