Gap junction inhibitors modulate S100B secretion in astrocyte cultures and acute hippocampal slices.

Astrocytes sense, integrate, and respond to stimuli generated by neurons or neural injury; this response involves gap junction (GJ) communication. Neuronal vulnerability to injury increased when cocultures of astrocytes and neurons were exposed to GJ inhibitors. However, GJ uncoupling could limit the extension of a lesion. We investigated a possible link between GJ communication and S100B secretion. S100B is a calcium-binding protein of 21 kDa that is predominantly expressed and secreted by astrocytes, which has trophic paracrine activity on neurite growth, glial proliferation, and neuronal survival. GJ inhibitors were analyzed in isolated astrocytes in primary cultures from hippocampus, acute hippocampal slices, and C6 glioma cells, which were used as a negative control. Our data indicate that GJ blocking stimulates S100B secretion in astrocyte cultures and acute hippocampal slices. Different assays were used to confirm cell integrity during exposure to GJ inhibitors. S100B secretion was observed with different types of GJ inhibitors; the resulting event was dependent on time, the nature of the inhibitor, its putative molecular target of GJ blocking, and/or the cell preparation used. Only carbenoxolone induced a fast and persistent increase in S100B secretion in both preparations. Endothelin-1 increased S100B secretion in astrocyte cultures at 1 hr, but a decrease was observed at 6 hr or in acute hippocampal slices. Physiologically, a local GJ closure associated with release of S100B in injury conditions favors the idea of a common mechanism available to limit the extension of lesion and increase the chances of cell survival.

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11beta-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11beta-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18beta-glycyrrhetinic acid (18beta-GA) derivatives (2-5) and their inhibitory activities against rat hepatic11beta-HSD1 and rat renal 11beta-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds' ability to inhibit human 11beta-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18beta-GA derivatives 2 and 3 with apparent selectivity for rat 11beta-HSD1 showed a high percentage inhibition for human microsomal 11beta-HSD1 at 10 microM and exhibited IC50 values of 400 and 1100 nM, respectively. The side chain modified 18beta-GA derivatives 4 and 5, although showing selectivity for rat 11beta-HSD1 inhibited human microsomal 11beta-HSD1 with IC50 values in the low micromolar range.

Anti-ulcer activity of carbenoxolone and ISF 3401 on PGE2 release in rat gastric mucosa.

We have investigated the ability of carbenoxolone and ISF 3041, a new carbenoxolone derivative, to protect the rat gastric and intestinal mucosa against lesions induced by acetylsalicyclic acid (ASA) and indomethacin. Moreover, we determined the capacity of the rat gastric mucosa to release PGE2 both in vitro and ex vivo, in the presence or absence of carbenoxolone or its analogs. These compounds are effective against lesions induced by ASA and intestinal damage induced by indomethacin. The amount of PGE2 obtained from incubated rat gastric mucosal pieces by in vitro and ex vivo indicate that carbenoxolone and ISF 3401 cause a concentration related increase of PGE2 with exception of the highest concentration. Increased prostaglandin content of gastric mucosa can partly explain the gastric and intestinal protection of these compounds and additional mechanisms could be involved in this action.

The effect of cicloxolone sodium on the replication of vesicular stomatitis virus in BSC-1 cells.

The effect of cicloxolone sodium (CCX) on the replication of vesicular stomatitis virus (VSV) was investigated. The drug was active during all stages of the virus replication cycle, indicating that it does not operate by the specific inhibition of any single essential virus gene product. The drug reduced the number of VSV particles assembled and released by 100- to 1000-fold. Infectious virus yield was reduced 1000- to 10000-fold, giving a 10-fold or greater increase in the particle/p.f.u. ratio. The reduced number of virus particles produced in the presence of CCX results from two superimposed effects: suppression of VSV secondary transcription and viral protein synthesis, and perturbation of virion assembly. The inhibition of VSV assembly is due to impairment of a Golgi apparatus function related to transport of VSV glycoprotein G to the cell surface, and is characterized by accumulation of viral G and M proteins within the cell. Incubation of VSV-infected cells in the presence of two glycosylation inhibitors, tunicamycin and monensin, similarly leads to intracellular accumulation of G and M proteins, suggesting a common mechanism of action affecting VSV virion assembly. The differential effect of CCX concentration on intracellular levels of the L, N and NS proteins was analysed. CCX also possesses a virucidal effect on mature infectious VSV particles in suspension, 300 microM reducing the VSV titre about 10-fold in 24 h at 4 degrees C or 37 degrees C. The mode of antiviral activity against VSV is compared with that against herpes simplex virus.

The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type 1, two bunyaviruses and Semliki Forest virus.

The effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (less than 50 microM) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (greater than or equal to 100 microM) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found.

The difference in sensitivity to cicloxolone sodium between herpes simplex virus types 1 and 2 maps to the locations of genes UL22 (gH) and UL44 (gC).

Cicloxolone sodium (CCX) is a broad spectrum antiviral agent which has a largely non-specific and complex mode of antiviral action. However, the experimental finding that herpes simplex virus type 2 (HSV-2) (strain HG52) is consistently more sensitive to inhibition by CCX than HSV-1 (117 syn+) additionally implies the specific involvement of HSV genes. HSV-1/HSV-2 intertypic recombinants have been utilized to investigate this genetic difference by comparing their CCX ED50 concentrations. No short stretch of HSV-2 DNA was associated with its greater sensitivity to CCX, implying that two or more non-contiguously located HSV genes are involved. Correlating CCX sensitivity with recombinant virus genome structures allowed separate evaluation of the gene regions encoding glycoproteins gB, gC, gD, gE, gG, gH and gI and this suggests that the gene locations encoding gH and gC determine the CCX sensitivity difference. The selective inhibitor of Golgi apparatus glycosylation, monensin, gave results analogous to those obtained with CCX.

Cardiovascular action of a new carbenoxolone derivative.

The circulatory effects of oleanoic acid sodium hydrogen succinate (OSS), an analogue of the anti-ulcer drug, carbenoxolone, were investigated. As carbenoxolone produces such adverse effects as sodium retention and a subsequent elevation of the arterial blood pressure in man, the present study was aimed at determining whether OSS is similar or different from it in this respect. Carbenoxolone, (43,3 mg/kg po) and OSS (66,6 mg/kg po) were given to rats twice daily for 4 weeks. The systolic blood pressure was elevated already after the first week of treatment. The hypertension was accompanied by bradycardia, which increased with the time of treatment. In the blood an increase in the creatinine level, a decrease in the urea level, and a slight elevation in sodium concentration were found after the treatment, while the potassium concentration during the whole treatment period (4 weeks) remained unchanged. Although the principal aldosterone-like effects of carbenoxolone were attributed to the oxygen presence in position 11 of the glycyrrhetinic acid ring, [8], the absence of an oxygen at that position in OSS did not cause the loss of the adverse circulatory effect.

The effect of triterpenoid compounds on uninfected and herpes simplex virus-infected cells in culture. III. Ultrastructural study of virion maturation.

Electron microscope studies been made of mock-infected or herpes simplex virus (HSV) type 1 (strain 17)- or HSV-2(strain HG52)-infected Flow 2002 cells grown in the absence or presence of various concentrations of cicloxolone sodium (CCX). Fifty non-serial, thin sections of mock-infected or HSV-infected cells, which contained a portion of the nucleus, were examined by transmission electron microscopy. With increasing drug concentration, counts of capsid structures both in the nucleus and cytoplasm showed a decrease in the number of virions per cell section, an increase in the ratio of nuclear to cytoplasmic virus, a relative reduction in the percentage of cytoplasmic enveloped virus particles, but no effect on the ratio of empty to core-containing capsid structures. The high particle to p.f.u. ratio induced by CCX treatment is thus not explained through failure to assemble morphologically mature core-containing capsids. In part it can be explained by non-envelopment, but in addition, specific effects on other virion proteins (tegument and envelope) must be involved. The extracellular virus particle yield was unaffected, indicating that CCX treatment enhances the egress of HSV. In the presence of CCX the encapsidation of HSV DNA into DNase-resistant structures was unaffected.

The antiviral activity against herpes simplex virus of the triterpenoid compounds carbenoxolone sodium and cicloxolone sodium.

Dose-response experiments show that the presence of 300 microM cicloxolone sodium (CCX) or 500 microM carbenoxolone sodium (CBX) during the HSV replication cycle reduced the infectious virus yield by 10,000- to 100,000-fold: CCX is the more potent anti-herpes agent. HSV-2 replication was consistently more severely restricted by either drug than was that of HSV-1. The ED50 values obtained for either drug against HSV-1 or HSV-2 correlate well with data from dose-response curves. CCX, and to a lesser extent CBX, can be cytotoxic but the degree of cytotoxicity varies between cell lines and is also affected by the physiological state of the cells. Triterpenoid drugs exhibit some activity against virus particles in suspension but the effect is small and contributes little to the overall antiviral effect. The drugs appear to be active throughout the replication cycle. In contrast to all other anti-herpesvirus agents in clinical use the triterpenoid compounds do not appear to act directly to block virus DNA synthesis. HSV mutants resistant to ACG and PAA, or lacking a thymidine kinase gene, appear as sensitive as wild-type virus to CCX inhibition. HSV growth in the presence of the drugs resulted in a lower number of assembled virus particles but reduced to a much greater extent the infectious virus yield: thus the progeny virus quality is greatly diminished. Thermolability of progeny virus correlated well with this diminution of quality in increasing CCX concentration. SDS PAGE analysis of the structural proteins of virus particles made in cells treated with 300 microM CCX revealed numerous differences in the relative intensities of protein bands, which is in keeping with the changed quality of the drug-produced virus. SDS PAGE analysis of the polypeptides induced in drug treated infected cells revealed two effects; some polypeptides were synthesised in reduced amounts and the nuclear/cytoplasmic distribution of certain proteins was affected. Post-translational processing by glycosylation and sulphation of both cellular and HSV induced proteins was strongly inhibited by the triterpenoid drugs, while phosphorylation of only a few polypeptides appeared to be affected.

The effect of triterpenoid compounds on uninfected and herpes simplex virus-infected cells in culture. II. DNA and protein synthesis, polypeptide processing and transport.

The effect of the triterpenoid drugs carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) on DNA and protein synthesis in uninfected and herpes simplex virus (HSV)-infected BHK-21 or Flow 2002 cells has been studied. No consistent effect of 500 microM-CBX or 300 microM-CCX on HSV DNA synthesis was identified. With 300 microM-CCX, some inhibition of cellular DNA synthesis was observed, but this was relatively slight. The restriction enzyme digest profiles of HSV DNA from drug-treated cells appeared normal. The synthesis of several HSV-specified polypeptides was much reduced by treatment with CBX or CCX and the transport of certain proteins between the nuclear and cytoplasmic compartments of infected cells was also affected. CBX or CCX treatment strongly inhibited post-translational glycosylation and sulphation of both host- and HSV-specified proteins, but the phosphorylation of only a few proteins appeared affected. The drugs induced quantitative changes in the synthesis of some BHK cell polypeptides, but these were not considered important. CCX treatment of Flow 2002 cells, however, induced the synthesis of several new polypeptides, some of which had the same apparent molecular weights as identified Flow 2002 cell stress proteins. When treated with concentrations greater than 50 microM-CCX, the plasma membranes of both uninfected and HSV-infected cells became increasingly leaky. The SDS-PAGE polypeptide profile of purified virus particles made in BHK cells treated with 300 microM-CCX differed markedly from that synthesized under drug-free conditions. The greatly reduced amount of infectious virus made in cells treated with 300 microM-CCX was more thermolabile at 42 degrees C than virus produced in the absence of the drug. Our results indicate that the antiviral activity of the triterpenoid drugs is non-specific and operates by interfering with or changing the normal function of cell membranes so that cells, although retaining their viability, can no longer assemble the virus components efficiently into infectious particles. As a consequence, the population of virus particles made is of inferior quality. While we have no evidence that there is also a specific anti-HSV effect, this possibility cannot yet be ruled out.

The effect of triterpenoid compounds on uninfected and herpes simplex virus-infected cells in culture. I. Effect on cell growth, virus particles and virus replication.

The related triterpenoid compounds carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) have been investigated in clinical trials for treatment of herpes simplex virus (HSV) infections. When the drugs were tested in vitro, two dose-related effects on BHK cells became apparent: the rate of cell growth was reduced and the drugs exhibited cytotoxicity at high concentrations. Flow 2002 cells, in contrast, were apparently unaffected by all drug concentrations tested. The effect of up to 3 days incubation with 100 microM-CCX on BHK cells was reversible. The presence of 500 microM-CBX or 300 microM-CCX during the HSV replication cycle reduced the infectious virus yield to less than 0.01%: CCX is the more potent anti-herpes agent. The contribution made by cytotoxicity to the overall antiviral effect (measured by 24 h yield) was negligible in Flow 2002 cells, and was relatively unimportant in BHK cells. The amount of HSV-1 or HSV-2 adsorbing to pretreated BHK cells was reduced by 20% and 40% respectively at the highest drug concentrations. Neither 500 microM-CBX nor 300 microM-CCX treatment for 24 h completely inhibited HSV-1 replication, but HSV-2 replication was abolished. The drugs appear to be continuously active throughout the infectious cycle. Infectious HSV particles appeared to become inactivated during or soon after egress from the cell. The two triterpenoid drugs lowered the number of virus particles made, and to a much greater extent reduced the infectious virus yield; thus, the progeny virus quality is greatly diminished. HSV-2 infections were more readily inhibited by either CCX or CBX than were HSV-1 infections.

Treatment of herpes genitalis with carbenoxolone and cicloxolone creams: a double blind placebo controlled clinical trial.

preliminary results in vitro have indicated that carbenoxolone and analogues possessed activity against herpes viruses. We undertook a double blind clinical study to compare the efficacy of carbenoxolone and cicloxolone creams with placebo in initial and recurrent herpes genitalis. Seventy-nine patients (21 of whom were entered in the trial more than once) received 105 courses of treatment, 83 of which were suitable for life table analysis. There were significant differences in the time to disappearance of pain (p = 0.044) and the healing of lesions (p = 0.023) in favour of cicloxolone compared with placebo. Carbenoxolone showed some beneficial effect compared with placebo, but this was not significant. Results on day 5 were similar. The only adverse reaction was mild erythema with irritation in one patient in each treatment group. We conclude that further trials with more extensive virological investigation are indicated to confirm the beneficial effect of cicloxolone.