Potent Analgesic Action of 2-acetoxy-5-(2-4 (trifluoromethyl)-phenethylamino)-benzoic Acid (Flusalazine) in Experimental Mice.

Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2 selective inhibitors are the most widely used drugs to treat pain. Conventional NSAIDs and COX-2 selective inhibitors, however, cause several side effects such as gastric damage, kidney damage, and cardiovascular problems. Our previous study showed that 2-acetoxy-5-(2-4-(trifluoromethyl)-phenethylamino)-benzoic acid ie, flusalazine (also known as ND-07), which exerts dual actions by serving both as an anti-inflammatory agent and a free radical scavenger, is an effective and safe treatment for severe inflammatory diseases in mice. The goal of the present study was to examine the potential analgesic action and safety of flusalazine in mice models of pain. Methods and Results: Flusalazine showed a significant analgesic effect in an acetic acid-induced abdominal constriction model. Likewise, total paw licking was reduced significantly in neurogenic (early stage) and inflammatory (late stage) pain induced by formalin in flusalazine-treated mice. In the tail immersion test, flusalazine significantly increased tail withdrawal time at 2 h after its administration. Also, the formation of paw edema in the flusalazine-treated group was significantly inhibited in a carrageenan-induced inflammatory pain model. Gastric damage was not induced by flusalazine even up to 1000 mg/kg, while aspirin and indomethacin caused critical gastric bleeding. Conclusion: These findings suggest that flusalazine's safety profile and analgesic effects have high translational potential for the clinical treatment of patients experiencing pain.

Prevention effects of ND-07, a novel drug candidate with a potent antioxidative action and anti-inflammatory action, in animal models of severe acute pancreatitis.

Oxidative stress and inflammation both play major roles in the development of the acute pancreatitis. Currently, a pancreatic enzyme inhibitor with limited efficacy is only clinically available in a few countries, and antioxidants or non-steroidal anti-inflammatory drugs (NSAIDs) provide only partial tissue protection in acute pancreatitis animal models. Here, we introduce a new drug candidate for treating acute pancreatitis named ND-07 [chemical name: 2-acetoxy-5-(2-4-(trifluoromethyl)-phenethylamino)-benzoic acid] that exhibits both potent antioxidative and anti-inflammatory activities. In an electron spin resonance (ESR) study, ND-07 almost blocked hydroxyl radical generation as low as 0.05 µM and significantly suppressed DNA oxidation and cell death in a lipopolysaccharide (LPS)-stimulated pancreatic cell line. In a cerulein plus LPS-induced acute pancreatitis model, ND-07 pretreatment showed significant tissue protective effects, with reductions of serum amylase and lipase levels and pancreatic wet weights. ND-07 not only diminished the plasma levels of malondialdehyde (MDA) and nitric oxide but also significantly decreased prostaglandin E2 (PGE2) and expression of tumor necrotizing factor-alpha (TNF-α) in the pancreatic tissue. In a severe acute necrotizing pancreatitis model induced by a choline deficient, ethionine-supplemented (CDE) diet, ND-07 dramatically protected the mortality even without any death, providing attenuation of pancreas, lung, and liver damages as well as the reductions in serum levels of lactate dehydrogenase (LDH), amylase and lipase, MDA levels in the plasma and pancreatic tissues, plasma levels of TNF-α, and interleukin-1 (IL-1ß). These findings suggest that current dual synergistic action mechanisms of ND-07 might provide a superior protection for acute pancreatitis than conventional drug treatments.

Role of the NMDA receptor and iron on free radical production and brain damage following transient middle cerebral artery occlusion.

Excess activation of ionotropic glutamate receptors and iron is believed to contribute to free radical production and neuronal death following hypoxic ischemia. We examined the possibility that both NMDA receptor activation and iron overload determine spatial and temporal patterns of free radical production after transient middle cerebral artery occlusion (tMCAO) in male Sprague-Dawley rats. Mitochondrial free radical (MFR) levels were maximally increased in neurons in the core at 1 h and 24 h after tMCAO. Early MFR production was blocked by administration of MK-801, an NMDA receptor antagonist, but not deferoxamine, an iron chelator. Neither MK-801 nor deferoxamine attenuated late MFR production in the core. Increased MFRs were observed in penumbral neurons within 6 h and gradually increased over 24 h after tMCAO. Slowly-evolving MFRs in the core and penumbra were accompanied by iron overload. Deferoxamine blocked iron overload but reduced MFR production only in the penumbra. Combined MK-801/deferoxamine reduced late MFR production in both core and penumbra in an additive manner. Combination therapy significantly ameliorated infarction compared with monotherapy. These findings suggest that the NMDA receptor activation and iron overload mediate late MFR production and infarction after tMCAO.

Effects of collagen-induced rheumatoid arthritis on amyloidosis and microvascular pathology in APP/PS1 mice.

BACKGROUND: Evidence suggests that rheumatoid arthritis (RA) may enhance or reduce the progression of Alzheimer's disease (AD). The present study was performed to directly explore the effects of collagen-induced rheumatoid arthritis (CIA) on amyloid plaque formation, microglial activation, and microvascular pathology in the cortex and hippocampus of the double transgenic APP/PS1 mouse model for AD. Wild-type or APP/PS1 mice that received type II collagen (CII) in complete Freund's adjuvant (CFA) at 2 months of age revealed characteristics of RA, such as joint swelling, synovitis, and cartilage and bone degradation 4 months later. Joint pathology was accompanied by sustained induction of IL-1ß and TNF-α in plasma over 4 weeks after administration of CII in CFA. RESULTS: CIA reduced levels of soluble and insoluble amyloid beta (Aß) peptides and amyloid plaque formation in the cortex and hippocampus of APP/PS1 mice, which correlated with increased blood brain barrier disruption, Iba-1-positive microglia, and CD45-positive microglia/macrophages. In contrast, CIA reduced vessel density and length with features of microvascular pathology, including vascular segments, thinner vessels, and atrophic string vessels. CONCLUSIONS: The present findings suggest that RA may exert beneficial effects against Aß burden and harmful effects on microvascular pathology in AD.

Neu2000, an NR2B-selective, moderate NMDA receptor antagonist and potent spin trapping molecule for stroke.

Excess activation of ionotropic glutamate receptors, primarily N-methyl-D-aspartate (NMDA) receptors and free radicals, evoke nerve cell death following hypoxic-ischemic brain injury in various animal models. However, clinical trials in stroke patients using NMDA receptor antagonists have failed to show efficacy primarily due to the limited therapeutic time window for neuroprotection and a narrow therapeutic index. In comparison, antioxidants prolonged the time window for neuroprotection in animal models of ischemic stroke and showed greater therapeutic potential in clinical trials for ischemic stroke. Excess activation of NMDA receptors and free radicals mediate the two separate pathways of nerve cell death in stroke and a safe and multifunctional drug that can block both routes in the brain will likely provide a better therapeutic outcome in patients with stroke. Derivatives of the lead structures of sulfasalazine and aspirin have led to the discovery of a new molecule, Neu2000, that has demonstrated excellent neuroprotection against NMDA- and free radical-induced cell death. Neu2000 is an NR2B-selective, moderate NMDA receptor antagonist with potent cell-permeable, spin trapping antioxidant action even at nanomolar concentrations. Nonclinical and human phase I studies demonstrated that Neu2000 can be translated to treat patients with stroke with better efficacy and therapeutic time window.

The functional and neuroprotective actions of Neu2000, a dual-acting pharmacological agent, in the treatment of acute spinal cord injury.

The goal of the present study was to examine the neuroprotective and functional significance of targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress using a dual-acting compound, Neu2000, in rat model of moderate spinal cord injury (SCI). An initial set of experiments was conducted in uninjured rats to study the pharmacokinetic profile of Neu2000 following intraperitoneal and intravenous administration. A second experiment measured free radical production in mitochondria isolated from sham or injured spinal cords of animals receiving vehicle or Neu2000 treatment. A third set of animals was divided into three treatment groups consisting of vehicle treatment, a single dose of Neu2000 (50 mg/kg) administered at 10 min following injury, or a repeated treatment paradigm consisting of a single bolus of Neu2000 at 10 min following injury (50 mg/kg) plus a maintenance dose (25 mg/kg) administered every 24 h for an additional 6 days. Animals were tested once a week for a period of 6 weeks for evidence of locomotor recovery in an open field and kinematic analysis of fine motor control using the DigiGait Image Analysis System. At the end of the testing period, spinal cord reconstruction was performed to obtain nonbiased stereological measures of tissue sparing. The results of this study demonstrate that Neu2000 treatment significantly reduced the production of mitochondrial free radicals and improved locomotor outcomes that were associated with a significant increase in the volume of spared spinal cord tissue.

Effects of estrogen on lifespan and motor functions in female hSOD1 G93A transgenic mice.

Amyotrophic lateral sclerosis (ALS) is a progressive disease which is caused by degeneration of motor neurons in the central nervous system. The incidence of ALS is higher in men than women, but the female advantage disappears with increased age. Here, we report evidence that the female advantage is due to the protective role of estrogen. In an ALS mouse model carrying the human Cu/Zn superoxide dismutase (hSOD1) G93A transgene, ovariectomy did not alter the onset age of the disease while reducing the female lifespan by 7 days and making it comparable to that of the male transgenic mice. Treatment of ovariectomized females with 17beta-estradiol (E2) did not delay the onset of disease, but prevented progression of ALS motor dysfunctions as shown by extension reflex test for a limited time window. Importantly, E2 treatment rescued the lifespans in overiectomized females. These findings will provide important new insights to interpretation of disease progression in post-menopausal female ALS patients.

Anti-hypertensive effect of the Dongchunghacho, Isaria sinclairii, in the spontaneously hypertensive rats.

The present study examined the effect of the methanol extract of Isaria sinclairii, a kind of Donchunghacho (Tochukaso), on blood pressure in spontaneously hypertensive rats (SHR). Blood pressure and heart rate were measured after treatment with the methanol extract of I. sinclairii by the indirect tail-cuff method and the direct in vivo model. Starting at 12 weeks of age, male SHR were treated with the extracts for 2 or 4 weeks. We found that, when compared to untreated control SHR, oral treatment with I. sinclairii methanol extract (30 mg/kg/day) remarkably decreased systolic blood pressure from 200 to 112 mmHg and decreased diastolic blood pressure from 114 to 88 mmHg. Furthermore, efficacy of methanol extract of I. sinclairii was superior to captopril (30 mg/kg/mL, positive control), an angiotensin-converting enzyme inhibitor, with a lowering effect that dropped systolic blood pressure from 201 to 130 mmHg and diastolic blood pressure from 102 to 92 mmHg. However, in normal Wistar Kyoto rats, I. sinclairii methanol extract did not significantly change the normal blood pressure, suggesting that this type of Dongchunghacho has a selective effect against hypertension. Therefore, methanol extract of I. sinclairii may be used as an anti-hypertensive food/agent. Furthermore, this extract also has multiple actions such as No production in endothelial cells, inhibiting thrombin-induced blood coagulation by thrombin and mildly decreasing in prostaglandin E2 levels in cultured macrophage cells, all of which might contribute to protection against atherogenesis and thrombus formation. HPLC and MS analysis of methanol extract of I. sinclairii revealed the presence of adenosine.

Concurrent administration of Neu2000 and lithium produces marked improvement of motor neuron survival, motor function, and mortality in a mouse model of amyotrophic lateral sclerosis.

The Fas pathway and oxidative stress mediate neuronal death in stroke and may contribute to neurodegenerative disease. We tested the hypothesis that these two factors synergistically produce spinal motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Levels of reactive oxygen species were increased in motor neurons from ALS mice compared with wild-type mice at age 10 weeks, before symptom onset. The proapoptotic proteins Fas, Fas-associated death domain, caspase 8, and caspase 3 were also elevated. Oral administration of 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid (Neu2000), a potent antioxidant, blocked the increase in reactive oxygen species but only slightly reduced activation of proapoptotic proteins. Administration of lithium carbonate (Li(+)), a mood stabilizer that prevents apoptosis, blocked the apoptosis machinery without preventing oxidative stress. Neu2000 or Li(+) alone significantly enhanced survival time and motor function and together had an additive effect. These findings provide evidence that jointly targeting oxidative stress and Fas-mediated apoptosis can prevent neuronal loss and motor dysfunction in ALS.

Marked prevention of ischemic brain injury by Neu2000, an NMDA antagonist and antioxidant derived from aspirin and sulfasalazine.

Excitotoxicity and oxidative stress mediate neuronal death after hypoxic-ischemic brain injury. We examined the possibility that targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress would result in enhanced neuroprotection against hypoxic-ischemia. 2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid (Neu2000) was derived from aspirin and sulfasalazine to prevent both NMDA neurotoxicity and oxidative stress. In cortical cell cultures, Neu2000 was shown to be an uncompetitive NMDA receptor antagonist and completely blocked free radical toxicity at doses as low as 0.3 micromol/L. Neu2000 showed marked neuroprotection in a masked fashion using histology and behavioral testing in two rodent models of focal cerebral ischemia without causing neurotoxic side effects. Neu2000 protected against the effects of middle cerebral artery occlusion, even when delivered 8 h after reperfusion. Single bolus administration of the drug prevented gray and white matter degeneration and spared neurologic function for over 28 days after MACO. Neu2000 may be a novel therapy for combating both NMDA receptor-mediated excitotoxicity and oxidative stress, the two major routes of neuronal death in ischemia, offering profound neuroprotection and an extended therapeutic window.

Effect of alpha-tocopherol and deferoxamine on methamphetamine-induced neurotoxicity.

Methamphetamine (MA)-induced dopaminergic neurotoxicity is believed to be associated with the increased formation of free radicals. This study examined the effect of alpha-tocopherol (alpha-TC), a scavenger of reactive oxygen species, and deferoxamine (DFO), an iron chelator, on the MA-induced neurotoxicity. Male rats were treated with MA (10 mg/kg, every 2 h for four injections). The rat received either alpha-TC (20 mg/kg) intraperitoneally for 3 days and 30 min prior to MA administration or DFO (50 mg/kg) subcutaneously 30 min before MA administration. The concentrations of dopamine (DA), serotonin and their metabolites decreased significantly after MA administration, which was inhibited by the alpha-TC and DFO pretreatment. alpha-TC and DFO attenuated the MA-induced hyperthermia as well as the alterations in the locomotor activity. The level of lipid peroxidation was higher and the reduced glutathione concentration was lower in the MA-treated rats. These changes were significantly attenuated by alpha-TC and DFO. This suggests that alpha-TC and DFO ameliorate the MA-induced neuronal damage by decreasing the level of oxidative stress.

Effect of Holotrichia diomphalia larvae on liver fibrosis and hepatotoxicity in rats.

Holotrichia diomphalia larvae, one of the most widely used Korean folk medicinal preparations, have long been used for the treatment of chronic liver cirrhosis. The present study was undertaken to clarify whether extract of Holotrichia diomphalia larvae could prevent acute liver damage and liver fibrosis in rats. A single administration of Holotrichia diomphalia protected rats from acute liver damage induced by carbon tetrachloride (200 micro l/kg, i.p.) and beta-D-galactosamine (600mg/kg, i.p.). This was evidenced by the lowered serum aminotransferase (ALT, AST) activities in rats treated with Holotrichia diomphalia. The hepatic cirrhosis was induced by 28 days of bile duct ligation/scission in rats. The four-week treatment with Holotrichia diomphalia reduced the serum ALT, AST, alkaline phosphatase activities, and hydroxyproline content in the liver and improved the histological appearance of the liver sections. The present results led us to conclude that Holotrichia diomphalia larvae can reduce the degree of hepatocellular damage and may become a promising antifibrotic agent for liver fibrosis/cirrhosis.

Effects of hibarimicins and hibarimicin-related compounds produced by Microbispora on v-Src kinase activity and growth and differentiation of human myeloid leukemia HL-60 cells.

We studied the effects of hibarimicins and hibarimicin-related compounds produced by Microbispora rosea subsp. hibaria [glycosides (hibarimicins A, B, C, D, E, G, H and I) and aglycon (hibarimicinone)] or compounds produced by its mutants [glycosides (HMP-P4 and -Y6), aglycons (HMP-P1 and -Y1) and shunt products (HMP-M1, M2, M3 and -M4)] on v-Src tyrosine kinase and growth and differentiation of human myeloid leukemia HL-60 cells. Among them, hibarimicin B was a strong and the most selective v-Src kinase inhibitor with differentiation inducing activity of HL-60 cells. Hibarimicin E similarly induced HL-60 cell differentiation but had no v-Src kinase inhibitory activity. Hibarimicinone was the most potent v-Src kinase inhibitor, although less selective, and did not induce differentiation of HL-60 cells. Hibarimicin B competitively inhibited ATP binding to the v-Src kinase, but hibarimicinone showed noncompetitive inhibition. These two compounds, however, showed similar mixed types of inhibition against a Src substrate binding to the v-Src kinase. Altogether, these results suggest that signaling molecules other than Src might be more important in the differentiation induction of HL-60 cells.