Hydrogen sulfide and sulfaceutic or sulfanutraceutic agents: Classification, differences and relevance in preclinical and clinical studies.

Hydrogen sulfide (H2S) has been extensively studied as a signal molecule in the body for the past 30 years. Researchers have conducted studies using both natural and synthetic sources of H2S, known as H2S donors, which have different characteristics in terms of how they release H2S. These donors can be inorganic salts or have various organic structures. In recent years, certain types of sulfur compounds found naturally in foods have been characterized as H2S donors and explored for their potential health benefits. These compounds are referred to as "sulfanutraceuticals," a term that combines "nutrition" and "pharmaceutical". It is used to describe products derived from food sources that offer additional health advantages. By introducing the terms "sulfaceuticals" and "sulfanutraceuticals," we categorize sulfur-containing substances based on their origin and their use in both preclinical and clinical research, as well as in dietary supplements.

Hydrogen sulfide dysfunction in metabolic syndrome-associated vascular complications involves cGMP regulation through soluble guanylyl cyclase persulfidation.

Here, by using in vitro and ex vivo approaches, we elucidate the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with metabolic syndrome (MetS). In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, we observe significant hallmarks of endothelial dysfunction, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, a genetic MetS model, we identify a downregulation of CBS and CSE expression in aorta, coupled with a diminished L-cysteine-induced vasorelaxation. Molecular mechanisms of eNOS/NO signaling impairment, dissected using pharmacological and molecular approaches, indicate an altered eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation and increased L-NIO-induced contraction. In vivo treatment with the H2S donor Erucin ameliorates vascular dysfunction observed in db/db mice without impacting eNOS, further highlighting a specific action on smooth muscle component rather than the endothelium. Analyzing the NO signaling pathway in db/db mice aortas, reduced cGMP levels were detected, implicating a defective sGC/cGMP signaling. In vivo Erucin administration restores cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation observed in sGC overexpressed cells, coupled with PDE5 inhibition. In conclusion, our study demonstrates a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MetS involving an impairment of both H2S and NO signaling. Exogenous H2S supplementation through Erucin represents a promising alternative in MetS therapy, targeting smooth muscle cells and supporting the importance of lifestyle and nutrition in managing MetS.

Development of inhalable hyaluronan/mannitol composite dry powders for flucytosine repositioning in local therapy of lung infections.

Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.

Phenotypic modifications of vascular smooth muscle cells could be responsible for vascular hyporeactivity to contracting agent in mechanically injured rat carotid artery.

Vascular smooth muscle cells (VSMCs) that accumulate in neointima after angioplastic injury show different phenotypic characteristics from those of medial layer and an impaired reactivity to contracting agents. The aim of the study was to correlate the vascular hyporesponsiveness to the changes in intracellular calcium concentration [Ca(2+)](i) and the expression of proteins necessary for its utilization in mechanically injured rat carotid arteries (IC) at 14 and 28 days after angioplastic balloon. IC showed a significant reduction (P<0.01) to PE- or KCl-induced contraction as compared to uninjured carotid (UC). Fura-2AM-loaded VSMCs isolated from IC revealed that this hyporeactivity to PE or KCl was accompanied by the impairment of the increase in [Ca(2+)](i) induced by contracting agents in both Ca(2+)-free or -containing medium. Similar results were observed following the ryanodine challenge in VSMC. Western blot analysis showed a significant (P<0.05) reduction in myosin heavy chain (MHC) and IP(3)-type III receptor expression in IC isolated at 14 days from injury compared to UC, while an improvement of these proteins expression was observed at 28 days after damage. On the other hand, in IC tissue, SERCA2 and alpha-actin expression, compared to UC was significantly higher at 14 days than at 28 days. These data indicate that vascular hyporeactivity induced by mechanical injury may be due to alterations of either [Ca(2+)](i) or contractile proteins. These modifications could be related to the changes of VSMC phenotypic characteristics, as supported by the observed modifications in MHC, SERCA2 and alpha-actin expression, proteins considered as biological markers of cellular differentiation.

Peripheral relaxant activity of apomorphine and of a D1 selective receptor agonist on human corpus cavernosum strips.

Apomorphine is used in the erectile dysfunction therapy and its action has been ascribed to the stimulation of central dopamine receptor. At the present stage, very little is known about the peripheral action of apomorphine on human corpus cavernosum (HCC). We have investigated the peripheral action of apomorphine and the role of dopamine receptors in HCC. We here demonstrate that both D1 and D2 receptors were expressed in the HCC, D1 receptors were two-fold more abundant than D2 and that both receptors were mainly localized on the smooth muscle cell component. Apomorphine in vitro exerted an anti-alpha1 adrenergic activity in human cavernosal strips since it prevented contraction induced by phenylephrine (PE), but not by U46619 or endothelin. Apomorphine elicited endothelium-independent and concentration-dependent relaxation of the strips contracted by PE, U46619 or endothelin. The EC50 values (microM) for apomorphine, in the presence and absence of endothelium, were 51.0+/-16 and 16.0+/-14, 120+/-19 and 150+/-18, 59.0+/-15 and 140+/-50 on PE-, U46619- or endothelin-induced contraction, respectively. Selective dopamine receptor agonist A-68930 (D1-like), but not quinpirole (D2-like), caused concentration-dependent relaxation of the cavernosal strips, which was partially prevented by endothelium removal or by treatment with an inhibitor of nitric oxide (NO) synthase. In conclusion, we show that (1) apomorphine has a peripheral relaxant direct effect as well as an antiadrenergic activity, (2) HCC possesses more D1-like (D1 and D5) than D2-like (D2, D3 and D4) receptors, (3) both D1- and D2-like receptors are mainly localized on smooth muscle cells and (4) the relaxant activity is most probably mediated by D1-like receptor partially through NO release from endothelium.

Crucial role of androgen receptor in vascular H2S biosynthesis induced by testosterone.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H2S) is a gaseous mediator strongly involved in cardiovascular homeostasis, where it provokes vasodilatation. Having previously shown that H2 S contributes to testosterone-induced vasorelaxation, here we aim to uncover the mechanisms underlying this effect. EXPERIMENTAL APPROACH: H2 S biosynthesis was evaluated in rat isolated aortic rings following androgen receptor (NR3C4) stimulation. Co-immunoprecipitation and surface plasmon resonance analysis were performed to investigate mechanisms involved in NR3C4 activation. KEY RESULTS: Pretreatment with NR3C4 antagonist nilutamide prevented testosterone-induced increase in H2S and reduced its vasodilator effect. Androgen agonist mesterolone also increased H2S and induced vasodilatation; effects attenuated by the selective cystathionine-γ lyase (CSE) inhibitor propargylglycine. The NR3C4-multicomplex-derived heat shock protein 90 (hsp90) was also involved in this effect; its specific inhibitor geldanamycin strongly reduced testosterone-induced H2S production. Neither progesterone nor 17-ß-oestradiol induced H2S release. Furthermore, we demonstrated that CSE, the main vascular H2S-synthesizing enzyme, is physically associated with the NR3C4/hsp90 complex and the generation of such a ternary system represents a key event leading to CSE activation. Finally, H2S levels in human blood collected from male healthy volunteers were higher than those in female samples. CONCLUSIONS AND IMPLICATIONS: We demonstrated that selective activation of the NR3C4 is essential for H2S biosynthesis within vascular tissue, and this event is based on the formation of a ternary complex between cystathionine-γ lyase, NR3C4and hsp90. This novel molecular mechanism operating in the vasculature, corroborated by higher H2S levels in males, suggests that the L-cysteine/CSE/H2S pathway may be preferentially activated in males leading to gender-specific H2S biosynthesis.

Involvement of ATP-sensitive potassium channels in a model of a delayed vascular hyporeactivity induced by lipopolysaccharide in rats.

We have investigated the role of ATP-sensitive potassium (K(ATP)) channels in an experimental model of a delayed phase of vascular hyporeactivity induced by lipopolysaccharide (LPS) in rats. After 24 h, from LPS treatment, in anaesthetized rats the bolus injection of phenylephrine (PE) produced an increase in mean arterial pressure (MAP) significantly (P<0.05) reduced in LPS-treated rats compared to the vehicle-treated rats. This reduction was prevented by pre-treatment of rats with glibenclamide (GLB), a selective inhibitor of K(ATP) channels. GLB administration did not affect the MAP in vehicle-treated rats but produced an increase of MAP in rats treated with LPS. Cromakalim (CRK), a selective K(ATP) channel opener, produced a reduction of MAP that was significantly (P<0.05) higher in LPS- than in vehicle-treated rats. In contrast, the hypotension induced by glyceryl trinitrate (GTN) in LPS-treated rats was not distinguishable from that produced in vehicle-treated rats. Experiments in vitro were conducted on aorta rings collected from rats treated with vehicle or LPS 24 h before sacrifice. The concentration-dependent curve to PE was statistically (P<0.005) reduced in aorta rings collected from LPS- compared to vehicle-treated rats. This difference was totally abolished by tetraethylammonium (TEA), a non-selective inhibitor of K+ channels. CRK produced a relaxation of PE precontracted aorta rings higher in rings from LPS- than in vehicle-treated rats. GLB inhibited CRK-induced relaxation in both tissues, abolishing the observed differences. In conclusion, our results indicate an involvement of K(ATP) channels to the hyporesponsiveness of vascular tissue after 24 h from a single injection of LPS in rats. We can presume an increase in the activity of K(ATP) channels on vascular smooth muscle cells but we cannot exclude an increase of K(ATP) channel number probably due to the gene expression activation.

Dexamethasone improves vascular hyporeactivity induced by LPS in vivo by modulating ATP-sensitive potassium channels activity.

(1) Septic shock represents an important risk factor for patients critically ill. This pathology has been largely demonstrated to be a result of a myriad of events. Glucocorticoids represent the main pharmacological therapy used in this pathology. (2) Previously we showed that ATP-sensitive potassium (KATP) channels are involved in delayed vascular hyporeactivity in rats (24 h after Escherichia coli lipopolysaccharide (LPS) injection). In LPS-treated rats, we observed a significant hyporeactivity to phenylephrine (PE) that was reverted by glybenclamide (GLB), and a significant increase in cromakalim (CRK)-induced hypotension. (3) We evaluated the effect of dexamethasone (DEX 8 mg kg-1 i.p.) whether on hyporeactivity to PE or on hyperreactivity to CRK administration, in vivo, in a model of LPS (8 x 106 U kg-1 i.p.)-induced endotoxemia in urethane-anaesthetised rats. (4) DEX treatment significantly reduced, in a time-dependent manner, the increased hypotensive effect induced by CRK in LPS-treated rats. This effect was significantly (P<0.05) reverted by the glucocorticoid receptor antagonist RU38486 (6.6 mg kg-1 i.p.). (5) GLB-induced hypertension (40 mg kg-1 i.p.), in LPS-treated rats, was significantly inhibited by DEX if administered at the same time of LPS. (6) Simultaneous administration of DEX and LPS to rats completely abolished the hyporeactivity to PE observed after 24 h from LPS injection. (7) In conclusion, our results suggest that the beneficial effect of DEX in endotoxemia could be ascribed, at least in part, to its ability to interfere with KATP channel activation induced by LPS. This interaction may explain the improvement of vascular reactivity to PE, mediated by DEX, in LPS-treated rats, highlighting a new pharmacological activity to the well-known anti-inflammatory properties of glucocorticoids.

Dexamethasone modulates hypotension induced by opioids in anaesthetised rats.

The effect of dexamethasone on hypotension induced by mu-, kappa- and delta-opioid receptor agonists was investigated in pentobarbital-anaesthetised rats. Morphine (nonselective opioid receptor agonist), DAGO (D-Ala2-N-methyl-[Phe4-Gly5-ol]enkephalin; mu-opioid receptor-selective agonist), U50-488H (trans(+/-)-3,4-dichloro-N-methyl-N-(2[1pyrrolidynyl]cyclohexyl)-benzeneacetamide; kappa-opioid receptor-selective agonist) and deltorphin II (delta-opioid receptor-selective agonist), given intravenously, 5 micromol/kg, induced hypotension in rats. This hypotension was characterised by a fall in mean arterial blood pressure in 1-2 min that recovered in 30 min for morphine and U50-488H and in 5 or 20 min for DAGO and deltorphin II, respectively. Dexamethasone per se at a dose of 7.5 micromol/kg, i.v. did not significantly modify the mean arterial blood pressure of animals. Dexamethasone administration 90 min, but not 30 or 60 min, before the opioid agonists injection, prevented the hypotension induced by morphine or U50-488H, but not that induced by DAGO or deltorphin II. Pretreatment with RU-38486 (mifepristone; 7.5 micromol/kg, i.v.), a glucocorticoid receptor antagonist, 15 min before the steroid, prevented dexamethasone inhibition of hypotension induced by morphine and U50-488H. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor (3.5 micromol/kg, i.v.), was also able to abolish the effects of dexamethasone on morphine- and U50-488H-induced hypotension. Results of the present study indicate that dexamethasone inhibited kappa-opioid receptor-mediated hypotension in rats, indicating a further important functional interaction between corticosteroids and the opioid system at kappa receptors. The ability of cycloheximide and RU-38486 to block dexamethasone effects indicates that steroid interference with kappa-opioid receptor-mediated hypotension involves a protein synthesis-dependent mechanism via glucocorticoid receptors.

Cloricromene in endotoxemia: role of NF-kappaB.

In this study we investigated, for the first time in vivo, the effect of cloricromene, a cumarine derivative, on NF-kappaB activation in endotoxin-treated rats. Endotoxemia was induced in male rats by the intravenous injection of Salmonella typhosa lipopolysaccharide (LPS; 2 mg/kg/i.v.). In vivo treatment with cloricromene (2 mg/kg/i.v.) 30 min before lipopolysaccharide administration reversed the LPS-induced loss in tone of the aortic rings, improved their reactivity to phenylephrine, decreased both nitric oxide (NO) and TNF-alpha serum levels by inhibiting LPS-induced inducible NO synthase and TNF-alpha mRNA expression, and interestingly inhibited LPS-induced NF-kappaB activation. Our data suggest that cloricromene protects rats from LPS by blocking LPS-induced NF-kappaB activation, leading to inhibition of NO and TNF-alpha overproduction and thereby reversing the LPS-induced vascular hyporeactivity.

A standardized procedure for using human corpus cavernosum strips to evaluate drug activity.

The main problem of using human corpus cavernosum (HCC) tissue to perform bioassay is linked to its limited availability further complicated by the heterogeneous source of the tissues used. Here, we show that gender reassignment is a reliable source of human tissue without major ethical problems. Indeed, the entire corpus cavernosum is obtained from the surgery procedure, which allows creating a standardized procedure to prepare HCC strip. In addition, human tissue, if kept in the fridge in the condition described, does not loose its ability to contract to phenylephrine (PE; alpha agonist), angiotensin II (AG II) and KCl up to 4 days. Furthermore, once contracted with PE, HCC relaxes to acetylcholine (endothelium-dependent mechanism); sodium nitroprusside (endothelium-independent mechanism); cromakalim (CRK), a K(ATP) channel opener; or alprostadil, a synthetic PGE2 (ALPR). In conclusion, we have standardized a procedure that allows the use of HCC strips to evaluate drug activity and/or to study pathophysiological mechanisms with an intact functional human tissue up to 4 days from the surgery procedure.

Improvement of gliquidone hypoglycaemic effect in rats by cyclodextrin formulations.

This study was carried out with the aim to optimize the pharmacological profile of gliquidone (GLI)--a poorly bioavailable hypoglycaemic agent sparingly soluble in water--through complexation with cyclodextrins. In order to increase the apparent solubility of GLI, two cyclodextrins, namely beta-cyclodextrin (betaCD) and hydroxypropyl-beta-cyclodextrin (HPbetaCD), were tested. The effect of cyclodextrin addition on the aqueous solubility of GLI was evaluated by the phase solubility method at different pH values. The amount of GLI in solution increased upon CD addition according to A type plots. The aqueous solubility of GLI was enhanced more at higher pH values and using HPbetaCD. On the basis of its performance, HPbetaCD was selected as host to prepare GLI oral formulations. GLI/HPbetaCD solid systems were prepared at 1:2 molar ratio by co-grinding, spray-drying and freeze-drying and characterized by DSC, FTIR and X-ray powder diffractometry. Powders were amorphous and showed an improved dissolution rate in comparison with GLI. GLI/HPbetaCD co-ground and freeze-dried products were the most interesting systems, since they dissolved 62 and 94% of total drug after 15 min, respectively. The hypoglycaemic effect of the most rapidly dissolving binary systems was evaluated after oral administration in fasted rats by measuring plasma glucose level in the time interval 0.5-36 h and compared to free GLI. Our findings indicate that cyclodextrin-containing formulations not only provide an onset of hypoglycaemic effect faster than GLI, but also enhance significantly the pharmacological effect due to improved biopharmaceutics. The association GLI/HPbetaCD allows a reduction of the oral dose and is expected to provide a better control over drug side effects, contributing to improve safety and efficacy of GLI.

OS064. Contribute of the L-cysteine/ H2S pathway in placenta homeostasisin hypertensive disorders.

INTRODUCTION: Hydrogen sulfide (H2S) is considered the third endogenous gas transmitter besides nitric oxide and carbon monoxide [1]. It is produced from L-cysteine or L-methionine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). H2S is involved in the control of vascular homeostasis, having either relaxant or contractant effect on smooth muscle cells. The H2S involvement in rat and human intrauterine tissues has also been shown [2]. OBJECTIVES: The aim of our study was to investigate the L-cysteine/ H2S pathway in rat and human placenta in hypertensive state. METHODS: Placental samples were collected from spontaneous hypertensive rats (SHR) and normotensive rat (Wistar Kyoto; WKY). In parallel, placental samples were collected from 10 pre-eclamptic women and 5 controls after caesarean sections. Pre-eclamptic women were divided into two subgroups: Group1 (women who developed Early Preeclampsia, n=4); Group2 (women who developed Late Preeclampsia, n=6). The expression of CBS and CSE was evaluated in sample tissues by Western blotting analysis. The enzymatic activity was assessed in basal and stimulated (L- cysteine) condiction by a colorimetric assay. Statistical analysis was performed by using Student's t test. P<0,05 was considered as statistically significant. RESULTS: The expression of CBS and CSE in placenta of SHR rats were significantly reduced (p<0.05) compared to WKY. The H2S production resulted significantly (p<0,05) lower in SHR than WKY rats. In human placenta, the basal H2S production was similar in the three groups; interestingly the H2S production by adding L-cysteine, was higher in Late Preeclampsia compared to control group. CONCLUSION: H2S was produced in rat and human placenta. CBS and CSE, the enzymes involved in the production of H2S, were down-regulated in SHR rats and, as a consequence the H2S production was significantly reduced. Starting from these data, we tried to analyze the role of hydrogen sulfide in preeclampsia to assess the contribute of this gas transmitter in the development of this condition. Unexpectedly, preliminary data demonstrated that in women developing Late Preeclampsia there was an higher production of H2S after stimulation with L-cysteine, not revealed in Early Preeclampsia or in healthy control group. Our results indicated that the L-cysteine/H2S pathway could contribute to the development of preeclampsia condition.