In vitro and in vivo comparison between poractant alfa and the new generation synthetic surfactant CHF5633.

BACKGROUND: CHF5633 is a new generation synthetic surfactant containing both SP-B and SP-C analogues developed for the treatment of respiratory distress syndrome. Here, the optimal dose and its performance in comparison to the animal-derived surfactant poractant alfa were investigated. METHODS: In vitro surfactant activity was determined by means of the Wilhelmy balance and the capillary surfactometer. The dose-finding study was performed in preterm rabbits with severe surfactant deficiency. CHF5633 doses ranging from 50 to 300 mg/kg were used. Untreated animals and animals treated with 200 mg/kg of poractant alfa were included for comparison. RESULTS: In vitro, minimum surface tension (γmin) was decreased from values above 70 to 0 mN/m by both surfactants, and they formed rapidly a film at the air-liquid interface. In vivo studies showed a clear dose-dependent improvement of lung function for CHF5633. The pulmonary effect of CHF5633 200 mg/kg dose was comparable to the pulmonary response elicited by 200 mg/kg of poractant alfa in preterm rabbits. CONCLUSION: CHF5633 is as efficient as poractant alfa in our in vitro and in vivo settings. A clear dose-dependent improvement of lung function could be observed for CHF5633, with the dose of 200 mg/kg being the most efficient one.

Combined simvastatin-manidipine protect against ischemia-reperfusion injury in isolated hearts from normocholesterolemic rats.

This study investigated whether oral simvastatin and manidipine interact in protecting the perfused rat heart from ischemia-reperfusion damage. Simvastatin (0.3 to 3 mg/kg) and manidipine (1 to 10 mg/kg) were given orally singly or together to normocholesterolemic rats once a day for seven consecutive days. At the end of treatment, systolic blood pressure and heart rate were measured in conscious rats, and the lipid profile and other biochemical markers, such as thromboxane B(2), nitrite/nitrates and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)) were determined in the plasma. Hearts were then isolated, perfused with Krebs-Henseleit, and subjected to low flow ischemia-reperfusion injury. Post-ischemic recovery of left ventricular function was measured as left ventricular developed pressure and left ventricular end-diastolic pressure. Creatine kinase, lactate dehydrogenase, tumor necrosis factor-alpha and 6-keto-PGF(1alpha) were measured in the heart effluents. In conscious animals, simvastatin alone increased plasma 6-keto-PGF(1alpha) release while manidipine alone reduced systolic blood pressure with a slight sympathetic reflex increase in heart rate, and increased plasma nitrite/nitrates. The combined treatment produced the same effects, but significantly more marked, and accompanied by a significant reduction of thromboxane B(2). Combined treatment was also significantly more effective than the single drugs in protecting the hearts from ischemia-reperfusion injury, with inhibition of creatine kinase, lactate dehydrogenase and tumor necrosis factor-alpha, and enhancement of 6-keto-PGF(1alpha) during reperfusion. These data show that simvastatin and manidipine interact positively in protecting the rat heart from ischemia-reperfusion injury, possibly through increased prostaglandin and nitric oxide formation by the vascular endothelial cells.

Effect of beta-adrenergic and renin-angiotensin system blockade on myocyte apoptosis and oxidative stress in diabetic hypertensive rats.

Diabetes aggravates the clinical severity and represents an additional independent risk factor of hypertension. Since both diseases separately concur to cardiomyocyte apoptosis, a mechanism at least partly involving unbalanced oxidative stress, we investigated whether the combination of diabetes and hypertension potentiated cardiac cell death in experimental models, compared to either disease alone. We also evaluated the short-term effects of different drugs in these models. Streptozotocin-induced diabetic normotensive (WKY) or hypertensive (SHR) rats were treated for one week with a DA(2)/alpha(2) agonist (CHF-1024), a selective beta1 adrenergic blocker (metoprolol), an angiotensin II-receptor blocker (valsartan) or a radical scavenger (tempol). In separate experiments, isolated cardiomyocytes were cultured in high glucose medium (25 mM) containing the same drugs. Although the number of apoptotic cardiomyocytes and the myocardial density of oxygen radicals were higher in non diabetic hypertensive than in normotensive controls, diabetes raised these variables to comparable absolute levels in both strains. All drugs except metoprolol significantly reduced apoptosis and oxidative stress in the diabetic animals of both strains and in the isolated myocytes cultured with high glucose. In conclusion, hypertensive rat is no more susceptible than its normotensive control to acute apoptosis induced by diabetes. Oxidative stress might be considered the common trigger for cardiac myocyte apoptosis in both conditions.

Positive interaction of the beta2-agonist CHF 4226.01 with budesonide in the control of bronchoconstriction induced by acetaldehyde in the guinea-pigs.

Pretreatment of anaesthetized guinea-pigs with either CHF 4226.01 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methylethyl]amino]ethyl] carbostyril hydrochloride), formoterol or budesonide reduced acetaldehyde (AcCHO)-evoked responses in the lungs with a rank order of potency CHF 4226.01 (ED(50) values, from 1.88 to 3.31 pmol) > formoterol (ED(50) values, from 3.03 to 5.51 pmol) >> budesonide (ED(50) values, from 335 to 458 nmol). The duration of action of CHF 4226.01 in antagonizing the airway obstruction elicited by AcCHO was also substantially longer than formoterol (area under the curve) at 10 pmol, 763+/-58 and 480+/-34, respectively; P<0.01). Continuous infusion of a subthreshold dose of AcCHO enhanced the intratracheal pressure (ITP) increases caused by subsequent challenges with substance P (from 9.7+/-0.8 to 27.5+/-1.6 cm H(2)O as a peak, P<0.001). Pretreatment with either CHF 4226.01 or formoterol prevented the sensitizing effect of AcCHO on substance P responses (ED(50) values, 2.85 and 6.11 pmol, respectively; P<0.01). The ED(50) value of budesonide (396 nmol) in preventing AcCHO-evoked ITP increase was reduced when this glucocorticoid was combined with 0.1 pmol CHF 4226.01 (ED(50) 76 nmol; P<0.001). CHF 4226.01/budesonide was two-fold more effective (P<0.01) than the formoterol/budesonide combination. These results suggest that CHF 4226.01/budesonide, by optimizing each other's beneficial potential in the control of pulmonary changes caused by AcCHO in the guinea-pigs, may represent a new fixed combination in asthma.

Evaluation of therapeutic pulmonary surfactants by thin liquid film methods.

An example of the application of the Black Foam Film (BFF) Method and the Wetting Film Method, using the Microinterferomertric and the Pressure Balance Techniques, for characterization interfacial properties of the animal derived therapeutic pulmonary surfactant preparations (TSP), is presented. BFF thickness, probability of black film formation, and disjoining pressure for foam films from TSP aqueous solutions are measured as well as the wetting properties of TSP solutions on solid surfaces with different hydrophobicity have been studied. Interfacial characteristics such as minimal surfactant concentration to obtain black film (critical concentration) and concentration at which a black film is 100% obtained (threshold concentration) are determined. An evaluation of the four widely used TSP ­ Curosurf, Infasurf, Survanta, and Alveofact ­ by these methods has been carried out. Thus the methods of the thin liquid films are useful tools for studying the interfacial properties of TSP solutions, as well as for their improvement.

A novel D2-dopaminergic and alpha2-adrenoceptor receptor agonist induces substantial and prolonged IOP decrease in normotensive rabbits.

The effects of a novel and selective D2-dopaminergic/alpha2-adrenoceptor agonist, CHF1035, and its metabolite CHF1024 on intraocular pressure (IOP) were determined in rabbits. Because CHF1035 is a mixture of two enantiomers, CHF1800 (+) and CHF1810 (-), pure enantiomers were also studied to determine possible differences in IOP-decreasing ability depending on the stereochemistry of the molecule. CHF1035, CHF1800 (+), CHF1810 (-), CHF1024, brimonidine and 0.9% NaCl were administered topically to rabbits and IOP was then measured at fixed time intervals. The dose-response profile (0.01-1.0% w/v) was determined for CHF1035. CHF1035 and its metabolite CHF1024 significantly lowered IOP in the treated eyes. CHF1035 showed a maximum IOP decrease (7.6 +/- 1.5 mmHg) 5 h post-dosing, whereas the metabolite CHF1024 showed a maximum decrease in IOP (7.0 +/- 0.8 mmHg) 3 h post-dosing. The maximum IOP decrease produced by CHF1035 in the treated eye was comparable with that produced by brimonidine (7.8 +/- 0.9 mmHg), but CHF1035 had a significantly longer duration of action. Unlike brimonidine, CHF1035 and CHF1024 did not decrease IOP in the untreated eye. CHF1810 (-) lowered the IOP more than CHF1800 (+). No irritation, evaluated as eyelid closure, was observed after topical administration of any of the compounds. Only in the case of CHF1035 1% solution, two rabbits out of six closed the eye for 30-45 s. In conclusion, CHF1035 and its metabolite CHF1024 significantly decreased the IOP in rabbits, and are potential novel IOP lowering agents. Especially, CHF1035 produced a substantial decrease in IOP for a prolonged period of time, and thus may prove useful in glaucoma therapy.

New surfactant with SP-B and C analogs gives survival benefit after inactivation in preterm lambs.

BACKGROUND: Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if P(a)O(2) dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals. CONCLUSIONS: For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.

Positive interaction of the novel beta2-agonist carmoterol and tiotropium bromide in the control of airway changes induced by different challenges in guinea-pigs.

This study evaluated the bronchodilating activity of the beta(2)-agonist carmoterol and the muscarinic M(3)-antagonist tiotropium, given intratracheally alone or in combination in anaesthetized artificially ventilated normal and actively sensitized guinea-pigs. Carmoterol (0.3-100pmol) and tiotropium (10-1000pmol) were superfused (0.01ml/min) for 5min before challenges with acetylcholine (20mug/kg i.v.), histamine (10mug/kg i.v.) or ovalbumin (5mg/kg i.v.). Both compounds given alone were markedly active against all the challenges. Tiotropium resulted more effective towards cholinergic challenge and carmoterol was very potent against histamine and ovalbumin-induced reaction, being effective already at 1pmol. In the presence of tiotropium, the bronchodilating activity of carmoterol was significantly augmented. The ED(50) value of carmoterol on the acetylcholine challenge was reduced by about 10 and 28 times (0.1 and 0.3pmol of tiotropium), that on the histamine one by 4.5 and 13 times (1 and 3pmol of tiotropium) and that on the ovalbumin-induced one by 8 and 25 times (10 and 30pmol of tiotropium). A positive interaction was also evident when other parameters were evaluated. The histamine-induced release of thromboxane B(2) was markedly reduced (56%, P<0.001) by combining completely ineffective doses of the two drugs (0.3 and 3pmol for carmoterol and tiotropium, respectively). In ovalbumin-challenged animals the time to death, amounting in control animals to 7.2+/-0.9min, was dose-dependently prolonged up to achieve complete protection from death with combination of 1 and 30pmol of carmoterol and tiotropium, respectively. The favorable interaction between carmoterol and tiotropium can represent a good option in the control of bronchopulmonary diseases marked by an increase of airway resistances.

Formoterol and beclomethasone dipropionate interact positively in antagonising bronchoconstriction and inflammation in the lung.

These studies were designed to assess the pharmacodynamic interaction between formoterol and beclomethasone dipropionate (BDP) in controlling the bronchoconstriction and inflammatory response induced by various challenges in guinea-pigs and rats. In anaesthetised guinea-pigs, superfusion of the formoterol/BDP combination into the tracheal lumen had significantly more effect than the single components in antagonising the bronchoconstricting and inflammatory responses to acetylcholine or ovalbumin in a standard model of airway hyper-responsiveness. After ovalbumin challenge, the combination completely protected animals from death at doses lower than those effective when given separately. The combination, at doses ineffective individually, even counteracted the development of lung oedema induced by sephadex in the rat. Finally, in tracheal strips from ovalbumin-sensitised guinea-pigs pre-treatment with BDP (30 mg kg(-1) i.m.) completely reversed the rightward shift of the formoterol dose-response curve due to beta(2)-receptor desensitisation. In conclusion, these results indicate that formoterol and BDP together induce a favourable pharmacodynamic interaction which can be considered more than additive, at least in these experimental settings.

Effects of the alpha2-adrenergic/DA2-dopaminergic agonist CHF-1024 in preventing ventricular arrhythmogenesis and myocyte electrical remodeling, in a rat model of pressure-overload cardiac hypertrophy.

Cardiac hypertrophy induces morpho-functional myocardial alterations favoring arrhythmogenesis, especially under specific conditions such as sympathetic stimulation. We analyzed whether the dopaminergic agent CHF-1024, given its effect in decreasing adrenergic drive and collagen deposition in hypertrophied hearts, can also reduce arrhythmia vulnerability. Eighty-one male Wistar rats with intrarenal aortic coarctation and 18 control animals were studied. Fifty-eight banded animals were treated with CHF-1024 at four different doses (6, 2, 0.67, or 0.067 mg/Kg/die). One month after aortic ligature, spontaneous and sympathetic-induced ventricular arrhythmic events (VAEs) were telemetrically recorded in conscious animals. After sacrifice, membrane capacitance (Cm) and action potential duration (APD) were measured in isolated left ventricular myocytes (patch-clamp). In all groups, spontaneous VAEs were negligible whereas they significantly increased during sympathetic activation (stress exposure). Banded untreated animals showed a higher number of stress-induced VAEs, longer action potentials, and larger values of Cm and cell width as compared with control group. The treatment with CHF-1024 exhibited an antiarrhythmic effect, abolished APD prolongation, and reduced cell width at all doses. The lowest dose also prevented Cm increase. In conclusion, we demonstrated that in this model of pressure-overload hypertrophy CHF-1024 reduces arrhythmogenesis and causes a recovery of cell excitable properties toward a normal phenotype.

The aminotetraline derivative (+/-)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024) displays cardioprotection in postischemic ventricular dysfunction of the rat heart.

To analyze the protective effects of the aminotetraline derivative (+/-)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024), a compound endowed with DA2-dopaminergic/alpha2-adrenergic receptor agonistic activity, in myocardial ischemia/reperfusion damage. A model of isolated and perfused (15 ml/min) electrically driven (300 beats/min) rat heart subjected to global ischemia (1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min) was followed. Cardiac mechanics changes were evaluated together with biochemical markers of cardiac ischemia in perfusate and tissue tumor necrosis factor-alpha (TNF-alpha). CHF-1024, perfused through the heart for 15 min before ischemia at different molar concentrations (1-100 nM), significantly improved left ventricle developed pressure during reperfusion, and normalized left ventricular end-diastolic pressure and coronary perfusion pressure. This anti-ischemic effect of CHF-1024 was associated to a decrease in creatine kinase and lactate dehydrogenase, both released during heart reperfusion. These events were concomitant with maintenance of a higher production of 6-keto-prostaglandin F1alpha The ability of CHF-1024 to improve postischemic ventricular dysfunction was correlated with a dose-dependent inhibition of the release of both norepinephrine (NE), from sympathetic nerve endings, and TNF-alpha from cardiac tissue. The effect of CHF-1024 on NE release was almost completely antagonized by specific antagonists of presynaptic inhibitory receptors domperidone and rauwolscine. The finding that this new aminotetraline derivative possesses anti-ischemic properties and limits NE release from cardiac nerve endings may bear some therapeutic potential in cardiovascular diseases.

Effect of nolomirole on monocrotaline-induced heart failure.

Neurohormonal activation has been shown to be a major factor in congestive heart failure progression and mortality. The beneficial effects obtained in clinical trials with angiotensin converting enzyme (ACE) inhibitors, beta-blockers and aldosterone antagonists have confirmed this hypothesis. 5,6-Diisobutirroyloxy-2-methyl-aminotetraline hydrochloride (nolomirole) is a selective agonist of prejunctional D(2)-dopaminergic and alpha(2)-adrenergic receptors. The stimulation of these receptors inhibits catecholamine release from sympathetic nerve endings. To confirm that this mechanism can be useful in congestive heart failure, we studied the effects of nolomirole on monocrotaline-induced congestive heart failure. The ACE inhibitor trandolapril was used as reference compound. Rats were given single intraperitoneal injection of either saline (control group; n=20) or monocrotaline (50 mg kg(-1)). Three days later, the monocrotaline-treated animals were randomly allocated (n=50 per group) to oral treatment with distilled water (vehicle group), nolomirole (0.25 mg kg(-1)) twice a day, or trandolapril (0.3 mg kg(-1)) once a day up to sacrifice. On the fourth week after monocrotaline injection, animals with signs of congestive heart failure were sacrificed for evaluation of heart hypertrophy and neuroendocrine alterations. Atrial natriuretic peptide (ANP) and alderosterone were determined by radioimmunoassay in plasma. Tissue norepinephrine concentration was quantified by high-pressure liquid chromatography. Nolomirole and trandolapril significantly reduced (a) hypertrophy of right atria and ventricles, (b) plasma levels of ANP and presence of pleural/peritoneal effusions and (c) norepinephrine depletion of right ventricle. These findings confirmed that nolomirole, like trandolapril, is able to attenuate the heart failure signs in the monocrotaline-induced congestive heart failure model.