Glucose-induced insulin secretion is potentiated by a new imidazoline compound.

Sulfonylureas stimulate insulin secretion independent of the blood glucose concentration. This can lead to hypoglycaemia in type 2 diabetic patients. Over the last years a number of imidazoline derivatives have been identified that stimulate insulin secretion in a more glucose-dependent way. In agreement with this, our aim was to generate imidazoline derivatives with a potential for the treatment of type 2 diabetic patients. We developed the compound 2-[4-(4-chlorophenyl)-3-(2-methoxyethoxy)-2-naphthalenyl]-4,5-dihydro-1-H-imidazole monohydrochloride (LY389382) with an imidazoline moiety and investigated its effects on glucose-dependent insulin secretion in a beta-cell line, isolated rat islets and in vivo. We could demonstrate that LY389382 induces insulin secretion in MIN6 cells and rat islets in a glucose-dependent manner (EC50=1.1 microM and 0.3 microM, respectively). Furthermore during hyperglycaemia LY389382 increased insulin secretion in a dose-dependent manner in healthy rats, whereas the compound had no effect at euglycemia in a tenfold higher dosage. After 7 days of treatment of Zucker Diabetic Fatty [ZDF/ (Gmi/fa)] rats with LY389382 with a dose of 15 mg/kg twice daily the blood glucose concentration was reduced from 22.7 +/- 1.7 mM to 16.6 +/- 2.3 mM. During the same time period the glucose concentration increased from 21.7+/-1.7 mM to 28.9 +/- 1.3 mM in the vehicle-treated group (P<0.05). The drop of the insulin level was also inhibited by LY389382 in ZDF rats. In contrast to other well-characterised imidazolines that have been shown to induce a glucose-dependent insulin secretion only within a limited range of concentrations, LY389382 stimulates insulin secretion over a concentration range of at least two log units in a glucose-dependent manner. These data suggest that this imidazoline compound has a potential for the treatment of type 2 diabetes.

Different modes of action of the imidazoline compound RX871024 in pancreatic beta-cells. Blocking of K+ channels, mobilization of Ca2+ from endoplasmic reticulum, and interaction with exocytotic machinery.

The imidazoline compound RX871024 glucose-dependently potentiates the release of insulin in pancreatic islets and beta-cell lines. This activity of the compound is not related to its action by stimulating alpha 2-adrenoceptors and I1- and I2-imidazoline receptors. There are at least three modes of action of RX871024 in beta-cells: (1) RX871024 blocks the ATP-dependent, Ca(2+)-activated, and delayed rectifier K+ channel activity; (2) RX871024 causes mobilization of Ca2+ from thapsigargin-sensitive intracellular stores, the effect probably controlled by cytochrome P450; and (3) the stimulatory activity of RX871024 on insulin release involves interaction of the compound with the exocytotic machinery, unrelated to the changes in membrane potential and cytoplasmic-free Ca2+ concentration, whereas protein phosphorylation plays an important role in this process. The maximal insulinotropic effect of RX871024 is much higher than that of the sulfonylurea glibenclamide. RX871024 stimulates insulin release and normalizes blood glucose levels in rats in vivo without affecting blood pressure and heart rate.

Regulation of glucagon-like peptide-1 synthesis and secretion in the GLUTag enteroendocrine cell line.

Glucagon-like peptide-1 (GLP-1) released from the intestine is a potent stimulator of glucose-dependent insulin secretion. To elucidate the factors regulating GLP-1 secretion, we have studied the enteroendocrine GLUTag cell line. GLP-1 secretion was stimulated in a dose-dependent fashion by activation of protein kinase A or C with forskolin or phorbol 12,13-dibutyrate, respectively (by 2.3 +/- 0.5-fold at 100 microM and 4.3 +/- 0.6-fold at 0.3 microM, respectively; P < 0.01-0.001). Of the regulatory peptides tested, only glucose-dependent insulinotropic peptide stimulated the release of GLP-1 (by 2.3 +/- 0.2-fold at 0.1 microM; P < 0.001); glucagon was without effect, and paradoxically, the inhibitory neuropeptide somatostatin-14 increased secretion slightly (by 1.6 +/- 0.3-fold at 0.01 microM; P < 0.05). In tests of several neurotransmitters, only the cholinergic agonists carbachol and bethanechol stimulated peptide secretion in a dose-dependent fashion (by 2.3 +/- 0.5- and 1.7 +/- 0.3-fold at 1000 microM; P < 0.05-0.001); the beta-adrenergic agonist isoproterenol and the chloride channel inhibitor gamma-aminobutyric acid did not affect release of GLP-1. Long chain monounsaturated fatty acids (18:1), but not saturated fatty acids (16:0), also stimulated the release of GLP-1 (by 1.7 +/- 0.1-fold at 150 microM; P < 0.001). Consistent with the presence of a cAMP response element in the proglucagon gene, activation of the protein kinase A-dependent pathway with forskolin increased proglucagon messenger RNA transcript levels by 2-fold (P < 0.05); glucose-dependent insulinotropic peptide and phorbol 12,13-dibutyrate were without effect. Therefore, by comparison with results obtained using primary L cell cultures or in vivo models, GLUTag cells appear to respond appropriately to the regulatory mechanisms controlling intestinal GLP-1 secretion.

Identification and pharmacological characterization of somatostatin receptors in rat lung.

1. [125I]-[LTT]SRIF-28 and [125I]-SMS 201-995 were used to identify and characterize somatostatin (SRIF) receptors localized in rat lung tissue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [125I]-[LTT]SRIF-28 without any significant specific binding of the sst2/sst5-receptor selective ligand [125I]-SMS 201-995. 2. In radioligand binding studies, specific binding of [125I]-[LTT]SRIF-28 to membranes of rat lung was linearly related to the concentration of membrane protein used with only a small portion of nonspecific binding. With [125I]-SMS 201-995 no specific binding could be observed up to a membrane concentration of 0.1 mg of protein/assay tube. 3. [125I]-[LTT]SRIF-28 bound rapidly to rat lung membranes with an apparent association rate constant (kapp) of 1.8 +/- 0.1 h-1 (n = 3). The equilibrium of specific binding was reached after an incubation period of approximately 90 min at room temperature and remained constant for the next 3 h. The association rate constant (k1) was calculated to be 3.7 x 10(10) M-1 h-1. The dissociation reaction followed first order kinetics with a dissociation rate constant (k-1) = 0.44 +/- 0.07 h-1 corresponding to a half-time of 95 +/- 15 min (n = 3). From these kinetic experiments an equilibrium dissociation constant (KD) for the binding of [125I]-[LTT]SRIF-28 was calculated to be 11.9 pM. 4. Saturation binding of [125I]-[LTT]SRIF-28 revealed an equilibrium dissociation constant (KD) of 50.1 pM (pKD = 10.3 +/- 0.1; n = 3) and a receptor density (Bmax) of 78 +/- 3 fmol mg-1 protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites. 5. Specific binding of [125I]-[LTT]SRIF-28 to rat lung membranes was inhibited by SRIF-14, SRIF-28 and different SRIF analogues. SRIF and different synthetic short chain SRIF analogues exhibited the following rank order of potency: SRIF-28 > SRIF-14 > CGP 23996 >> RC 160 > BIM 23014 > SMS 201- 995 > BIM 23056 > MK 678. 6. The binding affinities for SRIF and the various SRIF analogues determined using rat lung tissue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst, (r = 0.92) and sst4 (r = 0.95) receptors, respectively. 7. Reverse transcriptase--polymerase chain reaction (RT-PCR) showed the predominant expression of mRNA specific for sst4 receptors as well as some weak sst1 mRNA expression. 8. The findings suggest that sst4 receptor expression is the predominant form of the somatostatin receptors identified in rat lung tissue. In this study we demonstrated for the first time the existence of sst4 receptors in mammalian tissue.

Investigations of the mechanism of the positive inotropic action of BDF 9148: comparison with DPI 201-106 and the enantiomers.

The electromechanical and biochemical activities of the positive inotropic compounds BDF 9148 and DPI 201-106 were compared in guinea-pig myocardic preparations. Additionally, the properties of the BDF 9148 enantiomers were studied to compare their positive inotropic effects. In guinea pig papillary muscles, BDF 9148 exerted a concentration-dependent increase of force of contraction with a 50% effective concentration (EC50) value of 0.6 microM, compared with 1.3 microM for DPI 201-106. Like that of DPI, the inotropic effect of BDF 9148 was abolished by treatment with tetrodotoxin (TTX) but not affected by treatment with carbachol. Likewise, pretreatment of the papillary muscles with propranolol, cimetidine, and histamine did not affect the contractile effects of BDF 9148. In the left atria, both agents had a positive inotropic effect with an EC50 of 0.2 microM for BDF and 0.8 microM for DPI. Incubation of single concentrations of the respective drugs for a period of 90 min with guinea pig papillary muscles resulted in slightly differing parameters of isometric contraction. In contrast to DPI, BDF 9148 prolonged the contraction time transiently. Time to peak force was not markedly influenced by either drug. The functional refractory period was prolonged by both drugs to a similar extent. At 10 microM, BDF 9148 showed a biphasic effect on the action potential duration (APD) most evident at APD90, whereas DPI prolonged APD90 progressively until the 90 min. The positive inotropic effect of BDF 9148 could be demonstrated by the (S-), whereas the (R+)-enantiomer was without effect. Neither DPI nor BDF 9148 increased myocardial cyclic adenosine monophosphate (cAMP) in isolated rat cardiomyocytes and guinea pig papillary muscles. Additionally, neither BDF 9148 nor DPI showed an inhibitory effect on the guinea pig myocardic Na+/K(+)-ATP'ase activity in the concentration range with a positive inotropic effect in the guinea-pig papillary muscle.

Binding properties of somatostatin receptor subtypes.

In the past few years, five different somatostatin (SRIF) receptor subtypes (sst1.5) have been identified, which form a distinct group in the superfamily of G-protein-coupled receptors. The naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst1.5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201-995, RC-160, or BIM 23014, primarily interact with the sst2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst2 (MK 678), the sst3 (BIM 23056), or the sst5 (BIM 23052, L362-855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst1.5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst1, for SMS 201-995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst1, almost full sst2-like binding of SMS 201-995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pKi values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for [125I]Tyr3-SMS 201-995 was detected, since SMS 201-995 has low affinity for sst4. In contrast, specific binding of [125I]SRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst4 receptors.

Reproducibility and consistency of the responses to supine bicycle ergometry; evaluation in conjunction with beta 1-adrenoceptor occupancies.

A protocol is presented for supine bicycle ergometry in healthy subjects, which aims for a target increase of heart rate (HR = 140 beats min-1) after 4 min cycling under constant load. The required load is selected from a pre-study ergometry with stepwise increasing load. Repeated testing with this protocol was shown to yield highly reproducible ergometric HR-responses. Because of their high reliability, the ergometric endpoints and increments permit a highly sensitive comparison of beta 1-adrenoceptor antagonism across dose and time within a given compound. The relationship between the changes of the ergometric rise of HR and the beta 1-adrenoceptor occupancy (estimated from radioreceptor assay data) permits to evaluate the ergometric efficiency of different beta-adrenoceptor antagonists across time and doses and to identify eventual differences that do not relate primarily to the extent of beta 1-adrenoceptor antagonism itself.

Dose-effect and pharmacokinetic-pharmacodynamic relationships of the beta 1-adrenergic receptor blocking properties of various doses of carvedilol in healthy humans.

OBJECTIVE: To evaluate the pharmacodynamic properties of carvedilol across a broad range of doses in relation to its enantiospecific kinetics and adrenergic receptor occupancies, relative to placebo and propranolol. METHODS: Twelve healthy male subjects were investigated on six separate occasions at least 1 week apart when they received either a single peroral dose of 40 mg propranolol, 12.5, 25, 50, or 100 mg carvedilol, or placebo. The subjects were extensively profiled at supine rest, and they underwent supine bicycle ergometry before and at 2, 4, 6, 9, 12, and 22 hours after dosing. At these time points blood was drawn for the high performance liquid chromatographic determination of the enantiomers of carvedilol and for the radioreceptor assay determination of alpha 1- and beta 1-adrenergic receptor binding and related concentrations. RESULTS: Carvedilol was confirmed to bind to beta 1-adrenergic receptors and (albeit to a lesser extent) to alpha 1-adrenergic receptors. Carvedilol furthermore attenuated the ergometric increase in heart rate in a closely dose-related fashion, which exemplified its beta 1-adrenergic receptor blocking effects. However, the basal efferent adrenergic drive might have been too low to show consistent alpha 1-blocking properties. The radioreceptor and enantiomer kinetics were proportional with dose. There was no indication that the overall kinetic behavior of contributing active metabolites would differ from that of the S(-)-enantiomer. On average, there was a smooth linear relationship between the ergometric treatment responses and log-transformed dose, log-transformed concentrations of the S(-)-enantiomer, and the radioreceptor assay derived beta 1-adrenergic receptor occupancies. CONCLUSION: The relative complexity of the kinetics of carvedilol (enantiospecific kinetics and dynamics, protein binding, and involvement of active metabolites) does not preclude relatively simple and straight-forward dose-effect and kinetic-dynamic relationships.

Measurements of serum ACE activity in vitro after administration of enalapril in man cannot reflect inhibition of the enzyme in vivo.

We previously demonstrated the dependency of serum angiotensin converting enzyme (ACE) inhibition by the inhibitor enalaprilat in vitro on the concentration of substrate and enzyme present in the assay, according to a competitive tight-binding mechanism. In the present study, the relevance of these findings for ex vivo measurements after administration of enalapril has been confirmed in serum samples of four patients which were assayed twice using different substrate concentrations in vitro. The measured extent of ACE inhibition in the samples was markedly different depending on the substrate concentration added in vitro (in relation to its Km value: S/Km, respectively), suggesting lower inhibition over time when higher S/Km was used. When the dilution factor and the added S/Km were taken into account the measured values could successfully be predicted from the respective serum enalaprilat concentrations by means of the concentration-effect model previously evaluated for the in vitro relationship (Emax tight model). Considering conditions which probably better reflect the situation in vivo (no dilution, physiological substrate concentrations far below Km) we simulated a time course of in vivo serum ACE activity in these patients which suggests almost complete inhibition of serum ACE over time in contrast to the in vitro measurement. Thus, we conclude that the usual ex vivo measurements of ACE activity lead to an underestimation of the extent of inhibition because of sample dilution and high exogenous substrate in vitro, and therefore must fail to reflect enzyme inhibition in vivo.

The influence of hydrochlorothiazide on the pharmacokinetics of enalapril in elderly patients.

In a randomized, cross-over, single-dose study of 19 elderly hypertensive patients (aged 62-84 y, SBP greater than 160 mmHg, DBP greater than 100 mmHg, creatinine clearance 11-93 ml.min-1) we have studied the pharmacokinetics of the angiotensin converting enzyme (ACE) inhibitor enalapril after a single oral dose of either 10 mg enalapril or 10 mg enalapril + 25 mg hydrochlorothiazide. The pharmacokinetics of enalapril were unaffected by hydrochlorothiazide, but there was a significant reduction in renal clearance and a significant increase in AUC(0-24 h) of enalaprilat after hydrochlorothiazide, resulting in higher serum concentrations of the active drug. This was independent of the individual degree of renal impairment and might be due either to an initial reduction of GFR by hydrochlorothiazide or to interference with the tubular secretion of enalaprilat. The relationships between serum enalaprilat and serum ACE activity were similar after both treatments, both consistent with a value for Ki of enalaprilat of about 0.1 nmol.l-1. Thus, serum ACE activity was not affected by hydrochlorothiazide but completely reflected the pharmacokinetics of enalaprilat in both treatments.

Oral administration of carvedilol and prazosin inhibits the prostaglandin F2 alpha- and noradrenaline-induced contraction of human hand veins in vivo.

Carvedilol is a beta-blocker with additional vasodilating activity. This study was performed in order to determine whether the vasodilator action of orally administered carvedilol in man is based upon an alpha-adrenoceptor antagonism exclusively or if evidence for an additional mechanism could be confirmed. The influence of carvedilol (50 mg p.o.) and prazosin (2 mg p.o.) upon the vasoconstrictor effect of noradrenaline and prostaglandin F2 alpha, infused into superficial hand veins, was established in 8 healthy male volunteers. Increasing dosages of the vasoconstrictors below their threshold of systemic activity were employed in order to obtain dose-response curves of the hand veins congested at a venous occlusion pressure of 40 mmHg. These dose-response curves were repeated 1 and 3.5 h after oral administration of either carvedilol, prazosin, or placebo. The ex vivo, in vitro alpha 1-receptor occupancy in plasma was measured before and after each vasoconstrictor dose-response curve, using an alpha 1-radioreceptor binding assay. Washout periods of 48 h were kept between study days, investigating the influence of one orally administered drug upon one of the local vasoconstrictor dose-response curves at a time. In the alpha 1-radioreceptor assay, plasma concentrations from 0.9- to 1.7-fold the equilibrium dissociation constant (Ki) of carvedilol could be evaluated 1 as well as 3.5 h after medication, corresponding with a receptor occupancy of 44%-63%. After prazosin, 9-13 times the Ki values were determined, which amounts to an alpha 1-adrenoceptor occupation of about 90%-93%.(ABSTRACT TRUNCATED AT 250 WORDS)

Constriction of human dorsal hand veins in vivo with several vasoconstrictors and the influence of oral administration of carvedilol.

In vivo measurements of human dorsal hand vein diameters have become a valuable tool in investigating vasoactive agents. This study in 13 healthy volunteers was performed to establish the influence of locally infused clonidine, angiotensin II, norepinephrine, and prostaglandin F2 alpha (PGF2 alpha) on hand vein diameter in a nonsystemically active dose range. The study was intended to select agents suitable for venous preconstriction before and after oral administration of 50 mg carvedilol to obtain information on the mechanisms of vasodilation of this combined alpha- and beta-blocker. Infusions of norepinephrine (ED50, 20-40 ng/min) and PGF2 alpha (ED50, 480-1,170 ng/min) into the hand vein under investigation led to nearly complete constriction of the vessel. Infusions with clonidine and angiotensin II led to approximately 35% diameter reduction under the same conditions. A wide interindividual variety of hand vein dose response was observed for all vasoconstrictors. Oral administration of 50 mg carvedilol led to a rightward shift in the dose-response curves of angiotensin II, norepinephrine, and PGF2 alpha. On a 5% significance level, the venoconstrictive effects of norepinephrine and PGF2 alpha were attenuated by carvedilol compared with placebo. We conclude that a mechanism of vasodilation by carvedilol in addition to alpha-adrenoceptor antagonism could be responsible for the attenuation of venoconstriction induced by PGF2 alpha.

The role of enzyme and substrate concentration in the evaluation of serum angiotensin converting enzyme (ACE) inhibition by enalaprilat in vitro.

The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson equation: it is directly applicable without conversion of the data and avoids the stochastic dependency of the variables, allowing non-linear regression of all data points contributing with the same weight.

Characterization of muscarinic receptors of bovine coronary artery by functional and radioligand binding studies.

The nature of the muscarinic receptor subtype mediating contraction of the endothelium-denuded bovine coronary artery was investigated in vitro by functional measurements and radioligand binding studies. The acetylcholine (ACh)-induced isotonic contraction of circularly cut muscle strips was recorded and expressed as a percentage of the maximum contraction obtained with 80 mM K+. In order to distinguish between M1, M2 and M3 receptors, the potency of the five subtype-selective antagonists, 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), parafluor-hexahydro-siladifenidol (pFHHSiD), pirenzepine, AF-DX 116 and methoctramine, to block the ACh-induced contraction was estimated. All the antagonists competitively inhibited the responses induced by ACh, with one exception, namely, 4-DAMP, whose Schild plot had a slope greater than one. The low affinity of pirenzepine (pA2 7.14 +/- 0.14) excluded an action at the M1 subtype. The low affinity of AF-DX 116 (pA2 6.49 +/- 0.18) and methoctramine (pA2 5.88 +/- 0.07) suggest that the bovine coronary artery smooth muscle receptor is not of the M2 (cardiac) subtype. In contrast, 4-DAMP (pA2 9.04 +/- 0.03) and pFHHSiD (pA2 7.64 +/- 0.04) potently inhibited the ACh-induced contraction with affinities similar to those reported for the M3 (glandular) receptor. In addition, the muscarinic receptors mediating coronary artery contraction were characterized in antagonist/[3H]N-methyl-scopolamine ([3H]NMS) competition binding studies. With the exception of AF-DX 116, all antagonists bound to a homogeneous population of receptors with pseudo-Hill slopes not different from unity. The pKi values, albeit somewhat lower, essentially substantiated the functional affinity estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and converting enzyme inhibition after morning and evening administration of oral enalapril to healthy subjects.

Possible circadian changes in the pharmacokinetics and effect on serum angiotensin-converting enzyme (ACE) activity of the ACE inhibitor enalapril have been studied in 8 healthy subjects after oral ingestion of 10 mg enalapril maleate either at 08.00 h or 20.00 h. The time to peak serum concentration (tmax) of enalapril was increased after administration at 20.00 h compared to 08.00 h (2.4 h versus 1.3 h), whereas other kinetic parameters were not significantly altered. The 24 h-kinetics of the active metabolite enalaprilat did not differ significantly between the two treatments, but the area under the curve (AUC (0-24] and the peak serum concentration (Cmax) were slightly higher after intake at 20.00 h. The relationship between the measured serum enalaprilat level and the degree of inhibition of serum ACE was the same after both treatments. Overall, the evening and morning administration of enalapril did not differ markedly in the pharmacokinetics and the time course of ACE inhibition.

Measurement of carvedilol enantiomers in human plasma and urine using S-naproxen chloride for chiral derivatization.

The enantiospecific procedure for assaying carvedilol includes the extraction of the drug from plasma or urine with diisopropylether after alkalization of the sample with pH 9.8 buffer. After evaporation of the org. solvent a chiral derivatization is performed using S-(+)-naproxen chloride. The HPLC separation of the diastereomeric amides is possible on a silica gel stationary phase with a mixture of n-hexane, dichloromethane, and ethanol as mobile phase. Detection of the products is performed by fluorescence measurement at 285/355 nm. Preliminary pharmacokinetic studies after i.v. infusion of racemic compound to healthy volunteers showed that the concentrations of the R-(+)-enantiomer exceeded those of the S-(-)-enantiomer. Overall, both carvedilol enantiomers exhibited a high clearance with preference for the S-enantiomer. The difference was even more expressed after p.o. dosage indicating a stereoselective first-pass effect with higher extraction of the levorotatory enantiomer, which is more potent with respect to beta-adrenoceptor antagonistic activity.

The effect of oral cilazapril and prazosin on the constrictor effects of locally infused angiotensin I and noradrenaline in human dorsal hand veins.

The effects of the ACE inhibitor cilazapril (5 mg p.o.) and the alpha 1-adrenoceptor blocker prazosin (2 mg p.o.) were investigated on the dose-response curves to angiotensin I and to noradrenaline, administered locally in the hand veins in six healthy male volunteers in doses not producing systemic effects. Both angiotensin I and noradrenaline produced a dose-dependent constriction of the congested veins. The angiotensin I effects were completely abolished after the administration of cilazapril but not significantly altered after the administration of prazosin. The noradrenaline dose-response curves were shifted to the right (dose ratio about 10) by prazosin, but not by cilazapril. The data suggest that angiotensin I, after having been converted to angiotensin II exerts direct venoconstrictor effects which under resting conditions are not mediated by noradrenaline release.

Agonist binding at alpha 2-adrenoceptors of human platelets using 3H-UK-14,304: regulation by Gpp(NH)p and cations.

The agonist/alpha 2-adrenoceptor interactions at human platelet membranes have been examined in radioligand binding studies with the full agonist ligand 3H-UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] and the antagonist ligand 3H-yohimbine. From association kinetics of different concentrations of 3H-UK-14,304 (0.75-8.1 nmol/l) a KD-value of 2.37 nmol/l in agreement with the high-affinity KD-value (KDH = 1.60 +/- 0.15 nmol/l) obtained from equilibrium binding studies was derived. In the presence of Gpp(NH)p about 6% of specific radioligand binding was observed in the association reaction. Addition of Gpp(NH)p at equilibrium resulted in a rapid loss (t 1/2 less than 1 min) of approximately 80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 mumol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of 1/5 of rapidly (t 1/2 less than 2 min) and 4/5 of slowly dissociating ligand (k-1 = 0.033 +/- 0.004 min-1). Application of a sequential binding model resulted in KD-values from this approach also in agreement with KDH from equilibrium binding studies. The rank order of potency for different agonists and antagonists to compete for binding with 3H-UK-14,304 indicated an alpha 2-adrenoceptor interaction: (-)adrenaline greater than or equal to clonidine greater than (-)noradrenaline greater than (-)isoprenaline and yohimbine = rauwolscine greater than phentolamine greater than prazosin greater than or equal to corynanthine greater than timolol respectively. The analysis of competition isotherms of UK-14,304 versus 3H-yohimbine (Hill-coefficient = 0.59 +/- 0.03) showed that the agonist binds to two affinity states of the alpha 2-adrenoceptor, with high (KDH = 1.77 +/- 0.50 nmol/l) and low affinity (KDL = 71.2 +/- 11.6 nmol/l) respectively. From these experiments a fraction of 56.9% +/- 2.1% of the total number of alpha 2-adrenoceptors (Bmax = 198.4 +/- 8.0 fmol/mg of protein) in the high-affinity state was calculated. Similar results were obtained from 3H-UK-14,304 saturation isotherms according to a two-state binding model (KDH = 1.60 +/- 0.15 nmol/l; KDL = 66.2 +/- 10.7 nmol/l; BmaxH = 57.6% +/- 2.3%). Adrenoceptor agonists competed for specific binding of 3H-UK-14,304 and 3H-yohimbine in a manner that suggests that the 3H-UK-14,304 (approximately 3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the alpha 2-adrenoceptor.(ABSTRACT TRUNCATED AT 400 WORDS)