W. Soudijn

Relationships between structure and α-adrenergic receptor affinity of clonidine and some related cyclic amidines

The inhibition of specific [3H]clonidine binding to alpha-adrenergic receptors in rat brain has been studied for 43 compounds structurally related to clonidine. Oxymetazoline was found to be the most potent compound in displacing [3H]clonidine from its binding sites. 2-Methylimidazoline, 1,2,3,5-tetrahydroimidazo[2,1-b]quinazoline and heterocyclic N-methyl-substituted compounds showed no affinity at all for [3H]clonidine receptor sites. There was a good correlation between hypotensive activity and alpha 2-adrenergic receptor affinity to some 2-(phenylamino)imidazolidines. Parallelisms between Kis and pharmacological activities were also observed for other compounds. Apparent structural requirements for alpha 2-receptor affinity were the presence of an aromatic moiety and a N-hydrogen in the heterocyclic ring. Ortho substitution in the phenyl ring was necessary for high affinity. Compounds with an oxazolidine ring had approximately similar affinities for the [3H]clonidine binding sites compared with the corresponding imidazolines (except for the 2,6-dichlorophenyl-substituted compound) whereas the pyrrolidine derivatives showed a 10-fold weaker affinity. [3H]Clonidine sites showed a homogeneous character. KD values from saturation and displacement experiments were in good agreement with one another (2.6 and 2.7 nM, respectively) and with values in the literature.

Purification and characterization of enkephalin-degradating enzymes from calf-brain striatum

Enkephalinase A and B are extracted from Triton-X 100 washed calf-brain particles and purified by DEAE-cellulose chromatography. Both enzymes have identical Km values in their membrane-bound and soluble form. Enkephalinase A has a pH optimum at 6.9 and a Km for Leu-enkephalin of 20-25 microM, which hardly depends on the pH. Thiorphan and phosphate are purely competitive inhibitors of Enkephalinase A with Ki values of 3 nM and 1.5 mM respectively (pH = 6.85). Enkephalinase B is not affected by phosphate or thiorphan. It has a Km for Leu-enkephalin of 10 microM, a pH optimum of 7.0 and is inhibited by low concentrations of apolar dipeptides.

Inhibitors of calf-brain enkephalinase A and B

Enkephalinase A and B isolated from calf-brain striatum have comparable substrate specificity (Km for Leu-enkephalin = 1-3.10(-5) microM) but a quite different affinity for certain inhibitors: phosphate, Secobarbital and Thiorphan are effective inhibitors for Enkephalinase A (IC50 of 2.5 mM, 30 microM and 4 nM respectively), while Enkephalinase B does not react with any of these compounds. Both enzymes are inhibited by 1 mM EDTA and o-phenanthroline indicating the presence of a metal atom in or near their active sites. Although with different abilities, both enzymes recognize dipeptides having at least one hydrophobic amino-acid side chain. The potency of such dipeptides can be used for a description of the active site.

Opiate receptor binding of nicomorphine and its hydrolysis products in rat brain

Abstract In vitro receptor binding studies with paraoxon treated rat brain membranes, and stability studies using HPLC analysis, showed a negligible opiate receptor affinity for the morphinomimetic agent nicomorphine and its hydrolysis product 3-nicotinoylmorphine. Two other hydrolysis products, 6-nicotinoylmorphine and morphine, had a high affinity in the nanomolar range and are probably the active metabolites of nicomorphine.

The identification of flunitrazepam and its metabolites in urines samples

Abstract A thin-layer chromatographic method with fluorescence detection is described for the identification of flunitrazepam and its metabolites in urine. The method is based on the hydrolysis and subsequent formation of acridine derivatives by cyclization in dimethylformamide and is selective for benzodiazepines with a 2-fluorophenyl group. The method is applicable to urine samples even after an oral dose of only 2 mg of flunitrazepam. Flunitrazepam and flurazepam consumption can be distinguished.

Synthesis of enkephalinase B inhibitors, and their activity on isolated enkephalin-degrading enzymes.

Compounds in which a dipeptide moiety is linked to a metal chelating mercapto group were synthesized to obtain effective enkephalinase B inhibitors. Inhibitors containing two hydrophobic amino acid side-chains decrease enkephalinase B activity with a potency depending on the length of the spacer connecting the mercapto group and the dipeptide (IC50 values vary between 0.35 and 14 microM) and they also inhibit enkephalinase A and aminopeptidase activity. Compounds lacking the carboxy terminal side-chain are not recognized by enkephalinase B or aminopeptidase but are potent inhibitors of enkephalinase A. Our most potent enkephalinase B inhibitor is mercaptoacetyl-Phe-Phe (designated phelorphan), having an IC50 value of 0.35 microM for enkephalinase B. This compound also effectively inhibits enkephalinase A (IC50 = 0.02 microM) and aminopeptidase activity (IC50 = 13 microM). Phelorphan can therefore be considered as a complete inhibitor of enkephalin biodegradation.

Effects of halopemide on GABA receptor binding, uptake and release

The recently developed psychotropic agent halopemide (R 34301) has a unique pharmacological and clinical profile11,1~. Although structurally related to neuroleptics of the butyrophenone type and showing some affinity to neuroleptic binding sites in vitro, it is not preferentially accumulated in dopamine-rich areas such as the nucleus caudatus, the nucleus accumbens or the tuberculum olfactorium, all regions with a selective uptake of related neuroleptics14,15. Moreover, clinical investigations have shown, that halopemide does not cause extrapyramidal side-effects and seems to be a psychic energizer, of use in the treatment of withdrawn, inactive patients11,13. Neuroleptic agents are generally believed to exert their beneficial effects by the reduction of the activity of dopaminergic receptors. In addition, some other biochemical effects have been reported, which correlate more or less with their clinical potencies. Interference with GABAergic mechanisms is one of them. Certain antipsychotics are known to increase GABA turnover in discrete brain areas in vivog, 16. Inhibition of [aH]GABA uptake4,7,9, is receptor binding 10 and releasea, is in vitro by some, but not all neuroleptic agents has been noticed. Therefore, it may be possible, that modulation of GABA neuroregulatory function contributes to part of the (side-)effects of neuroleptic drugs. It is the aim of the present study to investigate the interference of halopemide and some of its putative metabolites with GABA uptake, receptor binding and release, and to compare their effects with those of related neuroleptics. Male Wistar rats of 200-250 g b.w. were sacrificed by decapitation. The brains were rapidly removed, cooled on ice and dissected. Unless stated otherwise all proceedings were performed at 0 °C. Protein was measured by the Lowry method. All data were given as mean 4S.E.M. To determine ICs0 values in binding experiments, log concentration-probit values were plotted and

Effect of various enkephalin analogs and dipeptides on the enzymatic activity of enkephalinase B isolated from calf-brain striatum

The inhibitory potency of several enkephalin analogs and dipeptides on the calf-brain enkephalinase B activity was established with the aim to characterize its active site. Highest potency was measured for dipeptides with a large side chain on both amino acids. The nature of the distal amino acid is of minor importance, provided it is not a glycine. Free carboxylic function is required for good interaction, whereas the stereochemical configuration of the dipeptide is less so. Enkephalinase B has only little affinity for D-Ala2-Leu-enkephalin. The data are to be used for the design of new enkephalinase B inhibitors.

Improved qualitative method for establishing flunitrazepam abuse using urine samples and column liquid chromatography with fluorimetric detection

The abuse of flunitrazepam (Rohypnol), a potent benzodiazepine-type hypnotic, cannot be established with the widely used immunoassays for urine analysis owing to lack of specificity. A simple method is described which is based on the formation of acridine derivatives and measures the sum of metabolites of flunitrazepam in urine samples using column liquid chromatography with fluorimetric detection.

The combined use of high-performance liquid chromatography and radioimmunoassay for the bioanalysis of nicomorphine and its metabolites

Methods have been developed for the determination of nicomorphine using reversed-phase HPLC with UV detection; for the simultaneous assay of morphine and mononicotinoylmorphine by a coupled normal-phase HPLC-radioimmunoassay method; and for conjugates of morphine and mononicotinoylmorphine by radioimmunoassay. The methods have been evaluated and applied to a pharmacokinetic study of nicomorphine administered intramuscularly.