Discovery of Novel Pyrazole-Based Selective Aldosterone Synthase (CYP11B2) Inhibitors: A New Template to Coordinate the Heme-Iron Motif of CYP11B2.

It is necessary for aldosterone synthase (CYP11B2) inhibitors to have both high potency and high selectivity over 11ß-hydroxylase (CYP11B1), a critical enzyme for cortisol synthesis. Previous studies have reported a number of CYP11B2 inhibitors, most of which have an imidazole or pyridine ring to coordinate the heme-iron motif of CYP11B2; however, highly selective inhibitors of human CYP11B2 are still needed. To expand the selectivity in humans, we explored alternative templates and found that pyrazoles were suitable templates for CYP11B2 inhibitors. Investigation of pyrazoles, especially N-alkyl pyrazoles, as a new template to coordinate the heme-iron motif led to a potent and highly selective CYP11B2 inhibitor 28 with an aldosterone-lowering effect at 1 mg/kg dosing in cynomolgus monkeys.

Partial deletion of the ROCK2 protein fails to reduce renal fibrosis in a unilateral ureteral obstruction model in mice.

Renal fibrosis is a well­known cause for the progression of chronic kidney disease. Rho/Rho­associated coiled­coil kinase (ROCK) signaling is involved in renal fibrotic processes. Non­selective ROCK1/2 inhibitors have been reported to reduce renal interstitial fibrosis in a rodent unilateral ureteral obstruction (UUO) model. To clarify the role and contribution of ROCK2 in renal fibrosis, the present study used ROCK2 heterozygous knockout (HKO) mice to assess collagen deposition and fibrosis­associated gene expression in the kidney of the UUO model. In the ROCK2 HKO mice, the expression level of ROCK2 in the normal kidney was half of that in the kidney of wild­type (WT) mice. The expression levels of ROCK1 in the ROCK2 HKO mice and WT mice were equivalent. Furthermore, in the ROCK2 HKO and the WT mice, the hydroxyproline content and the gene expression levels of collagen I and transforming growth factor­ß1 in the obstructed kidneys were augmented following UUO. By contrast, the mRNA expression of α­smooth muscle actin decreased in the ROCK2 HKO mice, compared with that in the WT mice. The activity of ROCK in the obstructed kidneys, indicated by the phosphorylation of myosin phosphatase target subunit­1, which is a non­selective substrate of ROCK1 and ROCK2, was equivalent among the ROCK2 HKO and WT mice. In conclusion, no differences in renal interstitial fibrosis or UUO­induced ROCK activity were identified between the ROCK2 HKO and WT mice, indicating that the genetic partial disruption of ROCK2 is insufficient for protecting against renal fibrosis.

Inhibitory effects of fasudil on renal interstitial fibrosis induced by unilateral ureteral obstruction.

Renal fibrosis is the major cause of chronic kidney disease, and the Rho/Rho-associated coiled-coil kinase (ROCK) signaling cascade is involved in the renal fibrotic processes. Several studies have reported that ROCK inhibitors attenuate renal fibrosis. However, the mechanism of this process remains to be fully elucidated. The present study assessed the inhibitory effect of fasudil, a ROCK inhibitor using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blot analyses, in vivo and in vitro, to elucidate the mechanisms underlying renal interstitial fibrosis. In mice induced with unilateral ureteral obstruction (UUO), collagen accumulation, the expression of fibrosis­associated genes and the content of hydroxyproline in the kidney increased 3, 7, and 14 days following UUO. Fasudil attenuated the histological changes, and the production of collagen and extracellular matrix in the UUO kidney. The expression of α­smooth muscle actin (α­SMA) and the transforming growth factor­ß (TGFß)­Smad signaling pathway, and macrophage infiltration were suppressed by fasudil in the kidneys of the UUO mice. The present study also evaluated the role of intrinsic renal cells and infiltrated macrophages using NRK­52E, NRK­49F and RAW264.7 cells. The mRNA and protein expression levels of collagen I and α­SMA increased in the NRK­52E and NRK­49F cells stimulated by TGF­ß1. Hydroxyfasudil, a bioactive metabolite of fasudil, attenuated the increase in the mRNA and protein expression levles of α­SMA in the two cell types. However, the reduction in the mRNA expression of collagen I was observed in the NRK­49F cells only. Hydroxyfasudil decreased the mRNA expression of monocyte chemoattractant protein­1 (MCP­1) induced by TGF­ß1 in the NRK­52E cells, but not in the NRK­49F cells. In the RAW264.7 cells, the mRNA expression levels of MCP­1, interleukin (IL)­1ß, IL­6 and tumor necrosis factor α were increased significantly following lipopolysaccharide stimulation, and were not suppressed by hydroxyfasudil. These data suggested that the inhibition of ROCK activity by fasudil suppressed the transformation of renal intrinsic cells into the myofibroblast cells, and attenuated the infiltration of macrophages, without inhibiting the expression or the activation of cytokine/chemokines, in the progression of renal interstitial fibrosis.

MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor, ameliorates ischemic brain damage in a non-human primate model.

Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) in response to DNA damage is considered to play a crucial role in the development of post-ischemic neuronal injury, such as ischemic stroke. The present study was undertaken to clarify the beneficial effects of MP-124, a novel PARP-1 inhibitor, on neurological deficits and cerebral infarcts following middle cerebral artery occlusion (MCAO) in the monkey. The effects of MP-124 on cerebral infarcts and neurological deficits in monkeys were investigated in permanent MCAO (pMCAO) and transient MCAO (tMCAO) models. In a dose-dependency study, the neurological deficits and cerebral infarct volume were assessed at 28h after pMCAO. MP-124 significantly reduced the total infarct volume, including that in the cortex/white matter and striatum, at doses of 0.3, 1 and 3mg/kg/h by 22, 54 and 64%, respectively. In addition, MP-124 at all doses significantly reduced the overall neurological deficits. Such ameliorative effects of MP-124 were observed in female as well as male monkeys. In the therapeutic time window (TTW) study, the neurological deficits and cerebral infarct volume were assessed at several time points after pMCAO or tMCAO. Treatment with MP-124 at 3 and 6h after MCAO significantly ameliorated not only the neurological deficits but also the infarct volume. MP-124 is thought to exhibit neuroprotective effects with a broad TTW regardless of sex in MCAO models. Such findings suggest that MP-124 may be beneficial for the treatment of acute ischemic stroke.

Neuroprotective effects of a novel water-soluble poly(ADP-ribose) polymerase-1 inhibitor, MP-124, in in vitro and in vivo models of cerebral ischemia.

Cerebral ischemia induces excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), leading to neuronal cell death and the development of post-ischemic dysfunction. Blockade of PARP-related signals during cerebral ischemia has become a focus of interest as a new therapeutic approach for acute stroke treatment. The purpose of the present study was to examine the pharmacological profiles of MP-124, a novel water-soluble PARP-1 inhibitor, and its neuroprotective effects on ischemic injury in vitro and in vivo. MP-124 demonstrated competitive inhibition of the PARP-1 activity of human recombinant PARP-1 enzyme (Ki=16.5nmol/L). In P388D(1) cells, MP-124 inhibited the LDH leakage induced by H(2)O(2) in a concentration-dependent manner. (IC(50)=20.8nmol/L). In rat primary cortical neurons, MP-124 also inhibited the NAD depletion and polymerized ADP-ribose formation induced by H(2)O(2) exposure. Moreover, we investigated the neuroprotective effects of MP-124 in rat permanent and transient stroke models. In the rat permanent middle cerebral artery occlusion (MCAO) model, MP-124 was administered intravenously for 24h from 5min after the onset of MCAO. MP-124 (1, 3 and 10mg/kg/h) significantly inhibited the cerebral infarction in a dose-dependent manner (18, 42 and 48%). In rat transient MCAO model, MP-124 was administered intravenously from 30min after the onset of MCAO. MP-124 (3 and 10mg/kg/h) significantly reduced the infarct volume (53% and 50%). The present findings suggest that MP-124 acts as a potent neuroprotective agent in focal ischemia and its actions can be attributed to a reduction in NAD depletion and PAR formation.

Edaravone, a free radical scavenger, protects components of the neurovascular unit against oxidative stress in vitro.

The concept of the neurovascular unit suggests that to be successful, stroke therapies must protect all neuronal, glial and endothelial components in brain. In this study, we tested the efficacy of the free radical scavenger edaravone in three cellular models of oxidative stress. HT22 neuronal cells were subjected to oxidative stress using the standard glutamate-induced glutathione depletion model. Primary rat astrocytes were exposed to H(2)O(2). Oxidative stress was induced in human brain endothelial cells with sodium nitroprusside (SNP). Edaravone significantly reduced oxidative cell death in both HT22 neuronal cells and primary rat astrocytes in a dose-dependent manner. SNP did not kill brain endothelial cells but instead reduced their production of brain-derived neurotrophic factor (BDNF). Edaravone significantly ameliorated this response. These data suggest that free radical scavengers are effective in all cell types of the neurovascular unit, and should still be considered as a potential therapeutic approach for stroke.

Combination therapy with normobaric oxygen (NBO) plus thrombolysis in experimental ischemic stroke.

BACKGROUND: The widespread use of tissue plasminogen activator (tPA), the only FDA-approved acute stroke treatment, remains limited by its narrow therapeutic time window and related risks of brain hemorrhage. Normobaric oxygen therapy (NBO) may be a useful physiological strategy that slows down the process of cerebral infarction, thus potentially allowing for delayed or more effective thrombolysis. In this study we investigated the effects of NBO started simultaneously with intravenous tPA, in spontaneously hypertensive rats subjected to embolic middle cerebral artery (MCA) stroke. After homologous clot injection, animals were randomized into different treatment groups: saline injected at 1 hour; tPA at 1 hour; saline at 1 hour plus NBO; tPA at 1 hour plus NBO. NBO was maintained for 3 hours. Infarct volume, brain swelling and hemorrhagic transformation were quantified at 24 hours. Outcome assessments were blinded to therapy. RESULTS: Upon clot injection, cerebral perfusion in the MCA territory dropped below 20% of pre-ischemic baselines. Both tPA-treated groups showed effective thrombolysis (perfusion restored to nearly 100%) and smaller infarct volumes (379 +/- 57 mm3 saline controls; 309 +/- 58 mm3 NBO; 201 +/- 78 mm3 tPA; 138 +/- 30 mm3 tPA plus NBO), showing that tPA-induced reperfusion salvages ischemic tissue and that NBO does not significantly alter this neuroprotective effect. NBO had no significant effect on hemorrhagic conversion, brain swelling, or mortality. CONCLUSION: NBO can be safely co-administered with tPA. The efficacy of tPA thrombolysis is not affected and there is no induction of brain hemorrhage or edema. These experimental results require clinical confirmation.

Angiotensin II type 2 receptor-mediated inhibition of norepinephrine release in isolated rat hearts.

We investigated whether endogenous and exogenous angiotensin II (Ang II) regulates norepinephrine (NE) release from cardiac sympathetic nerves via both Ang II type 2 receptors (AT2Rs) and Ang II type 1 receptors (AT1Rs). Using isolated rat hearts, sympathetic nerves were electrically stimulated. Ang II with PD-123319 (AT2R antagonist) but not Ang II alone produced a significant increase in nerve stimulation-induced NE overflow, which was abolished by the addition of AT1R antagonist losartan. In contrast, NE overflow was markedly decreased by losartan with or without Ang II. This decrease was abolished by the combination with PD-123319, nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (NOARG), icatibant (bradykinin B2 receptor antagonist), or PKSI-527 (kininogenase inhibitor). CGP-42112A (AT2R agonist) suppressed nerve stimulation-induced NE overflow in the same way as the combination of Ang II and losartan, and this suppression was abolished by PD-123319, NOARG, icatibant, or PKSI-527. There were significant increases in NOx (NO2/NO3) contents in coronary effluent under conditions where NE overflow was suppressed. Ang II seems to function as an inhibitory modulator of cardiac noradrenergic neurotransmission via AT2Rs and well-known AT1R-mediated stimulatory actions. The inhibitory mechanism may involve local bradykinin production, its B2 receptor activation, and NO as a downstream effector.

Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma.

PURPOSE: Rho-associated coiled-coil-forming protein kinase (ROCK) is pivotally involved in invasion by tumor cells and their evolution to metastasis. We have developed a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride]. In the present study, we investigated its effect on in vitro invasion and in vivo pulmonary metastasis of B16 melanoma. METHODS: The following were evaluated: the anti-invasive effect of Wf-536 against the motility of mouse B16BL6 melanoma cells through a culture insert layered with reconstituted basement membrane (Matrigel); the cytotoxic effect of Wf-536 in the same cell line; the antimetastatic effect of Wf-536, administered by osmotic pump, on spontaneous pulmonary metastasis following subcutaneous injection of B16BL6 melanoma in mice; and the inhibitory effect of orally administered Wf-536, alone or in combination with the antineoplastic drug paclitaxel, on pulmonary metastasis of intravenously injected B16F10 melanoma in mice. RESULTS: Wf-536 inhibited in vitro invasion by B16BL6 cells significantly and in a concentration-dependent manner and displayed an anti-invasive effect under conditions of both chemotaxis and chemokinesis. No cytotoxic effect was observed at any of the concentrations used. In vivo, Wf-536 administration suppressed tumor colony formation on the lung surface in a dose-dependent manner (0.3-3 mg/kg per day), with a metastasis inhibition rate of 95% at 3 mg/kg per day. In experimental metastasis of B16F10 melanoma, oral administration of Wf-536 significantly decreased tumor colony formation in the lung, with an inhibition rate of 41% at 3 mg/kg per day. The inhibition rate of paclitaxel (5 mg/kg per day) was 27%. The combination of Wf-536 and paclitaxel produced a synergistic effect on B16F10 metastasis and a 68% inhibition rate. Wf-536 administration at the doses used did not alter body weight, blood pressure or the health of treated animals as compared to vehicle-treated controls. CONCLUSION: The results suggest that Wf-536 is a potentially valuable drug for preventing tumor metastasis both in monotherapy and in combination with an antineoplastic drug.

Wf-536 prevents tumor metastasis by inhibiting both tumor motility and angiogenic actions.

The signaling pathway of Rho and Rho-associated coiled-coil forming protein kinase (ROCK) is involved in tumor metastasis. In the present study, we investigated the suppressive effect of a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride], on spontaneous tumor metastasis in vivo and analyzed its action on tumor cell motility and angiogenesis to clarify its action mechanism. Wf-536 (0.3-3 mg/kg/day) was found to inhibit Lewis lung carcinoma (LLC) metastasis and LLC-induced angiogenesis in orally treated mice; in vitro, it inhibited both invasion and migration by LLC cells and invasion, migration, and formation of capillary-like tubes on Matrigel by endothelial cells, without cytotoxicity or anti-proliferative action in either cell type. We conclude that Wf-536 has tumor anti-metastatic activity which may depend on inhibition of tumor motility and angiogenesis. The findings support its further clinical development as an anti-metastatic agent.