I(1) imidazoline receptors involved in cardiovascular regulation: where are we and where are we going?

Clonidine-like drugs (hybrid drugs) reduce blood pressure by acting centrally at both alpha(2)-adrenergic receptors (alpha(2)AR) and I(1) receptors (I(1)R). Some attempts at cloning I(1)R have failed, probably because of the lack of selectivity of the ligands. Recently, compounds acting exclusively at I(1)R were synthesized: LNP 911, LNP509, and S23515. For example, LNP911 has a K(d) value of 1.7 nmol/L at I(1)R. LNP509 and S23515 reduce blood pressure when injected centrally in anesthetized animals, whereas S23757 behaves as an antagonist of hypotensive imidazolines. LNP509 reduces blood pressure even in genetically engineered mice lacking functional alpha(2)AR. An exclusive action at central I(1)R is therefore sufficient to modify blood pressure. With the help of drugs selective for I(1)R and alpha-methylnoradrenaline, selective for alpha(2)AR, we showed that imidazoline and alpha(2)-adrenergic mechanisms interact synergistically in controlling the blood pressure. Such a synergism may explain the very powerful hypotensive effects of hybrid drugs. The new ligands selective for I(1)R will be very helpful to investigate the molecular features and the signaling system of I(1)R.

Synthesis and biological evaluation of pyrrolinic isosteres of rilmenidine. Discovery of cis-/trans-dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H-pyrrol-2-yl)-amine (LNP 509), an I1 imidazoline receptor selective ligand with hypotensive activity.

To find new compounds selective for purported I1 imidazoline receptors (I1Rs) over I2 imidazoline binding sites (I2BS) and alpha2-adrenoceptors (alpha2ARs), a series of pyrrolinic isosteres of rilmenidine has been prepared and their biological activity at I1Rs, I2BS, and alpha2ARs evaluated. This isosteric replacement provided us with compounds which still bound to I1Rs but not to I2BS nor to alpha2ARs. A limited structure-affinity relationship was generated around the heterocyclic moiety of these ligands. One compound in this series, LNP 509 (1e) [cis-/trans-dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H-pyrrol-2-yl)-amine], had no detectable affinity at alpha2ARs yet was capable of lowering blood pressure after central administration. These pyrrolinic analogues constitute a new chemical class of imidazoline related compounds with high selectivity for the I1Rs. They could be used as new tools in the study of I1Rs and in the conception of new centrally acting hypotensive drugs.

Participation of imidazoline receptors and alpha(2-)-adrenoceptors in the central hypotensive effects of imidazoline-like drugs.

The central hypotensive effect of imidazoline-like drugs (IMs) involves non-adrenergic imidazoline receptors (IRs). IMs cause hypotension irrespective of their affinity and selectivity for one or the other alpha-adrenoceptor subtypes. LNP 509, which binds to I1Rs (Ki = 5.10(-7) M) but roughly not to alpha 2-adrenoceptors (A2Rs) (Ki > 10(-5) M), causes hypotension when injected alone into the brainstem. As far as hybrid drugs, that is, those with mixed binding profiles (I1/alpha 2), are concerned, a significant correlation was reported between their central hypotensive effect and their affinity for IRs. Imidazoline antagonists such as idazoxan competitively antagonized the centrally induced hypotensive effect of IMs. Yohimbine, an A2Rs antagonist, blocks the hypotensive effect of hybrids but usually in a noncompetitive manner. Mutation of A2Rs prevented the hypotensive effects of drugs highly selective for A2Rs, but also that of hybrids such as clonidine. These data indicate that triggering of the hypotensive effects of IMs (1) needs implication of IRs; (2) appears to be facilitated by additional activation of A2Rs; and (3) requires integrity of A2Rs along the sympathetic pathways.

Binding of new cirazoline derivative to imidazoline receptors from human brain.

Imidazoline compounds are known to interact with alpha 2-adrenoceptors as well as with specific non-adrenergic binding sites. Such binding sites are present in the brain and in peripheral tissues. Hypotensive effects of imidazolines were shown to be related to specific interaction with imidazoline binding sites within the brainstem. Heterogeneity of these sites based on differences in selectivities was reported. In order to facilitate the characterization of human brain imidazoline receptors, we synthetized new ligands by substitutions on the cirazoline phenyl ring. Affinities of these cirazoline derivatives were determined in two imidazoline binding site models, namely the human brain and the rabbit kidney. Interaction of these compounds with imidazoline binding sites from the human brain appeared more sensitive to structural variations of the imidazoline than those with rabbit kidney sites. Moreover, no correlation was found between affinities for imidazoline binding sites and those for alpha 2-adrenoceptors of the rat brain. Arylazide derivative of 2-(5-amino-2-methyl-phenoxymethyl)-imidazoline exhibited a higher affinity for human brain imidazoline binding sites than for human brain alpha 2-adrenoceptors. Photoincorporation of this azido-compound in human brain imidazoline binding sites was achieved and blockade of [3H]idazoxan imidazoline specific binding observed. These new tools may allow fine characterization of the different subtypes of imidazoline binding proteins.

[Erythrocyte/plasma ratio of lithium. Determination method and individual stability]. / Le rapport érythroplasmatique du lithium. Méthode de mesure et stabilité individuelle.

Lithium is a very efficient drug for the treatment of maniac-depressive psychosis, but its therapeutic index is narrow. Moreover, even with lithium plasmatic concentrations in the therapeutic range, some patients showed toxic signs when others seemed not to be efficiently treated. The measurement of erythrocyte lithium concentration might provide a more rational method than the measurement of plasma lithium for monitoring the course of lithium therapy, since the mechanisms of lithium transfer across red blood cells resemble those for neurons. Toxic effects could then be related to high lithium erythrocyte concentrations or a high erythrocyte/plasma lithium ratio (EPR). The intracellular lithium concentration depends mainly on the Li(+)-Na+ countertransport, which is genetically determined. We wondered therefore whether the EPR is a specific characteristic of each patient and how long it requires to stabilise when lithium therapy is started. The EPR was determined by directly measuring the plasma and erythrocyte concentrations with a carefully standardised assay (lithium carbonate administered at 7 pm, blood sample taken at 7 am the next day). Potassium edetate was preferred as anticoagulant to sodium heparinate, which gave an overestimation of EPR. Plasma trapped in the red cell column, with polyfructosan as marker, was found to average 4% in our assay conditions (centrifugation at 3,200 g, 15 min, 4 degrees C). This standardised assay makes it possible to determine the EPR very accurately with an inter-assay variation coefficient of about 2.4%. Following the start of lithium therapy, the EPR stabilised very quickly, mainly within two days (three out of four patients), at a time when lithium erythrocyte and plasma concentrations were still increasing.(ABSTRACT TRUNCATED AT 250 WORDS)

gamma-Hydroxybutyrate ligands possess antidopaminergic and neuroleptic-like activities.

The action of agonists or antagonists at the gamma-hydroxybutyrate (GHB) receptor represents a possibility to modulate dopaminergic activities in brain. In the present study, GHB and six structural analogs were tested for their ability to displace [3H] GHB binding from striatal membranes. All the analogs tested exhibited higher affinity for GHB as compared with GHB itself. Parallel experiments were carried out on striatal slices in order to determine IC50 values for inhibition of dopamine release in the presence of these compounds. All substances inhibited dopamine release with higher potency as compared with GHB itself. These antidopaminergic activities were confirmed in several neuropharmacological tests, usually used to predict neuroleptic activities in vivo. There appears to be a relationship between the affinity for the GHB striatal low-affinity receptor and the inhibition of dopamine release on one hand, and the antidopaminergic activity (as revealed by the in vivo tests) on the other hand. Thus, it is suggested that GHB agonists possessing antidopaminergic activities, may represent potential drugs endowed with neuroleptic properties.

Negative-ion mass spectra of methylated diuretics.

The negative-ion mass spectra of 19 methylated diuretics are presented and correlations between these spectra and the chemical structures of the compounds are indicated. The spectra are of interest as the basis of an analytical method for the identification of small quantities of diuretics in the urine of hypokalemic patients.

Synthesis and ocular antihypertensive activity of new imidazolidine derivatives containing a beta-blocking side chain.

The syntheses of new phenylimidazolidine derivatives (3-6)1 containing a propanolamine oxime or an oxypropanolamine moiety attached either to the aromatic or to the imidazolidine ring are described. These compounds were evaluated for potential ocular antihypertensive activity in alpha-chymotrypsin-induced ocular hypertension in rabbits. These compounds represent a unique series of effective ocular antihypertensive agents that despite possessing structural characteristics of beta-blockers and of imidazolidine derivatives, exhibit weak alpha- and beta-adrenergic agonist and antagonist activities. These findings may be of significant therapeutic importance in the medical management of glaucoma.

Metabolism and vascular effects of gamma-L-glutamyl-L-dopa on the isolated rat kidney.

Gamma-L-glutamyl-L-dopa (or gludopa), a dopamine (DA) prodrug, is selectively metabolized in vivo by the kidney through the sequential action of two renal enzymes, gamma-glutamyl transpeptidase (gamma-GT) and aromatic L-amino acid decarboxylase (AADC). This study was designed to analyze, in vitro, the factors regulating gludopa metabolism and its renal vascular effects. Rat kidneys were perfused in closed circuit with a cell-free perfusion buffer containing 6% bovine serum albumin (BSA). Adding gludopa (final concentration 10(-5) M in the perfusate) led to the release of DA both into urine and perfusate (0.53 +/- 0.21 and 1.38 +/- 0.28 nmol/min/g kidney wt, respectively, during the first 5 min after substrate addition, N = 5, mean +/- SEM). Total DA release (urine plus perfusate) was 73.7 +/- 15.8 nmol/g kidney wt within 30 minutes of recirculation. In non-filtering kidneys, total DA release in the recirculating medium was lower (12.5 +/- 1.4 nmol/g kidney wt, P less than 0.01). Glomerular filtration and access to the gamma-GT on the brush border membrane of proximal tubular cells are therefore required for the maximal conversion rate of gludopa. On filtering kidneys, L-dopa was also converted to DA, but at a higher rate than gludopa (total DA formed within 30 min of recirculation = 131.2 +/- 31.9 nmol/g kidney wt) and this rate was not reduced in non-filtering kidneys (224.2 +/- 41.7 nmol/g kidney wt DA formed within 30 min). Metabolic conversion of L-dopa by AADC is thus preserved in the case of an approach via the basolateral side of the proximal tubular cells. The renal vascular effects of gludopa were studied after vascular tone had been restored by continuous perfusion of PGF2 alpha and after the inhibition of alpha- and beta-adrenoceptors. Gludopa (3.10(-6) to 4.10(-5) M) elicited concentration-dependent renal vasodilatation.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal dopamine synthesis from precursors.

In the Wistar rat in vivo L-dopa (10 mg/kg, subcutaneously) was shown to have the characteristics of a kidney-directed dopamine (DA) prodrug: two daily injections increased 24 h urinary DA excretion 450-fold but had no systemic effects on blood pressure and heart rate. In inactin-anesthesized rats, L-dopa increased natriuresis, diuresis and renal blood flow; these effects were linked to endorenal DA synthesis and to DA-1 receptor stimulation since they were suppressed by both carbidopa and SCH 23390. In the isolated perfused rat kidney, DA was synthesized from L-dopa with a greater yield than from gludopa. In nonfiltering kidneys, L-dopa metabolism was not limited when the access to dopa decarboxylase was restricted to the basolateral membrane. The same was not true for gludopa, for which the basolateral metabolism was low.

Improved radioreceptor assay for the determination of plasma levels of dihydropyridine calcium channel blockers in humans.

Ways of improving sensitivity of the radioreceptor assay to determine the plasma levels of dihydropyridine calcium antagonists were investigated. Extraction of the drug from plasma with organic solvent was found to enhance the sensitivity of the assay (method 1). Alternatively, the inhibitory effect observed when plasma is added directly to the binding assay can be counteracted by increasing the amount of membranes in the assay (method 2). Plasma levels after single oral doses of nitrendipine and nicardipine were followed with method 1. Plasma levels of isradipine were measured with methods 1 and 2 and by mass fragmentography. The data confirm that nitrendipine plasma level kinetics vary widely from patient to patient, whereas for nicardipine the drug level profile is more homogeneous. The similarity of the data obtained from the radioreceptor assay and from mass fragmentography suggests the absence of any active metabolite of isradipine.

[Metabolism and vascular effects of gammaglutamyl L-dopa on the isolated kidney of the rat]. / Métabolisme et effets vasculaires de la gammaglutamyl L-dopa sur le rein isolé de rat.

The gammaglutamyl L-dopa (or gludopa), a dopamine (DA) prodrug, may be usefull in antihypertensive therapy as an orally active specific renal vasodilator. Indeed, gludopa is selectively metabolized in vivo by the kidney. This is the consequence of the sequential action of two renal enzymes, gamma-glutamyl transpeptidase (gamma-GT) and aromatic L-amino acid decarboxylase. The aim of this work was to elucidate, in vitro, the factors regulating its metabolism and to characterize its renal vascular effects. The rat kidney was isolated and perfused at constant flow in a closed circuit with a modified Krebs-Henseleit solution (BSA 6g/100 ml). Gludopa injection (10(-5) M) led to generation of DA (measured by gaz chromatography/mass spectrometry) in the venous effluent (134 +/- 39 ng/ml, n = 3) and in the urine (257 +/- 107 ng/mn/g). In non filtering kidneys, the level of DA in recirculating medium was depressed (47 +/- 5 ng/ml, n = 5; p less than 0.05). Glomerular filtration and access to the gamma-GT localized on the brush border membrane of proximal tubular cells are thus important for optimal metabolism of gludopa. Vascular effects of gludopa were studied on the isolated rat kidney after reestablishing vascular tone by continuous perfusion with prostaglandin F2 alpha (10(-8) M/mn) and after inhibition of alpha- and beta-adrenoceptors. Gludopa (3 X 10(-6) to 4 X 10(-5) M) induced dose-dependent renal vasodilation. At 4 X 10(-5) M, the renal response (30 +/- 3 p. 100 of the relaxation induced by 10(-4) M of papaverine) was similar to that elicited by DA at 20 fold lower concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Negative ion mass spectra of dihydropyridine calcium-channel blockers.

The negative ion mass spectra of some dihydropyridine analogues of nifedipine are studied; they show a fragmentation which is highly dependent on the position of the nitro group on the phenyl ring: 3'-nitro derivatives give essentially the molecular anion, whereas 2'-nitro derivatives lose successively H2O, RO and O. In addition, (2,1,3-benzoxadiazol-4-yl) derivatives show essentially a [M-ROH]- peak. Possible pathways for these fragmentations are given.

A rapid and sensitive method for the determination of gamma-hydroxybutyric acid and trans-gamma-hydroxycrotonic acid in rat brain tissue by gas chromatography/mass spectrometry with negative ion detection.

gamma-Hydroxybutyric acid (GHB) and trans-gamma-hydroxycrotonic acid (HCA) together with their respective internal standards were derivatized to give the pentafluorobenzyl esters of the N-tert-butyldimethylsilyl derivatives. These compounds give, under electron capture conditions, very simple negative ion mass spectra. A very sensitive and specific assay for GHB and HCA in brain tissue (detection limit of about 5 pg per injection) using gas chromatography/negative ion mass spectrometry is described. The average levels measured in the whole brain were 1.10 +/- 0.18 nmol GHB/g wet weight and 0.18 +/- 0.02 nmol HCA/g wet weight.

Pharmacokinetics of rilmenidine.

Rilmenidine, an alpha 2-adrenoceptor agonist, was studied (1 mg single dose) in order to determine the effects of pathology on its basic pharmacokinetic parameters. Because of the mainly renal elimination of rilmenidine, studies involved hypertensive, elderly hypertensive, renal insufficient and hepatic insufficient patients. Hypertension was found to influence neither the absorption, the distribution nor the elimination processes; the linearity in the range of 1 to 2 mg and the absence of accumulation in long-term treatment were confirmed. In contrast, in the elderly, the absorption phase was delayed. The slight decrease in the apparent volume of distribution (-12%), with a notable decrease in the apparent total clearance (-50%) led to a prolonged elimination half-life (+50%). In renal failure, linear relations between the degree of renal impairment and the elimination parameters were shown. These relations allow the evaluation of the predicted steady-state level of rilmenidine for a given degree of renal failure. In hepatic insufficiency, the modification of rilmenidine disposition concerned exclusively the elimination phase in which apparent clearance was decreased approximately 20%. In conclusion, these results lead to a decreased dosage regimen in patients with severe renal failure.

Gamma hydroxybutyrate distribution and turnover rates in discrete brain regions of the rat.

Gamma-hydroxybutyric acid and trans-gamma-hydroxycrotonic acid levels have been determined in 24 regions of the rat brain after sacrifice by microwave irradiation. Concentration ranges are from 4 pmol/mg protein (frontal cortex) to 46 pmol/mg protein (substantia nigra) for gamma-hydroxybutyric acid and from 0.4 pmol/mg protein (striatum) to 11 pmol/mg protein (hypothalamus) for trans-gamma-hydroxycrotonic acid. It appears that gamma-hydroxybutyric acid levels correlate well with GABA distribution in the same region. However this correlation is not evident with regard to the distribution of the gamma-hydroxybutyric acid synthesizing enzyme, specific succinic semialdehyde reductase. Using the antiepileptic drug, valproate which strongly inhibits gamma-hydroxybutyric acid release and degradation, we estimated the turnover rate of this compound in six regions of the rat brain. Turnover numbers ranged from 6.5 h(-1) in hippocampus to 0.76 h(-1) in cerebellum.

Gas chromatographic negative ion mass spectrometric assay of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug.

The quantification in plasma and urine of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug is described using a sensitive gas chromatographic negative ion mass spectrometric method with ammonia as moderating gas. After a two-step extraction, derivatization is carried out with 3,5-bis(trifluoromethyl)benzoyl chloride and the abundance of the molecular ion (m/z 420) obtained is compared with that of the tetradeuterated standard (m/z 424). The low background due to the high mass and negative ion detection provides a detection limit of about 1 pg per injection. Oral administration of 1 or 2 mg S-3341 to patients gives a maximum concentration of 3.3 +/- 0.7 ng ml-1 and 7.6 +/- 2.0 ng ml-1 at 1.8 +/- 0.6 h and 1.4 +/- 0.7 h and an average elimination half-life of 6.7 h.

Postjunctional alpha-adrenoceptors. Alpha 1 and alpha 2 subtypes in rat vasculature in vitro and in vivo.

The postsynaptic alpha-adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine alpha-adrenergic agonists and to the selective antagonists yohimbine (alpha 2) and prazosin (alpha 1) and the nonselective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [3H] yohimbine and [3H] prazosin. On rat aorta, prazosin is 1,000 times more potent than yohimbine against each alpha-adrenoceptor agonist, whether alpha 1- or alpha 2-selective. Rat aorta probably contains only alpha 1-adrenoceptors. Pressor effects in pithed rats are mediated by post-junctional alpha 1- and alpha 2-adrenoceptors. The dose-response curve for alpha-methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed alpha 1- and alpha 2-adrenoceptors, respective proportions being 80.5 and 19.5%.