Phase 2 study of upacicalcet in Japanese haemodialysis patients with secondary hyperparathyroidism: an intraindividual dose-adjustment study.

Background: Upacicalcet is a novel small-molecule calcimimetic agent developed for intravenous injection. Here, we evaluated the long-term efficacy and safety of upacicalcet treatment via intraindividual dose adjustment in haemodialysis patients with secondary hyperparathyroidism (SHPT). Methods: A phase 2, multicentre, open-label, single-arm study was conducted. Upacicalcet was administered for 52 weeks; the starting dose was 50 µg thrice a week, and then adjusted to 25, 50, 100, 150, 200, 250, or 300 µg, according to the dose-adjustment method set in the protocol. The primary endpoint was the percentage of patients with serum intact parathyroid hormone (iPTH) level achieving a target range of 60-240 pg/mL (target achievement rate) at week 18. Results: A total of 58 patients were administered upacicalcet. The target achievement rate of serum iPTH level at week 18 was 57.9%, which increased to 80.8% at week 52. The serum-corrected calcium (cCa) level decreased immediately after upacicalcet administration, but no further decrease was observed. Adverse events were observed in 94.8% of patients, and adverse drug reactions (ADRs) occurred in 20.7% of patients. The most common ADR was decreased adjusted calcium in eight patients; dizziness occurred as a serious ADR in one patient. The serum cCa level of patients who interrupted upacicalcet treatment at a serum cCa level of <7.5 mg/dL recovered to ≥7.5 mg/dL immediately after the interruption. Conclusions: In haemodialysis patients with SHPT, upacicalcet doses of 25-300 µg for 52 weeks were found to be highly effective and well-tolerated, with minor safety concerns.

Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier.

Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy.

Elevated neprilysin activity in vitreous of patients with proliferative diabetic retinopathy.

PURPOSE: Diabetic retinopathy (DR) is the leading cause of blindness in the industrialized world. Hyperglycemia induces retinal hypoxia, which upregulates a range of vasoactive factors that may lead to macular edema and/or angiogenesis, and hence potentially to sight-threatening retinopathy. The control of signal-peptide activity by cell-surface proteases is one of the main factors regulating the development and behavior of organisms. In mammals, neprilysin is known to play a key role in these processes, and its inactivation can initiate cellular disorganization. Neprilysin is a rate-limiting peptidase involved in the physiological degradation of amyloid beta (Abeta) in the brain. In this study, we measured both the enzymatic activity of neprilysin and the concentration of Abeta in patients with proliferative DR (as compared to their levels in patients with macular hole), and we analyzed their association. METHODS: In vitreous samples collected from patients who underwent vitrectomy, an HPLC-fluorometric system (recently established by us), and sensitive and specific enzyme-linked immunosorbent assays were used to determine the enzymatic activity of neprilysin and the concentration of Abeta. RESULTS: By comparison with the levels in the control (macular-hole) patients, there was a significant increase in neprilysin activity level and a significant decrease in Abeta level in proliferative DR patients. There was a significant inverse correlation between neprilysin and Abeta among all subjects. CONCLUSIONS: Neprilysin activity and Abeta concentrations displayed converse changes in patients with proliferative DR.

Vitreous fluid levels of beta-amyloid((1-42)) and tau in patients with retinal diseases.

PURPOSE: A decrease in beta-amyloid(1-42) (Abeta42) and an increase in tau in the cerebrospinal fluid are reported to be characteristic phenomena in Alzheimer's disease patients. To test the idea that Abeta42 and tau contribute to the development of retinal diseases, we measured Abeta42 and tau concentrations in the vitreous fluid from patients with macular hole (n = 13), diabetic retinopathy (n = 15), or glaucoma concurrent with other ocular diseases (n = 8). METHODS: Vitreous samples were collected from patients who underwent vitrectomy, and sensitive and specific enzyme-linked immunosorbent assays were used to determine the concentrations of Abeta42 and tau. RESULTS: By comparison with the levels in the control macular-hole patients (33.9 +/- 7.1 pg/ml for Abeta42; 3.3 +/- 3.2 pg/ml for tau), there was a significant decrease in the Abeta42 level and a significant increase in the tau level in patients with diabetic retinopathy (1.8 +/- 1.9 pg/ml for Abeta42, P = 0.002; 153.7 +/- 71.6 pg/ml for tau, P = 0.041) or glaucoma concurrent with other ocular diseases (2.8 +/- 1.8 pg/ml for Abeta42, P = 0.006; 113.6 +/- 43.1 pg/ml for tau, P = 0.023). CONCLUSIONS: Our findings indicate the possibility of a role for Abeta42 and tau in the pathogenesis of some retinal diseases.

Comparison of the therapeutic indexes of different molecular forms of botulinum toxin type A.

Botulinum toxin is produced by Clostridium botulinum in three different molecular-weight forms: LL toxin, 900 kDa; L toxin, 500 kDa; and M toxin, 300 kDa. We isolated the M toxin, then compared its muscle-weakening efficacy with those of L+LL toxin and BOTOX both in vitro and in vivo. The twitch tension of the mouse isolated phrenic nerve-hemidiaphragm was used for the in vitro study. For the in vivo study, grip strength was measured in the toxin-injected legs. Undesirable muscle weakening was evaluated by grip-strength measurement in the contralateral leg. Concentration-response curves for effects on the phrenic nerve-hemidiaphragm showed that M toxin was 10 times more potent than L+LL toxin. The therapeutic index in vivo was 3- to 5-times higher for M toxin than for L+LL toxin or BOTOX, indicating a greater separation for M toxin between doses with local efficacy and systemic toxicity. These findings indicate that the M toxin preparation may have a better pharmacological profile than the conventional preparation.

Neuroprotective effects of beta-secretase inhibitors against rat retinal ganglion cell death.

Beta-secretase, an enzyme participating in amyloid beta-peptide generation, is thought to be involved in the pathogenesis of Alzheimer's disease. We examined the effects of beta-secretase inhibitors such as N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) and H-EVNstatineVAEF-NH2 (GL-189) on glutamate-induced retinal ganglion cell (RGC) death in vitro. In cultures of purified RGCs from neonatal rats, 2-day exposure to 25 microM glutamate induced RGC death, and Z-VLL-CHO (100 nM) and GL-189 (1 microM) had neuroprotective effects. We also found out that Z-VLL-CHO showed a neuroprotective effect on retinal damage induced by optic nerve crush in vivo. Thus, beta-secretase could be a potential target for therapy of neurodegenerative retinal diseases.

Effects of poly(ADP-ribose) polymerase inhibitor on NMDA-induced retinal injury.

PURPOSE: Excessive activation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme that is activated by DNA damage, leads to neuronal cell death through depletion of ATP. The purpose of this study was to determine whether inhibition of PARP has some neuroprotective effects on the N-methyl-D-aspartate (NMDA)-induced functional and morphological injury to the rabbit retina. METHODS: Visually evoked potentials (VEPs) were recorded at different times after an intravitreal injection of NMDA (200, 660, and 2000 nmol) alone, or NMDA with 3-aminobenzamide (ABA, 200 nmol), a PARP inhibitor, or with MK-801 (200 nmol), an NMDA antagonist. The physiological changes were followed for 2 weeks, after which the eyes were enuculeated and prepared for histological examinations. RESULTS: Intravitreal injections of NMDA reduced the amplitudes of rabbit VEPs and the number of cells in the retinal ganglion cell layer in a dose-dependent manner. No significant changes could be detected in the bright-flash electroretinograms (ERGs). Simultaneous injection of MK-801 (200 nmol) significantly diminished the changes induced by intravitreal NMDA. 3-Aminobenzamide (ABA) (200 nmol) also suppressed these changes, but its effects were less than those of MK-801. CONCLUSIONS: NMDA-induced retinal damage can be detected by VEPs, and PARP inhibition has some neuroprotective effects on the NMDA-induced retinal damage.

Adenosine protects cultured retinal neurons against NMDA-induced cell death through A1 receptors.

PURPOSE: To determine whether adenosine can protect cultured retinal neurons, consisting mainly of amacrine cells, from N-methyl-D-aspartate (NMDA)-induced neurotoxicity, and to determine whether agonists and antagonists of adenosine receptors also have a protective effect. METHODS: Cultured retinal neurons obtained from fetal Wistar rats (gestational age 18-19 days) were maintained for 10-11 days. Neurons were exposed to NMDA (1.0 mM) for 10 min with or without adenosine or to NMDA with adenosine receptor agonists or antagonists. Neuronal death was assessed by the trypan-blue exclusion method 24 hr after the exposure. RESULTS: Adenosine at doses of 0.01 microM and higher significantly protected (p < 0.05, Dunnett) primary cultured fetal rat retinal neurons from apoptotic and/or necrotic death induced by NMDA (1.0 mM). The protective effect of adenosine (10 microM) against NMDA-induced neuronal death was lost by simultaneous exposure to selective A1 receptor antagonist but not to A2a receptor antagonist. Selective A1 receptor agonists had similar effects as adenosine, but A2a receptor agonists and 8-Br-cyclic AMP had no effect on cell viability. CONCLUSIONS: Adenosine can protect cultured retinal neurons against NMDA-induced cell death via the A1 receptor.

Topiramate reduces excitotoxic and ischemic injury in the rat retina.

The effects of topiramate, a drug used clinically as an anti-epileptic, were investigated in excitotoxin-induced neurotoxicity models involving two different retinal primary cultures and in a rat model of retinal ischemic injury. For the in vitro studies, we used retinal-neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the retinal-neuron cultures, neurotoxicity was induced by a 10-min exposure to 1 mM glutamate or (+/-)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). In RGCs, neurotoxicity was induced by incubation for 3 days in a culture medium containing 25 microM glutamate. For the in vivo study, retinal ischemia was induced by elevating intraocular pressure to 130 mmHg for 45 min, and topiramate was administered intraperitoneally before and after the ischemia. Retinal damage was evaluated by measuring the number of cells in the ganglion cell layer (GCL) and the thickness of the inner plexiform layer (IPL), and by examining the a- and b-waves of the electroretinogram (ERG). Topiramate (> or =1 microM) markedly reduced the neuronal cell death induced by each of the excitotoxins in rat retinal-neuron cultures and in RGCs. Ischemia caused a decrease in GCL cells and in IPL thickness, and a diminution of the ERG waves. Histopathologic and functional analyses indicated that systemic treatment with topiramate prevented ischemia-induced damage in a dose-dependent manner. In conclusion, topiramate was protective against excitotoxic and ischemic retinal-neuron damage in vitro and in vivo, respectively. Therefore, it may be useful for treatment of the retina-related diseases such as central retinal artery occlusion, diabetic retinopathy, and glaucoma.