In vitro and in vivo pharmacological profile of OPC-61815, a water-soluble phosphate ester pro-drug of tolvaptan.

Tolvaptan is an orally active vasopressin V2 receptor antagonist and used for the treatment of volume overload in some disease as an aquaretic. Tolvaptan sodium phosphate (OPC-61815) is a pro-drug of tolvaptan that was designed to improve water solubility and enable intravenous use. The conversion of OPC-61815 to tolvaptan was evaluated for in vitro and in vivo pharmacokinetic studies. The pharmacodynamics of OPC-61815 were evaluated for in vitro receptor binding affinity, in vivo aquaretic and anti-edematous action. The solubility of OPC-61815 in water at 25 °C was 72.4 mg/mL and more than 100,000 times the solubility of tolvaptan. OPC-61815 was hydrolyzed to tolvaptan by human tissue S9 fractions and main enzyme of hydrolysis was alkaline phosphatase. After intravenous administration of OPC-61815 to rats and dogs, tolvaptan was detected in plasma within 5 min and the bioavailability of tolvaptan was 57.7% and 50.9%, respectively. Binding affinity of OPC-61815 for the human V2 receptor was 1/14 weaker than that of tolvaptan. OPC-61815 exerted dose-dependent aquaretic action in rats and dogs and a corresponding anti-edematous action in rat edema models. These results suggest that OPC-61815, a water-soluble phosphate ester pro-drug of tolvaptan, is an effective aquaretic by converting to tolvaptan after intravenous administration.

Tolvaptan delays the onset of end-stage renal disease in a polycystic kidney disease model by suppressing increases in kidney volume and renal injury.

Tolvaptan, a selective vasopressin V2 receptor antagonist, slows the increase in total kidney volume and the decline in kidney function in patients with the results of the Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Outcome (TEMPO) 3:4 trial. However, it was unclear which dose of tolvaptan was optimal or whether tolvaptan was able to delay progression to end-stage renal disease (ESRD). Here we examined the relationship with aquaresis and the inhibitory effect on cyst development in short-term treatment and mortality as an index of ESRD in long-term treatment with tolvaptan using DBA/2FG-pcy mice, an animal model of nephronophthisis. With short-term treatment from 5 to 15 weeks of age, tolvaptan (0.01-0.3% via diet) dose-dependently enhanced aquaresis, prevented increases in kidney weight and cyst volume, and was associated with significant reductions in kidney cAMP levels and extracellular signal-regulated kinase activity. Maximal effects of tolvaptan on aquaresis and the prevention of development of polycystic kidney disease (PKD) were obtained at 0.1%. Interestingly, tolvaptan also dose-dependently reduced urinary neutrophil gelatinase-associated lipocalin levels in correlation with the kidney volume. With long-term treatment from 5 to 29 weeks of age, tolvaptan significantly attenuated the increase in kidney volume by up to 50% and reduced urinary albumin excretion. Furthermore, tolvaptan significantly reduced the mortality rate to 20%, compared with 60% in the control group. These data indicate that tolvaptan may delay the onset of ESRD in PKD by suppressing the increases in kidney volume and renal injury, providing a promising treatment for PKD.

[Tolvaptan, a vasopressin V2 receptor antagonist, is the world's first approved drug for treatment of autosomal dominant polycystic kidney disease (ADPKD)].

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease. Fluid-filled cysts develop and enlarge in both kidneys, eventually leading to kidney failure. Tolvaptan is a selective vasopressin V2 receptor antagonist and the first and only drug approved for treatment of ADPKD. It blocks binding of arginine vasopressin (AVP) to V2 receptors in the collecting duct of kidney, thereby inducing water diuresis (aquaresis) without losing electrolytes. Therefore, tolvaptan was originally developed and approved as the first oral aquaretic agent for treatment of hyponatremia and fluid volume overload in heart failure and cirrhosis. During the development of tolvaptan as aquaretics, efficacy of V2 antagonist in polycystic kidney animal model was reported and then the development of tolvaptan for ADPKD was also initiated. Cyclic adenosine monophosphate (cAMP) plays an important role in cyst growth by promoting cell proliferation and fluid secretion. Tolvaptan showed suppression of cyst growth through inhibiting AVP-induced cAMP production and delayed the onset of end-stage renal disease in an animal model. In the phase 3 clinical trial in ADPKD patients (TEMPO 3:4 trial), 3-year treatment with tolvaptan slowed the disease progression including increase of kidney volume and decline in renal function. Efficacy of tolvaptan in patients with late-stage ADPKD was confirmed in another 1-year phase 3 REPRISE trial. Tolvaptan is approved for treatment of ADPKD in more than 40 countries and we expect it can contribute to more ADPKD patients worldwide. We also expect that drugs with new mechanisms will be available in the near future.

Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy.

Influenza-associated encephalopathy (IAE) is characterized by persistent high fever, febrile convulsions, severe brain edema, and high mortality in otherwise apparently healthy individuals. We have reported that a large proportion of patients suffering from disabling or fatal IAE, with transiently elevated serum acylcarnitine during high fever, exhibit a thermolabile phenotype of compound homo-/heterozygous variants of carnitine palmitoyltransferase II (CPT II, gene symbol CPT2). We characterized the enzymatic properties of five single and three compound CPT II variants in patients with IAE. The kinetic characteristics of WT and variant CPT IIs, expressed in COS-7 cells, indicated that the variants exert a dominant-negative effect on the homotetrameric protein of the enzyme. Among the variants, three compound variations found in patients with severe encephalopathy; [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile)], [c.1511C>T (p.Pro504Leu); c.1813G>C (p.Val605Leu)], and [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile); c.1813G>C (p.Val605Leu)], showed reduced activities, thermal instability, and short half-lives compared with the WT. Like other disease-causing mutant proteins, these variant proteins were poly-ubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome pathway. COS-7 cells transfected with the compound variants had their fatty acid beta-oxidation decreased to 30-59% and intracellular ATP levels to 48-79%, and a marked reduction of mitochondrial membrane potential at 41 degrees C, compared with control cells transfected with WT at 37 degrees C. The unstable CPT II variants with decreased enzymatic activities may bring mitochondrial fuel utilization below the phenotypic threshold during high fever, and thus may play an important etiopathological role in the development of brain edema of IAE.

The influence of lower extremity postures on kinematics and injuries of cyclists in vehicle side collisions.

OBJECTIVE: A cyclist assumes various cyclic postures of the lower extremities while pushing the pedals in a rotary motion while pedaling. In order to protect cyclists in collisions, it is necessary to understand what influence these postures have on the global kinematics and injuries of the cyclist. METHOD: Finite element (FE) analyses using models of a cyclist, bicycle, and car were conducted. In the simulations, the Total Human Model of Safety (THUMS) occupant model was employed as a cyclist, and the simulation was set up such that the cyclist was hit from its side by a car. Three representative postures of the lower extremities of the cyclist were examined, and the kinematics and injury risk of the cyclist were compared to those obtained by a pedestrian FE model. The risk of a lower extremity injury was assessed based on the knee shear displacement and the tibia bending moment. RESULTS: When the knee position of the cyclist was higher than the hood leading edge, the hood leading edge contacted the leg of the cyclist, and the pelvis slid over the hood top and the wrap-around distance (WAD) of the cyclist's head was large. The knee was shear loaded by the hood leading edge, and the anterior cruciate ligament (ACL) ruptured. The tibia bending moment was less than the injury threshold. When the cyclist's knee position was lower than the hood leading edge, the hood leading edge contacted the thigh of the cyclist, and the cyclist rotated with the femur as the pivot point about the hood leading edge. In this case, the head impact location of the cyclist against the car was comparable to that of the pedestrian collision. The knee shear displacement and the tibia bending moment were less than the injury thresholds. CONCLUSION: The knee height of the cyclist relative to the hood leading edge affected the global kinematics and the head impact location against the car. The loading mode of the lower extremities was also dependent on the initial positions of the lower extremities relative to the car structures. In the foot up and front posture, the knee was loaded in a lateral shear direction by the hood leading edge and as a result the ACL ruptured. The bicycle frame and the struck-side lower extremity interacted and could influence the loadings on lower extremities.

Protective effects of XBP1 against oxygen and glucose deprivation/reoxygenation injury in rat primary hippocampal neurons.

The accumulation of misfolded and unfolded proteins in the endoplasmic reticulum (ER) induces ER stress, activating the unfolded protein response (UPR). One of the effectors of the UPR is XBP1, a critical transcriptional factor for genes responsible for cell survival. ER stress is also known to play a vital role in mediating ischemic reperfusion damage in the brain. In this study, we investigated the role of XBP1 in rat primary hippocampal neurons subjected to oxygen and glucose deprivation followed by reoxygenation (OGD/R) stress, an in vitro model of ischemia/reperfusion (I/R) injury. Primary neurons subjected to OGD had increased levels of spliced XBP1 (XBP1s) mRNA. Interestingly, the level of XBP1s decreased during the initial reoxygenation stress period. The combination of OGD and the subsequent 20-h reoxygenation stress period significantly increased the apoptotic death of primary cells. Overexpression of XBP1s suppressed cell death induced by OGD/R stress. These results suggest that suppression of XBP1 activation accelerates neuronal cell death after I/R and that activation of the XBP1 pathway may provide a therapeutic approach for the treatment of cerebral I/R injury.

Protective effect against Parkinson's disease-related insults through the activation of XBP1.

The accumulation of misfolded and unfolded proteins in endoplasmic reticulum (ER) induces ER stress, activating the unfolded protein response (UPR). Recent evidence has suggested the relationship between UPR and dopaminergic neuronal cell death in Parkinson's disease (PD); however, it remains unclear whether it makes sense to modulate UPR, to mitigate the progression of PD. In this study, we investigated a role of the IRE1 alpha-XBP1 pathway in the survival of dopaminergic cells, under stress induced by PD-related insults. The exogenous expression of the active-form XBP1 (XBP1s) protein had protective effects against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and proteasome inhibitors. Moreover, adenoviral XBP1s expression significantly suppressed the degeneration of dopaminergic neurons in the mouse model of PD, as induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These results demonstrate that the enhancement of XBP1 could be a novel PD therapeutic strategy.