Safety and Efficacy of Empagliflozin as Add-On Therapy to GLP-1 Receptor Agonist (Liraglutide) in Japanese Patients with Type 2 Diabetes Mellitus: A Randomised, Double-Blind, Parallel-Group Phase 4 Study.

INTRODUCTION: Empagliflozin, a highly selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, improves glycaemic control in patients with type 2 diabetes mellitus (T2DM) by inducing urinary glucose excretion. Combination therapy with empagliflozin and glucagon-like peptide-1 (GLP-1) receptor agonists had not previously been assessed, so we investigated the safety, tolerability and efficacy of empagliflozin as an add-on therapy to liraglutide, a GLP-1 receptor agonist. METHODS: This was a randomised, double-blind, parallel-group phase 4 trial of empagliflozin (10 mg or 25 mg) for 52 weeks as an add-on therapy to liraglutide (0.9 mg/day) in Japanese patients with T2DM insufficiently controlled by liraglutide alone. RESULTS: 59.4% (19/32) and 66.7% (22/33) of patients in the empagliflozin 10 mg and 25 mg groups, respectively, reported at least one adverse event (AE). 9.4% (3/32) and 21.2% (7/33) of patients, respectively, reported drug-related AEs (primary endpoint). From baseline to week 52, adjusted mean changes with empagliflozin 10 mg and 25 mg, respectively, were: - 0.55 (standard error: 0.15) and - 0.77 (0.14)% for glycated haemoglobin; - 32.5 (4.6) and - 36.0 (4.5) mg/dL for fasting plasma glucose; - 2.6 (0.4) and -3.1 (0.3) kg for body weight; - 6.7 (2.2) and - 8.4 (2.1) mmHg for systolic blood pressure; and - 3.0 (1.2) and - 4.7 (1.1) mmHg for diastolic blood pressure. CONCLUSION: Empagliflozin as an add-on to liraglutide for 52 weeks was well tolerated and led to clinically meaningful and sustained improvements in glycaemic control, body weight and blood pressure in Japanese patients with T2DM. TRIAL REGISTRATION: ClinicalTrials.gov with the identifier NCT02589626. FUNDING: Nippon Boehringer Ingelheim Co. Ltd.

Efficacy of tiotropium/olodaterol on lung volume, exercise capacity, and physical activity.

Purpose: This study evaluated the efficacy of tiotropium/olodaterol vs tiotropium on lung function, exercise capacity, and physical activity in patients with COPD. Patients and methods: A total of 184 patients aged ≥40 years with COPD (Global Initiative for Chronic Obstructive Lung Disease stage II-IV) received tiotropium/olodaterol for 6 weeks, then tiotropium for 6 weeks, or vice versa. The primary endpoint was inspiratory capacity (IC) at peak post-dose. Results: Adjusted mean IC after 6-week treatment was 1.990 L with tiotropium/olodaterol vs 1.875 L with tiotropium (difference: 115 mL; 95% CI: 77, 153; p<0.0001). Forced expiratory volume in 1 s (difference: 105 mL; 95% CI: 88, 123), forced vital capacity (difference: 163 mL; 95% CI: 130, 197), and slow vital capacity (difference: 134 mL; 95% CI: 91, 176) improved with tiotropium/olodaterol (all p<0.0001). Adjusted mean 6-min walk distance was similar between treatments in the overall population but was significantly increased with tiotropium/olodaterol in the subgroup with Global Initiative for Chronic Obstructive Lung Disease stage III/IV at baseline (difference: 18.1 m; 95% CI: 2.3, 33.9; p=0.0254). In a post hoc analysis, tiotropium/olodaterol improved the values for ≥2.0 metabolic equivalents (difference: 5.0 min; 95% CI: 0.4, 9.7; p=0.0337). Conclusion: Tiotropium/olodaterol significantly improved IC compared with tiotropium and potentially enhanced the exercise capacity in COPD patients. A slight improvement in physical activity of relatively more than moderate intensity was also seen with tiotropium/olodaterol.

Study Design of VESUTO®: Efficacy of Tiotropium/Olodaterol on Lung Hyperinflation, Exercise Capacity, and Physical Activity in Japanese Patients with Chronic Obstructive Pulmonary Disease.

INTRODUCTION: The superiority of tiotropium/olodaterol is demonstrated in improvement of lung function, dyspnea, lung hyperinflation, and quality of life compared with either monotherapy in patients with chronic obstructive pulmonary disease (COPD). Japanese Respiratory Society Guidelines for COPD management include improvement of exercise tolerance and daily physical activity as the treatment goals; however, there is limited evidence in Japanese patients with COPD. METHODS: A protocol is developed for the VESUTO® study that investigates the efficacy of tiotropium/olodaterol fixed-dose combination (FDC) compared with tiotropium alone on inspiratory capacity (IC, volume from functional residual capacity to total lung capacity), exercise capacity, and daily physical activity in Japanese patients with COPD. RESULTS: A total of 180 Japanese patients with COPD, aged ≥40 years will be enrolled into the double-blind, multicenter, active-controlled, crossover study (NCT02629965) and will be randomized to receive either tiotropium/olodaterol FDC or tiotropium for 6 weeks each [two puffs via RESPIMAT® (Boehringer Ingelheim, Ingelheim, Germany) inhaler in the morning]. The primary endpoint is IC at rest measured at 60 min post-dose after 6 weeks treatment. The secondary endpoints include the 6-min walk distance (6MWD) at 90 min post-dose and physical activity measured by the activity monitor in the last 2 weeks of the 6-week treatment periods. Lung function tests will also be assessed after 6 weeks treatment. A mixed-effects model repeated measures approach will be used for the primary and secondary endpoints. CONCLUSION: The VESUTO® study is the first randomized interventional study to investigate exercise capacity (6MWD) and physical activity measured by a 3-axis accelerometer in Japanese patients with COPD. The study could provide additional evidence of long-acting muscarinic antagonist (LAMA) + long-acting ß2-agonist (LABA) combination therapy on patients' physical activities as well as lung function. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02629965 (registered on December 1, 2015). FUNDING: The VESUTO study was funded by Nippon Boehringer Ingelheim Co., Ltd., Tokyo, Japan.

Isolation and identification of novel neutrophil-activating cryptides hidden in mitochondrial cytochrome C.

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on ß-hexosaminidase (ß-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced ß-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

Mitocryptide-2, a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like 1.

Peptides simultaneously produced during maturation and degradation of peptidergic hormones and functional proteins have recently become a great interest because they display unpredictably different biological roles than the parent proteins. Namely, we discovered two novel functional cryptic peptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial cytochrome c oxidase and cytochrome b, that efficiently induced neutrophilic migration and activation at nanomolar concentrations. We named these functional "cryptic" peptides hidden in protein structures as "cryptides." In this study, we investigated the receptor molecules and cellular signaling mechanisms for neutrophil-activating N-formylated cryptide MCT-2. In order to identify the receptor molecules, we established HEK-293 cells stably expressing either formyl-peptide receptor (FPR) or its homologue FPR-like 1 (FPRL1), because neutrophilic cells express these receptor molecules which recognize N-formylated peptides. We observed that MCT-2 directly bound to FPRL1 and promoted an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and neither interacted with nor activated FPR, demonstrating that MCT-2 is a specific agonist for FPRL1. Moreover, MCT-2 induced not only [Ca(2+)](i) increase and phosphorylation of extracellular signal-regulated protein kinases 1 and 2, but also ß-hexosaminidase release in neutrophilic/granulocytic cells differentiated from HL-60 cells. Such signaling events were diminished by pretreatment with pertussis toxin, indicating that MCT-2-promoted neutrophilic function is a consequence of G(i)- or G(o)-type G protein-dependent intracellular signaling events via FPRL1 activation. These findings suggest that MCT-2, a cryptide derived from mitochondrial cytochrome b, is a specific endogenous agonist for FPRL1 which is proposed to play key roles in inflammatory responses but whose physiological agonists are equivocal.

Mitocryptide-2: purification, identification, and characterization of a novel cryptide that activates neutrophils.

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide "mitocryptide-2" (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide. Synthetic MCT-2 and its human homolog hMCT-2 induced beta-hexosaminidase release in and chemotaxis of HL-60 cells differentiated into neutrophilic/granulocytic cells. The induction of beta-hexosaminidase release, chemotaxis, and the increase in the intracellular free Ca(2+) concentration by hMCT-2 were completely suppressed by pertussis toxin, indicating the involvement of G(i)- or G(o)-type G proteins in the signaling pathways. Moreover, MCT-2 and hMCT-2 also stimulated beta-hexosaminidase secretion in human neutrophils isolated from peripheral blood in a concentration-dependent manner. Additionally, these peptides partially competed with [(3)H]formyl-Met-Leu-Phe binding to HL-60 cells differentiated into neutrophilic/granulocytic cells, presenting the possibility that the receptor for MCT-2 and hMCT-2 is one of the formyl peptide receptors. These results demonstrate that MCT-2 and its human homolog hMCT-2 are cryptides that activate neutrophils, thus suggesting the presence of regulatory mechanisms involving such mitocryptides in innate immunity.

Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart.

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing beta-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and G(i2)-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce beta-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.

Stable expression and characterization of human PN1 and PN3 sodium channels.

Nociceptive transduction in inflammatory and neuropathic pain involves peripherally expressed voltage-gated sodium channels, such as tetrodotoxin (TTX)-sensitive PN1 and TTX-resistant PN3. We generated recombinant cell lines stably expressing the human PN1 and PN3 sodium channels in Chinese hamster ovary (CHO) cells using inducible expression vectors. The PN1 and PN3 cDNAs were isolated from human adrenal gland and heart poly(A)+ RNAs, respectively. The recombinant human PN1 currents exhibited rapid activation and inactivation kinetics and were blocked by TTX with a half-maximal inhibitory concentration (IC50) of 32.6 nM. The human PN3 channel expressed in stable transfectants showed TTX-resistant inward currents with slow inactivation kinetics. The IC50 value for TTX was 73.3 microM. The voltage-dependence of activation of the PN3 channel was shifted to the depolarizing direction, compared to that of the PN1 channel. Lidocaine and mexiletine exhibited tonic and use-dependent block of PN1 and PN3 channels. The PN1 channel was more susceptible to inhibition by mexiletine than PN3. These results suggest that stable transfectants expressing the human PN1 and PN3 sodium channels will be useful tools to define subtype selectivity for sodium channel blockers.