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Our previous studies have shown that ginsenoside Rg1 (Rg1) exerts antidepressant-like effects in animal models of depression, accompanied by an improvement of astrocytic gap junction functions. However, whether connexin 43 (Cx43), the major connexin forming gap junctions between astrocytes, is the key regulator of Rg1-induced antidepressant-like effects is still unknown. In this study, we examine in vitro and in vivo the involvement of Cx43 in the antidepressant effects of Rg1. Corticosterone was used to establish an in vitro rat model of depression. Treatment with Rg1 1 h prior to corticosterone significantly improved the cell viability of astrocytes, which was significantly inhibited by carbenoxolone, a widely used gap junction inhibitor. Moreover, Rg1 treatment significantly ameliorated antidepressant-sensitive behaviours induced by infusion of carbenoxolone or Gap26, a selective inhibitor of Cx43, into the prefrontal cortex of the animals. Rg1 treatment increased the expression of Cx43 compared with Gap26 group. According to these results, the antidepressant-like effects of Rg1 were mainly mediated by Cx43-formed gap junctions., Competing Interests: Declaration of competing interest All the authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Peptic ulcer disease, the most common gastrointestinal disorder, is currently treated with several types of drugs, but all have severe side effects. The aim of the present study was to evaluate the gastroprotective activity of juanislamin, isolated from Calea urticifolia , in a rat model of ethanol-induced gastric lesions. Thirty minutes after orally administering a given dose of juanislamin (from 1 to 30 mg/kg) or carbenoxolone (the reference drug, at 1-100 mg/kg) to rats, 1 mL of ethanol was applied, and the animals were sacrificed 2 h later. The stomachs were removed and opened to measure the total area of lesions in each. To examine the possible participation of prostaglandins, nitric oxide and/or sulfhydryl groups in the mechanism of action of juanislamin, the rats received indomethacin, NG-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or N -ethylmaleimide pretreatment, respectively, before being given juanislamin and undergoing the rest of the methodology. Juanislamin inhibited gastric lesions produced by ethanol in a non-dose-dependent manner, showing the maximum gastroprotective effect (100%) at 10 mg/kg. The activity of juanislamin was not modified by pretreatment with indomethacin, l-NAME or N -ethylmaleimide. In conclusion, juanislamin protected the gastric mucosa from ethanol-induced damage, and its mechanism of action apparently does not involve prostaglandins, nitric oxide or sulfhydryl groups.

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone., (© 2018 John Wiley & Sons, Ltd.)

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., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.

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 β hydroxysteroid dehydrogenase type1 (11β-HSD1) was reported to have an anti-obesity effect by reducing fat mass. However, the link between the role of 11β-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 11β-HSD1 inhibitors both in vitro and in vivo. In hypothalamic neurones, carbenoxolone (a non selecitve 11β-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 11β-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 11β-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 11β-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., (© 2018 British Society for Neuroendocrinology.)

Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11β-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11β-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11β-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. 11β-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 11β-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11β-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., (Copyright © 2018 Endocrine Society.)

Metastatic spread is the leading cause for cancer-related mortality, with the lungs being a major site for metastatic seeding. Available therapies for patients with metastatic disease are extremely limited. Therefore, there is a desperate need for new strategies to prevent or limit metastatic dissemination and treat existing metastases. The metastatic cascade is highly complex and is affected by multiple factors related to both tumor cells themselves and the microenvironment in the future site of metastasis. We hypothesized that modifying the lung microenvironment by blocking central ubiquitous signals may affect metastatic seeding in the lungs. Given the high basal levels of the Receptor for Advanced Glycation End products (RAGE) in the pulmonary tissue, and its pro-inflammatory properties, we investigated the consequences of interfering with its ligand; High Mobility Group Box 1 (HMGB1). To this end, we tested the effect of Carbenoxolone, an HMGB1 antagonist, on primary tumor growth and metastatic progression in several murine tumor models. We show that antagonizing HMGB1 prevents the adhesion and colonization of cancer cells in the lungs through the reduction of their adhesion and cell-cell interaction both in vitro and in vivo. We demonstrated that these activities are mediated by downregulation of the adhesion molecule Intercellular Adhesion Molecule 1 (ICAM1) and ultimately result in reduced metastatic burden. Carbenoxolone decreases significantly lung metastases formation and can be used potentially as prophylactic therapy for metastatic diseases.

The theta rhythm discovered in the posterior hypothalamus area (PHa) differs from theta observed in the hippocampal formation. In comparison to hippocampal spontaneous theta, the theta recorded in the PHa is rarely registered, has lower amplitude, often disappears, and sometimes returns after a few minutes. These features indicate that spontaneous theta recorded in the PHa is not an appropriate experimental model to search for the correlation between PHa cell discharges and local field potential. In this paper we present standard experimental conditions necessary to record theta-related cells in the PHa in anesthetized rats. Three pharmacological agents were used in the experiments to induce PHa theta rhythm in urethanized rats: carbachol (CCH), carbenoxolone and kainic acid, which are potent enough to induce well-synchronized PHa theta. However, CCH was found to be the best pharmacological tool to induce PHa theta oscillations, due to its longest duration of action and lack of preliminary epileptogenic effects. It seems that CCH-induced theta can be the most suitable pharmacological model for experiments with the use of protocol of long-lasting recordings of PHa theta-related cell discharges., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hypothalamic endoplasmic reticulum (ER) stress is known to be increased in obesity. Induction of ER stress on hypothalamic neurons has been reported to cause hypothalamic neuronal apoptosis and malfunction of energy balance, leading to obesity. Carbenoxolone is an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor that converts inactive glucocorticoid into an active form. In addition to its metabolic effect via enzyme inhibitory action, carbenoxolone has shown anti-apoptotic activity in several studies. In this study, the direct effects of carbenoxolone on ER stress and cell death in hypothalamic neurons were investigated. Carbenoxolone attenuated tunicamycin induced ER stress-mediated molecules such as spliced XBP1, ATF4, ATF6, CHOP, and ROS generation. In vivo study also revealed that carbenoxolone decreased tunicamycin-induced ER stress in the hypothalamus. In conclusion, the results of this study show that carbenoxolone has protective effects against tunicamycin induced-ER stress and apoptosis in hypothalamic neurons, suggesting its direct protective effects against obesity. Further study is warranted to clarify the effects of carbenoxolone on hypothalamic regulation of energy balance in obesity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to suppressive negative feedback by glucocorticoids. The abnormal expression of 11beta-hydroxysteroid dehydrogenase (11HSD), which is a cortisol metabolic enzyme, is found in human and murine corticotroph adenomas. Our recent studies demonstrated that canine corticotroph adenomas also have abnormal expression of 11HSD. 11HSD has two isoforms in dogs, 11HSD type1 (HSD11B1), which converts cortisone into active cortisol, and 11HSD type2 (HSD11B2), which converts cortisol into inactive cortisone. It has been suggested that glucocorticoid resistance in corticotroph tumors is related to the overexpression of HSD11B2. Therefore it was our aim to investigate the effects of carbenoxolone (CBX), an 11HSD inhibitor, on the healthy dog's pituitary-adrenal axis. Dogs were administered 50 mg/kg of CBX twice each day for 15 days. During CBX administration, no adverse effects were observed in any dogs. The plasma adrenocorticotropic hormone (ACTH), and serum cortisol and cortisone concentrations were significantly lower at day 7 and 15 following corticotropin releasing hormone stimulation. After completion of CBX administration, the HSD11B1 mRNA expression was higher, and HSD11B2 mRNA expression was significantly lower in the pituitaries. Moreover, proopiomelanocortin mRNA expression was lower, and the ratio of ACTH-positive cells in the anterior pituitary was also significantly lower after CBX treatment. In adrenal glands treated with CBX, HSD11B1 and HSD11B2 mRNA expression were both lower compared to normal canine adrenal glands. The results of this study suggested that CBX inhibits ACTH secretion from pituitary due to altered 11HSD expressions, and is potentially useful for the treatment of canine Cushing's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by akinesia, bradykinesia, resting tremors and postural instability. Although various models have been developed to explain basal ganglia (BG) pathophysiology in PD, the recent reports that dominant beta (β) oscillations (12-30Hz) in BG nuclei of PD patients and parkinsonian animals coincide with motor dysfunction has led to an emerging idea that these oscillations may be a characteristic of PD. Due to the recent realization of these oscillations, the cellular and network mechanism(s) that underlie this process remain ill-defined. Here, we postulate that gap junctions (GJs) can contribute to β oscillations in the BG of hemiparkinsonian rats and inhibiting their activity will disrupt neuronal synchrony, diminish these oscillations and improve motor function. To test this, we injected the GJ blockers carbenoxolone (CBX) or octanol in the right globus pallidus externa (GPe) of anesthetized hemiparkinsonian rats and noted whether subsequent changes in β oscillatory activity occurred using in vivo electrophysiology. We found that systemic treatment of 200mg/kg CBX attenuated normalized GPe β oscillatory activity from 6.10±1.29 arbitrary units (A.U.) (pre-CBX) to 2.48±0.87 A.U. (post-CBX) with maximal attenuation occurring 90.0±20.5min after injection. The systemic treatment of octanol (350mg/kg) also decreased β oscillatory activity in a similar manner to CBX treatment with β oscillatory activity decreasing from 3.58±0.89 (pre-octanol) to 2.57±1.08 after octanol injection. Next, 1μl CBX (200mg/kg) was directly injected into the GPe of anesthetized hemiparkinsonian rats; 59.2±19.0min after injection, β oscillations in this BG nucleus decreased from 3.62±1.17 A.U. to 1.67±0.62 A.U. Interestingly, we were able to elicit β oscillations in the GPe of naive non-parkinsonian rats by increasing GJ activity with 1μl trimethylamine (TMA, 500nM). Finally, we systemically injected CBX (200mg/kg) into hemiparkinsonian rats which attenuated dominant β oscillations in the right GPe and also improved left forepaw akinesia in the step test. Conversely, direct injection of TMA into the right GPe of naive rats induced contralateral left forelimb akinesia. Overall, our results suggest that GJs contribute to β oscillations in the GPe of hemiparkinsonian rats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Morphine produces a state dependent learning. The hippocampus is involved in this kind of learning. Gap junctions (GJs) are involved in some of the effects of morphine and exist in different areas of the hippocampus. We investigated the effects of blocking GJ channels of the hippocampal CA1 area, by means of pre-test bilateral injection of carbenoxolone (CBX), on morphine state dependent learning, using a passive avoidance task. Post-training subcutaneous administrations of morphine (0.5, 2.5, 5 and 7.5 mg/kg) dose-dependently impaired memory retrieval. Pre-test administration of morphine (0.5, 2.5, 5 and 7.5 mg/kg) induced a state-dependent retrieval of the memory acquired under post-training morphine influence. Pre-test injections of CBX (25, 75 and 150 nM) dose dependently prevented memory retrieval by post-training (7.5 mg/kg) and pre-test (0.5, 2.5, 5, 7.5 mg/kg) injections of morphine. The results suggest that intercellular coupling via GJ channels of the hippocampal CA1 area modulates morphine state dependent learning., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Spinal glial gap junctions may play an important role in dorsal horn neuronal sensitization and neuropathic pain. In rats after an L5 spinal nerve ligation (SNL), we examined the effects of intrathecal injection of carbenoxolone (CBX), a gap junction decoupler, on neuropathic pain manifestations and on wide-dynamic range (WDR) neuronal activity in vivo. Intrathecal injection of CBX dose-dependently (0.1-50 μg, 10 μl) inhibited mechanical hypersensitivity in rats at 2-3 weeks post-SNL. However, the same doses of glycyrrhizic acid (an analogue of CBX that does not affect gap junctions) and mefloquine hydrochloride (a selective neuronal gap junction decoupler) were ineffective. Intrathecal CBX (5μg) also attenuated heat hypersensitivity in SNL rats. Further, rats did not develop tachyphylaxis to CBX-induced inhibition of mechanical hypersensitivity after repetitive drug treatments (25 μg/day) during days 14-16 post-SNL. Electrophysiological study in SNL rats showed that spinal topical application of CBX (100 μg, 50 μl), which mimics intrathecal drug administration, attenuated WDR neuronal responses to mechanical stimuli and to repetitive intracutaneous electrical stimuli (0.5 Hz) that induce windup, a short-form of activity-dependent neuronal sensitization. The current findings suggest that the inhibition of neuropathic pain manifestations by intrathecal injection of CBX in SNL rats may involve an inhibition of glial gap junctions and an attenuation of WDR neuronal activity in the dorsal horn., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Insulin resistance and associated metabolic sequelae are common in chronic kidney disease (CKD) and are positively and independently associated with increased cardiovascular mortality. However, the pathogenesis has yet to be fully elucidated. 11β-Hydroxysteroid dehydrogenase type 1 (11βHSD1) catalyzes intracellular regeneration of active glucocorticoids, promoting insulin resistance in liver and other metabolic tissues. Using two experimental rat models of CKD (subtotal nephrectomy and adenine diet) which show early insulin resistance, we found that 11βHSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11βHSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11βHSD inhibitor carbenoxolone to uremic rats for 2 wk improved glucose tolerance and insulin sensitivity, improved insulin signaling, and reduced hepatic expression of gluconeogenic and lipogenic genes. Furthermore, 11βHSD1(-/-) mice and rats treated with a specific 11βHSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11βHSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11βHSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.

Glial cells are being increasingly implicated in mechanisms underlying pathological pain, and recent studies suggest glial gap junctions involving astrocytes may contribute. The aim of this study was to examine the effect of a gap junction blocker, carbenoxolone (CBX), on medullary dorsal horn (MDH) nociceptive neuronal properties and facial mechanical nociceptive behavior in a rat trigeminal neuropathic pain model involving partial transection of the infraorbital nerve (p-IONX). p-IONX produced facial mechanical hypersensitivity reflected in significantly reduced head withdrawal thresholds that lasted for more than 3weeks. p-IONX also produced central sensitization in MDH nociceptive neurons that was reflected in significantly increased receptive field size, reduction of mechanical activation threshold, and increases in noxious stimulation-evoked responses. Intrathecal CBX treatment significantly attenuated the p-IONX-induced mechanical hypersensitivity and the MDH central sensitization parameters, compared to intrathecal vehicle treatment. These results provide the first documentation that gap junctions may be critically involved in orofacial neuropathic pain mechanisms., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Background: In models of temporal lobe epilepsy and in patients with this pathology, high frequency oscillations called fast ripples (FRs, 250-600 Hz) can be observed. FRs are considered potential biomarkers for epilepsy and, in the light of many in vitro and in silico studies, we thought that electrical synapses mediated by gap junctions might possibly modulate FRs in vivo., Methods: Animals with spontaneous recurrent seizures induced by pilocarpine administration were implanted with movable microelectrodes in the right anterior and posterior hippocampus to evaluate the effects of gap junction blockers administered in the entorhinal cortex. The effects of carbenoxolone (50 nmoles) and quinine (35 pmoles) on the mean number of spontaneous FR events (occurrence of FRs), as well as on the mean number of oscillation cycles per FR event and their frequency, were assessed using a specific algorithm to analyze FRs in intracranial EEG recordings., Results: We found that these gap junction blockers decreased the mean number of FRs and the mean number of oscillation cycles per FR event in the hippocampus, both during and at different times after carbenoxolone and quinine administration., Conclusion: These data suggest that FRs may be modulated by gap junctions, although additional experiments in vivo will be necessary to determine the precise role of gap junctions in this pathological activity associated with epileptogenesis.

Background: The exact mechanisms of morphine-induced dependence and withdrawal symptoms remain unclear. In order to identify an agent that can prevent withdrawal syndrome, many studies have been performed. This study was aimed to evaluate the effect of gap junction blockers; carbenoxolone (CBX) or mefloquine (MFQ); on morphine withdrawal symptoms in male rat. Adult male Wistar rats (225 - 275 g) were selected randomly and divided into 10 groups. All groups underwent stereotaxic surgery and in order to induce dependency, morphine was administered subcutaneously) Sc) at an interval of 12 hours for nine continuous days. On the ninth day of the experiment, animals received vehicle or CBX (100, 400, 600 μg/10 μl/rat, icv) or MFQ (50, 100 and 200 μg/10 μl/rat, icv) after the last saline or morphine (Sc) injection. Morphine withdrawal symptoms were precipitated by naloxone hydrochloride 10 min after the treatments. The withdrawal signs including: jumping, rearing, genital grooming, abdomen writhing, wet dog shake and stool weight, were recorded for 60 minutes., Results: Results showed that CBX and MFQ decreased all withdrawal signs; and the analysis indicated that they could attenuate the total withdrawal scores significantly., Conclusion: Taking together it is concluded that gap junction blockers prevented naloxone-precipitated withdrawal symptoms.

The pathogen- and damage-associated molecular patterns (for example, bacterial endotoxin and adenosine 5'-triphosphate [ATP]) activate the double-stranded RNA-activated protein kinase R (PKR) to trigger the inflammasome-dependent high mobility group box 1 (HMGB1) release. Extracellular ATP contributes to the inflammasome activation through binding to the plasma membrane purinergic P2X7 receptor (P2X7R), triggering the opening of P2X7R channels and the pannexin-1 (panx-1) hemichannels permeable for larger molecules up to 900 daltons. It was previously unknown whether panx-1 channel blockers can abrogate lipopolysaccharide (LPS)-induced PKR activation and HMGB1 release in innate immune cells. Here we demonstrated that a major gancao (licorice) component (glycyrrhizin, or glycyrrhizic acid) derivative, carbenoxolone (CBX), dose dependently abrogated LPS-induced HMGB1 release in macrophage cultures with an estimated IC50 ≈ 5 μmol/L. In an animal model of polymicrobial sepsis (induced by cecal ligation and puncture [CLP]), repetitive CBX administration beginning 24 h after CLP led to a significant reduction of circulating and peritoneal HMGB1 levels, and promoted a significant increase in animal survival rates. As did P2X7R antagonists (for example, oxidized ATP, oATP), CBX also effectively attenuated LPS-induced P2X7R/panx-1 channel activation (as judged by Lucifer Yellow dye uptake) and PKR phosphorylation in primary peritoneal macrophages. Collectively, these results suggested that CBX blocks LPS-induced HMGB1 release possibly through impairing PKR activation, supporting the involvement of PKR in the regulation of HMGB1 release.

Carbenoxolone (CEX), a semi-synthetic derivative of glycyrrhetinic acid, has previously been used as a disodium salt for the management of dyspepsia and peptic ulcer because of its anti-inflammatory properties. Although glycyrrhetinic acid is available in pharmaceutical and personal care products for skin care, the topical use of the free acid form of CEX, has not previously been reported. In this work we investigated the percutaneous penetration of CEX. Solubility and permeability studies were conducted using a range of solvents or skin permeation enhancers (SPEs) commonly used for skin delivery. Binary combinations of dimethyl isosorbide (DMI) and Transcutol™ (TC) with isopropyl myristate (IPM) were effective in promoting skin permeation of CEX although individual solvents were not. Alternative fatty acid esters to IPM were subsequently investigated with the most promising formulation consisting of TC and propylene glycol laurate (PGL). Interestingly, propylene glycol monolaurate (PGML) did not demonstrate comparable efficacy when combined with TC. A ternary formulation consisting of TC, PGL and IPM demonstrated the best permeation enhancement of CEX compared with all other vehicles. The findings confirm (i) the feasibility of promoting CEX penetration across the skin (ii) the synergistic effect of combinations of solvents and SPEs on dermal and transdermal delivery (iii) the necessity for more fundamental studies to explain the differential effects of fatty acid esters on the skin barrier., (Copyright © 2013 Elsevier B.V. All rights reserved.)

1. One of the major causes of metabolic syndrome is elevated 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in the liver and adipose tissue. High 11β-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 11β-HSD1 inhibition by carbenoxolone in db/db mice, a genetic model of diabetes. 2. Inhibition of 11β-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 11β-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 11β-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., (© 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.)

Gap junctions play a key role in maintaining the functional integrity of the vascular wall. Using carbenoxolone (CBX) as a gap junction blocker, we aimed to assess the contribution of gap junctions in the vascular wall to flow-mediated vasodilatation (FMD) in healthy adults. Percentage FMD (%FMD) and circulating vasoactive molecules/activity, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), aldosterone, cortisol, plasma renin activity (PRA), and endothelin (ET-1), were measured in 25 healthy volunteers (mean age: 30.1 ± 5.4 yr; 14 males) before and after oral administration of CBX (100 mg). %FMD decreased after ingestion of CBX (9.71 ± 3.1 vs. 3.40 ± 2.0%; P < 0.0001). The levels of ANP, BNP, cortisol, and ET-1 remained stationary, while both PRA and aldosterone decreased (P < 0.005) after CBX ingestion. Blood pressure and heart rate were minimally changed by CBX. Inhibition of gap junctional communication by CBX impairs FMD in healthy persons, suggesting that physiologically, vascular gap junctions participate in the maintenance of FMD. CBX does not induce the release of vasoconstricting molecules or enhance vasoconstriction, suggesting that inhibition of gap junctional communication by CBX underlies the impairment of FMD. Therefore, administering CBX in FMD examination can be a way to follow the effect of gap junctions on endothelial function, but further work remains to verify the specificity of CBX effect.

The objective of this study was to prepare and evaluate the pectin-based dosage form for buccal adhesion. Carbenoxolone sodium, which is used for the treatment of aphthous ulcers in oral cavity, was used as a model drug. The pectin buccal discs were prepared by direct compression. The water uptake and erosion of pectin disc increased progressively with the swelling time. The bioadhesion of dried pectin discs decreased when either the discs were hydrated or the buccal tissue was wet with a small volume of medium. The influencing factors such as pectin type, pectin to lactose ratio, and sweetener type on the formulations were investigated. The results demonstrated that buccal discs prepared from pectin with a high degree of esterification (DE) showed a weaker and more friable characteristic than that with low DE. Decreasing pectin to lactose ratio resulted in the high dissolution rate with low bioadhesive properties. Addition of sweetener in the formulations also affected the hardness, friability, and bioadhesive properties of the discs. The pectin discs containing sweetening agent showed a higher drug release than those without sweetener. The results suggested that pectin-based bioadhesive discs could be used to deliver carbenoxolone sodium in oral cavity.

Gap junction inhibition has been demonstrated to reverse the vascular contraction that follows experimental subarachnoid hemorrhage. This study hypothesizes that the use of established gap junction inhibitors: octonal and carbenoxolone, to interrupt cell to cell communication will provide neuroprotection against early brain injury after SAH. The filament perforation model of SAH was performed in male Sprague-Dawley rats weighing between 300 and 380 g. Octanol (260.46 mg or 781.38 mg/kg), carbenoxolone (100 mg/kg), or vehicles were given via intraperitoneal injection 1 h after SAH. Neurologic deficits and cerebral apoptosis were assessed 24 and 72 h after SAH. In addition, Western blot analysis was performed to confirm the in vivo inhibition of CNS gap junctions. The administration of octanol and carbenoxolone both failed to attenuate the neurological deficits induced by SAH, and they did not reduce neuronal apoptosis. Additionally, carbenoloxone increased post SAH mortality and exacerbated SAH-induced apoptosis. Despite previous studies that show gap junction inhibitors reverse vasospasm following experimental SAH, they failed to improve clinical outcomes or provide neuroprotection in this study., (2009 Elsevier B.V. All rights reserved.)

Previous experimental studies have shown the protective effects of CBX on brain ischemic injures in global and in vitro models of ischemia. However, effects of CBX in temporary model of focal cerebral ischemia are not clear. Hence, the aim of this study was to investigate the effects of central microinjection of CBX on post-ischemic reperfusion injuries in a temporary model of focal cerebral ischemia. Transient focal cerebral ischemia was induced in rats by 60 min middle cerebral artery occlusion (MCAO), followed by 23 h reperfusion. CBX was administered into the right ventricle at doses of 1, 12, 25, 50 and/or 100 microg/kg at the beginning of MCAO. Cortical and striatal infarct volumes and motor dysfunctions were assessed 24 h after MCAO. Administration of CBX at doses of 1, 12, 25 and/or 50 microg/kg significantly reduced cortical infarct volumes by 35%, 49%, 41% and 43%, respectively (P<0.001). In addition, CBX only at dose of 25 microg/kg significantly reduced striatal infarct volume and improved neurological dysfunctions (P<0.01). Our findings indicated that central microinjection of CBX has protective effect on against ischemic reperfusion injuries in a transient model of focal cerebral ischemia.

The nucleus accumbens (Acb) is a part of the striatum which integrates information from cortical and limbic brain structures, and mediates behaviors which reinforce reward. Previous work has suggested that neuronal synchrony mediated by gap junctions in Acb-related areas is involved in brain pleasure and reward. In order to gain insight into functional aspects of the neural information processing at the level of the striatum, we explored the possible role of Acb gap junctional communication and chemical synapses on reward self-stimulation in rats using positive reinforcement. Rats were trained to press a lever that caused an electrical current to be delivered into the hypothalamus, which is recognized to cause pleasure/reward. Intracerebral infusion into the Acb of the gap junctional blocker carbenoxolone (CBX) decreased the lever-pressing activity. Considering that the net effect of blocking gap junctions is a reduced synchronized output of the cellular activities, which at some level represents a decrease in excitability, two other inhibitors of neuronal excitability, carbamazepine (CBZ) and tetrodotoxin (TTX), were infused into the Acb and their effects on lever-pressing assessed. All manipulations that diminished excitability in the Acb resulted in reduced lever-pressing activity. CBX and TTX were also infused into motor cortex mediating forelimb lever-pressing with no effect. However, a manipulation that has the net effect of increasing excitation, the infusion of the opiate antagonist naloxone, also decreased significantly brain self-stimulation. We conclude that reward behaviors depend to a great extent on both excitability and gap junction-mediated mechanisms in Acb neuronal networks. Thus, the Acb provides a site for the study of pleasure/reward, addiction and conscious experience.

Carbenoxolone (CBX) has a neuroprotective effect in experimental models of brain ischemia and trauma. However, systemic effect of CBX on ischemic reperfusion injuries has not been investigated in a temporary model of focal cerebral ischemia. Male Wistar rats (n = 32) were divided into control and CBX-treated (100, 200, or 400 mg/kg, intraperitoneally) groups. Transient focal cerebral ischemia was induced by 60-minute middle cerebral artery occlusion by filament method, followed by 23-hour reperfusion. At the end of 24-hour ischemia, neurologic deficit score was tested and infarct volumes were determined using triphenyltetrazolium chloride staining. Administration of CBX (100, 200, or 400 mg/kg) at the beginning of ischemia significantly reduced cortical infarct volumes by 48%, 58%, and 63%, and striatal infarct volumes by 34%, 63%, and 63%, respectively. Nevertheless, CBX has no effect on neurologic dysfunction. Our findings indicated that peripheral administration of CBX has a neuroprotective effect on postischemic damage in a temporary model of focal cerebral ischemia in rat.

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., (2008 S. Karger AG, Basel.)

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).

We have examined the metabolic effects of daily administration of carbenoxolone (CBX), a naturally occurring 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) inhibitor, in mice with high fat diet-induced insulin resistance and obesity. Eight-week-old male Swiss TO mice placed on a synthetic high fat diet received daily intraperitoneal injections of either saline vehicle or CBX over a 16-day period. Daily administration of CBX had no effect on food intake, but significantly lowered body weight (1.1- to 1.2-fold) compared to saline-treated controls. Non-fasting plasma glucose levels were significantly decreased (1.6-fold) by CBX treatment on day 4 and remained lower throughout the treatment period. Circulating plasma corticosterone levels were not significantly altered by CBX treatment. Plasma glucose concentrations of CBX-treated mice were significantly reduced (1.4-fold) following an intraperitoneal glucose load compared with saline controls. Similarly, after 16-day treatment with CBX, exogenous insulin evoked a significantly greater reduction in glucose concentrations (1.4- to 1.8-fold). 11beta-HSD1 gene expression was significantly down-regulated in liver, whereas glucocorticoid receptor gene expression was increased in both liver and adipose tissue following CBX treatment. The reduced body weight and improved metabolic control in mice with high fat diet-induced obesity upon daily CBX administration highlights the potential value of selective 11beta-HSD1 inhibition as a new route for the treatment of type 2 diabetes and obesity.

Amygdala plays an important role in induction and control of limbic seizures. There is a network of gap junctional communications in basolateral amygdala (BLA) as well. We compared the effect of intra-BLA infusion of the typical gap junction (GJ) blocker carbenoxolone (CBX), with the effect of putative GJ blocker all-trans retinoic acid (ATRA), on the afterdischarge (AD) recorded from BLA in amygdala-kindled rats. Both CBX and ATRA showed a rapid anti-seizure effect. Conversely, intra-BLA infusion of the known GJ opener, trimethylamine (TMA), enhanced seizure susceptibility by prolonging the duration of AD and generalized behavioral seizures. ATRA administered intra-BLA prevented the proconvulsant effect of TMA. The reduction of gap junctional conductance might be involved in the observed anticonvulsant effect of ATRA.

The protective effect of beef and pig collagen hydrolysates and their fractions were tested as anti-ulcerogenic agents in rats (weighing 250-350 g) against ulcerative lesions caused by ethanol. Beef and pig collagen hydrolysates were fractionated by ultrafiltration into different molecular weight fractions. The protocol employed a negative and a positive control and a single dose of the experimental samples given by intragastric intubation. The beef collagen did not present a dose-response correlation in the ethanol model, whereas pig collagen showed a logarithmic dose-response relationship. Beef collagen hydrolysate decreased the ulcerative lesion index of 55% versus a 61% decrease for pig collagen hydrolysate at the same dosage (750 mg/kg of body weight). No significant differences were found (P > .05) between the hydrolysates and their fractions.

Context: The pathophysiological importance of glucocorticoids (GCs) is exemplified by patients with Cushing's syndrome who develop hypertension, obesity, and insulin resistance. At a cellular level, availability of GCs to the glucocorticoid and mineralocorticoid receptors is controlled by the isoforms of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). In liver and adipose tissue, 11beta-HSD1 converts endogenous, inactive cortisone to active cortisol but also catalyzes the bioactivation of the synthetic prednisone to prednisolone., Objective: The objective of the study was to compare markers of 11beta-HSD1 activity and demonstrate that inhibition of 11beta-HSD1 activity limits glucocorticoid availability to adipose tissue., Design and Setting: This was a clinical study., Patients: Seven healthy male volunteers participated in the study., Intervention: Intervention included carbenoxolone (CBX) single dose (100 mg) and 72 hr of continuous treatment (300 mg/d)., Main Outcome Measures: Inhibition of 11beta-HSD1 was monitored using five different mechanistic biomarkers (serum cortisol and prednisolone generation, urinary corticosteroid metabolite analysis by gas chromatography/mass spectrometry, and adipose tissue microdialysis examining cortisol generation and glucocorticoid-mediated glycerol release)., Results: Each biomarker demonstrated reduced 11beta-HSD1 activity after CBX administration. After both a single dose and 72 hr of treatment with CBX, cortisol and prednisolone generation decreased as did the urinary tetrahydrocortisol+5alpha-tetrahydrocortisol to tetrahydrocortisone ratio. Using adipose tissue microdialysis, we observed decreased interstitial fluid cortisol availability with CBX treatment. Furthermore, a functional consequence of 11beta-HSD1 inhibition was observed, namely decreased prednisone-induced glycerol release into adipose tissue interstitial fluid indicative of inhibition of GC-mediated lipolysis., Conclusion: CBX is able to inhibit rapidly the generation of active GC in human adipose tissue. Importantly, limiting GC availability in vivo has functional consequences including decreased glycerol release.

Purpose: Epilepsy is the most common serious neurologic disease, and current treatments are ineffective for or=1 h before drug infusion and for >or=3 h afterward. No ill effects were observed., Results: An immediate and marked reduction in percentage of seizure time was seen in rats receiving carbenoxolone (baseline, 69.4%+/- 7.0% (SEM); maximum effect, 9.3%+/- 3.5%, p

The present study was undertaken to investigate whether gap junctional communication could be involved in morphine-induced antinociceptive response using blockers of the gap junctional channel, carbenoxolone and Gap27. Intrathecal pretreatment with either carbenoxolone or Gap27 caused a dose-dependent attenuation of morphine-induced antinociception. Furthermore, the dose-response line for morphine-induced antinociception was shifted to the right by 2.53-fold following intrathecal treatment with carbenoxolone. These findings suggest that gap-junctional-dependent communication in the mouse spinal cord may play, at least in part, a role in the expression of morphine-induced antinociception.

Background: Carbenoxolone (CBX) is a widely used gap junctional blocker. Considering several reports indicating that transient gap junctional blockade could be a favourable intervention following injuries to central nervous tissue, and some current enthusiasm in studies using systemic injections of CBX, it is imperative to consider the penetration of CBX into central nervous tissue after systemic administrations. So far, only very indirect evidence suggests that CBX penetrates into the central nervous system after systemic administrations. We thus determined the amounts of CBX present in the blood and the cerebrospinal fluid of rats after intraperitoneal administration, using high performance liquid chromatography., Results: CBX was found in the blood of the animals, up to 90 minutes post-injection. However, the cerebrospinal fluid concentration of CBX was negligible., Conclusion: Thus, we conclude that, most likely, CBX does not penetrate the blood brain barrier and therefore recommend careful consideration in the manner of administration, when a central effect is desired.

As carbenoxolone, a semisynthetic derivative of glycyrrhizinic acid, has a free radical scavenging property, thus the effects of carbenoxolone during ischemia-reperfusion was evaluated on an animal model of ischemia-reperfusion injury in the rat hind limb and hippocampus. Peripheral and central ischemia were induced by free-flap surgery in skeletal muscle and four-vessel-occulation (4VO) of rat, respectively. Carbenoxolone (50-200 mg/kg) and normal saline (10 ml/ kg) were administered intraperitoneally. In peripherlal ischemia, during preischemia, ischemia and reperfusion conditions the electromyographic (EMG) potentials in the muscles were recorded. The malondialdehyde (MDA) was measured by the thiobarbituric acid (TBA) test after reperfusion in peripheral and central ischemia. In peripheral ischemia, the average peak-to-peak amplitude during ischemic-reperfusion was found to be significantly larger in carbenoxolone group (100-200mg/kg) in comparison to control group. The MDA levels were recovered significantly upon carbenoxolone (100-200 mg/kg) therapy in the skeletal muscle and hippocampus of ischemic rats. These results suggest that carbenoxolone can salvage the skeletal muscle and hippocampus from acute ischemia-reperfusion injury.

This investigation evaluated the gastroprotective activity of Astragaloside IV, a cycloartane-type triterpene glycoside isolated from Astragalus zahlbruckneri. Gastric mucosal damage was induced in rats by intragastric ethanol (1 mL/rat). Rats treated orally with Astragaloside IV suspended in Tween 80 at 3, 10 and 30 mg kg(-1), showed 15, 37 and 52% gastroprotection, respectively. The gastroprotection observed at 30 mg kg(-1) for this compound was attenuated in rats pretreated with N(G)-nitro-L arginine methyl ester (70 mg kg(-1), i.p), a nitric oxide (NO)-synthase inhibitor, suggesting that the gastroprotective mechanism of this glycoside involves, at least in part, the participation of NO. The gastroprotective effect of Astragaloside IV was not affected by the inhibition of prostaglandin synthesis with indometacin (10 mg kg(-1), s.c.) nor by the block of endogenous sulfhydryls with N-ethylmaleimide (NEM, 10 mg kg(-1), s.c.). Carbenoxolone was used as a gastroprotective model drug and showed a dose-dependent gastroprotective effect (25, 43 and 88% of gastroprotection, at 3, 10 and 30 mg kg(-1), respectively). The partial participation of prostaglandins, sulfhydryls and NO was observed in the gastroprotective mechanism of carbenoxolone.

Background: High-frequency oscillations (ripples) are proposed to be involved in memory consolidation. Ripple oscillations persist in the absence of chemical synaptic transmission and inhibitors of gapjunctions were shown to block high-frequency oscillations in hippocampal slices. In this study we investigated the effects of carbenoxolone, as a gap-junction blocker, on spatial learning in the rat. Cognitive performance was assessed using the Morris water maze (MWM)., Material/methods: Adult male rats were bilaterally cannulated into the CA1 region of their hippocampus. Carbenoxolone (1.23, 2.46, 4.92 microg/side), scopolamine (3 microg/side), and saline were injected through the cannulas before 4 consecutive training trials and one test trial that conducted 24 h after the training trials with the platform removed., Results: The results showed that carbenoxolone could significantly reduce the percentages of time spent, distance traveled, and entry into Q2 (the target quadrant) compared with the control group. The carbenoxolone group failed to use a selective search strategy during the final test trial, in which the platform was removed., Conclusion: The results of this study suggest that carbenoxolone as a gap-junction channel blocker can decelerate the learning performances of rats in a spatial memory task, such as the MWM, that requires the integrity of the hippocampus.

Purpose: The human eye is an important target tissue for steroid hormones, and glucocorticoids have been implicated in the pathogenesis of ocular disease, including glaucoma. In peripheral tissues, corticosteroid hormone action is regulated at a prereceptor level through the activity of the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) isozymes: an oxo-reductase (11 beta-HSD1) that activates cortisol (F) from cortisone (E) and a dehydrogenase (11 beta-HSD2) that inactivates F to E. The purpose of this study was to analyze the expression and putative role of 11 beta-HSD within the human eye., Methods: Immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) studies were performed on sections of human ocular tissues, surgical trabecular meshwork (TM) specimens and a ciliary nonpigmented epithelial (NPE) cell-line. Free F and E concentrations in aqueous humor were determined by gas chromatography-mass spectrometry (GC/MS). IOP was measured in eight male volunteers before and after oral ingestion of carbenoxolone (CBX), a known inhibitor of 11 beta-HSD., Results: 11 beta-HSD1 was expressed in the basal cells of the corneal epithelium and the NPE. 11 beta-HSD2 was restricted to the corneal endothelium. RT-PCR revealed mRNA for only the glucocorticoid receptor (GR) in the TM specimens, whereas GR, mineralocorticoid receptor and 11 beta-HSD1 mRNAs were all present in the NPE cell line. The demonstration of free F in excess of E (F/E 14:1) in the aqueous humor suggested predominant 11 beta-HSD1 activity. Compared with baseline (14.7 +/- 1.06 mm Hg, mean +/- SD), the IOP decreased significantly on both the third and seventh days of CBX ingestion (12.48 +/- 1.11 mm Hg, P < 0.0001 and 11.78 +/- 1.50 mm Hg, P < 0.0001, respectively)., Conclusions: These results suggest that the 11 beta-HSD1 isozyme may modulate steroid-regulated sodium transport across the NPE, thereby influencing IOP.

The fetal environment is now recognized as a key determinant of the adult phenotype, being linked to development of diseases, including hypertension, as well as the timing of puberty. Such links may be related, in part, to the level of fetal exposure to maternal glucocorticoids in utero, which is normally regulated by placental expression of the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). The present study examined whether manipulation of fetal glucocorticoid exposure, either directly or indirectly via 11beta-HSD inhibition, influences the subsequent timing of puberty. Administration of dexamethasone acetate at low (LDEX, 0.25 microg/ml drinking water) or high doses (HDEX, 1 microg/ml) or carbenoxolone (CBX, 2 x 10 mg/day, sc; an inhibitor of 11beta-HSD) to pregnant rats from day 13 to term (day 23) reduced offspring birthweight (LDEX: 9%; HDEX: 27%; CBX: 8%) and resulted in a subsequent delay in the onset of puberty in females (control: 41.4 +/- 0.5; LDEX: 44.8 +/- 0.7; HDEX: 48.5 +/- 0.4; CBX: 43.6 +/- 0.5 days). Importantly, the effects of CBX were not observed in the absence of maternal adrenals, indicating that they were mediated by increased fetal exposure to endogenous maternal glucocorticoids. In contrast, maternal treatment with metyrapone (MET; an inhibitor of glucocorticoid synthesis; 500 microg/ml drinking water from day 13) increased birthweight by 5% and advanced puberty onset in male offspring (control: 48.8 +/- 1.0; MET: 45.7 +/- 0.8 days). Changes in the timing of puberty onset were not attributable to changes in either bodyweight at puberty or peripubertal plasma leptin concentrations. Peripubertal plasma LH was also unaffected in animals with delayed puberty but was elevated in male offspring of MET-treated mothers. Collectively, these results demonstrate that fetal glucocorticoid exposure is an important determinant of the timing of puberty onset in postnatal life, and that this effect is operable within the normal physiological range of glucocorticoid concentrations.

Recent human epidemiological studies have linked low birth weight with a substantially increased risk of non-insulin-dependent diabetes mellitus in later life. These data suggest that the intrauterine environment plays a crucial role in determining later glucose homeostasis, but the mechanism is unknown. We have proposed that exposure of the fetus to excess maternal glucocorticoids may underpin the epidemiological findings. Normally placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD-2) protects the fetus from the normally higher maternal levels of glucocorticoids by inactivating corticosterone and cortisol to inert 11-keto products. Here we show that administration of carbenoxolone, an inhibitor of placental 11 beta-HSD 2, to pregnant rats, leads to a significant reduction in average birth weight (20% fall). At 6 months of age, the male offspring of carbenoxolone-treated pregnancies had similar weights to controls, but showed significantly higher fasting plasma glucose (6.0 +/- 0.3 vs 4.8 +/- 0.2 mmol/l; p < 0.01) and exhibited significantly greater plasma glucose (10% higher) and insulin (38% higher) responses to an oral glucose load. These effects of carbenoxolone require intact maternal adrenal glands suggesting that inhibition of feto-placental 11 beta-HSD 2 is key. These data support the notion that deficiency of placental 11 beta-HSD, by exposing the fetus to excess maternal glucocorticoids, reduces growth and predisposes to hyperglycaemia in later life.

There is strong evidence from different types of studies, including the discrete infusion of agonists and antagonists and ablation of specific brain areas or transmitter-type neurons, that mineralocorticoids, in excess, act in the brain to elevate blood pressure. Aldosterone enhances the entry of Na+ through amiloride-sensitive Na+ channels in some mineralocorticoid-sensitive transport epithelial cells. To define possible cellular mechanisms involved in central mineralocorticoid action, benzamil, an amiloride analogue with selective affinity for the Na+ channel, was continuously infused intracerebroventricularly in three mineralocorticoid-dependent hypertension models in Sprague-Dawley rats, the continuous subcutaneous infusion of aldosterone, the intracerebroventricular infusion of aldosterone, and the ingestion of carbenoxolone, a synthetic licorice analogue. The intracerebroventricular infusion of 0.3 and 0.5 micrograms/h of benzamil, doses that did not have an adverse effect on growth and that had no effect on the blood pressure when infused subcutaneously, prevented the increase in blood pressure in these models. The infusion of these levels of benzamil had no effect on urine volume even in those animals in which it prevented an increase in blood pressure. These data suggest that the central effects of mineralocorticoids on blood pressure are mediated, at least in part, by the effects of mineralocorticoids on amiloride-sensitive sodium transport.

Pseudohypoaldosteronism (PHA), characterized by congenital resistance to aldosterone and excessive salt loss, has been traditionally treated with salt replacement. Although the mineralocorticoid receptor (MR) has been suggested as a potential locus of the defect in this disease, no such abnormality has been identified as yet. We studied a 17-yr-old male patient with congenital multifocal target organ resistance to aldosterone. Both carbenoxolone, an 11 beta-hydroxysteroid dehydrogenase inhibitor, and a high dose of fludrocortisone normalized the patient's serum electrolyte concentrations and decreased his urinary excretion of sodium, suggesting that this patient's resistance was partial and could be overcome by high concentrations of endogenous or exogenous mineralocorticoids. We hypothesized that the beneficial effect of these treatments was mostly mediated by the MR, because the administration of dexamethasone, while this patient was receiving a therapeutic dose of carbenoxolone, caused its reversal. These findings convinced us that there was functional, albeit possibly defective, MR in this patient and led us to perform molecular studies. Both alleles of the MR gene were expressed in the patient and his clinically and biochemically normal father. A conservative heterozygous mutation (A760-->G760, Ileu180-->Val180) and a nonconservative homozygous mutation (C944-->T944, Ala241-->Val241) were identified in the complementary DNA of both the patient and his father. The first untranslated exon and 0.9 kilobase of the 5'-regulatory region were also identical in the two men. It appears that the mutations causing amino acid substitutions represent polymorphisms, as we found high frequencies of both in the general population. We conclude that carbenoxolone and fludrocortisone may help define the presence of functional MR in patients with PHA and that the former could be used in the long term therapy of this disease. We hypothesize that the defect causing PHA in this patient might be in a post-MR step of aldosterone action.

Carbenoxolone (CX), the succinyl ester of glycyrrhetinic acid, causes hypokalemia and hypernatremia. Its pharmacological effects are believed to be due to its inhibition of 11 beta-hydroxysteroid dehydrogenase (11-HSD). There was a marked inhibition of this enzyme in the liver, kidney, pituitary, hippocampus, hypothalamus and amygdala 1 h after intraperitoneal administration of CX (100 mg kg-1) to intact male rats. Intracerebral injection of CX (1.5 mg kg-1) into the 3rd ventricle inhibited the oxidation of corticosterone to 11-dehydrocorticosterone by 11-HSD in the pituitary and hippocampus and produced marked behavioral hyperactivity but had no effect in the liver or kidney. Lower amounts of CX (10-50 micrograms/rat) given intracerebroventricularly (i.c.v) were without significant effect on 11-HSD in the pituitary or amygdala 1 h after infusion but inhibited this enzyme differentially in the hippocampus and hypothalamus. Inhibition of 11-HSD activity in the hippocampus and hypothalamus was observed up to 6 h after i.c.v. administration of CX (50 micrograms/rat) together with some decrease in activity of this enzyme in the pituitary at 3 h. The findings that low doses of CX given i.c.v. can alter the activity of 11-HSD in specific brain regions without affecting its activity in peripheral tissues, and only marginally in the pituitary, provides a method to study the central role of this enzyme independently of systemic effects.

The in vivo effect(s) of carbenoxolone (CS) on renal 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD), hepatic 11 beta-OHSD, and 5 beta-reductase enzymatic activity was investigated, under conditions previously shown to confer mineralocorticoid (MC)-like activity on the glucocorticoids cortisol and corticosterone; it has been suggested that this Na+ retention is linked to inhibition of renal 11 beta-OHSD. The results show that acute administration of CS [2.5 mg/rat for 0.5 or 2 hours; and 10 or 25 mg/rat for 2 hours subcutaneously (sc)] to rats caused no inhibition of 11 beta-OHSD activity in kidney homogenates, minces, and microsomes when compared with controls. However, addition of 50 nM CS to the incubation medium completely inhibited the 11 beta-OHSD activity in kidney homogenates and microsomes (from controls or CS-injected rats). In contrast, hepatic microsomal 11 beta-OHSD was significantly inhibited after in vivo treatment with CS (P < 0.05) using 2 microM and 50 microM corticosterone, as was 5 beta-reductase (P < 0.05) using 4 microM corticosterone as substrate. However, chronic glycyrrhizin administration (15 mg/rat/day sc for 14 days) significantly inhibited renal 11 beta-OHSD activity when assayed in minces or homogenates. Thus, it appears that when CS is administered acutely, its effects are primarily on hepatic 11 beta-OHSD and 5 beta-reductase with no inhibition of renal 11 beta-OHSD.(ABSTRACT TRUNCATED AT 250 WORDS)

The normal urothelium is covered by a glycosaminoglycan (GAG) layer which acts as a barrier to the adhesion of crystals. Destruction of the GAG layer increases the number of adhered crystals, and it is therefore assumed that it promotes crystal growth and stone formation. Intravesical instillation of pentosanpolysulfate, an exogenous glycosaminoglycan, after destruction of this layer reduces the adhesion of crystals to the urothelium. Intramuscular administration of carbenoxolone sodium following the experimental destruction of the GAG layer increases the rate of healing of the layer and reduces the number of adhered crystals.

The effect of carbenoxolone on the output of salivary electrolytes stimulated by a nasogastric tube was studied in healthy volunteers. Mean +/- SE values for volume and electrolyte concentrations in control studies were volume 121 +/- 19 ml/hr, pH7.39, Na+19 +/-5, K+20 +/- U, Cl-20 +/- 3, HCO3-18 MEq/liter and osmolality 71 +/- 8 mOsm/kg. Mean +/- electrolyte outputs were quite large, Na+ 2.4, k+2.4, Cl-26, and bicarbonate 2.2 mEq/hr. Our control data indicate that in the course of gastric secretory testing the indwelling gastric tube stimulates salivary electrolyte output sufficient to interfere with accurate assessment of gastric ionic fluxes and gastric acid analysis. Therapeutic doses of carbenoxolone (100 mg three times daily) given orally for 3 weeks had no effect on salivary volume, electrolyte output, or concentration. We conclude that carbenoxolone has no minerallocorticoid effect on mixed salivary secretion. Attention to salivary secretion is recommended in all gastric secretory studies and is mandatory where low acid output is suspected.

Carbenoxolone sodium has been shown to accelerate the rate of healing of both gastric and duodenal ulcers, but its overall value in duodenal ulcer is probably less because of the high rate of natural remission of duodenal ulcers. Further studies are required to decide whether it should be used prophylactically to delay ulcer recurrence. Carbenoxolone may act by affecting both the proliferative activity of gastric epithelium and the differentiation of the epithelial cells to produce mucus (as well as favourably altering the physicochemical properties of mucus and by reducing peptic activity), factors which may be relevant ot the prevention of acute gastric ulcers. Some studies suggest that carbenoxolone adds to the effect of hospitalisation and bed rest on ulcer healing. Whether bed rest confers additional benefit to the drug's ulcer healing effect in outpatients is also uncertain. There is no evidence that accelerated healing by carbenoxolone is associated with improved overall prognosis. Carbenoxolone is of greatest benefit in accelerating the healing of gastric ulcers in patients for whom hospitalisation is not possible or desirable, but it should only be used in the ambulatory patient when careful and regular observation of serum electrolytes (particularly potassium), blood pressure and weight is possible and when it is known that the patient will attend regular follow-up. Patient must be educated in the proper use of the drug. If severe mineralocorticoid-like toxic effects such as sodium and water retention and hypokalaemia appear, as they do in a variable proportion of patients but most frequently in those receiving excessive doses, carbenoxolone should be stopped and the complication treated; they respond to thiazide diuretics and potassium supplements, and probably to amiloride given in conjunction with a low dose of a thiazide diuretic. Treatment with carbenoxolone can continue with concurrent diuretic therapy in patients with less severe side-effects. Optimum therapeutic effect in gastric ulcer with the least side-effects is achieved with a dosage of 100mg carbenoxolone tablets 3 times daily for the first week followed by 50mg 3 times daily thereafter, best taken before meals. A lower dosage is desirable in the elderly and in those with liver, cardiac or renal disease. Barium meal or preferably endoscopic examinations should be performed regularly and therapy continued until the ulcer is healed. Dosage for duodenal ulcer is 50mg 4 times daily, in special positioned-release capsules. These are best taken about 20 minutes before meals.