Inhibition of Schwann cell pannexin 1 attenuates neuropathic pain through the suppression of inflammatory responses.

BACKGROUND: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. METHODS: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. RESULTS: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. CONCLUSION: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.

Comparative effect of vitamin D3 and carbenoxolone treatments in metabolic syndrome rats.

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including central obesity, hypertension, insulin resistance, dyslipidemia, and hyperglyemia. MetS is found to be a positive predictor of cardiovascular morbidity and mortality. The present study was planned to test the efficacy of vitamin D3 supplementation as compared with cortisol inhibition on MetS parameters. Wistar rats were allocated into four groups: control, untreated MetS, and MetS treated with either vitamin D3 (10 µg/kg) or carbenoxolone (50 mg/kg). MetS was induced by combination of high-fat diet and oral fructose. After the induction period (8 weeks), MetS was confirmed, and treatment modalities started for a further 4 weeks. Compared with untreated MetS, vitamin D3- and carbenoxolone-treated rats showed significant reduction in blood pressure, body mass index, Lee index, waist circumference, retroperitoneal fat, and improvement of dyslipidemia. Meanwhile, treatment with carbenoxolone significantly lowered the elevated liver enzymes, and vitamin D3 resulted in improved insulin sensitivity, enhanced glucose uptake by muscles, and replenished glycogen content in the liver and muscles near control levels. In conclusion, although treatment with vitamin D3 or carbenoxolone reduced the risk factors associated with MetS, vitamin D3 was effective in ameliorating insulin resistance which is the hallmark of MetS.

The Anti-Epileptic Effects of Carbenoxolone In Vitro and In Vivo.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

The role of connexin43 in neuropathic pain induced by spinal cord injury.

Neuropathic pain is caused by the damage or dysfunction of the nervous system. In many neuropathic pain models, there is an increase in the number of gap junction (GJ) channels, especially the upregulation of the expression of connexin43 (Cx43), leading to the secretion of various types of cytokines and involvement in the formation of neuropathic pain. GJs are widely distributed in mammalian organs and tissues, and Cx43 is the most abundant connexin (Cx) in mammals. Astrocytes are the most abundant glial cell type in the central nervous system (CNS), which mainly express Cx43. More importantly, GJs play an important role in regulating cell metabolism, signaling, and function. Many existing literatures showed that Cx43 plays an important role in the nervous system, especially in the CNS under normal and pathological conditions. However, many internal mechanisms have not yet been thoroughly explored. In this review, we summarized the current understanding of the role and association of Cx and pannexin channels in neuropathic pain, especially after spinal cord injury, as well as some of our own insights and thoughts which suggest that Cx43 may become an emerging therapeutic target for future neuropathic pain, bringing new hope to patients.

TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice.

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

HIS-388, a novel orally active and long-acting 11ß-hydroxysteroid dehydrogenase type 1 inhibitor, ameliorates insulin sensitivity and glucose intolerance in diet-induced obesity and nongenetic type 2 diabetic murine models.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is considered a potential therapeutic target in the treatment of type 2 diabetes mellitus. In this study, we investigated the pharmacological properties of HIS-388 (N-[(1R,2s,3S,5s,7s)-5-hydroxyadamantan-2-yl]-3-(pyridin-2-yl) isoxazole-4-carboxamide), a newly synthesized 11ß-HSD1 inhibitor, using several mouse models. In cortisone pellet-implanted mice in which hypercortisolism and hyperinsulinemia occur, single administration of HIS-388 exhibited potent and prolonged suppression of plasma cortisol and lowered plasma insulin levels. These effects were more potent than those achieved using the same dose of other 11ß-HSD1 inhibitors (carbenoxolone and compound 544 [3-[(1s,3s)-adamantan-1-yl]-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine]), indicating that HIS-388 potently and continuously suppresses 11ß-HSD1 enzyme activity in vivo. In diet-induced obese mice, HIS-388 significantly decreased fasting blood glucose, plasma insulin concentration, and homeostasis model assessment-insulin resistance score, and ameliorated insulin sensitivity. In addition, HIS-388 significantly reduced body weight and suppressed the elevation of blood glucose during the pyruvate tolerance test. In nongenetic type 2 diabetic mice with disease induced by a high-fat diet and low-dose streptozotocin, HIS-388 also significantly decreased postprandial blood glucose and plasma insulin levels and improved glucose intolerance. The effects of HIS-388 on glucose metabolism were indistinguishable from those of an insulin sensitizer, pioglitazone. Our results suggest that HIS-388 is a potent agent against type 2 diabetes. Moreover, amelioration of diabetic symptoms by HIS-388 was at least in part attributable to an antiobesity effect or improvement of hepatic insulin resistance. Therefore, potent and long-lasting inhibition of 11ß-HSD1 enzyme activity may be an effective approach for the treatment of type 2 diabetes and obesity-associated disease.

The gastroprotective effects of Eugenia dysenterica (Myrtaceae) leaf extract: the possible role of condensed tannins.

We applied a taxonomic approach to select the Eugenia dysenterica (Myrtaceae) leaf extract, known in Brazil as "cagaita," and evaluated its gastroprotective effect. The ability of the extract or carbenoxolone to protect the gastric mucosa from ethanol/HCl-induced lesions was evaluated in mice. The contributions of nitric oxide (NO), endogenous sulfhydryl (SH) groups and alterations in HCl production to the extract's gastroprotective effect were investigated. We also determined the antioxidant activity of the extract and the possible contribution of tannins to the cytoprotective effect. The extract and carbenoxolone protected the gastric mucosa from ethanol/HCl-induced ulcers, and the former also decreased HCl production. The blockage of SH groups but not the inhibition of NO synthesis abolished the gastroprotective action of the extract. Tannins are present in the extract, which was analyzed by matrix assisted laser desorption/ionization (MALDI); the tannins identified by fragmentation pattern (MS/MS) were condensed type-B, coupled up to eleven flavan-3-ol units and were predominantly procyanidin and prodelphinidin units. Partial removal of tannins from the extract abolished the cytoprotective actions of the extract. The extract exhibits free-radical-scavenging activity in vitro, and the extract/FeCl3 sequence stained gastric surface epithelial cells dark-gray. Therefore, E. dysenterica leaf extract has gastroprotective effects that appear to be linked to the inhibition of HCl production, the antioxidant activity and the endogenous SH-containing compounds. These pleiotropic actions appear to be dependent on the condensed tannins contained in the extract, which bind to mucins in the gastric mucosa forming a protective coating against damaging agents. Our study highlights the biopharmaceutical potential of E. dysenterica.

Adenosine triphosphate release inhibitors targeting pannexin1 improve recovery after spinal cord injury.

Traumatic spinal cord injury is characterized by immediate and irreversible tissue loss at the lesion site and secondary tissue damage. Secondary injuries should, in principle, be preventable, although no effective treatment options currently exist for patients with acute spinal cord injury. Traumatized tissues release excessive amounts of adenosine triphosphate and activate the P2X purinoceptor 7/pannexin1 complex, which is associated with secondary injury. We investigated the neuroprotective effects of the blue dye Brilliant Blue FCF, a selective inhibitor of P2X purinoceptor 7/pannexin1 that is approved for use as a food coloring, by comparing it with Brilliant Blue G, a P2X7 purinoceptor antagonist, and carbenoxolone, which attenuates P2X purinoceptor 7/pannexin1 function, in a rat spinal cord injury model. Brilliant Blue FCF administered early after spinal cord injury reduced spinal cord anatomical damage and improved motor recovery without apparent toxicity. Brilliant Blue G had the highest effect on this neurological recovery, with Brilliant Blue FCF and carbenoxolone having comparable improvement. Furthermore, Brilliant Blue FCF administration reduced local astrocytic and microglial activation and neutrophil infiltration, and no differences in these histological effects were observed between compounds. Thus, Brilliant Blue FCF protects spinal cord neurons after spinal cord injury and suppresses local inflammatory responses as well as Brilliant Blue G and carbenoxolone.

Inhibition of 11ß-hydroxysteroid dehydrogenase 1 by carbenoxolone affects glucose homeostasis and obesity in db/db mice.

1. One of the major causes of metabolic syndrome is elevated 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) in the liver and adipose tissue. High 11ß-HSD1 expression contributes significantly to the diabetic phenotype in db/db mice. The purpose of the present study was to test the effect of the pharmacological inhibition of 11ß-HSD1 inhibition by carbenoxolone in db/db mice, a genetic model of diabetes. 2. Inhibition of 11ß-HSD1 by carbenoxolone was evaluated in liver homogenates obtained from untreated mice. At 0.4, 0.8, 1.6 and 3.2 µmol/L, carbenoxolone reduced the conversion of cortisone to cortisol by 21%, 48%, 82% and 95%, respectively. 3. In another series of experiments in which female db/db mice were dosed orally with carbenoxolone (10, 25 and 50 mg/kg, twice daily) for 10 days, dose-dependent decreases were observed in 11ß-HSD1 activity in the brain, adipose and liver. In the case of 10 mg/kg carbenoxolone, the effects were not significant. In addition, the bodyweight of female db/db mice was reduced by 10% and 13% following treatment with 10 and 50 mg/kg carbenoxolone, respectively. Carbenoxolone treatment dose-dependently improved fat mass, energy expenditure, the serum lipid profile, serum leptin and insulin and glucose tolerance. Furthermore, 50 mg/kg carbenoxolone reduced both phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) activity in the liver by 75% and 52%, respectively. These decreases were associated with increased glucokinase protein expression and activity in the liver. 4. Carbenoxolone inhibition of 11ß-HSD1 in the liver, adipose and brain significantly improves the symptoms of metabolic syndrome in db/db mice. These improvements can be attributed to increased energy expenditure, decreased activity of the gluconeogenic enzymes PEPCK and G6Pase in the liver and improved glucokinase function in the liver and pancreas.

Gastroprotection of suaveolol, isolated from Hyptis suaveolens, against ethanol-induced gastric lesions in Wistar rats: role of prostaglandins, nitric oxide and sulfhydryls.

Hyptis suaveolens is a medicinal plant that is, according to traditional medicine, considered useful in the treatment of gastric ulcers. Although its gastroprotective activity was reported, the active compounds have not been identified. Therefore, the aim of the present study was to identify at least one active compound potentially responsible for the gastroprotective activity of H. suaveolens by using a bioassay guided study with an ethanol-induced gastric ulcer experimental model in rats. The results show that the hexane extract had protective activity (close to 70% when using doses between 10 and 100 mg/kg), and that the compound suaveolol, isolated from this extract, was one of the active gastroprotective agents. This is the first report about the gastroprotective activity of suaveolol. Rats treated with this compound at 3, 10, 30 and 100 mg/kg showed 12.6, 21.3, 39.6 and 70.2% gastroprotection respectively. The effect elicited by suaveolol (at 100 mg/kg) was attenuated by pretreatment with either N(G)-nitro-L-arginine methyl ester (70 mg/kg, i.p.), a nitric oxide (NO) synthase inhibitor, indomethacin (10 mg/kg, s.c.), a blocker of prostaglandin synthesis, or N-ethylmaleimide (10 mg/kg, s.c.), a blocker of sulfhydryl groups. This suggests that the gastroprotective mechanism of action of this compound involves NO, prostaglandins and sulfhydryl groups.

Inhaled carbenoxolone prevents allergic airway inflammation and airway hyperreactivity in a mouse model of asthma.

BACKGROUND: Asthma is a chronic respiratory disease, which needs a safer medication preferably in inhalation form. In view of this, we have evaluated the effect of inhaled carbenoxolone (CBX), a herbal-derived compound, on asthma in a mouse model. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) to develop certain characteristic features of asthma such as airway hyperreactivity (AHR), airway eosinophilia, lung inflammation and mucus hypersecretion. To evaluate the effect of CBX on the above asthmatic features, CBX (2.5, 5 and 10 mg/ml, 3 ml) or vehicle (water) was given by inhalation. AHR was determined using whole-body plethysmography. Infiltration of eosinophils was estimated by microscopy. Lung inflammation and mucus hypersecretion were assessed using hematoxylin and eosin, and periodic acid-Schiff staining, respectively. Th-2 cytokines, IL-4 and IL-5 were measured in bronchoalveolar lavage (BAL) fluid and IgE in sera. To identify the possible mode of CBX action, we measured corticosterone levels in the BAL fluid and 5-lipoxygenase (5-LO) expression in the lungs. RESULTS: CBX (5 mg/ml) inhalation markedly alleviated AHR (p = 0.0032) and reduced lung inflammation and mucus hypersecretion. Also, it prevented the increase in IL-4 (p = 0.0192), IL-5 (p = 0.0116) and eosinophils (p < 0.0005) in the BAL fluid, and OVA-specific IgE levels (p = 0.00061) in sera. 5-LO expression was also markedly reduced. However, corticosterone levels were not affected. CONCLUSIONS: Inhaled CBX alleviates the asthmatic features in mice and could be a potent nebulized therapy in clinical asthma.

Effect of gap junction inhibition on intracerebral hemorrhage-induced brain injury in mice.

It has been reported that gap junction contributes to ischemic brain injury and gap junction inhibitors improve neurological outcome in ischemic brain injury models. In the present study, we investigated the effects of gap junction inhibitor, carbenoxolone, on mortality, neurological deficits and brain edema in mice with intracerebral hemorrhage. A total of 80 male CD-1 mice were divided into two parts with two end-points for this study. In part one, animals were divided into four groups: sham, vehicle treatment following intracerebral hemorrhage induction, low-dose carbenoxolone (33 mg/kg) treatment 1 hour after intracerebral hemorrhage induction and high-dose carbenoxolone (100 mg/kg) treatment 1 hour after intracerebral hemorrhage induction groups. Animals were euthanized after 24 hours. In part two, animals were divided into four groups: sham, vehicle treatment 1 hour after intracerebral hemorrhage induction, single high-dose of carbenoxolone treatment at 1 hour after intracerebral hemorrhage induction and three high-doses of carbenoxolone treatment 1, 24 and 48 hours respectively after intracerebral hemorrhage induction. Animals were euthanized after 72 hours. Intracerebral hemorrhage was induced by collagenase injection. Neurological deficits were evaluated using modified Garcia's neurological test, wire hanging and beam balance tests. Brain edema was measured by brain water content. Our results showed that intracerebral hemorrhage produced brain edema and neurological deficits in mice. Carbenoxolone treatment failed to reduce brain edema and neurological deficits. In fact, the high dose of carbenoxolone aggravated neurological deficits and increased mortality 72 hours after the treatment. In conclusion, inhibition of gap junction has no short-term neuroprotective effect on intracerebral hemorrhage-induced brain injury. Further studies are required to assess the long-term effects of gap junction inhibitors in intracerebral hemorrhage models.

Effects of gap junctional blockers on cerebral vasospasm after subarachnoid hemorrhage in rabbits.

The role of gap junctional communication in the coordination of vascular behavior has been well established experimentally. The aim of this study was to investigate whether the gap junctional blockers would inhibit cerebral vasospasm after experimental subarachnoid hemorrhage (SAH). We used the double-hemorrhage model of SAH with injection of autologous arterial blood into the cisterna magna on days 1 and 2. Octanol or carbenoxolone was administered intracisternally on day 3, and repeated on days 4 and 5. Angiography was performed before (day 0) and 7 days after (day 7) SAH, and the diameter of the basilar artery was measured. Paraffin blocks of brain tissues were prepared and sectioned for hematoxylin and eosin staining for morphologic analysis. Arterial narrowing in SAH + octanol group and SAH + carbenoxolone group at day 7 was significantly less than that in SAH-only group and each SAH + vehicle group, respectively. Less morphologic changes in SAH + octanol group and SAH + carbenoxolone group were observed when compared to that in SAH-only group and each SAH + vehicle group. Western blotting showed that carbenoxolone down-regulated Cx43 protein expression in the BA, which in the SAH-only group was significantly higher than that of the normal group. Gap junction blockers attenuate the experimental cerebral vasospasm and down-regulate the increase in expression of Cx43 protein after SAH in rabbits. These data suggest that gap junctions play an important role in the development of cerebral vasospasm.

Inhibitory effect of gap junction blockers on cerebral vasospasm.

OBJECT: The gap junction is important in the propagation of dilation/constriction signals along vessels for coordinated behavior in control of vascular tone. The authors hypothesized that gap junctions might play a role in cerebral vasospasm following subarachnoid hemorrhage (SAH). The aims of the present study were to investigate the role of gap junctions and to observe the potential therapeutic efficacy of gap junction blockers in cerebral vasospasm in vitro and in vivo. METHODS: For the in vitro investigation, the effect of heptanol on the oxyhemoglobin (HbO(2))-induced contraction of isolated rabbit basilar arteries (BAs) was observed by using an isometric tension-recording method. For the in vivo experiments, the potential therapeutic efficacy of heptanol and carbenoxolone was surveyed after it was given intravenously in the rabbit double-hemorrhage model. Light microscopy was performed to assess the morphological changes in the arteries examined. RESULTS: For the in vitro method, heptanol significantly inhibited the sustained contraction induced both by HbO(2) and K(+) in the BA rings. The magnitude of the heptanol-induced relaxation was dose dependent. The inhibitory effect of heptanol on the K(+)-induced vasoconstriction was weaker than that on the HbO(2)-induced constriction. After arterial rings were pretreated for 10 minutes, heptanol significantly decreased their responses to the HbO(2)-induced contraction. For the in vivo method, heptanol and carbenoxolone significantly decreased the narrowing of BAs when given intravenously in the rabbit double-hemorrhage model. In both treated groups, the diameters of the arteries had not changed significantly on Day 7 compared with those of the arteries in the SAH + vehicle and the SAH-only group. CONCLUSIONS: Heptanol and carbenoxolone significantly inhibited the experimental cerebral vasospasm both in vitro and in vivo. Blockage of gap junctions is a probable candidate for a new approach in the treatment of cerebral vasospasm. Gap junctions may play a pathophysiological role in cerebral vasospasm.

[Studies on analgesic effects and sites of oxymatrine-carbenoxolone sodium complex].

OBJECTIVE: To study the analgesic effects and sites of oxymatrine-carbenoxolone sodium complex (OCSC). METHOD: Adopting formalin test, warm water tail-flick test and intracerebroventricularly (icv) injection to observe the analgesic effects of OCSC in mice. RESULT: Intraperitoneally injecting (ip) OCSC (75, 150 mg x kg(-1)) remarkedly inhibited the pain of mice in the formalin test and prolonged latent phases of tail-shrinking of mice, icy OCSC (1.875, 3.75, 7.5 mg x kg(-1)) significantly prolonged latent phases of tail-shrinking of mice, it had dose-dependent effect with concentration. CONCLUSION: The result indicated that OCSC has obvious analgesic effects and its mechanism may be involved in central nervous system (CNS).

Quinine and carbenoxolone enhance the anticonvulsant activity of some classical antiepileptic drugs.

Objective Quinine (QUIN) and carbenoxolone (CNX) elicit anticonvulsant effects typically characterized by the reduction of the epileptiform activity as well as changes in behavioral parameters related to seizures. Therefore, the aim of this study was to analyze the effects of these molecules on the anticonvulsant activity of some classical antiepileptic drugs. Methods Male Wistar rats were used. Valproate (VPA), phenytoin (PHT), or carbamazepine (CBZ) was administered at sub-therapeutic doses for intraperitoneal via. Subsequently, animals were administered with a single dose of QUIN or CNX. The anticonvulsant activity was evaluated with the maximal electroshock (MES) test and pentylenetetrazole (PTZ) administration. Additionally, the plasma levels of CBZ were determined using an HPLC method. Results All the control rats presented generalized tonic-clonic seizures after the MES test or the administration of PTZ. For the MES test, all of the antiepileptic drugs increased their anticonvulsant activity when were co-administered with QUIN. For the PTZ test, only the combination CBZ plus QUIN significantly increased the percentage of protection against the generalized tonic-clonic seizures. The co-administration of CBZ plus QUIN resulted in an augmented concentration of CBZ in plasma. Discussion The present study shows that QUIN and CNX enhance the anticonvulsant activity of some classical antiepileptic drugs. However, only the combination CBZ/QUIN had significant effects on both MES and PTZ models. Such anticonvulsant activity could be attributed to increased levels of CBZ in plasma. We propose that these molecules could improve the pharmacological actions of antiepileptic drugs administered at sub-therapeutic doses.

Carbenoxolone Could Deteriorate Streptozotocin-induced Diabetes through Induction of Heat Shock Protein 70 and IFN-γ in C57BL/6 Mice.

Type 1 diabetes (T1D), a spontaneous autoimmune disease, is associated with destruction of insulin-producing ß-cells in the pancreas. Since some heat shock proteins (HSP), such as HSP70 exert a protective effect in both tissues and cells, the present study was conducted to elucidate the effects of carbenoxolone (CBX) as an HSP70 inducer on T1D. The disease was induced in male C57BL/6 mice using streptozotocin (STZ) and subjects were allocated to therapeutic 1 and therapeutic 2 groups, as well as negative and positive control groups. The treated mice (therapeutic 1 and therapeutic 2 groups) received 50 mg/kg CBX intraperitoneally every 24 hours, in the therapeutic 1  group the drug was injected before and after disease induction whereas in the therapeutic 2 group the drug was injected only after disease induction. Serum fasting blood sugar (FBS) level, cytokines production (Interferon-gamma (IFN-γ), Interleukin 10 (IL-10), and IL-17), serum HSP70 level and CD4+CD25+Foxp3+ regulatory T cell (Treg) frequency measurements were outperformed 14 days after the last STZ injection. Our results showed that in the treated groups, serum HSP70, IFN-γ, and IL-17 levels were increased in contrast to the untreated groups. The IL-10 level was markedly decreased in comparison to untreated diabetic mice (p<0.05). Moreover, it was found that the frequency of Tregs in treated mice was lower in comparison to the untreated mice but the difference was not significant (p>0.05). Our results confirm that CBX might through HSP70 induction, followed by increasing IFN-γ level leads to suppression of IL-10 production in diabetic mice resulted in toxic effects on pancreatic islet beta cells and deteriorating of disease.

Carbenoxolone as a novel therapy for attenuation of cancer-induced bone pain.

Pain is a major complication for patients with cancer significantly compromising their quality of life. Current treatment is far from optimal and particularly bone-related cancer pain poses an increasing clinical and socioeconomical problem. Connexins, key proteins in cell-cell communication, have the potential to affect cancer-induced bone pain at multiple levels, including nociceptive signaling and bone degradation. This study tested the analgesic potential of carbenoxolone, a broad-acting connexin blocker, in a mouse model of cancer-induced bone pain. In addition, a pharmacological approach was used to elucidate the underlying mechanisms using the 2 specific blockers Gap27 and Gap26. Compared with vehicle treatment, chronic systemic administration of 20 or 40 mg/kg carbenoxolone caused a significantly later onset and attenuation of movement-evoked and on-going pain, assessed with limb use and weight bearing, respectively. In addition, the carbenoxolone-treated groups demonstrated a significant delay in time to reach the humane endpoint. Acute intrathecal administration of Gap27 significantly attenuated both limb use and weight bearing, whereas Gap26 had a less pronounced effect. Carbenoxolone treatment had a minor effect on the bone degradation in the early phase of disease progression, whereas no effect was observed in the late phase. Surprisingly, connexin43 was downregulated in the cancer-bearing animals compared with shams. The results suggest that connexins are involved in cancer-induced bone pain, and that carbenoxolone could be a novel analgesic treatment for the pain state.

Acute anti-obesity effects of intracerebroventricular 11ß-HSD1 inhibitor administration in diet-induced obese mice.

The hypothalamus is the regulatory centre of both appetite and energy balance and endoplasmic reticulum (ER) stress in the hypothalamus is involved in the pathogenesis of obesity. Recently, inhibition of 11 ß hydroxysteroid dehydrogenase type1 (11ß-HSD1) was reported to have an anti-obesity effect by reducing fat mass. However, the link between the role of 11ß-HSD1 in the hypothalamus and obesity has yet to be determined. In the present study, embryonal primary hypothalamic neurones and high-fat diet (HFD) fed mice were used to investigate the anorexigenic effects of 11ß-HSD1 inhibitors both in vitro and in vivo. In hypothalamic neurones, carbenoxolone (a non selecitve 11ß-HSD inhibitor) alleviated ER stress and ER stress-induced neuropeptide alterations. In HFD mice, i.c.v. administration of carbenoxolone or KR67500 (nonselective and selective 11ß-HSD1 inhibitors, respectively) was associated with less weight gain compared to control mice for 24 hours after treatment, presumably by reducing food intake. Furthermore, glucose regulated protein (Grp78), spliced X-box binding protein (Xbp-1s), c/EBP homologous protein (chop) and ER DnaJ homologue protein (Erdj4) expression was decreased in the hypothalami of mice administrated 11ß-HSD1 inhibitors compared to controls. Conversely, the phosphorylation of protein kinase B (PKB/Akt), signal transducer and activator of transcription 3 (Stat3), mitogen-activated protein kinase (MAPK/ERK) and S6 kinase1 (S6K1) in the hypothalamus was induced more in mice treated using the same regimes. In conclusion, acute 11ß-HSD1 inhibition in the hypothalamus could reduce food intake by decreasing ER stress and increasing insulin, leptin, and mammalian target of rapamycin complex 1 (mTORC1) signalling.

Topical 11ß-Hydroxysteroid Dehydrogenase Type 1 Inhibition Corrects Cutaneous Features of Systemic Glucocorticoid Excess in Female Mice.

Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11ß-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11ß-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11ß-HSD1 inhibition by carbenoxolone (CBX) in mice treated with corticosterone (CORT) or vehicle for 6 weeks. Mice were treated bidaily with topical CBX or vehicle (VEH) 7 days before wounding and during wound healing. CORT mice displayed skin thinning and impaired wound healing but also increased epidermal integrity. 11ß-HSD1 activity was elevated in unwounded CORT skin and was inhibited by CBX. CORT mice treated with CBX displayed 51%, 59%, and 100% normalization of wound healing, epidermal thickness, and epidermal integrity, respectively. Gene expression studies revealed normalization of interleukin 6, keratinocyte growth factor, collagen 1, collagen 3, matrix metalloproteinase 9, and tissue inhibitor of matrix metalloproteinase 4 by CBX during wound healing. Importantly, proinflammatory cytokine expression and resolution of inflammation were unaffected by 11ß-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11ß-HSD1 inhibition can limit the cutaneous effects of GC excess, which may improve the safety profile of systemic steroids and the prognosis of chronic wounds.