Peripheral and central H1 histamine receptor occupancy by levocetirizine, a non-sedating antihistamine; a time course study in the guinea pig.

BACKGROUND AND PURPOSE: The H(1) receptor occupancy (H1RO) in brain is an indicator of central side effects of antihistamines. Here, we determined the kinetics of central and peripheral H1RO by levocetirizine in relation to its brain and plasma concentration, and investigated the role of the blood-brain barrier in any delay in brain H1RO. EXPERIMENTAL APPROACH: Concentration-time profiles in plasma and brain were obtained after 0.1 and 1 mg kg(-1) oral doses of levocetirizine in guinea pigs. H1RO in brain was measured ex vivo using [3H]-mepyramine and, in the periphery, by measuring the degree of inhibition of histamine-induced contractions of isolated guinea pig ileum. KEY RESULTS: The concentration-time profile of levocetirizine indicated lower levels (partition coefficient, K(p)=0.06-0.08), higher t(max) (2-4 h vs 1-1.5 h) and longer terminal half-life (4-5.6 h vs 2.1-2.8 h) in brain than plasma. The H1RO at 0.1 and 1 mg kg(-1) were 75% and 97%, respectively, at 1 hr in the periphery and, in the brain, were <20% and 28-67% respectively, at all time points studied. Brain H1RO vs plasma concentrations profile showed a delay, but not when compared to brain concentrations. CONCLUSIONS AND IMPLICATIONS: This study demonstrates an effective peripheral antihistamine effect of levocetirizine without central adverse effects at the dose close to human therapeutic dose. The slow increase in H1RO in the brain with time was caused by slow blood-brain barrier transport of levocetirizine. This demonstrates the importance of measuring time course of brain H1RO in relation to brain concentrations of drugs.

Contractile properties of various histaprodifen derivatives in guinea pig isolated ileum and trachea.

We characterized the histamine H(1) receptor agonism of various histaprodifen derivatives in guinea pig isolated ileum and trachea in comparison with histamine. Based on their affinity (calculated pK(A) values for ileum and trachea, respectively), the compounds were ranked as follows: suprahistaprodifen (8.31/8.08) > N(alpha)-(4-phenylbutyl)histaprodifen (7.22/5.93) >or= histamine (5.79/5.19) approximately methylhistaprodifen (5.57/6.07). Based on their efficacy (calculated tau values for ileum and trachea, respectively), the compounds were ranked as follows: methylhistaprodifen (37.67/2.50) > histamine (5.64/1.80) > suprahistaprodifen (1.63/1.42) >or= N(alpha)-(4-phenylbutyl)histaprodifen (0.083/1.54). In the ileum, histamine and methylhistaprodifen showed a high histamine H(1) receptor reserve while suprahistaprodifen and N(alpha)-(4-phenylbutyl)histaprodifen are devoid of any histamine H(1 )receptor reserve. On the trachea, no histamine H(1 )receptor reserve was demonstrable with the four tested agonists. The kinetic of contraction/relaxation of the ileum was faster with histamine and methylhistaprodifen than with suprahistaprodifen and N(alpha)-(4-phenylbutyl)histaprodifen. Histamine contracted the trachea faster than histaprodifen derivatives. Levocetirizine antagonized contractions induced by histamine and histaprodifen derivatives in both tissues. The differences observed in the calculated pA(2) (7.60-8.29) and/or pD'(2) values (6.28-7.90) depending on the tissue and/or the agonist are discussed.

A new series of M3 muscarinic antagonists based on the 4-amino-piperidine scaffold.

A series of 4-amino-piperidine containing molecules have been synthesized and structure-affinity relationship toward the M3-muscarinic receptor has been investigated. Chemical modulations provided molecules with K(i) for the human M3-R up to 1 nM with variable selectivity (3- to 40-fold) over the human M2-R. Compounds 2 (pA(2)=8.3, 8.6) demonstrates in vitro on guinea pig bladder and ileal strips potent anticholinergic properties and tissue selectivity.

Design and synthesis of substituted compounds containing the 1, 4-benzodioxin subunit. New potential calcium antagonists.

New compounds possessing 1,4-benzodioxin or its saturated analogous heterocyclic system were synthesized and tested for calcium antagonist activity. Biological differences were seen between the different modifications applied. These compounds have been shown to be representative of a novel series of calcium channel antagonists.

New substituted 1,4-benzoxazine derivatives with potential intracellular calcium activity.

Substituted 1,4-benzoxazines bearing an amino side chain at the 2-position were prepared and were found to have a moderate activity on intracellular calcium. Of the compounds studied it was found that those which possess a homoveratrylamino moiety exhibited superior potency. The chain length and the nature of the amine (4-fluorophenylpiperazine, 4-fluorobenzhydryloxyethylamine, N-substituted homoveratrylamine) is discussed. The 4-benzyl-3, 4-dihydro-2-[3-[[2-(3,4-dimethoxyphenyl)ethyl]amino]propyl]-2H-1, 4-benzoxazine (3c) is the most potent derivative of the series with a ratio of IC50 values against PE (phenylephrine) and K+ of 2.1. Under these test conditions a ratio near 1 indicates potential intracellular calcium activity while a ratio greater than 100 an action on extracellular calcium influx.

Comparison of amitriptyline metabolism in hepatocytes from streptozocin-induced diabetic rats and from non-diabetic rats.

Biotransformation of amitriptyline (AMI) was studied at different intervals in freshly isolated hepatocytes from healthy or streptozocin-induced diabetic rats in order to investigate the influence of the diabetic state. Levels of free and conjugated AMI, demethylated and hydroxylated metabolites, were assessed by HPLC analysis. In hepatocytes isolated from diabetic rats, AMI was less completely metabolized and the demethylation reaction became more important than in non-diabetic rat hepatocytes. Although the proportions of hydroxylated metabolites decreased in diabetic rats, it always remained predominant. Furthermore, glucuronidation of metabolites was greater, especially for (Z)-10-hydroxynortriptyline in diabetic animals.

In vitro calcium antagonistic and antioxidant effects of Org 13061 and its enantiomers, new potential antiatherosclerotic compounds.

The calcium antagonistic and antioxidant properties of a new potential antiatherosclerotic agent, Org 13061 were compared with those of its (-) and (+) enantiomers (Org 13471 and Org 13581) In vitro and with appropriate reference drugs. Org 13061 antagonized contractions induced by potassium in rabbit aortic rings with an IC50 value of 0.50 microM and reduced the maximum rate of phase 0 depolarization (Vmax) of the 'slow' calcium-mediated transmembrane action potentials in cardiac tissue (IC25 = 0.82 microM). Similarly to reference drugs, Org 13061 was more selective in reducing vascular compared to cardiac contraction. In concentrations overlapping those exerting vasorelaxant actions, Org 13061 inhibited copper ion-induced human low density lipoprotein (LDL) peroxidation (0.1-1 microM) and inhibited lipid accumulation by rat aortic smooth muscle cells in culture (1-3 microM). Higher concentrations (3 microM) modestly inhibited proliferation of these cells. The (-) enantiomer was ten times more potent than the (+) enantiomer as a vasorelaxant but was equipotent in inhibiting lipid accumulation and LDL peroxidation (eg, lag phase of conjugated dienes formation increased by 29 and 61 min and by 22 and 56 min in response to 0.3 and 1 microM (-) and (+) enantiomers, respectively). The antioxidant probucol was approximately three times more potent than Org 13061 in inhibiting lipid accumulation but was 30 times less potent in antagonizing LDL peroxidation. The classical calcium channel blocking agents were totally ineffective on lipid accumulation (1-10 microM), whereas human LDL peroxidation was slightly reduced by nifedipine (0.1-3 microM) but unaltered by diltiazem (0.1-30 microM) and verapamil (0.1-3 microM). In conclusion, the racemic Org 13061 selectively blocks voltage-operated calcium channels (VOCs) in concentrations that also exert marked antioxidant activity. The (-) enantiomer is largely responsible for calcium channel block but as antioxidants, the enantiomers are equipotent. This mixed pharmacological profile of Org 13061, not shared by known calcium channel blocking agents, may be potentially useful in the treatment of atherosclerosis.

Failure of calcium channel blockade to reduce platelet-mediated cyclic flow variations in dogs with coronary stenosis and endothelial injury.

Experimental canine coronary artery stenosis associated with endothelial injury results in a typical pattern of coronary flow characterized by gradual decreases in blood flow to almost zero values followed by abrupt restorations to original levels. Cyclic flow variations (CFVs) are the consequence of recurrent platelet aggregation at the site of the stenosis and subsequent dislodgement of the thrombus. The present study was designed to test the efficacy of diltiazem, nifedipine, and verapamil in inhibiting in vivo platelet aggregation as compared with that of aspirin and ketanserin, two potent reference compounds effective in this model. Except for aspirin, compounds were given as a slow intravenous infusion (i.v.) for 60 min to avoid hemodynamic changes. Diltiazem (0.01 mg/kg/min), nifedipine (3 micrograms/kg/min), and verapamil (0.01 mg/kg/min) were totally inactive against CFVs. A higher dose of verapamil (0.02 mg/kg/min) abolished CFVs in 3 of 4 dogs, but serious side effects were observed [atrioventricular (AV) block and death of 2 animals]. Aspirin (10 mg/kg bolus) caused complete inhibition of CFVs in 4 of 4 dogs, and ketanserin (0.01 mg/kg/min) abolished CFVs in 4 of 5 dogs. These data suggest that calcium channel blockade alone in contrast to cyclooxygenase inhibition or 5-HT2 antagonism cannot inhibit thrombus formation in this model.

Effects of the 5-hydroxytryptamine1A receptor agonists, 8-OH-DPAT, buspirone and flesinoxan, upon brain damage induced by transient global cerebral ischaemia in gerbils.

The effects of the 5-hydroxytryptamine1A agonists, 8-OH-DPAT, buspirone and flesinoxan, on the delayed hyperactivity and on the ensuing neuronal degeneration induced by transient global cerebral ischaemia, were studied. In normothermic, male Mongolian gerbils, subjected to 3 min bilateral carotid artery ligation, the locomotor activity was measured 1 day after ischaemia. The neuronal damage was quantified 7 days later using an image analysis system. Buspirone (3 and 10 mg/kg, i.p.) and flesinoxan (1 and 3 mg/kg, i.p.), administered twice a day for 3 days both in pre- and post-ischaemic conditions, failed to significantly protect the CA1 zone of the hippocampus against neuronal damage. In contrast, 8-OH-DPat (1 and 3 mg/kg, i.p.) significantly reduced the neuronal degeneration. All compounds abolished the hyperactivity but there was no correlation between this parameter and the extent of the reduction in neuronal damage. The ineffectiveness of buspirone and flesinoxan was not the result of too low a dose - as evidenced by the complete inhibition of hyperactivity with both compounds and by the appearance of a serotonin behavior syndrome with flesinoxan - but is possibly related to a partial agonist activity at the 5-hydroxytryptamine1A receptor, as reported for buspirone. Further studies are necessary to explain the differences between these agonists.

Lack of efficacy of 5-HT2A receptor antagonists to reduce brain damage after 3 minutes of transient global cerebral ischaemia in gerbils.

Stimulation of the 5-HT2A receptors by serotonin has been reported to exert an excitatory effect on neocortical neurons in rats and mice, to facilitate ischaemia-induced release of excitatory amino acids and to mediate the vasomotor constrictor component of the response of blood vessels to 5-HT. 5-HT2A receptor antagonists have, therefore, been proposed as potential protectants against the effects of cerebral ischaemia. The aim of this study was to evaluate the effects of two relatively selective 5-HT2A receptor antagonists, ketanserin and ritanserin, on delayed hyperactivity and the ensuing neuronal degeneration induced by 3 minutes of bilateral carotid artery ligation in Mongolian gerbils. Effects were compared to that of flunarizine, which blocks calcium overload and served as a positive control in this paradigm. Temporal and/or rectal temperatures were measured and strictly controlled during the ischaemia and the early reperfusion phase. Locomotor activity was measured one day after the ischaemia and neuronal degeneration quantified 7 days later using an image analysis system (Quantimet 570, Leica). Global ischaemia in gerbils elicits hyperactivity associated with a delayed neuronal degeneration predominantly in the CA1 zone of the hippocampus. Ketanserin and ritanserin (3 and 10 mg/kg ip, twice daily for 3 days, pre- and postischaemia) did not protect the CA1 neurons against ischaemic damage. The postischaemic hyperactivity was inhibited only with the higher dose of ketanserin. As previously reported, flunarizine (30 mg/kg po) markedly reduced neuronal degeneration (-44.2%, p < 0.01) and totally abolished the ischaemia-induced hyperactivity. These data demonstrate that ketanserin and ritanserin are not effective protectants of the gerbil hippocampus against ischaemic damage when the body temperature of the animals is controlled, thus suggesting that 5-HT2A receptors are not directly implicated in the pathogenesis of global cerebral ischaemia in this model.

Paracetamol potentiates isaxonine toxicity in vitro.

The toxicity of isaxonine alone and in combination with the known glutathione depletor, paracetamol, was evaluated using rat hepatocyte primary cultures in vitro by measuring morphometric parameters and the leakage of intracellular lactate dehydrogenase into the culture medium. No cytotoxicity was observed with isaxonine at concentrations up to 10(-3) M, whereas paracetamol was cytotoxic at concentrations above 0.6 x 10(-3) M in the culture medium. Paracetamol cytotoxicity (0.6-3.3 x 10(-3) M) was enhanced in the presence of a non-cytotoxic concentration of isaxonine (10(-7) M). Furthermore cytotoxicity was observed when cells were exposed to a combination of non-cytotoxic concentrations of the paracetamol (0.3 x 10(-3) M) and isaxonine (10(-7) M). These findings demonstrate that isaxonine has no direct cytotoxic effect even at high concentrations. However co-administration of isaxonine with paracetamol greatly potentiates cytotoxicity. We suggest that this effect may be related to glutathione depletion within the cell but additional studies are required to verify this hypothesis.

Electrophysiological effects of Org 7797 in the closed-chest anaesthetized dog.

1. The intravenous electrophysiological effects of a new antifibrillatory agent, Org 7797, were studied in closed chest anaesthetized dogs. Effects of fast sodium and slow calcium-mediated action potentials were also examined in guinea-pig isolated papillary muscle. 2. The major effects of a known antifibrillatory dose of Org 7797 (0.5 mg kg-1) were a protracted slowing of AV nodal conduction (for at least 20 min) and prolongation of the AV nodal functional refractory period. Conduction in the atria and His-Purkinje system (reflected by the St-A and HV intervals) were not significantly modified whilst ventricular conduction (reflected by the QRS interval) and the ventricular functional refractory period were only transiently prolonged. No other electrophysiological changes were seen. 3. A higher dose of Org 7797 (1.5 mg kg-1) slowed conduction at all levels of the myocardium (as evidenced by increases in the St-A, AH, HV and QRS intervals), slightly shortened cardiac repolarization (as assessed from JTc) and decreased Wenckebach rate. Atrial refractory periods were increased whereas effects on ventricular refractory periods were modest. 4. Neither heart rate nor sinus node recovery time were modified by either dose of Org 7797. 5. Org 7797, at a concentration (20 microM) which reduced Vmax of fast sodium-mediated action potentials in isolated papillary muscle by 83%, did not modify Vmax of slow calcium-mediated action potentials. It prolonged duration of the latter but did not modify that of the former. However, the plateau phase of both the 'fast' and especially the 'slow' action potentials was prolonged. 6. It is concluded that the main electrophysiological effects of a known antifibrillatory dose of Org 7797 in dogs with normal cardiac function are seen at the level of the AV node, actions which are unlikely to be explained by calcium channel block. Higher doses display a class Ic profile. This preferential action on the AV node may contribute to the control of ventricular rate during atrial fibrillation in the absence of infra-nodal conduction disturbances.7. These results contrast with those previously obtained in infarcted dogs and might further suggest that myocardial infarction enhances the Class I action of Org 7797.

An in vitro investigation of the relationships between potency, lipophilicity, cytotoxicity and chemical class of representative calcium antagonist drugs.

Interrelationships that might exist between potency, lipophilicity and cytotoxicity of the chemically diverse calcium antagonist group of drugs have been examined in the present study. The potency of 11 representative calcium antagonists in inhibiting KCl-induced contractions in rabbit isolated aortic rings and their relative lipophilicity was determined using reversed phase HPLC. Their cytotoxicity in rat hepatocyte primary cultures was also determined. Cytotoxicity failed to correlate with potency, except for the highly lipophilic, non-selective, diphenylalkylamines (DPAs), suggesting that cytotoxicity was not caused by blockade of plasmalemmal voltage-operated calcium channels. Cytotoxicity moderately correlated with relative lipophilicity, the most lipophilic drugs also being the most cytotoxic. Relative lipophilicity may partly determine the cytotoxicity and pharmacological potency of Ca++ antagonists in a broad sense, but this correlation was not valid in each individual chemical series. We suggest that the higher cytotoxicity of the DPAs is at least partly due to a greater incorporation of the drugs into the hepatocyte plasmalemma compared to compounds in other chemical classes investigated. Further studies are required to elucidate the particular cytotoxic mechanisms involved.

Calcium antagonists can be classified using in vitro toxicity and potency indices.

The toxicity of eleven calcium antagonists from different chemical families was determined in rat hepatocyte primary cultures. The calcium antagonist potency of the same compounds was also determined in isolated rabbit aortic rings contracted with high K+. The hepatocytotoxicity of the calcium antagonists was not directly linked to blockade of voltage-operated calcium channels, since there was no correlation between the rank order of hepatotoxicity and that for calcium antagonist potency. The toxicity and calcium antagonist potency of each calcium antagonist examined were used to calculate an in vitro therapeutic index value for each compound. It was observed that therapeutic indices fell into three distinct groups and we therefore propose that the in vitro therapeutic index can be used to subclassify the calcium antagonist group of drugs. The proposed classification corresponds very closely with one already suggested by Spedding on pharmacological grounds. In conclusion, the in vitro therapeutic index may provide a useful tool in the characterization and subclassification of novel calcium antagonist compounds.

Acetylcholine-induced relaxation of the rabbit aorta is preload-independent but markedly dependent upon the degree of excitatory agonist-induced tone.

1. The aim of the present study was to investigate the relationship between endothelium-dependent relaxation evoked by acetylcholine, tissue preload and the degree of noradrenaline-induced tone in the isolated rabbit aorta. 2. In the aorta preload-response curves were bell-shaped with increasing preload augmenting responses to noradrenaline up to a certain point (optimal value) and then declining. Removal of the endothelium significantly increased responses to low concentrations of noradrenaline (less than 0.1 microM) but did not significantly affect the maximum response of the aorta to this amine or the preload-response curves generated at several concentrations of noradrenaline. 3. The optimal preload value was around 10 g for the aorta and changes in preload did not influence the sensitivity of the tissue to noradrenaline as assessed by pEC50 values to this agonist. 4. Acetylcholine (0.01-10 microM) evoked endothelium-dependent relaxations which in absolute terms increased as the tissue preload was increased. This relationship was much less evident when acetylcholine responses were measured in terms of the percentage inhibition of the respective noradrenaline contraction, when little or no change in the acetylcholine responses was noted. 5. When acetylcholine relaxations were expressed in terms of a percentage of the maximum noradrenaline-induced response evoked at each preload setting, the results confirmed that preload changes had little or no influence upon acetylcholine responses. 6. In contrast, tissue sensitivity to, and the extent of acetylcholine-induced relaxation, were markedly affected by the level of excitatory agonist-induced tone. As noradrenaline-induced tone increased, maximum responses and pIC50 values to acetylcholine were reduced.(ABSTRACT TRUNCATED AT 250 WORDS)