Roberta d'Emmanuele di Villa Bianca

Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers

Although few experimental studies have been handled so far to exploit the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in the production of dry powders for antibiotic inhalation, there has been no comprehensive study on the role played by NP composition. In this work, we try to shed light on this aspect by designing and developing a pulmonary delivery system for antibiotics, such as tobramycin (Tb), based on PLGA NPs embedded in an inert microcarrier made of lactose, referred to as nano-embedded micro-particles (NEM). At nanosize level, helper hydrophilic polymers were used to impart the desired surface, bulk and release properties to PLGA NPs prepared by a modified emulsion-solvent diffusion technique. Results showed that poly(vinyl alcohol) (PVA) and chitosan (CS) are essential to optimise the size and modulate the surface properties of Tb-loaded PLGA NPs, whereas the use of alginate (Alg) allows efficient Tb entrapment within NPs and its release up to one month. Optimized formulations display good in vitro antimicrobial activity against P. aeruginosa planktonic cells. Furthermore, spray-drying of the NPs with lactose yielded NEM with peculiar but promising flow and aerosolization properties, while preserving the peculiar NP features. Nonetheless, in vivo biodistribution studies showed that PVA-modified Alg/PLGA NPs reached the deep lung, while CS-modified NPs were found in great amounts in the upper airways, lining lung epithelial surfaces. In conclusion, PLGA NP composition appears to play a crucial role in determining not only the technological features of NPs but, once processed in the form of NEM, also their in vitro/in vivo deposition pattern.

Hydrogen sulfide as a mediator of human corpus cavernosum smooth-muscle relaxation

Hydrogen sulfide (H2S) is synthesized by 2 enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). l-Cysteine (l-Cys) acts as a natural substrate for the synthesis of H2S. Human penile tissue possesses both CBS and CSE, and tissue homogenates efficiently convert l-Cys to H2S. CBS and CSE are localized in the muscular trabeculae and the smooth-muscle component of the penile artery, whereas CSE but not CBS is also expressed in peripheral nerves. Exogenous H2S [sodium hydrogen sulfide (NaHS)] or l-Cys causes a concentration-dependent relaxation of strips of human corpus cavernosum. l-Cys relaxation is inhibited by the CBS inhibitor, aminoxyacetic acid (AOAA). Electrical field stimulation of human penile tissue, under resting conditions, causes an increase in tension that is significantly potentiated by either propargylglycine (PAG; CSE inhibitor) or AOAA. In rats, NaHS and l-Cys promote penile erection, and the response to l-Cys is blocked by PAG. Our data demonstrate that the l-Cys/H2S pathway mediates human corpus cavernosum smooth-muscle relaxation.

Insulin-loaded PLGA/cyclodextrin large porous particles with improved aerosolization properties: In vivo deposition and hypoglycaemic activity after delivery to rat lungs

The aim of the present work is to develop large porous particles (LPP) of poly (lactide-co-glycolide) (PLGA) containing insulin with optimal aerodynamic properties and to test their in vivo potential, in pulmonary delivery. Insulin-loaded LPP were fabricated by a double emulsion method by aid of hydroxypropyl-beta-cyclodextrin (HPbetaCD). Conceiving this system for the controlled release of insulin to the lungs, the aerosolization properties and the release features in simulated lung fluids of PLGA/HPbetaCD/insulin LPP were investigated in depth. The technological results show that the combination of appropriate amounts of insulin and HPbetaCD plays a crucial role to achieve PLGA/HPbetaCD/insulin LPP with the desired bulk and aerodynamic properties, that is a highly porous structure, a very low density (0.1 g/ml), an experimental mass mean aerodynamic diameter (MMAD(exp)) ranging from 4.01 to 7.00 and a fine particle fraction (FPF) estimated to be 26.9-89.6% at the different airflow rates tested (i.e. 30-90 l/min). Confocal microscopy studies, performed after administration of labeled PLGA/HPbetaCD/insulin LPP to the rat lung by means of a low-scale dry powder inhaler (DPI), suggest that particles reach alveoli and remain in situ after delivery. The pharmacological effect of PLGA/HPbetaCD/insulin LPP was confirmed by dose-response studies performed on both normoglycaemic and streptozotocin-induced diabetic rats. While insulin solutions administered via pulmonary route are unable to cause a significant hypoglycaemic effect, insulin delivered through PLGA/HPbetaCD/insulin LPP at the same doses (0.5-4.0 IU/kg) significantly reduces blood glucose level as a function of the administered dose in both animal models. The developed LPP, tested in hyperglycaemic rats at evident pathological conditions, exerts a very significant and longer hypoglycaemic effect even at insulin doses as low as 0.5 IU/kg (about 0.5 mg of PLGA/HPbetaCD/insulin LPP per rat) as compared to a insulin solution. Taken together, our results support the viability of a dry powder formulation based on biodegradable LPP for the controlled release of insulin to the lungs. In vivo data show that PLGA/HPbetaCD/insulin LPP are able to reach alveoli, release insulin, which is absorbed in its bioactive form.

Involvement of β3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function

The β3-adrenoreceptor plays a major role in lipolysis but the role and distribution of β3-receptors in other specific sites have not been extensively studied. β3-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, β3-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO–cGMP signal transduction pathway. Here we show that β3-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective β3-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific β3-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal β3-receptor-mediated vasorelaxant tone and that β3-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that β3-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.

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 (KATP) 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 KATP 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 KATP 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 KATP 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 KATP channels on vascular smooth muscle cells but we cannot exclude an increase of KATP channel number probably due to the gene expression activation.

Sildenafil effect on the human bladder involves the L-cysteine/hydrogen sulfide pathway: a novel mechanism of action of phosphodiesterase type 5 inhibitors.

BACKGROUND Phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H(2)S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). OBJECTIVE To evaluate whether the L-cysteine/H(2)S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome. DESIGN, SETTING, AND PARTICIPANTS Samples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H(2)S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30 μM), 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP; 100 μM) or dibutyryl-cyclic adenosine monophosphate (dibutyryl-cAMP; 100 μM). A curve concentration effect of sodium hydrosulfide (NaHS), H(2)S donor (0.1 μM to 10mM), L-cysteine (0.1 μM to 10mM), and sildenafil (0.1-10 μM) was performed on precontracted detrusor dome strips. To investigate H(2)S signaling in a sildenafil effect, CBS and CSE inhibitors were used. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Analysis of variance was used, followed by the Bonferroni post hoc test. RESULTS AND LIMITATIONS CBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H(2)S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H(2)S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H(2)S production. CONCLUSIONS The sildenafil relaxant effect on the human bladder involves the H(2)S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H(2)S pathway in the human bladder may open new therapeutic approaches.

Hydrogen Sulfide-Induced Dual Vascular Effect Involves Arachidonic Acid Cascade in Rat Mesenteric Arterial Bed

Hydrogen sulfide (H2S), a novel gaseous transmitter, is considered a physiological regulator of vascular homeostasis. Recent evidence suggests H2S as an endothelium-hyperpolarizing factor (EDHF) candidate. To address this issue, we evaluated the vascular effect of sodium hydrogen sulfide (NaHS), an H2S donor, on the rat mesenteric arterial bed. NaHS concentration-response curve was performed on preconstricted mesenteric arterial bed. To assess the contribution of EDHF, we performed a pharmacologic dissection using indomethacin, NG-nitro-l-arginine methyl ester (l-NAME), or apamin and charybdotoxin as cyclooxygenase, nitric-oxide synthase, and calcium-dependent potassium channel inhibitors, respectively. In another set of experiments, we used 4-(4-octadecylphenyl)-4-oxobutenoic acid, baicalein, or proadifen as phospholipase A2 (PLA2), lipoxygenase, and cytochrome P450 inhibitors, respectively. Finally, an immunofluorescence study was performed to support the involvement of PLA2 in mesenteric artery challenged by NaHS. NaHS promoted a dual vascular effect (i.e., vasoconstriction and vasodilation). l-NAME or baicalein administration affected neither NaHS-mediated vasodilation nor vasoconstriction, whereas apamin and charybdotoxin significantly inhibited NaHS-induced relaxation. Pretreatment with PLA2 inhibitor abolished both the contracting and the relaxant effect, whereas P450 cytochrome blocker significantly reduced NaHS-mediated relaxation. The immunofluorescence study showed that NaHS caused a migration of cytosolic PLA2 close to the nucleus, which implicates activation of this enzyme. Our data indicate that H2S could activate PLA2, which in turn releases arachidonic acid leading, initially, to vasoconstriction followed by vasodilation mediated by cytochrome P450-derived metabolites. Because EDHF has been presumed to be a cytochrome P450 derivative of the arachidonic acid, our results suggest that H2S acts through EHDF release.

Hydrogen sulfide and erectile function: a novel therapeutic target

Hydrogen sulfide (H2S) is a gaseous transmitter involved in the control of vascular homeostasis. H2S is formed endogenously from L-cysteine or L-methionine by two enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), and normally circulates in blood. Studies from the past few years have demonstrated the involvement of H2S in erectile mechanisms in animal and human tissues. Exogenous H2S relaxes human and animal tissues in vitro and increases intracavernous pressure in experimental animal models. Electrical field stimulation studies on animal and human tissues have demonstrated that endogenous H2S is involved in the physiological control of penile tone. In humans, both CBS and CSE are widely expressed on trabecular muscle, implying that the smooth muscle component is the major source of H2S. Thus, the L-cysteine–H2S pathway may represent a promising target for development of new therapeutics for erectile dysfunction.

A superoxide dismutase mimetic with catalase activity (EUK-8) reduces the organ injury in endotoxic shock

Reactive oxygen species contribute to the multiple organ failure in endotoxic shock. Here, we investigate the effects of a salen-manganese complex, which exhibits both superoxide dismutase and catalase activity (EUK-8), on the circulatory failure, renal and liver injury and dysfunction caused by endotoxin in the anaesthetised rat. Endotoxaemia (6 mg/kg i.v., Escherichia coli lipopolysaccharide) for 6 h caused hypotension, renal dysfunction and liver injury. Treatment of rats with EUK-8 (0.3 or 1 mg/kg bolus injection followed by an infusion of 0.3 or 1 mg/kg/h) attenuated the renal and liver injury and dysfunction in a dose-related fashion. In addition, the higher dose of EUK-8 attenuated the delayed hypotension caused by endotoxin in the rat. Thus, an enhanced formation of reactive oxygen species importantly contributes to the circulatory failure, as well as the organ injury and dysfunction associated with endotoxic shock. We propose that small molecules, which have the catalytic activity of both superoxide dismutase and catalase, may represent a novel therapeutic approach for the therapy of endotoxic shock.

Melatonin prevents lipopolysaccharide‐induced hyporeactivity in rat

Abstract:  Melatonin (MT) is the principal secretory product of the pineal gland and its role as an immumo‐modulator is well established. Recent evidence shows that MT exerts protective effects in septic shock, hemorrhagic shock and inflammation. Lipopolysaccharide (LPS), from Escherichia coli, administered to animals directly stimulates a number of cells and systems to produce various inflammatory mediators. LPS‐induced septic shock is characterized by hypotension and vascular hyporeactivity to contracting agents. In particular, the reactive oxygen species such as superoxide and nitric oxide (NO) contribute to the pathophysiology of septic shock. In this study, we demonstrate that MT pretreatment prevents the hyporeactivity to phenylephrine in vivo and in aorta rings collected from rats treated with the endotoxin. The beneficial effect of MT seems related to its antioxidant properties and with inhibition of inducible nitric oxide synthase (iNOS) protein expression, reduction of NO production and nitrotyrosine formation, in aorta, preventing vascular, and endothelial injury. Additionally, we first demonstrate, that MT inhibited nuclear enzyme poly (ADP‐ribose) synthetase activation in vascular tissue. The current study underlined the protective effect of MT on the vascular dysfunction associated with septic shock, data that could support the clinical use of MT in human endotoxemia.