European Journal of Pharmacology (v.688, #1-3)

The effects of combined menthol and borneol on fluconazole permeation through the cornea ex vivo by Jingjing Liu; Shaoying Fu; Nan Wei; Yongsheng Hou; Xiaoning Zhang; Hao Cui (1-5).
The bioavailabilities of eye drops are very low, so it is eager to find a safe and effective penetration enhancer to improve drug bioavailability. In our study, the corneas of New Zealand albino rabbit were mounted in the improved Franz diffusion cells for the measurement of fluconazole diffusion across the corneal permeation barrier. The fluconazole concentrations and the cumulative osmolalities were calculated to investigate the changes of permeation parameters of fluconazole through the ex vivo cornea in the presence of different penetration enhancers. Compared with the control group, 0.05% and 0.1% menthol, as well as menthol combined with borneol were considered as the penetration enhancer significantly promoted the permeation of fluconazole through the cornea ex vivo (P  < 0.05), but the same trend was not found in borneol groups. Furthermore, the combination of borneol and menthol significantly enhanced the permeation rate in the same concentration, compared with their effects when used alone (P  < 0.01). In order to investigate the feasibility and safety of the mixture, the corneal hydration level or wink frequency over 5 min were detected. So there were no significant differences between the mixture group and the control one. Consequently, menthol combined with borneol can highly enhance fluconazole permeation through the ex vivo cornea. Because of its low irritation, it may be a new efficient and safe penetration enhancer with a good development and application potential.
Keywords: Menthol combined with borneol; Fluconazole; Cornea; Penetration enhancer;

MMPT, (5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone), a thiazolidin compound, was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. A previous study showed that MMPT inhibited cell growth, and induced apoptosis in H1792 cells. In this study, the antiproliferative activity of MMPT was investigated. The results showed that MMPT was able to inhibit A549 cell growth in a time- and dose-dependent manner by blocking cell cycle progression in the G2 phase and inducing apoptosis. MMPT induced DNA fragmentation and caspase activation in A549 cells, both of which are hallmarks of apoptosis. The apoptotic process was accompanied by the generation of reactive oxygen species, depletion of glutathione (GSH), and reduction the GSH/GSSG ratio, suggesting that MMPT may induce apoptosis in A549 cells through a reactive oxygen species dependent pathway. Treatment with a thiol antioxidant, NAC, showed the recovery of GSH depletion and the reduction of reactive oxygen species levels in MMPT-treated cells, which were accompanied by the inhibition of apoptosis. In contrast, L-buthionine sulfoximine (BSO), a well-known inhibitor of GSH synthesis, aggravated GSH depletion and cell death in MMPT-treated cells. In conclusion, we have demonstrated that MMPT inhibits the growth of A549 cells by inducing a G2 arrest of the cell cycle and by triggering apoptosis accompanied with the depletion of GSH.
Keywords: A549 cells apoptosis; Caspase; GSH; MMPT; Reactive oxygen species;

The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells by Kotamballi N. Chidambara Murthy; Guddadarangavvanahally K. Jayaprakasha; Bhimanagouda S. Patil (14-21).
In the current paper, berberine, an isoquinoline alkaloid, was tested for its chemopreventive potential in colon cancer (SW480) cells. Berberine inhibited proliferation of colon cancer cells in a dose‐ and time‐dependent manner. Interestingly, this compound exhibited minimum toxicity in normal cells at 200 μM. Berberine arrested SW480 cell cycle at G2/M phase, which was supported by induction of p21 expression. Induction of a series of biochemical events, including loss of mitochondrial membrane potential, release of cytochrome-c to cytosol, induction of Bcl-2 family proteins, caspases and cleavage of poly (ADP-ribose) polymerase (PARP), by berberine suggests its ability to induce apoptosis. In addition, berberine also inhibited inflammation, as evidenced by induction of expression of NFκB and Cox2. Furthermore, berberine inhibited caspase-8 mediated angiogenesis, as confirmed through expression of tumor necrotic factor related apoptosis-inducing ligand (TRAIL), vascular endothelial growth factor (VEGF) and survivin. The results of the current study demonstrated that berberine has the ability to cause cell cycle arrest, induce apoptosis and inhibit inflammation in colon cancer cells. The magnitude of the effects observed suggests that berberine may be worth considering for further studies of its potential applications for improving health, either as a preventative or a potential treatment.
Keywords: Berberine; Colon cancer; SW480 cells; Anti-inflammatory; Apoptosis; Caspases;

Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3β4 and αβεδ acetylcholine receptors in COS cells by Jiří Lindovský; Konstantin Petrov; Jan Krůšek; Vladimir S. Reznik; Eugeny E. Nikolsky; František Vyskočil (22-26).
The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2–4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10− 7  M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors.
Keywords: Nicotinic ACh receptor; α3β4; αβεδ; C-547; Anti-cholinesterase;

Chronic administration of the selective P2X3, P2X2/3 receptor antagonist, A-317491, transiently attenuates cancer-induced bone pain in mice by Rikke Rie Hansen; Arafat Nasser; Sarah Falk; Signe B. Baldvinsson; Pernille H. Ohlsson; Justyna M.C. Bahl; Michael F. Jarvis; Ming Ding; Anne-Marie Heegaard (27-34).
The purinergic P2X3 and P2X2/3 receptors are in the peripheral nervous system almost exclusively confined to afferent sensory neurons, where they are found both at peripheral and central synapses. The P2X3 receptor is implicated in both neuropathic and inflammatory pain. However, the role of the P2X3 receptor in chronic cancer-induced bone pain is less known. Here we investigated the effect of systemic acute and chronic administration of the selective P2X3, P2X2/3 receptor antagonist (5-[[[(3-Phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid sodium salt hydrate) (A-317491) in a murine model of cancer-induced bone pain. Chronic administration of A-317491 (30 μmol/kg s.c., b.i.d.) resulted in a transient attenuation of pain related behaviours in the early stage of the bone cancer model, but had no effect in the late and more progressed stage of bone cancer. Also, acute administration of A-317491 (100 μmol/kg s.c.) had no effect in the progressed stage of the bone cancer pain model. Thus, systemically administered A-317491 did not demonstrate a robust effect in the present mouse model of cancer-induced bone pain.
Keywords: Bone cancer pain; P2X3 receptor; P2X2/3 receptor; A-317491 (5‐[[[(3‐Phenoxyphenyl)methyl][(1S)‐1,2,3,4‐tetrahydro‐1‐naphthalenyl]amino]carbonyl]‐1,2,4‐benzenetricarboxylic acid sodium salt hydrate);

The α1-, but not α2-, adrenoceptor in the nucleus accumbens plays an inhibitory role upon the accumbal noradrenaline and dopamine efflux of freely moving rats by Tadashi Saigusa; Yuri Aono; Takuya Uchida; Koji Takada; Michel M.M. Verheij; Noriaki Koshikawa; Alexander R. Cools (35-41).
In vivo microdialysis was used to analyse the role of the α1- and α2-adrenoceptor subtypes in the regulation of noradrenaline and dopamine efflux in the nucleus accumbens of freely moving rats. Intra-accumbal infusion of α1-adrenoceptor agonist methoxamine (24 pmol) failed to alter the noradrenaline efflux, but decreased the dopamine efflux. The intra-accumbal infusion of α1-adrenoceptor antagonist prazosin (6, 600 and 6000 pmol) produced a dose-related increase and decrease of the noradrenaline and dopamine efflux, respectively. An ineffective dose of prazosin (6 pmol) counteracted the methoxamine (24 pmol)-induced decrease of dopamine efflux. The prazosin (6000 pmol)-induced increase of noradrenaline efflux, but not the decrease of dopamine efflux, was suppressed by the co-administration of an ineffective dose of methoxamine (0.024 pmol). Neither the α2-adrenoceptor agonist clonidine (300 pmol) and UK 14,304 (300 pmol) nor the α2-adrenoceptor antagonist RX 821002 (0.6, 3, 600 and 6000 pmol) significantly affected the accumbal noradrenaline and dopamine efflux. The doses mentioned are the total amount of drug over the 60-min infusion period. The present results show that (1) accumbal α1-adrenoceptors which are presynaptically located on noradrenergic nerve terminals inhibit the accumbal noradrenaline efflux, increasing thereby the accumbal dopamine efflux, (2) accumbal α1-adrenoceptors which are postsynaptically located on dopaminergic nerve terminals inhibit the accumbal dopamine efflux, and (3) accumbal α2-adrenoceptors play no major role in the regulation of accumbal efflux of noradrenaline and dopamine.
Keywords: α1-Adrenoceptor; α2-Adrenoceptor; Nucleus accumbens; Noradrenaline; Dopamine; Microdialysis; (Rat);

The gender difference of selective serotonin reuptake inhibitor, fluoxetine in adult rats with stress-induced gastric ulcer by Khaled A. Abdel-Sater; Wafaa M. Abdel-Daiem; Mohamad Sayyed Bakheet (42-48).
We investigated the gender difference of selective serotonin reuptake inhibitor, fluoxetine in adult rats with stress-induced gastric ulcer. The rats were randomly divided into six groups: Group I, control males and group II, control females; group III, acute cold restraint stressed males and group IV, acute cold restraint stressed females; group V, fluoxetine-treated stressed males and group VI, fluoxetine-treated stressed females. Acute cold restraint stress was established by fixing the four limbs of the rat and placing it in a refrigerator at 4 °C for 3 h. Fluoxetine was given intraperitoneal in a single dose of 10 mg/kg/day. After 2 weeks, stomach and brain tissues were collected for the assay of gastric malonaldehyde (MDA), catalase, nitric oxide (NO) and cortical gamma aminobutyric acid (GABA). Stressed animals exhibited increased total acidity in association with decreased gastric secretion volume. Gastric MDA was increased while gastric catalase, NO, and cortical GABA were decreased in stressed male rats when compared to stressed females. However, fluoxetine administration attenuated these stress-induced changes especially in stressed male animals. Stressed male rats were more responsive to the antiulcer effect of fluoxetine more than stressed females. However, fluoxetine might be considered to be the first-choice drug in depressive patients with gastric ulcers in the future.
Keywords: Cold restraint stress; Fluoxetine; Gastric stress ulcer; Oxidative stress; Nitric oxide; Cortical gamma aminobutyric acid;

Vas deferens smooth muscle responses to the nitric oxide-independent soluble guanylate cyclase stimulator BAY 41‐2272 by Fábio H. da Silva; Mário A. Claudino; Fernando R. Báu; Julio A. Rojas-Moscoso; Fabíola Z. Mónica; Gilberto De Nucci; E. Antunes (49-55).
The nitric oxide–cGMP signaling pathway modulates the ejaculatory functions. The nitric oxide (NO)-independent soluble guanylate cyclase haem-dependent stimulator BAY 41‐2272 potently relaxes different types of smooth muscles. However, no study investigated its effects in vas deferens smooth muscle. Therefore, we designed experiments to evaluate the in vitro relaxing responses of vas deferens to BAY 41‐2272. The effects of prolonged oral intake with BAY 41‐2272 in vas deferens contractions of rats treated chronically with the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) were also investigated. BAY 41‐2272 (0.001–100 μM) produced concentration-dependent relaxations in the prostatic and epididymal portions of vas deferens, an effect markedly reduced by the soluble guanylate cyclase inhibitor ODQ (100 μM). BAY 41‐2272 significantly increased cGMP levels that were fully prevented by ODQ. In separate protocols, rats received L-NAME (20 mg/rat/day) concomitantly with BAY 41‐2272 (10 mg/kg/day, 4 weeks), after which vas deferens contractions to electrical-field stimulation and noradrenaline were achieved. Electrical-field stimulation (1–32 Hz) evoked frequency-dependent contractions that were significantly enhanced in L-NAME-treated rats. Co-treatment with BAY 41‐2272 fully reversed the increased contractile responses in L-NAME group. Noradrenaline (0.01–100 μM)-induced contractions were also greater in L-NAME-treated rats, and that was normalized by BAY 41‐2272. In conclusion, BAY 41‐2272 potently relaxes in vitro rat vas deferens smooth muscle and elevates the cGMP levels in an ODQ-sensitive manner. Moreover, prolonged oral intake with BAY 41‐2272 restores the enhanced contractile vas deferens activity in rats treated with L-NAME. NO-independent soluble guanylate cyclase stimulators may be an alternative treatment for premature ejaculation.
Keywords: Ejaculatory functions; Cyclic GMP; Adrenoceptor; Neurogenic contractions;

Mycophenolate mofetil inhibits macrophage infiltration and kidney fibrosis in long-term ischemia–reperfusion injury by Shuai Jiang; Qunye Tang; Ruiming Rong; Liangfeng Tang; Ming Xu; Jidong Lu; Yichen Jia; YitReen Ooi; Jun Hou; Jianming Guo; Bin Yang; Tongyu Zhu (56-61).
Immunosuppressants have been widely used in renal transplantation, in which ischemia–reperfusion injury is inevitable. Mycophenolate mofetil (MMF) is a relative novel immunosuppressant and also attenuates ischemia–reperfusion injury in the acute phase, but its long-term effects are still obscure. Unilateral renal ischemia–reperfusion injury model was established in Sprague–Dawley rats and 30 mg/kg/day MMF or natural saline was administered a day before the surgery. Renal function was monitored, and histological changes and fibrosis in the kidney were evaluated in both short and long terms. TGF-β1 secretion and MCP-1 expression were determined by immunohistochemistry and real-time PCR respectively. The infiltration of macrophages in renal tissues was also assessed by fluorescence activated cell sorting (FACS). MMF treatment significantly improved renal function in ischemia–reperfusion injury rats in the short and long-term and also effectively prevented interstitial fibrosis. TGF-β1 secretion and MCP-1 expression in the renal tissue of MMF-treated rats were much lower than those in natural saline-treated rats, with much less macrophage infiltration as well. MMF treatment effectively prevented the deterioration of renal function and interstitial fibrosis in ischemia–reperfusion injury rats, which may be associated with decreased TGF-β1, MCP-1 and macrophages. These results provide evidence for the choice of MMF in the renal transplant patients not only for acute renal injury but also for long-term survival of renal allograft.
Keywords: Mycophenolate mofetil; Ischemia–reperfusion injury; TGF-β1 (transforming growth factor beta‐1); MCP-1 (monocyte chemotactic protein‐1); Macrophage;

Flutamide, an androgen receptor antagonist, improves heatstroke outcomes in mice by Chian-Yuh Lin; Chuan-Chih Hsu; Mao-Tsun Lin; Sheng-Hsien Chen (62-67).
Flutamide has been used as an adjunct for decreasing the mortality from subsequent sepsis. Heatstroke resembles septic shock in many aspects. We hypothesized that heat-induced multiple organ dysfunction syndromes and lethality could be reduced by flutamide therapy. In heatstroke groups, mice were exposed to whole body heating (41.2°C, for 1 h) in a controlled-environment chamber. The heat-stressed mice were returned to normal room temperature (24 °C) after whole body heating. Mice still alive on day 4 of WBH treatment were considered survivors. Physiological and biochemical parameters were monitored for 2.5 h post-WBH. Heatstroke mice were subcutaneously treated with flutamide (12.5–50 mg/kg body weight in 0.05 ml) or vehicle solution (0.05 ml/kg body weight) once daily for 3 consecutive days post-WBH. We evaluated the effect of flutamide in heatstroke mice and showed that flutamide significantly (i) attenuated hypothermia, (ii) reduced the number of apoptotic cells in the hypothalamus, the spleen, the liver, and the kidney, (iii) attenuated the plasma index of toxic oxidizing radicals (e.g., nitric oxide metabolites and hydroxyl radicals), (iv) diminished the plasma index of the organ injury index (e.g., lactate dehydrogenase), (v) attenuated plasma systemic inflammation response molecules (e.g., tumor necrosis factor-α and interleukin-6), (vi) reduced the index of infiltration of polymorphonuclear neutrophils in the lung (e.g., myeloperoxidase activity), and (vii) allowed three times the fractional survival compared with vehicle. Thus, flutamide appears to be a novel agent for the treatment of mice with heatstroke or patients in the early stage of heatstroke.
Keywords: Heat stroke; Flutamide; Multiple organ injury; Cytokines;

Splitomicin, is a cell-permeable lactone derived from naphthol and known to be a potent selective inhibitor of Sir2 (silent information regulator 2). Previous studies have demonstrated that naphtholic compounds possess an inhibitory effect on neutrophils. Here, we present our investigation on the inhibitory effects of splitomicin in human neutrophils. The primary goal of our study was to locate a possible candidate on inflammatory reactions and to hopefully develop a novel anti-inflammatory therapy. Neutrophils were prepared following standard procedures. Neutrophils induced by either fMLP (1 μM) or PMA (100 nM) were observed using a flow cytometer and the intracellular production of superoxide anions was investigated at different splitomicin concentrations. The cytosolic Ca++ influx concentration was measured using a fluorescence spectrophotometer, and Mac-1 expression was detected with a flow cytometer. The MAP kinases were measured using western blotting. Our results showed that splitomicin inhibited superoxide anion production by fMLP (1 μM) and NaF (20 mM) in a concentration-dependent manner (37.5–450 μM). Splitomicin (300 and 450 μM) also suppressed fMLP-induced intracellular calcium ion mobilization and extracellular-signal regulated kinase (ERK) phosphorylation. Moreover, splitomicin could inhibit fMLP-induced Mac-1 expression and increase cAMP levels in human neutrophils. Our data demonstrated that splitomicin exhibits a noticeable inhibitory effect on superoxide anion production in human neutrophils. This negative effect was well-correlated with increased cAMP levels via PKA activity and the subsequent inhibition of ERK (p42/p44) phosphorylation to decrease superoxide anion production.Display Omitted
Keywords: Splitomicin; Neutrophil; Superoxide anion; cAMP (3′-5′-cyclic adenosine monophosphate); ERK (extracellular-signal regulated kinase);

Interleukin-6 (IL-6) is considered to be an early marker of severe sepsis that is associated with increased morbidity and mortality. Therefore, we pretreated male ICR mice with IL-6 small interfering RNA (siRNA) before cecal ligation and puncture (CLP) and observed the changes in their survival in response to down regulation of IL-6, as well as the role of Th subsets during sepsis. In addition, sham and CLP operated mice were sacrificed at different time points to determine the serum IL-6 levels during early and late sepsis. IL-6 siRNA pretreated septic mice showed markedly extended survival (23.3%) up to 10 days and significantly reduced serum IL-6 levels at day 5 (209.90 ± 0.50 pg/ml; P < 0.0001) when compared to CLP mice at day 1. Furthermore, IL-6 mRNA and protein were highly expressed during early sepsis in CLP mice at day 1 and mRNA was not detected in IL-6 siRNA treated CLP mice at days 1 or 5 and serum level of IL-6 was also decreased significantly (P < 0.01). In addition, the mRNA expression of C5aR, ROR-γt, PU.1 and protein expression of IL-17 were high at day 5 in IL-6 siRNA treated mice. Taken together, the results of the present study demonstrate that pretreatment with IL-6 siRNA improved CLP induced septic mice survival. Furthermore, the IL-6 level was down-regulated and the transcription factors ROR-γt and PU.1 were up-regulated by IL-6 siRNA at late sepsis. The results presented herein also suggest that IL-6 siRNA could be a potential molecular therapeutic strategy for the treatment of sepsis.
Keywords: Cecal ligation puncture; Sepsis; Interleukin-6; siRNA; Th subset;

Screening for novel lead compounds increasing insulin expression in medullary thymic epithelial cells by Xiu-ying Yang; Dina Levi; Houria Ounissi-Benkalha; Xiao-yan Yu; Hui-qi Qu; Constantin Polychronakos; Guan-hua Du (84-89).
Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance to pancreatic beta-cells. Thymic insulin expression modulation might be an important drug target for preventing type 1 diabetes. We performed a high-throughput screening to identify compounds with such activity. A reporter plasmid was constructed with the human insulin promoter sequence including a short allele of the upstream variable number tandem repeat (VNTR) sequence (32 repeats), subcloned into the pGL4.17 vector. The plasmid was stably transfected into an insulin-transcribing medullary thymic epithelial cell (mTEC) line. Primary high-throughput screening assays were carried out by stimulating with candidate compounds for 24 h, and the activity of luciferase was measured. Positive compounds were further validated by real-time PCR. Of 19,707 compounds, we identified one compound that could enhance mTEC insulin expression, as confirmed by real-time PCR. We also observed that transfection with the autoimmune regulator (AIRE) increased endogenous AIRE expression in mTECs. Our insulin-VNTRI-promoter reporter system is consistent with the insulin expression regulation in mTECs, and one compound that was identified could increase insulin expression in mTECs. A positive feedback effect of AIRE in mTECS was observed. Whether these efforts in murine thymus cells apply to humans remains to be determined.
Keywords: Type 1 diabetes; High-throughput screening assay; Medullary thymic epithelial cell; Insulin; Immune tolerance; Autoimmune regulator;