[Studies of the Various Chronic Kidney Failure Rat Models and Hemodialysis Mini-pig Model for the Evaluation of Anti-hyperphosphatemia Drugs].

Animal models of chronic kidney failure (CKF) have been developed for the pharmacodynamic evaluation of various phosphate binders that are used clinically to treat hyperphosphatemia in patients with chronic kidney disease. However, these models represent different disease states and severities, depending on the experimental conditions and are not clearly defined for pharmacological evaluation. In addition, experimental models have not yet been established for artificial dialysis. The purpose of this study was to confirm the utility of the various rat models of CKF and the mini-pig model of hemodialysis as models of hyperphosphatemia for pharmacodynamic evaluation. Various rat models of pre-dialysis CKF (oral adenine dosing, 5/6 resection, and ligation nephrectomy model) were evaluated through determinations of serum and urinary parameters (osmolality, creatinine, and phosphorus), pathological observations of kidney, and the phosphorus-absorbing properties of lanthanum carbonate (La) formulations. The rat and mini-pig models were compared based on each evaluation index. In the oral adenine dosing model, serum phosphorus increased markedly and the area under the serum phosphorus concentration-time curve (phosphorus AUC) decreased in a dose-dependent manner with the administration of La formulations. In contrast, a significant decrease in serum phosphorus AUC, a prolongation of the dialysis interval, and an improvement in dialysis efficiency were observed after administration of La formulations to the mini-pig hemodialysis model. Furthermore, the results of bioequivalence studies between two La formulations (Fosrenol and SW670, a generic formulation) suggested that the rat and mini-pig models are useful and precise as pre-dialysis and dialysis models, respectively.

Whole cell-based surface plasmon resonance measurement to assess binding of anti-TNF agents to transmembrane target.

We developed a technique for the measurement of surface plasmon resonance (SPR) to detect interactions of anti-tumor necrosis factor (TNF) agents with transmembrane TNF-α (mTNF-α) on living whole cells. The injection of a suspension of mTNF-α expressing Jurkat cells, used as an analyte, gave a clear binding response to anti-TNF agents, such as etanercept, infliximab and adalimumab, immobilized on sensorchip. The binding response of the analyte cells increased in a concentration-dependent manner and was competitively reduced by adding soluble TNF receptors to the analyte cell suspension. Treatment of analyte cells with free anti-TNF agent before injection reduced the binding response between the analyte cells and immobilized-etanercept on sensorchip, and the inhibitory effect of free anti-TNF agent was concordant with the affinity of anti-TNF agent for soluble TNF-α. These findings indicate that the SPR response arises from specific binding between anti-TNF agent and its target on cell membrane.

Protective effect of N-acetylcysteine against nicardipine hydrochloride-induced autophagic cell death of human vascular endothelial cells.

Nicardipine hydrochloride (NIC) injection has been widely used for emergency treatment of abnormally high blood pressure. However, NIC injection often causes severe peripheral vascular injury. The purpose of the present study was to reduce the NIC-induced cell injury in human vascular endothelial cells by use of clinical agents. The mechanism of NIC-induced cell injury was evaluated by time-lapse microscopic imaging, autophagosome staining with monodansylcadaverine, immunostaining of light chain 3 isoform B (LC-3B) and assessment of cell viability after exposure to NIC with or without an inhibitor of autophagosome formation (3-methyladenine, 3-MA). Results from autophagosome labeling and immunostaining of LC-3B revealed an increase of autophagosomes and LC-3B in NIC-treated cells. NIC-mediated reduction of cell viability was inhibited by 3-methyladenine. Moreover, we found that N-acetylcysteine (NAC) reduced NIC-induced cell injury in human vascular endothelial cells. These findings suggest that NIC causes severe peripheral venous irritation via induction of autophagic cell death and that inhibition of autophagy with NAC could contribute to the reduction of NIC-induced vascular injury.

Characterization of nicardipine hydrochloride-induced cell injury in human vascular endothelial cells.

Nicardipine hydrochloride (NIC), a dihydropyridine calcium-channel blocking agent, has been widely used for the treatment of hypertension. Especially, nicardipine hydrochloride injection is used as first-line therapy for emergency treatment of abnormally high blood pressure. Although NIC has an attractive pharmacological profile, one of the dose-limiting factors of NIC is severe peripheral vascular injury after intravenous injection. The goal of this study was to better understand and thereby reduce NIC-mediated vascular injury. Here, we investigated the mechanism of NIC-induced vascular injury using human dermal microvascular endothelial cells (HMVECs). NIC decreased cell viability and increased percent of dead cells in a dose-dependent manner (10-30 µg/mL). Although cell membrane injury was not significant over 9 hr exposure, significant changes of cell morphology and increases in vacuoles in HMVECs were observed within 30 min of NIC exposure (30 µg/mL). Autophagosome labeling with monodansylcadaverine revealed increased autophagosomes in the NIC-treated cells, whereas caspase 3/7 activity was not increased in the NIC-treated cells (30 µg/mL). Additionally, NIC-induced reduction of cell viability was inhibited by 3-methyladenine, an inhibitor of autophagosome formation. These findings suggest that NIC causes severe peripheral venous irritation via induction of autophagic cell death and that inhibition of autophagy could contribute to the reduction of NIC-induced vascular injury.