Repeated otilonium bromide administration prevents neurotransmitter changes in colon of rats underwent to wrap restraint stress.

Otilonium bromide (OB) is a spasmolytic drug successfully used for the treatment of irritable bowel syndrome (IBS). Its efficacy has been attributed to the block of L- and T-type Ca2+ channels and muscarinic and tachykinin receptors in the smooth muscle. Furthermore, in healthy rats, repeated OB administration modified neurotransmitter expression and function suggesting other mechanisms of action. On this basis, we investigated whether repeated OB treatment prevented the functional and neurochemical changes observed in the colon of rats underwent to wrap restrain stress (WRS) a psychosocial stressor considered suitable to reproduce the main IBS signs and symptoms. In control, WRS and OB/WRS rats functional parameters were measured in vivo and morphological investigations were done ex vivo in the colon. The results showed that OB counteracts most of the neurotransmitters changes caused by WRS. In particular, the drug prevents the decrease in SP-, NK1r-, nNOS-, VIP-, and S100ß-immunoreactivity (IR) and the increase in CGRP-, and CRF1r-IR. On the contrary, OB does not affect the increase in CRF2r-IR neurons observed in WRS rats and does not interfere with the mild mucosal inflammation due to WRS. Finally, OB per se increases the Mr2 expression in the muscle wall and decreases the number of the myenteric ChAT-IR neurons. Functional findings show a significantly reduction in the number of spontaneous abdominal contraction in OB treated rats. The ability of OB to block L-type Ca2+ channels, also expressed by enteric neurons, might represent a possible mechanism through which OB exerts its actions.

Stimulation of platelet nitric oxide production by nebivolol prevents thrombosis.

OBJECTIVE: dl-Nebivolol, a selective ß1-adrenergic receptor antagonist, besides its hypotensive activity exerts vasodilatory and platelet inhibitory effects in vitro by a mechanism involving nitric oxide (NO). Our aim was to evaluate whether nebivolol exerts in vivo antithrombotic effects, to unravel the mechanism of this action and to clarify the relative roles of its 2 enantiomers: d- and l-nebivolol. METHODS AND RESULTS: In wild-type mice, dl-nebivolol, l-nebivolol, and d-nebivolol, but not bisoprolol, reduced mortality consequent to platelet pulmonary thromboembolism induced by the intravenous injection of collagen plus epinephrine (-44%, -45%, -29%, respectively; P<0.05), whereas in eNOS(-/-) mice only dl-nebivolol and d-nebivolol were effective. dl-Nebivolol, l- and d-nebivolol reduced photochemical damage-induced femoral artery thrombosis in wild-type mice, whereas in eNOS(-/-) mice only dl-nebivolol and d-nebivolol were active. Moreover, dl-nebivolol and l-nebivolol increased plasma, urinary-, and platelet-derived nitrites and nitrates (NOx), NO degradation products, in wild-type but not in eNOS(-/-) mice. In vivo platelet activation, assessed by platelet P-selectin expression, was reduced by dl-nebivolol and l- and d-nebivolol in wild-type mice but only by dl-nebivolol and d-nebivolol in eNOS(-/-) mice. In bone marrow-transplanted, chimeric mice with only blood cells, and not the endothelium, producing NO dl-nebivolol and l-nebivolol maintained their antithrombotic activity, whereas they lose it in chimeras with only endothelium, and not blood cells, producing NO. In vitro, with isolated platelets, dl-nebivolol and l-nebivolol, but not d-nebivolol and bisoprolol, increased platelet cGMP and NOx formation. Treatment with dl-nebivolol and l-nebivolol increased phophorylated eNOS in platelets. CONCLUSIONS: Our data show that dl-nebivolol exerts an antithrombotic activity by stimulating the formation of NO by platelets, and that this effect is generated by its l-enantiomer, whereas the d-enantiomer exerts a weak antiplatelet effect because of ß-adrenergic receptor-independent stimulation of adenyly cyclase. These results confirm that platelet-derived NO plays a role in thrombosis prevention and it may represent a target of pharmacological intervention.

Capsaicin receptor as target of calcitonin gene-related peptide in the gut.

Calcitonin gene-related peptide (CGRP), a 37 aminoacid-residue peptide, is a marker of afferent fibers in the upper gastrointestinal tract, being almost completely depleted following treatment with the selective neurotoxin capsaicin that targets these fibers via transient receptor potential vanilloid type-1 (TRPV-1). It is widely distributed in the peripheral nervous system of mammals where it is present as alpha isoform, while intrinsic neurons of the enteric nervous systems express predominantly CGRP-beta. Many gastrointestinal functions involve CGRP-containing afferent fibers of the enteric nervous system such as defense against irritants, intestinal nociception, modulation of gastrointestinal motility and secretion, and healing of gastric ulcers. The main effects on stomach homeostasis rely on local vasodilator actions during increased acid-back diffusion. In humans, release of CGRP through the activation of TRPV-1 has been shown to protect from gastric damage induced by several stimuli and to be involved in gastritis. In both dyspepsia and irritable bowel syndrome the repeated stimulation of TRPV-1 induced an improvement in epigastric pain of these patients. The TRPV-1/CGRP pathway might be a novel target for therapeutics in gastric mucosal injury and visceral sensitivity.

T-type Ca(2+) channel modulation by otilonium bromide.

Antispasmodics are used clinically to treat a variety of gastrointestinal disorders by inhibition of smooth muscle contraction. The main pathway for smooth muscle Ca(2+) entry is through L-type channels; however, there is increasing evidence that T-type Ca(2+) channels also play a role in regulating contractility. Otilonium bromide, an antispasmodic, has previously been shown to inhibit L-type Ca(2+) channels and colonic contractile activity. The objective of this study was to determine whether otilonium bromide also inhibits T-type Ca(2+) channels. Whole cell currents were recorded by patch-clamp technique from HEK293 cells transfected with cDNAs encoding the T-type Ca(2+) channels, Ca(V)3.1 (alpha1G), Ca(V)3.2 (alpha1H), or Ca(V)3.3 (alpha1I) alpha subunits. Extracellular solution was exchanged with otilonium bromide (10(-8) to 10(-5) M). Otilonium bromide reversibly blocked all T-type Ca(2+) channels with a significantly greater affinity for Ca(V)3.3 than Ca(V)3.1 or Ca(V)3.2. Additionally, the drug slowed inactivation in Ca(V)3.1 and Ca(V)3.3. Inhibition of T-type Ca(2+) channels may contribute to inhibition of contractility by otilonium bromide. This may represent a new mechanism of action for antispasmodics and may contribute to the observed increased clinical effectiveness of antispasmodics compared with selective L-type Ca(2+) channel blockers.

ACE-inhibition ameliorates vascular reactivity and delays diabetes outcome in NOD mice.

Recently, we have demonstrated a direct correlation among hyperglycaemia, vascular dysfunction and eNOS post-translational regulation in non non-obese diabetic mice (NOD). Here, we evaluate the impact of two ACE-inhibitors therapy, zofenopril and enalapril in NOD mice. Insulin-dependent diabetes mellitus (IDDM) development was monitored weekly through glycosuria measurement. Zofenopril and enalapril were dosed at 0.5 mg/kg/die orally. Animals were sacrificed at different points and aortas used for western blotting or for tissue bath experiments. Bovine aortic endothelial cells in high glucose medium are treated with zofenoprilat or enalaprilat. Cells and supernatant were utilised for western blot analysis and for nitrite/nitrate determination, respectively. In ex-vivo experiments chronic administration of both drugs restored PE-induced contraction but not Isop-induced vasodilatation, however only zofenopril reduced caveolin-1 expression. In vitro, both drugs inhibited caveolin-1 expression and increased NOx production. However, zofenopril caused inhibition of both parameters at a concentration 200 fold lower than enalalpril. In vivo, zofenopril delays the onset of diabetic conditions of about 50%, and ameliorates polyuria. In conclusion our data suggest that ACE-inhibitor therapy may be useful in IDDM, in particular sulphydrylated inhibitor would display a better efficacy especially if administered early on the development of diabetes.

Different effects of angiotensin converting enzyme inhibitors on endothelin-1 and nitric oxide balance in human vascular endothelial cells: evidence of an oxidant-sensitive pathway.

Angiotensin converting enzyme inhibitors (ACE-I) are able to reduce the formation of the potent vasoconstrictor endothelin-1 and increase nitric oxide bioavailability in human vascular endothelial cells (HUVECs). We tested the effects of two sulfhydryl-containing ACE-I, zofenoprilat, and captopril, and two nonsulfhydryl containing ACE-I, enalaprilat and lisinopril, on endothelin-1/nitric oxide balance and oxidative stress in HUVECs. All the four tested ACE-I reduced endothelin-1 secretion and increased nitric oxide metabolite production by HUVECs. However, zofenoprilat (-42% after 8 hours of incubation) was more effective (P < .05) than enalaprilat (-25%), lisinopril (-21%), and captopril (-30%) in reducing endothelin-1 secretion. Similarly, zofenoprilat (+110% after 8 hours of incubation) was more effective (P < .05) than enalaprilat (+64%), lisinopril (+63%), and captopril (+65%) in increasing nitric oxide metabolite production. The effect of ACE-I on endothelin-1 and nitric oxide metabolite production is mediated by the activation of bradykinin B(2) receptor being counteracted, at least in part, by a specific antagonist. Zofenoprilat and, to a lesser extent, captopril also reduced oxidative stress in HUVECs. In conclusion, among the four tested ACE-I, zofenoprilat was more effective in improving endothelin-1/nitric oxide balance in HUVECs likely because of its greater antioxidant properties.

Effects of nebivolol on endothelial gene expression during oxidative stress in human umbilical vein endothelial cells.

The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs) following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L) for 24 hours and oxidative stress was induced by the addition of oxidized (ox)-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin); proteins linked to inflammation (IL-6 and TNFalpha), thrombotic state (tissue factor, PAI-1 and uPA), hypertension such as endothelin-1 (ET-1), and vascular remodeling such as metalloproteinases (MMP-2, MMP-9) and protease inhibitor (TIMP-1). The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

Experimental Models of Irritable Bowel Syndrome and the Role of the Enteric Neurotransmission.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal diseases in humans. It is characterized by visceral pain and/or discomfort, hypersensitivity and abnormal motor responses along with change in gut habits. Although the etio-pathogenesis of IBS is only partially understood, a main role has been attributed to psychosocial stress of different origin. Animal models such as neonatal maternal separation, water avoidance stress and wrap restraint stress have been developed as psychosocial stressors in the attempt to reproduce the IBS symptomatology and identify the cellular mechanisms responsible for the disease. The study of these models has led to the production of drugs potentially useful for IBS treatment. This review intends to give an overview on the results obtained with the animal models; to emphasize the role of the enteric nervous system in IBS appearance and evolution and as a possible target of drug therapies.

Otilonium Bromide: A Drug with a Complex Mechanism of Action.

Otilonium bromide (OB) is a drug with spasmolytic activity belonging to quaternary ammonium derivatives and extensively used to treat patients affected by the Irritable Bowel Syndrome (IBS). Thanks to its peculiar pharmacokinetic, OB concentrates in the large bowel wall and acts locally. From the pharmacodynamics point of view, OB is able to inhibit i) the main patterns of human colonic motility in vitro; ii) the contractility caused by excitatory motor neurons stimulation (pre-synaptic action) and iii) the contractility caused by the direct action of excitatory neurotransmitters (post-synaptic action). Interestingly, these effects derive from a complex interaction between the drug and several cellular targets. The main action consists in the blockade of Ca2+ entry through L-type Ca2+ channels and interference with intracytoplasmatic Ca2+ mobilization necessary for SMC contraction, thus preventing excessive bowel contractions and abdominal cramps. Further, OB blocks the T-type Ca2+ channels and interferes with the muscarinic responses; it interacts, directly or indirectly, with the tachykinin receptors on SMC and on primary afferent neurons whose combined effects may result in the reduction of motility and abdominal pain. In summary, a revision of this complex picture of OB activity could help to better address its therapeutic use.

Long-term nebivolol administration reduces renal fibrosis and prevents endothelial dysfunction in rats with hypertension induced by renal mass reduction.

OBJECTIVES: D/L-Nebivolol is a lypophilic beta1-adrenergic antagonist which is devoid of intrinsic sympathomimetic activity and can increase nitric oxide (NO) bioavailability with its subsequent vasodilating properties. The purpose of the present work was to assess the effect of long-term nebivolol administration on both renal damage and endothelial dysfunction induced by renal mass reduction (RMR) in rats. Atenolol, which does not increase NO bioavailability, was included in the study as a comparative beta-adrenoceptor antagonist. METHODS: Rats were subjected to both right nephrectomy and surgical removal of two-thirds of the left kidney in order to retain approximately one-sixth of the total renal mass. One week after ablation, rats were distributed randomly according to the following experimental groups: control group containing RMR rats without treatment; RMR rats treated daily with nebivolol for 6 months (drinking water, 8 mg/kg per day); and RMR rats treated daily with atenolol for 6 months (drinking water, 80 mg/kg per day). A group of sham-operated animals was also included. RESULTS: Administration of either nebivolol or atenolol similarly reduced arterial pressure in comparison with RMR untreated animals; however, animals receiving nebivolol presented lower levels of collagen type I expression as well as lower glomerular and interstitial fibrosis than those receiving atenolol. Urinary excretion of oxidative stress markers were also lower in animals receiving nebivolol than in rats treated with atenolol. Furthermore, nebivolol prevented RMR-induced endothelial dysfunction more efficiently than atenolol. CONCLUSIONS: Nebivolol protects against renal fibrosis, oxidative stress and endothelial dysfunction better than equivalent doses, in terms of arterial pressure reduction, of atenolol in a hypertensive model of renal damage induced by RMR.

Drug evaluation: Pumosetrag for the treatment of irritable bowel syndrome and gastroesophageal reflux disease.

Dynogen Pharmaceuticals Inc, under license from Mitsubishi Pharma Corp, is developing pumosetrag (MKC-733, DDP-733), an orally available gastroprokinetic agent and locally acting 5-HT3 partial agonist, for the potential treatment of irritable bowel syndrome (IBS) with constipation and nocturnal gastroesophageal reflux disease (GERD). In September 2005, Dynogen commenced a phase II proof-of-concept trial of pumosetrag in IBS with constipation; positive results were reported in February 2007 and a phase IIb trial was to start in the fourth quarter of 2007. In September 2006, the company had initiated a phase Ib trial in nocturnal GERD.

Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot lysis models: sensitivity to the inhibitory action of thrombin activatable fibrinolysis inhibitor (TAFI).

In this study, the in-vitro fibrinolytic efficacy of Tenecteplase, Amediplase and scu-PA was investigated in different external lysis models by measuring the lysis of human plasma clots after the addition of the plasminogen activators (PAs) to the surrounding plasma. The effect of TAFI was examined for each PA by neutralising TAFIa with potato carboxypeptidase inhibitor (PCI). The lytic efficacy of Amediplase was lower than that of Tenecteplase at low PA concentrations but slightly higher at therapeutic concentrations. The activity of scu-PA was clearly lower than that of either Tenecteplase or Amediplase. The TAFI system inhibited external clot lysis mediated by all the PAs when thrombomodulin was present in the model. In the therapeutic range (5-10 mug/ml) however, the TAFIa effect was negligible for both Amediplase and Tenecteplase. At lower PA concentrations the effect of TAFI on Amediplase was slightly stronger than that on Tenecteplase. Under static conditions the lysis rates were lower than with stirring. The role of TAFI was similar under both conditions. In conclusion, at therapeutic concentrations Amediplase was slightly more active than Tenecteplase and scu-PA under all conditions used. Therefore, Amediplase might possibly be a more potent thrombolytic agent at these concentrations and increase the efficacy of thrombolysis. The potential of TAFI for inhibiting thrombolytic therapy is probably low. However in conditions where the local PA concentrations are sub-optimal TAFI might affect the lysis rate.

Zofenopril Protects Against Myocardial Ischemia-Reperfusion Injury by Increasing Nitric Oxide and Hydrogen Sulfide Bioavailability.

BACKGROUND: Zofenopril, a sulfhydrylated angiotensin-converting enzyme inhibitor (ACEI), reduces mortality and morbidity in infarcted patients to a greater extent than do other ACEIs. Zofenopril is a unique ACEI that has been shown to increase hydrogen sulfide (H2S) bioavailability and nitric oxide (NO) levels via bradykinin-dependent signaling. Both H2S and NO exert cytoprotective and antioxidant effects. We examined zofenopril effects on H2S and NO bioavailability and cardiac damage in murine and swine models of myocardial ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: Zofenopril (10 mg/kg PO) was administered for 1, 8, and 24 hours to establish optimal dosing in mice. Myocardial and plasma H2S and NO levels were measured along with the levels of H2S and NO enzymes (cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and endothelial nitric oxide synthase). Mice received 8 hours of zofenopril or vehicle pretreatment followed by 45 minutes of ischemia and 24 hours of reperfusion. Pigs received placebo or zofenopril (30 mg/daily orally) 7 days before 75 minutes of ischemia and 48 hours of reperfusion. Zofenopril significantly augmented both plasma and myocardial H2S and NO levels in mice and plasma H2S (sulfane sulfur) in pigs. Cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and total endothelial nitric oxide synthase levels were unaltered, while phospho-endothelial nitric oxide synthase(1177) was significantly increased in mice. Pretreatment with zofenopril significantly reduced myocardial infarct size and cardiac troponin I levels after I/R injury in both mice and swine. Zofenopril also significantly preserved ischemic zone endocardial blood flow at reperfusion in pigs after I/R. CONCLUSIONS: Zofenopril-mediated cardioprotection during I/R is associated with an increase in H2S and NO signaling.

Talnetant GlaxoSmithKline.

Talnetant (SB-223412) is a selective, orally active NK3 antagonist based on 4-quinolinecarboxamide, and is under development by GlaxoSmithKline (formerly SmithKline Beecham) for the potential treatment of several disorders, including urinary incontinence, irritable bowel syndrome and schizophrenia. By November 2004, the compound had completed phase II trials.

Involvement of nitric oxide in both central and peripheral haemodynamic effect of D/L-nebivolol and its enantiomers in rats.

The cardiovascular profile of the racemate D/L-nebivolol and its enantiomers administered by intravenous (i.v.) or by intracerebroventricular (i.c.v.) route was investigated in anaesthetized normotensive rats. D/L-Nebivolol (0.1-0.5 mg/kg) induced a dose-related reduction in blood pressure when administered by i.c.v. route. These hypotensive effects were more marked as compared to those achieved by peripheral administration of D/L-nebivolol (0.1-1 mg/kg i.v.). Both enantiomers contributed to the hypotensive effect of D/L-nebivolol by i.c.v. route, while the effects of the drug on blood pressure by i.v. route were due to the d-enantiomer. The bradycardic effect of the racemic form given i.v. was dose-related and, at the highest dose (1 mg/kg), was more pronounced as compared to i.c.v. route. D-Nebivolol was responsible for chronotropic effects by both the i.v. and i.c.v. route, although by i.c.v. route L-nebivolol also induced a reduction in heart rate. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) administered at 5 mg/kg i.v. bolus + 0.1 mg/kg/min infusion or at 2.5 mg/kg i.c.v. counteracted the effects of D/L-nebivolol (either 1 mg/kg i.v. or 0.5 mg/kg i.c.v.) on blood pressure, while it did not inhibit the cardiovascular changes induced by isoprenaline (300 ng/kg i.v.) or calcitonin gene-related peptide (CGRP; 400 ng/kg i.v.). In addition, i.c.v. effects of D/L-nebivolol on blood pressure and heart rate were not affected by pre-treatment with atropine (2 mg/kg i.v.). The present findings demonstrate that D/L-nebivolol produced haemodynamic changes following both peripheral and central administration; these latter findings are mainly due to its L-enantiomer and these effects involve the L-arginine/nitric oxide pathway.

Novel therapeutics in the field of capsaicin and pain.

Capsaicin, a pharmacologically active agent found in chili peppers, causes burning and itching sensation due to binding at the transient receptor potential vanilloid-1 (TRPV-1) receptor, a polymodal receptor critical to the sensing of a variety of stimuli (e.g., noxious heat, bidirectional pH), and subsequent activation of polymodal C and A-δ nociceptive fibers. Acutely, TRPV-1 activation with peripheral capsaicin produces pronociceptive effects, which extends to the development of hyperalgesia and allodynia. However, capsaicin has been reported to display antinociceptive properties as well, largely through TRPV-1-dependent mechanisms. Local application of high concentration of capsaicin is used for neuropathic pain and repeated stimulation of TRPV-1 induced an improvement of epigastric pain in irritable bowel syndrome and dyspepsia patients by desensitization of nociceptive pathways. New TRPV-1 agonists are currently under preclinical study and TRPV-1 antagonists are in early clinical development as analgesics. The TRPV-1 pathway might be a novel target for therapeutics in pain sensitivity.

The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.

Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species.

Hypertension and coronary artery disease are intimately connected. The migration of circulating monocytes into the subendothelial occurs through the expression of some adhesion molecules on endothelial cells. The nuclear factor (NF)-kappaB, a redox-sensitive element, plays a key role in adhesion molecule gene induction. In this study we have compared the effects of two different angiotensin converting enzyme (ACE) inhibitors, one possessing an active sulfhydryl group (zofenopril) and one lacking this group (enalapril) on the cellular redox state (monitored by measuring intracellular reactive oxygen species and thiol status), expression of adhesion molecules, and activation of NF-kappaB in human umbilical vein endothelial cells (HUVECs). Zofenoprilat, the active form of zofenopril, significantly and dose dependently reduced the intracellular reactive oxygen species (ROS) and superoxide formation induced by oxidized low-density lipoprotein (ox-LDL) (P <.001) and tumor necrosis factor-alpha (TNF-alpha) (P <.001). Enalaprilat, the active form of enalapril, was ineffective. Zofenoprilat but not enalaprilat also decreased the consumption of the intracellular GSH induced by ox-LDL (P <.01) and TNF-alpha (P <.01). Although zofenoprilat significantly and dose dependently reduced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and E-selectin induced by ox-LDL (P <.01) and TNF-alpha (P <.01) on HUVECs, enalaprilat did not. Ox-LDL and TNF-alpha increased the activation of NF-kappaB and the preincubation of HUVECs with zofenoprilat, but not with enalaprilat, dose dependently reduced its activation (P <.001). The conclusion is that the sulfhydryl (SH)-containing ACE inhibitors may be useful in inhibiting foam cell formation and thus slow the development of atherosclerosis.

Extended analysis of a double-blind, placebo-controlled, 15-week study with otilonium bromide in irritable bowel syndrome.

BACKGROUND/OBJECTIVE: In order to follow the most recent developments and recommendations in trial methodology for drug evaluation in patients with irritable bowel syndrome, we performed an extended analysis of a large clinical trial from a previously published study of otilonium bromide, using an assessment that integrates the key symptoms of irritable bowel syndrome. MATERIALS AND METHODS: A large-scale clinical trial with a double-blind, placebo-controlled, parallel-group study design was conducted in 378 patients, treated for 15 weeks with the recommended standard dose of 40 mg otilonium bromide or placebo three times daily. The study was based on the collection of 12 single efficacy endpoints. The new efficacy assessment was based on the data reported by the patients. Rather than demonstrating score differences between the treatment groups of the study, we carried out an assessment that integrates the most frequent symptoms reported (pain frequency and intensity, presence of meteorism and distension) by the patient. RESULTS: The rate of response to treatment within 2-4 months (the primary efficacy outcome measure) was significantly higher in the otilonium bromide group (36.9%) than in the placebo group (22.5%; P = 0.007). In each month of treatment, the rate of monthly response was higher in the otilonium bromide group as compared to the placebo group (P < 0.05). The total monthly and weekly responses to the single endpoints (intensity and frequency of pain and discomfort, meteorism/abdominal distension, severity of diarrhoea or constipation and mucus in the stool) were significantly more frequent in the group treated with otilonium bromide than in the placebo-treated group, with differences ranging from 10% to 20%. The subgroup analysis of the intestinal habits endpoint indicates that patients with diarrhoea have an additional benefit. CONCLUSION: The present re-evaluation of a previously published study confirms that otilonium bromide is more effective than placebo for the treatment of irritable bowel syndrome, being very efficient in relieving pain and discomfort.

Hydrogen sulfide accounts for the peripheral vascular effects of zofenopril independently of ACE inhibition.

AIMS: Therapeutic use of sulfhydrylated inhibitor S-zofenopril has raised different hypotheses regarding the role played by its thiol group in the beneficial clinical effects exerted compared with other angiotensin-converting enzyme (ACE) inhibitors. Here, we investigated hydrogen sulfide (H2S) pathway as accountable for extra-beneficial effects in vascular function. METHODS AND RESULTS: Spontaneously hypertensive rat (SHRs) and control Wistar Kyoto (WKY) rats were treated with either S-zofenopril or enalapril in vivo. Aorta and carotid were harvested and ex vivo vascular reactivity to acetylcholine (Ach) and L-cysteine (L-cys) assessed. Cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptosulfur-transferase (3MST) expression, as well as H2S levels, were evaluated in both vascular tissues. The vascular response to Ach in both carotid and aorta was impaired in SHR (~30%, P < 0.001). S-zofenopril, but not enalapril, restored this response, while L-cys-induced relaxation was enhanced. CSE expression in vessels and tissue/plasma H2S levels were restored to WKY values in SHRs receiving S-zofenopril. In contrast, CBS and 3MST expression were not modified by treatments. S-zofenoprilat, an active metabolite of S-zofenopril, releases H2S in a 'cell-free' assay and it directly relaxed vessels in vitro in a concentration-dependent manner (P < 0.001). In vivo administration of R-zofenoprilat diasteroisomer, which does not inhibit ACE, did not modify blood pressure; nonetheless, it retained the beneficial effect on SHR vascular function as well as restored plasma/tissue H2S levels. CONCLUSION: Our findings establish that S-zofenopril improves vascular function by potentiating the H2S pathway in a model of spontaneous hypertension. This novel mechanism, unrelated to ACE inhibition and based on H2S release, could explain the beneficial effects of sulfhydrylated ACE inhibitors reported in the clinical literature.