Spectroscopic analyses on interaction of Naphazoline hydrochloride with bovine serum albumin.

The fluorescence and ultraviolet spectroscopy were explored to study the interaction between Naphazoline hydrochloride (Naphcon) and bovine serum albumin (BSA) at three different temperatures (292, 301, and 310 K) under imitated physiological conditions. The quenching mechanism of BSA by Naphacon was interpreted using the Stern-Volmer mechanism, and a combined quenching (dynamic and static quenching). The binding constants, binding sites and the corresponding thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction system were calculated at different temperatures. According to Förster non-radiation energy transfer theory, the binding distance between BSA and Naphcon was found to be 4.71 nm. Synchronous fluorescence spectroscopy showed the conformation of BSA changed in the presence of Naphacon. In addition, the effect of some common metal ions (Mg(2+), Ca(2+), Ni(2+), Cu(2+), and Fe(2+)) on the binding constant between Naphcon and BSA was examined.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

Reduced high-affinity alpha 1-adrenoceptors in liver of senescent rats: implications of assessment at various temperatures.

1. We investigated the changes occurring as a result of aging in alpha 1-adrenoceptors in the livers of Fisher 344 females rats. For comparison, we also measured beta-adrenoceptors in this tissue. Three age groups were studied, including young adults (aged 6 months), mature adults (aged 16 months) and senescent animals (aged 25 months). 2. The density of alpha 1-receptors was measured by use of [3H]-prazosin and was found to be reduced 39% (P less than 0.01) at 25 months compared with 6 months. The percentage of alpha 1-receptors displaying high affinity for adrenaline was also reduced from 85.6% at 6 months to 51.6% at 25 months (P less than 0.02). 3. In contrast, the density of beta-receptors, which was measured with [125I]-iodocyanopindolol, was increased 104% between 6 months and 25 months. The affinity of both alpha 1- and beta-adrenoceptors for antagonists was unchanged with age. 4. We found that receptor affinity for agonists may be measured accurately in binding studies conducted at 4 degrees C or 25 degrees C, but that the apparent affinity for agonist was artifactually reduced in studies conducted at 37 degrees C. This effect is poorly reversible, in that reduced agonist-affinity is also observed in tissue which has been incubated at 37 degrees C and then cooled to 4 degrees C before performing the binding studies. 5. It is concluded that liver alpha 1-adrenoceptor function is reduced and beta-adrenoceptor function increased in senescence.

The metabolism of nafimidone hydrochloride in the dog, primates and man.

1. The biotransformation of nafimidone, an imidazole-substituted anticonvulsant, has been studied by characterization of urinary metabolites in dogs, cynomolgus monkeys, baboons and man. 2. The biotransformation of nafimidone in these laboratory animals and man is initially very similar, in each case proceeding by reduction to the aliphatic alcohol metabolite, nafimidone alcohol or 1-[2-hydroxy-2-(2-naphthyl)ethyl]imidazole. 3. Further transformation of this metabolite involves oxidation in the naphthyl and imidazole functions, and/or conjugation. 4. The dog differs from the higher primates in that no metabolic modification of the naphthyl group takes place, the major metabolite in the dog being the O-beta-glucuronide of nafimidone alcohol. 5. In higher primates and man two isomers involving dihydroxylation in the naphthyl ring--1-[2-hydroxy-2-(5,6- or 7,8-dihydroxydihydro-2-naphthyl)ethyl]imidazole--were tentatively identified. These species alone showed evidence of an imidazole linked N-glucuronide of nafimidone alcohol. 6. The possible occurrence of stereoselective metabolism by the introduction of a chiral centre at C-9 in nafimidone alcohol was indicated in human urine by the presence of both epimers of the O-beta-glucuronide of nafimidone alcohol in a 2:1 ratio.

Relationship between alpha-adrenoceptor occupancy and contractile response in rat vas deferens. Experimental and theoretical analysis.

The rat vas deferens has been considered to be the tissue of choice to study alpha-adrenergic drugs. However, some of these agonists have elicited complex responses in this organ. Therefore, detailed characterization of alpha-adrenoceptor-mediated responses of the rat vas deferens was the aim of this work. Experiments were designed to study the contractile response of this tissue to phenylethanolamine (noradrenaline) and to two imidazolines (oxymetazoline and naphazoline). These responses were related to receptor occupancy and to other parameters of drug action, i.e. dissociation constants, relative efficacies, ED50 and maximal responses. A theoretical model was used to check the experimental data. There was a non-linear relationship between response and receptor occupancy with all three agonists. The dissociation constants for noradrenaline, oxymetazoline and naphazoline were 11.06, 0.15 and 0.10 microM, respectively. The rat vas deferens had 75-80% spare receptors for noradrenaline. Oxymetazoline and naphazoline were shown to be partial agonists with low relative efficacies as compared to noradrenaline (0.0063 and 0.0056 respectively). The dose-response curves generated by the model for partial agonists were similar to the curves obtained experimentally in vitro.

Pharmacokinetics of nafimidone in patients with chronic intractable epilepsy.

Nafimidone is a new antiepileptic drug which may be effective in partial onset seizures. We studied the pharmacokinetics of nafimidone and its metabolite, nafimidone alcohol, in 12 patients already taking phenytoin and/or carbamazepine. The half-life of nafimidone was 1.34 +/- 0.48 hours after a 100mg single dose and 1.69 +/- 0.91 hours after a 300mg single dose. However, the half-life of nafimidone alcohol increased from 2.84 +/- 0.72 hours after a 100mg single dose to 4.22 +/- 1.09 hours after a 300mg single dose (p less than 0.02). The clearance of nafimidone was 43.56 +/- 22.11 L/h/kg after a 100mg single dose and 35.51 +/- 28.93 L/h/kg after the 300mg single dose. The respective apparent volumes of distribution of nafimidone after single 100 and 300mg doses were 80.78 +/- 46.11 L/kg and 71.01 +/- 36.86 L/kg. After short term (9 to 10 weeks) and long term (127 to 152 weeks) maintenance therapy on nafimidone 600mg per day the half-life of nafimidone alcohol was 2.23 +/- 0.36 hours and 2.16 +/- 0.60 hours, respectively. No nafimidone could be detected in urine but from 4 to 7% of the daily nafimidone dose was recovered as nafimidone alcohol. Thus, it appears that over 90% of the administered dose of nafimidone is metabolised by pathways other than glucuronidation of nafimidone alcohol and urinary excretion.

Affinities of adrenergic drugs for alpha 2-adrenoceptors in dog saphenous vein in comparison with those in rat brain or human platelets.

The affinities of drugs for the postsynaptic alpha 2-adrenoceptors in the dog saphenous vein were examined and compared with those in human platelets or in rat brain. The pA2 or pD2 values in the vein were determined in the presence of phenoxybenzamine. The pKi values (dissociation constant) of drugs for binding to alpha 2-adrenoceptors in human platelets or in rat brain were determined by radioligand binding assay using 3H-yohimbine (YOH) or 3H-clonidine (CLO) as ligands. The binding of agonists to 3H-YOH binding sites was carried out in the presence of GTP. The pA2 or pD2 values in the vein had a good correlation with pKi values for YOH sites but not with those for CLO sites in rat brain. The pKi values for YOH sites in human platelets had a good correlation with those for YOH sites in the brain or with pA2 or pD2 values in the vein. These results suggested that the affinities of adrenergic drugs for postsynaptic alpha 2-adrenoceptors in the dog saphenous vein are similar to those in brain and in platelets, and it is probable that the adrenergic drugs produce their alpha 2-adrenoceptor-mediated effects on vascular tissues primarily through binding to a low-affinity form of the alpha 2-adrenoceptors.