Analysis of Onset Mechanisms of a Sphingosine 1-Phosphate Receptor Modulator Fingolimod-Induced Atrioventricular Conduction Block and QT-Interval Prolongation.

Fingolimod, a sphingosine 1-phosphate (S1P) receptor subtype 1, 3, 4 and 5 modulator, has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular conduction block and/or QT-interval prolongation have been reported in some patients after the first dose. In this study, we directly compared the electropharmacological profiles of fingolimod with those of siponimod, a modulator of sphingosine 1-phosphate receptor subtype 1 and 5, using in vivo guinea-pig model and in vitro human ether-a-go-go-related gene (hERG) assay to better understand the onset mechanisms of the clinically observed adverse events. Fingolimod (0.01 and 0.1mg/kg) or siponimod (0.001 and 0.01mg/kg) was intravenously infused over 10min to the halothane-anaesthetized guinea pigs (n=4), whereas the effects of fingolimod (1µmol/L) and siponimod (1µmol/L) on hERG current were examined (n=3). The high doses of fingolimod and siponimod induced atrioventricular conduction block, whereas the low dose of siponimod prolonged PR interval, which was not observed by that of fingolimod. The high dose of fingolimod prolonged QT interval, which was not observed by either dose of siponimod. Meanwhile, fingolimod significantly inhibited hERG current, which was not observed by siponimod. These results suggest that S1P receptor subtype 1 in the heart could be one of the candidates for fingolimod- and siponimod-induced atrioventricular conduction block since S1P receptor subtype 5 is localized at the brain, and that direct IKr inhibition may play a key role in fingolimod-induced QT-interval prolongation.

EF6265, a novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor, protects against sepsis-induced organ dysfunction in rats.

OBJECTIVE: Although thrombin-activatable fibrinolysis inhibitor (TAFI) has been implicated as a negative regulator of fibrinolysis, its pathophysiological significance remains to be unveiled. We performed the pharmacologic study to assess the effect of EF6265, a specific inhibitor of activated form of TAFI (TAFIa) on sepsis-induced organ dysfunction models. DESIGN: A controlled, in vivo laboratory study. SETTING: Company research laboratory. SUBJECTS: Wistar and Sprague-Dawley rats. INTERVENTIONS: Endotoxemia and sepsis models were induced by intravenous injection of lipopolysaccharide and Pseudomonas aeruginosa, respectively. MEASUREMENTS AND MAIN RESULTS: In the endotoxemia model, posttreatment (1 hour) with EF6265 reduced fibrin deposits in the kidney and liver accompanied by no significant changes in platelet count and fibrinogen concentration in plasma. This compound also significantly decreased levels of plasma lactate dehydrogenase and aspartate aminotransferase, markers of organ dysfunction. In the sepsis model, EF6265, simultaneously administered with ceftazidime (CAZ) 2 hours after Pseudomonas aeruginosa injection, showed no influence on the antibiotic activity of CAZ. Meanwhile, it dramatically potentiated the interleukin-6-reducing effect of CAZ in plasma, suggesting that inhibition of TAFIa leads to the reduction in systemic inflammatory response associated with bacterial infection. This combined treatment also lowered plasma lactate dehydrogenase and blood urea nitrogen more potently than single treatment with CAZ. CONCLUSIONS: These results clearly suggest that TAFI plays an important role in the deterioration of organ dysfunction in sepsis and the inhibitor of TAFIa protects against sepsis-induced tissue damage through regulation of fibrinolysis and inflammation.

Bile acid-induced elevated oxidative stress in the absence of farnesoid X receptor.

The major function of farnesoid X receptor (FXR) is to maintain bile acid and lipid homeostasis. Fxr-null mice, in which the levels of hepatic bile acid and lipid have been elevated, develop spontaneous liver tumors. We evaluated differences in hepatic bile acid and triglyceride concentrations, and in generation of oxidative stress between wild-type mice and Fxr-null mice. The hepatic levels of 8-hydroxy-2'-deoxyguanosine (8OHdG), thiobarbituric acid-reactive substance (TBARS) and hydroperoxides, oxidative stress-related genes, and nuclear factor (erythroid-2 like) factor 2 (Nrf2) protein in Fxr-null mice were significantly higher than those in wild-type mice. An increase in the hepatic bile acid concentration in Fxr-null mice fed a cholic acid (CA) diet resulted in an increase in the hepatic levels of hydroperoxides, TBARS and 8OHdG, whereas a decrease in the hepatic concentration in mice fed a diet containing ME3738 (22beta-methoxyolean-12-ene-3beta,24(4beta)-diol) resulted in a decrease in these oxidative stress marker levels. A good correlation was observed between the hepatic bile acid concentrations and the hepatic oxidative stress marker levels, although there was no significant correlation between the hepatic triglyceride concentrations and oxidative stress. The results show that oxidative stress is spontaneously enhanced in Fxr-null mice, which may be attributable to a continuously high level of hepatic bile acids.

EF6265, a novel plasma carboxypeptidase B inhibitor, protects against renal dysfunction in rat thrombotic glomerulonephritis through enhancing fibrinolysis.

BACKGROUND/AIM: Plasma carboxypeptidase B is a physiological fibrinolysis inhibitor. In the present study, the effects of EF6265, a novel specific plasma carboxypeptidase B inhibitor, on renal dysfunction in a rat thrombotic glomerulonephritis model were examined. METHODS: The model was induced by injection of anti-glomerular basement membrane serum and lipopolysaccharide to rats. Renal microthrombosis was histologically evaluated by phosphotungstic acid-hematoxylin staining for fibrin thrombi. Renal dysfunction was evaluated on the basis of plasma levels of blood urea nitrogen as well as renal edemas and urine volume. RESULTS: The glomerular microthrombi observed in a positive control group were significantly reduced after a short-term treatment (4 h) with EF6265 at a dose which enhanced fibrinolysis. The elevation of blood urea nitrogen and renal edema formation decreased, and the reduction of the urine volume improved after a long-term treatment (21 h) with EF6265. In addition, EF6265 had a protective activity against multiple organ dysfunction, because it reduced plasma lactate dehydrogenase and alanine aminotransferase levels and mortality in this model. CONCLUSION: EF6265, which inhibits plasma carboxypeptidase B, showed a protective effect on thrombotic renal dysfunction in thrombotic glomerulonephritis through enhancing the fibrinolysis.

Enhancement of fibrinolysis by EF6265 [(S)-7-amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl] methyl]heptanoic acid], a specific inhibitor of plasma carboxypeptidase B.

Plasma procarboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is converted by thrombin into the active enzyme, carboxypeptidase B (CPB)/activated TAFI. Plasma CPB down-regulates fibrinolysis by removing carboxy-terminal lysines, the ligands for plasminogen and tissue-type plasminogen activator (tPA), from partially degraded fibrin. To target thrombosis in a new way, we have identified and optimized a phosphinic acid-containing inhibitor of CPB, EF6265 [(S)-7-amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino) propyl]hydroxyphosphinoyl]methyl]heptanoic acid] and determined both the pharmacological profile and pathophysiological role of CPB in rat thrombolysis. EF6265 specifically inhibited plasma CPB activity with an IC(50) (50% inhibitory concentration) of 8.3 nM and enhanced tPA-mediated clot lysis in a concentration-dependent manner. EF6265 decreased detectable thrombi (percentage of glomerular fibrin deposition; control, 98 +/- 1.1; EF6265, 0.1 mg/kg, 27 +/- 9.1) that had been generated by tissue factor in a rat microthrombosis model with concomitant increases in plasma D-dimer concentration (control, <0.5 microg/ml; EF6265, 0.1 mg/kg, 15 +/- 3.5 microg/ml). EF6265 reduced plasma alpha2-antiplasmin activity to a lesser extent than tPA. In an arteriovenous shunt model, EF6265 (1 mg/kg) enhanced exogenous tPA-mediated thrombolysis under the same conditions that neither EF6265 nor tPA (600 kIU/kg) alone reduced thrombi. EF6265 (1 and 30 mg/kg) did not affect the bleeding time in rats. Moreover, it did not prolong the bleeding time evoked by tPA (600 kIU/kg). These results confirm that circulating procarboxypeptidase B functions as a fibrinolysis inhibitor's zymogen and validates the use of CPB inhibitors as both an enhancer of physiological fibrinolysis in microcirculation and as a novel adjunctive agent to tPA for thromboembolic diseases while maintaining a small effect on primary hemostasis.

Carboxypeptidase B inhibitors reduce tissue factor-induced renal microthrombi in rats.

Procarboxypeptidase B (also known as thrombin-activatable fibrinolysis inhibitor) is a recently described plasma zymogen known to be activated by thrombin in plasma. Carboxy-terminal lysine residues from partially degraded fibrin are important for the binding and activation of plasminogen, and carboxypeptidase B, an active form of procarboxypeptidase B, has been shown to inhibit fibrinolysis by eliminating these residues. The present paper investigates the effects of carboxypeptidase B inhibitors, DL-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGPA) and potato-derived carboxypeptidase inhibitor (CPI), on tissue factor (TF)-induced microthrombosis in rats. Intravenous injection of MGPA (3 mg/kg and higher) or CPI (0.3 mg/kg and higher) after microthrombi formation dramatically attenuated TF-induced glomerular fibrin deposition with an increase in plasma levels of D-dimer. These results indicate that carboxypeptidase B inhibitors can enhance endogenous fibrinolysis and reduce thrombi in the TF-induced microthrombosis model after systemic administration even after thrombi formation.