Pharmacokinetics of a new extended-release nifedipine formulation following a single oral dose, in human volunteers.

This study aimed to define the pharmacokinetics of nifedipine following oral administration of a new extended-release formulation. Twelve healthy volunteers of both sexes, aged 39 +/- 4 years, were treated with a single oral tablet of a new extended-release formulation containing 40 mg of nifedipine. Samples of venous blood were taken before dosing, after 30 min and at 1, 2, 4, 8, 12, 16, 20 and 24 h after administration. Nifedipine concentration was measured by means of a high-performance liquid chromatography method. Noncompartmental pharmacokinetics parameters were then calculated. The plasma concentration of nifedipine increased slowly and in seven subjects biphasic peaks occurred. The mean values were as follows: t(max): 8.5 +/- 1.2 h; C(max): 36.55 +/- 6.76 ng/ml; AUC: 347.06 +/- 51.61 ng/h/ml; AUC 409.99 +/- 61.08 ng/h/ml; A(half-life): 2.26 +/- 0.36 h; D(half-life): 2.43 +/- 0.44 h; E(half-life): 4.62 +/- 0.79 h. Twenty-four hours after administration nifedipine was still detectable (3.17 +/- 0.67 ng/ml). Arterial blood pressure decreased and heart rate increased concurrently and proportionally to the increase in nifedipine concentration. Extended-release nifedipine formulations have better tolerability profiles than immediate-release formulations, which are at present not recommended in the treatment of hypertension, hypertensive crises or myocardial infarction. This new extended-release formulation has interesting pharmacokinetic parameters and may be effective in conditions in which dihydropyridine calcium channel blockers are indicated.

Protective effect of melanocortin peptides in rat myocardial ischemia.

The influence of the melanocortin peptide ACTH-(1-24) (adrenocorticotropin) on the consequences of short-term coronary ischemia (5 min) followed by reperfusion, and the effect of the long-acting melanocortin [Nle(4),D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-MSH) on the damage induced by a permanent coronary occlusion, were investigated in anesthetized rats. Ischemia was produced by ligature of the left anterior descending coronary artery. Reperfusion-induced arrhythmias [ventricular tachycardia (VT), ventricular fibrillation (VF)] and survival rate within the 5 min following reperfusion, blood levels of free radicals detected 2 min after reperfusion by electron spin resonance spectrometry, and amount of healthy myocardial tissue, measured 72 h after permanent coronary occlusion on immunohistologically stained serial sections, were evaluated. Postischemic reperfusion induced VT in all saline-treated rats, and VF and death in a high percentage of animals (87%). In rats treated i.v. (2.5 min after coronary occlusion) with ACTH-(1-24) (0.16-0.48 mg/kg) there was a significantly dose-dependent reduction in the incidence of arrhythmias and lethality. Ischemia/reperfusion caused a large increase in free radical blood levels; treatment with ACTH-(1-24) (0.48 mg/kg i.v.) almost completely prevented this increase. In rats subjected to permanent coronary occlusion, the amount of healthy myocardial tissue was much reduced in saline-treated rats, while in rats treated s.c. with NDP-MSH (0.27 mg/kg every 12 h) it was significantly higher. The present data demonstrate, for the first time, an unforeseen property of melanocortin peptides, i.e., their ability to significantly reduce both heart ischemia/reperfusion injury and size of the ischemic area induced by permanent coronary occlusion.

Adrenocorticotropin inhibits nitric oxide synthase II mRNA expression in rat macrophages.

During hemorrhagic shock there is a massive overproduction of nitric oxide (NO). In such conditions, the intravenous (i.v.) injection of melanocortin peptides in nanomolar amounts produces a long-lasting restoration of cardiovascular and respiratory functions associated with the normalization of NO blood levels. To clarify the mechanism of such melanocortin-induced inhibition of NO overproduction, the influence of the adrenocorticotropin fragment 1-24 [ACTH-(1-24)] on the NO synthesizing activity of rat macrophages was studied in vitro. Nitrite production, an indicator of NO synthesis, was measured in the supernatant of rat macrophages whose inducible NO synthase (NOS II, iNOS) had been stimulated by the addition of S. enteritidis lipopolysaccharide (LPS, 50 microg/ml). ACTH-(1-24) (25, 50 and 100 nM) inhibited nitrite production when incubated together with LPS, but had no effect when applied 6 h after LPS. Further, the effect of ACTH-(1-24) on the expression of iNOS mRNA in rat macrophages activated with LPS was studied by means of a reverse transcriptase-polymerase chain reaction assay. ACTH-(1-24) (25, 50 and 100 nM), applied together with LPS, dose-dependently suppressed iNOS gene activation. The present data suggest that the melanocortin-induced normalization of NO blood levels during hemorrhagic shock is due, at least in part, to a direct inhibition of iNOS induction, at the level of mRNA transcription.

Evidence that melanocortin 4 receptor mediates hemorrhagic shock reversal caused by melanocortin peptides.

Melanocortin peptides are known to be extremely potent in causing the sustained reversal of different shock conditions, both in experimental animals and humans; the mechanism of action includes an essential brain loop. Three melanocortin receptor subtypes are expressed in brain tissue: MC(3), MC(4,) and MC(5) receptors. In a volume-controlled model of hemorrhagic shock in anesthetized rats, invariably causing the death of control animals within 30 min after saline injection, the i.v. bolus administration of the adrenocorticotropin fragment 1-24 (agonist at MC(4) and MC(5) receptors) at a dose of 160 microg/kg i.v. (54 nmol/kg) produced an almost complete and sustained restoration of cardiovascular and respiratory functions. An equimolar dose of gamma(1)-melanocyte stimulating hormone (selective agonist at MC(3) receptors) was completely ineffective. The selective antagonist at MC(4) receptors, HS014, although having no influence on cardiovascular and respiratory functions per se, dose-dependently prevented the antishock activity of adrenocorticotropin fragment 1-24, with the effect being complete either at the i.v. dose of 200 microg/kg or at the i.c.v. dose of 5 microg/rat (17-20 microg/kg). We concluded that the effect of melanocortin peptides in hemorrhagic shock is mediated by the MC(4) receptors in the brain.

Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock.

1. Tumour necrosis factor (TNF-alpha) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF-alpha is an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock. We therefore investigated the effects of the melanocortin peptide ACTH-(1 - 24) (adrenocorticotropin fragment 1 - 24) on the vascular failure induced by SAO shock. 2. SAO-shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham-shocked rats survived for more than 4 h), enhanced serum TNF-alpha concentrations (755+/-81 U ml-1), decreased mean arterial blood pressure, leukopenia, and increased ileal leukocyte accumulation, as revealed by means of myeloperoxidase activity (MPO=9.4+/-1 U g-1 tissue). Moreover, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM - 10 microM) (Emax and ED50 in shocked rats=7.16 mN mg-1 tissue and 120 nM, respectively; Emax and ED50 in sham-shocked rats=16.31 mN mg-1 tissue and 100 nM, respectively), reduced responsiveness to acetylcholine (ACh, 10 nM-10 microM) (Emax and ED50 in shocked rats=30% relaxation and 520 nM, respectively; Emax and ED50 in sham-shocked rats=82% relaxation and 510 nM, respectively) and increased staining for intercellular adhesion molecule-1 (ICAM-1). 3. ACTH-(1 - 24) [160 microg kg-1 intravenously (i.v.), 5 min after SAO] increased survival rate [SAO+ACTH-(1 - 24)=80% at 4 h of reperfusion], reversed hypotension, reduced serum TNF-alpha (55+/-13 U ml-1), ameliorated leukopenia, reduced ileal MPO (1.2+/-0.2 U g-1 tissue), restored the reactivity to PE, improved the responsiveness to ACh and blunted the enhanced immunostaining for ICAM-1 in the aorta. 4. Adrenalectomy only in part - but not significantly - reduced the ACTH-induced shock reversal, the survival rate of SAO+ACTH-(1 - 24) adrenalectomized rats being 60% at 4 h of reperfusion; and methylprednisolone (80 mg-1 i.v., 5 min after SAO) had a non-significant effect (10% survival) at 4 h of reperfusion. 5. The present data show that melanocortins are effective also in SAO shock, their effect being, at least in part, mediated by reduced production of TNF-alpha. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin-induced reversal of vascular failure and leukocyte accumulation.

High blood levels of nitric oxide in rats subjected to prolonged respiratory arrest and their modulation during adrenocorticotropin-induced resuscitation.

Anaesthetized rats, endotracheally intubated and mechanically ventilated with room air, were subjected to a 5-min period of asphyxia by turning off the ventilator. The ventilator was then turned back on and, simultaneously, the animals were treated with either the adrenocorticotropin fragment 1-24 [ACTH-(1-24), 160 microg/kg in a volume of 1 ml/kg i.v.] or an equivalent volume of saline. Nitric oxide (NO)-haemoglobin formation was detected ex vivo in arterial blood by electron spin resonance spectrometry; arterial blood pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for a 60-min observation period, or until prior death. During asphyxia, there was massive formation of NO (red cell concentrations 40-80 microM), associated with a dramatic fall in mean arterial pressure and pulse pressure, marked bradycardia and ECG signs of ischaemic damage, as well as an isoelectric EEG. Treatment with ACTH-(1-24) produced a prompt (within 15 min) and long-lasting drop in NO blood levels, associated with an almost immediate (within 1 min) restoration of cardiovascular function and with a more gradual recovery of EEG, which became normal after 3040 min; all parameters remained stable throughout the 60-min observation period. In saline-treated rats, on the other hand, there was a further increase in NO blood levels, as detected 3 min after treatment, and all died within 5-8 min. Moreover, pretreatment and treatment with S-methylisothiourea sulphate (SMT, 3 mg/kg i.v.), a relatively specific inhibitor of inducible NO synthase, inhibited NO formation, but did not affect the mortality rate (100% within 5-8 min). The present results provide the first evidence that prolonged asphyxia is associated with high blood concentrations of NO, and that the life-saving effect of melanocortin peptides in severe hypoxic conditions is associated with a complete normalization of NO blood levels. However, the lack of SMT protection in this experimental model seems to rule out the possibility that the ACTH-(1-24)-induced resuscitation is due to an effect on NO overproduction.

Tumour necrosis factor-alpha as a target of melanocortins in haemorrhagic shock, in the anaesthetized rat.

The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved (mostly through the activation of inducible nitric oxide synthase) in the pathogenesis of circulatory shock. On the other hand, melanocortin peptides are potent and effective in reversing haemorrhagic shock, both in animals (rat, dog) and in humans. This prompted us to study the influence of the melanocortin peptide ACTH-(1-24) on the blood levels of TNF-alpha in haemorrhage-shocked rats and on the in vitro production of TNF-alpha by lipopolysaccharide (LPS)-activated macrophages. Plasma levels of TNF-alpha were undetectable before starting bleeding and greatly increased 20 min after bleeding termination in saline-treated rats. In rats treated with ACTH-(1-24) the almost complete restoration of cardiovascular function was associated with markedly reduced levels of TNF-alpha 20 min after bleeding termination. On the other hand, ACTH-(1-24) did not influence TNF-alpha plasma levels in sham-operated, unbled rats. In vitro, ACTH-(1-24) (25-100 nM) dose-dependently reduced the LPS-stimulated production of TNF-alpha by peritoneal macrophages harvested from untreated, unbled rats. These results indicate that inhibition of TNF-alpha overproduction may be an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock.

Adrenocorticotropin counteracts the increase in free radical blood levels, detected by electron spin resonance spectrometry, in rats subjected to prolonged asphyxia.

We investigated the influence of the adrenocorticotropic fragment 1-24 [ACTH-(1-24)] on the blood levels of highly-reactive free radicals in a rat model of prolonged asphyxia. Anesthetized animals were endotracheally intubated and mechanically ventilated with room air; after a 10 min stabilization period, the ventilator was turned off to induce asphyxia for 5 min; then, the ventilator was turned back on, and, simultaneously, the rats were intravenously treated with either ACTH-(1-24) (160 microg/kg in a volume of 1 ml/kg) or equivolume saline. Free radicals were detected in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent employed (alpha-phenyl-N-tert-butylnitrone). Arterial pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for the 60 min observation period, or until prior death. At the end of the 5 min period of respiratory arrest, blood levels of free radicals were about four times higher than those of the basal, pre-asphyxia condition, arterial pressure had dramatically decreased, ECG showed marked bradycardia and signs of ischemic damage and the EEG had become isoelectric. Treatment with ACTH-(1-24) produced an immediate normalization of the blood levels of free radicals, associated with a restoration of cardiovascular function and full recovery of EEG within 30-45 min; all the saline-treated rats, on the other hand, died within 6.89 +/- 0.96 min. These results provide direct evidence that in a severe condition of prolonged asphyxia there is a rapid and massive production of highly-reactive free radicals and suggest that the resuscitating effect of adrenocorticotropin fragments in severe hypoxic conditions may be largely due to the inhibition of free radical overproduction during tissue reoxygenation.

Adrenocorticotropin normalizes the blood levels of nitric oxide in hemorrhage-shocked rats.

Anesthetized rats were subjected to volume-controlled hemorrhagic shock by stepwise bleeding. Besides cardiovascular and respiratory functions, nitric oxide (NO)-hemoglobin formation in arterial blood was directly evaluated by means of electron spin resonance spectroscopy. During hemorrhagic shock there was a massive increase in NO-hemoglobin, associated with a fall in mean arterial pressure, pulse pressure, respiratory rate and heart rate, and there was a further increase in NO-hemoglobin 15 min after intravenous (i.v.) treatment with saline. All rats died within 30 min. The reversal of the shock condition induced by the i.v. injection of the adrenocorticotropin (ACTH) fragment 1-24 (160 microg/kg, 5 min after bleeding termination) was associated with a prompt disappearance of NO-hemoglobin. Also S-methylisothiourea (3 mg/kg i.v.), a selective inhibitor of inducible NO synthase, provoked a disappearance of NO-hemoglobin and reversal of the shock condition. The present results provide a direct demonstration that volume-controlled hemorrhagic shock is associated with highly increased blood levels of NO, as indicated by increased NO-hemoglobin, and indicate that ACTH-induced reversal of the shock condition is associated with the normalization of NO blood levels, and a parallel improvement of cardiovascular and respiratory functions. This occurs probably through the inhibition of inducible NO synthase, as suggested by the fact that S-methylisothiourea, a selective inhibitor of this NO synthase isoform, produced the same results.

The reversal of experimental hemorrhagic shock induced by nicotine and dimethylphenylpiperazinium is adrenal-dependent.

In a rat model of volume-controlled hemorrhagic shock causing the death of all control animals within 30 min, the intravenous injection of either nicotine (50 micrograms/kg) or dimethylphenylpiperazinium (DMPP) (0.5 micrograms/kg) produced a rapid and sustained reversal of the shock condition, with 100% survival 2 h after treatment. Bilateral adrenalectomy completely prevented the anti-shock effect of the two drugs, even though administered at higher doses (150 micrograms/kg in the case of nicotine; 10 micrograms/kg in the case of DMPP). It is concluded that stimulation of adrenaline release plays a fundamental role in the mechanism of action of nicotine- and DMPP-induced shock reversal.