Alpha-adrenergic receptor blocking effect of Cleistanthus collinus (Roxb.) Benth. and Hook f. leaf extract on guinea pig isolated smooth muscle preparations.

Aqueous extract of C. collinus leaves inhibited norepinephrine induced contraction in guinea pig vas deferens and aortic strip in a dose-dependent manner. Inhibition of acetylcholine induced contraction in ileum was dose independent. C. collinus extract per se had no effect on isolated guinea pig vas deferens and aortic strip, but inhibited norepinephrine induced contraction in a dose-dependent manner probably by its antagonist action on alpha-adrenergic receptor. It had inconsistent effect on guinea pig ileum in vitro preparation.

Nonaqueous capillary electrophoresis conditions for the simultaneous separation of eight alpha-adrenergic blocking agents.

The analysis is described for the first time for separating eight alpha-adrenergic blocking agents (oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378) by capillary electrophoresis (CE) with UV detection. Optimum separation of the analytes was obtained on a 50 cm × 75 µm i.d. capillary using a buffer containing 20% acetonitrile, 60 mM ammonium acetate, and 1.0% glacial acetic in methanol medium, with applied voltage and capillary temperature of 23 kV and 25 °C, respectively. The relative standard deviations of the migration times and the peak areas of the eight analytes were in the ranges of 0.12-1.29% and 1.02-2.53%, respectively. Detection limits of oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378 were 0.5-1.0 µg mL(-1). In the tested concentration range, good linear relationships (correlation coefficients >98%) between peak areas and concentrations of the analytes were observed. This method has been successfully applied for determination of prazosin and phentolamine with recoveries of 97.30% and 98.12%, respectively. The proposed method was successfully applied for the rapid CE determination of the frequently applied alpha-adrenergic blocking compounds phentolamine and prazosin in general pharmaceutical preparation.

An in vivo chemical library screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis and lymphangiogenesis.

Angiogenesis and lymphangiogenesis are essential for organogenesis but also play important roles in tissue regeneration, chronic inflammation, and tumor progression. Here we applied in vivo forward chemical genetics to identify novel compounds and biologic mechanisms involved in (lymph)angiogenesis in Xenopus tadpoles. A novel 2-step screening strategy involving a simple phenotypic read-out (edema formation or larval lethality) followed by semiautomated in situ hybridization was devised and used to screen an annotated chemical library of 1280 bioactive compounds. We identified 32 active compounds interfering with blood vascular and/or lymphatic development in Xenopus. Selected compounds were also tested for activities in a variety of endothelial in vitro assays. Finally, in a proof-of-principle study, the adenosine A1 receptor antagonist 7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine, an inhibitor of blood vascular and lymphatic development in Xenopus, was shown to act also as a potent antagonist of VEGFA-induced adult neovascularization in mice. Taken together, the present chemical library screening strategy in Xenopus tadpoles represents a rapid and highly efficient approach to identify novel pathways involved in (lymph)angiogenesis. In addition, the recovered compounds represent a rich resource for in-depth analysis, and their drug-like features will facilitate further evaluation in preclinical models of inflammation and cancer metastasis.

Molecularly imprinted polymers for the clean-up of a basic drug from environmental and biological samples.

A molecularly imprinted polymer (MIP) was synthesized and evaluated to selectively extract an alpha-blocker, i.e. alfuzosin, from human plasma. The synthesis of the MIP was performed in dichloromethane with methacrylic acid as monomer and the target drug as template. A first series of experiments was carried out in dichloromethane to estimate the potential of the MIP in its specific recognition medium, i.e. dichloromethane, by developing a selective procedure and by measuring the capacity of the sorbent. An optimized procedure was developed for the selective extraction of alfuzosin with a recovery close to 100% in this medium and a specific capacity of 1.3 micromol g(-1) of MIP was measured. A study in aqueous media was also carried out by a comprehensive approach of the retention mechanism in order to build a selective procedure of extraction. The effects of the amount and of the charge of cations were studied and an optimal pH value was defined to limit matrix effects. Then, the alfuzosin MIP was then directly used to selectively extract the target drug from human plasma with an extraction recovery of 60%. Lastly, a soil was extracted by a pressurized solvent and the resulting extract was cleaned up on the MIP, showing the possibility to use this selective sorbent for the sample treatment of various complex matrices.

[Chiral separation and preparation of three new antagonists of alpha 1-adrenoceptors by chiral mobile phase HPLC].

AIM: To establish new methods for the chiral separation and preparation of three new drugs, alfuzosin, terazosin and doxazosin. METHODS: By optimizing factors which affect the chiral separation, modifier of solvent, chiral additive, pH of mobile phase, modifier of organic base and stationary phase, the optimum condition for chiral separation were selected. The preparation of enantiomers was carried out on semi-preparative reverse phase column (7.8 mm x 250 mm C4 5 microns). Acetonitrile-water modified by the addition of carboxymethyl-beta-cyclodextrin (2%-5%, w/v) was applied as chiral mobile phase. RESULTS: The enantiomers of three new drugs were base-line-separated and milligram-scale samples of enantiomer were obtained. CONCLUSION: The newly established method can be used in research and development of the enantiomers of three new drugs.

Continuous free flow electrophoresis for preparative chiral separations of piperoxan using sulfated beta-cyclodextrin.

Continuous free flow electrophoresis was investigated as a tool for the preparative chiral separation of piperoxan using a sulfated cyclodextrin chiral additive. In the absence of chiral additive, the sample stream was deflected cathodically. However, the presence of sulfated cyclodextrin in the run buffer caused anodic deflection and splitting of the sample stream into two streams, each enriched in one enantiomer. Although the sulfated cyclodextrin used was comprised of a mixture of homologues and isomers, this polydispersity did not seem to significantly impact band dispersion. Sample introduction rates ranged from approximately 0.9-7.2 mg h-1. Maximum resolution was 0.53, using an applied voltage of 220 V, buffer composition of 0.075% sulfated cyclodextrin, 7.6 mM citrate (pH 3), 4.5 degrees C.

[Separation of enantiomeric labetalol by reversed-phase high performance liquid chromatography].

A reversed-phase high performance liquid chromatographic method for the separation of labetalol enantiomers was developed. In the method, 2, 3, 4, 6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC) was chosen as the reagent for pre-column chiral derivatization of labetalol to give four diastereomeric thiourea derivatives. These derivatives were efficiently separated on a Nova-Pak C18 column using V(MeOH):V(0.01 mol.L-1(pH 7.00) H2PO4(-)-HPO(4)2- buffer) = 51:49 as the mobile phase and detected by UV detector at a wavelength of 250 nm or fluorescence detector at lambda ex = 340 nm and lambda em = 440 nm. The effects of the pH of mobile phase on the retention and fluorescence absorbance are also discussed.

Capillary electrophoretic and high-performance liquid chromatographic studies of the enantioselective separation of alpha1-adrenoreceptor antagonists.

Capillary electrophoresis (CE) and chiral stationary phase (CSP) HPLC methods were investigated for the determination of enantiomeric purity of alpha1-adrenoreceptor antagonists related to WB 4101. In the CE study, the enantioseparation of the analytes was performed by studying the effect of different types of cyclodextrin in the buffer, namely heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DMCD), hydroxypropyl-beta-cyclodextrin (HPCD) and beta-cyclodextrin (beta-CD). HPCD was found to be the most effective chiral selector in the enantioseparation of all the compounds, with high resolution values. A HPLC method, using immobilised serum protein columns, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), was also investigated. Two benzodioxane racemates were well resolved on a mixed tpe (50% HSA and 50% AGP) column, with enantioselective binding on AGP column.

Chiral recognition in liquid chromatography utilising chargeable cyclodextrins for resolution of doxazosin enantiomers.

The chromatographic resolution of rac-doxazosin using reversed-phase high performance liquid chromatography (HPLC) with the chargeable chiral mobile phase additive, carboxymethyl-beta-cyclodextrin (CM-beta-CD), is described. The effects of different modifiers (acetonitrile, methanol and tetrahydrofuran), pH, temperature, and cyclodextrin concentration were investigated to a) assess the key chromatographic parameters for subsequent chemometric optimisation, and b) explore the enantioselective mechanism. Assuming a 1:1 complex between each doxazosin enantiomer and CM-beta-CD, studies of the relationship between the capacity factors (k') and functions of CM-beta-CD concentration indicate that the mechanisms for retention and chiral selectivity are comparable with those proposed earlier by Sybilska et al. Stability constants (KG) calculated for rac-doxazosin complexed with CM-beta-CD (647 +/- 55 and 594 +/- 45 M-1 for each enantiomer respectively) are significantly larger than those calculated for the barbiturates complexed with beta-CD (ca. 101-108 M-1). Investigations on pH indicate an ionic or ino-pair interaction between the anionic CM-beta-CD and the cationic doxazosin enantiomers. A central composite design was used to optimise the key chromatographic parameters: pH, methanol (v/v) and CM-beta-CD concentration. The Kaiser peak separation index, Pi, was used for the response function. The predicted response for this chiral separation has been compared with that observed experimentally and samples of the four-dimensional response surface have been assessed for their value in showing robustness.

Pharmacological evaluation of N-methyl-actinodaphnine, a new vascular alpha-adrenoceptor antagonist, isolated from Illigera luzonensis.

The pharmacological activity of N-methyl-actinodaphnine, isolated from Illigera luzonensis, was determined by functional and binding experiments with peripheral tissues. In a functional study, N-methyl-actinodaphnine was a simple competitive antagonist of contractions elicited by phenylephrine (pA2 = 7.11) in rat thoracic aorta; it also competitively antagonised the clonidine-induced inhibition of the twitch response of rat vas deferens (pA2 = 5.01). In addition, [3H]inositol monophosphate formation caused by noradrenaline (3 microM) in rat isolated thoracic aorta was concentration dependently inhibited by N-methyl-actinodaphnine (1 and 10 microM); however, it had no effect on cyclic AMP and cyclic GMP contents. Additionally, N-methyl-actinodaphnine had extremely low affinity for thromboxane receptors, prostaglandin receptors, Ca2+ channels, muscarinic receptors, histamine receptors, beta-adrenoceptors, neurokinin and leukotriene receptors in vitro. However, N-methyl-actinodaphnine also possessed 5-hydroxytryptamine (5-HT) receptor blocking activity. Its potency for blocking 5-HT receptors was about 14 times less than that for blocking alpha 1-adrenoceptors. In binding experiments, N-methyl-actinodaphnine displaced biphasically the binding of 0.2 nM [3H]prazosin to cultured A10 cells. The selectivity for alpha 1-adrenoceptor subtypes was also investigated in rat vas deferens and spleens. The contractile response in rat vas deferens to noradrenaline was competitively inhibited by N-methyl-actinodaphnine with a pA2 value of 6.58; N-methyl-actinodaphnine also competitively antagonized the phenylephrine-induced contraction in rat spleen with a pA2 value of 7.38. These results indicate that N-methyl-actinodaphnine is a selective alpha 1-adrenoceptor antagonist. Furthermore, it is more selective for the alpha 1B- than for the alpha 1A-adrenoceptor subtype.

Effect of leaf extract from Clerodendron colebrookianum on blood pressure in rats.

Hypotensive effect of the extract of leaves of C. colebrookianum was investigated. An extremely bitter fraction was isolated, which gave positive test for alkaloids. The fraction produced fall of blood pressure in a dose dependent manner. The fall of blood pressure was blocked by atropine. Hypertensive effects of adrenaline and noradrenaline were slightly antagonised by the alkaloidal extract.

Pharmacological activity of (-)-discretamine, a novel vascular alpha-adrenoceptor and 5-hydroxytryptamine receptor antagonist, isolated from Fissistigma glaucescens.

1. The pharmacological activity of (-)-discretamine, isolated from Fissistigma glaucescens, was determined in rat isolated thoracic aorta, cardiac tissues and ventricular myocytes and guinea-pig isolated trachea. 2. (-)-Discretamine was found to be an alpha 1-adrenoceptor blocking agent in rat thoracic aorta as revealed by its competitive antagonism of noradrenaline (pA2 = 7.20 +/- 0.10)- or phenylephrine (pA2 = 7.60 +/- 0.09)-induced vasoconstriction. It was as potent as phentolamine (pA2 = 7.51 +/- 0.10), but was more potent than yohimbine (pA2 = 6.18 +/- 0.06). Removal of endothelium significantly increased the antagonistic potency of (-)-discretamine on noradrenaline (pA2 = 7.52 +/- 0.09)- or phenylephrine (pA2 = 7.90 +/- 0.09)-induced vasoconstriction. 3. (-)-Discretamine was also an alpha 2-adrenoceptor blocking agent (pA2 = 6.30 +/- 0.15) and a 5-hydroxytryptamine antagonist (pA2 = 6.87 +/- 0.06), both in rat aorta denuded of endothelium. 4. (-)-Discretamine protected alpha-adrenoceptors from alkylation by the irreversible blocking agent, phenoxybenzamine. 5. [3H]-inositol monophosphate formation caused by noradrenaline (3 microM) in rat thoracic aorta was suppressed by (-)-discretamine (10 and 30 microM) and prazosin (3 microM). 6. A high concentration of (-)-discretamine (30 microM) did not affect the contraction induced by the thromboxane receptor agonist U-46619, prostaglandin F2 alpha (PGF2 alpha), angiotensin II, high K+ or endothelin in rat aorta denuded of endothelium. Neither cyclic AMP nor cyclic GMP content of rat thoracic aorta was changed by (-)-discretamine. 7. Contraction of guinea-pig trachea caused by histamine, leukotriene C4 or carbachol was not affected by (-)-discretamine (30 microM). (-)-Discretamine also did not block beta l- or beta2-adrenoceptor-mediated responses induced by isoprenaline in rat right atria and guinea-pig trachea.8. A voltage clamp study in rat ventricular single myocytes revealed that sodium inward current, slow inward Ca2+ current or transient (ItO) and steady state (I800) outward current was not affected by(-)-discretamine (30 microM).9. It is concluded that (-)-discretamine is a selective x-adrenoceptor and 5-HT receptor antagonist in vascular smooth muscle.

Stereoselectivity at alpha-adrenoreceptor subtypes: observations with the enantiomers of WB 4101 separated through their amides of N-tosyl-(S)-proline.

We present a chromatographic method for the separation and determination of the optical purity of the enantiomers of WB 4101 [(+/-)-1], one of the most potent and selective alpha 1-adrenoreceptor antagonists. (+/-)-1 was converted into the amide of N-tosyl-(S)-proline. The two diastereoisomers were separated on silica gel and analysed by HPLC reversed phase. The analytical method described is both accurate and sensitive and allows the optical purity to be determined at very low concentrations and to obtain WB 4101 enantiomers with a purity of more than 99.95%.

Resolution and pharmacological properties of the enantiomers of the potent alpha-adrenoceptor antagonist 1-[2-ethoxy-2-(3'-pyridyl)ethyl]-4-(2'-methoxy-phenyl)piperazine.

Resolution of the optical isomers of the alpha-adrenoceptor antagonist IP-66 (1-[2-ethoxy-2-(3'-pyridyl)ethyl]-4-(2'-methoxy-phenyl)piperazine) and its intermediate IP-30 (1-[2-hydroxy-2-(3'-pyridyl)ethyl]-4- (2'-methoxy-phenyl)piperazine have been carried out. Optical purity was assayed by high-pressure liquid chromatography. The antagonistic potencies of racemic mixtures and stereoisomers toward phenylephrine- and norepinephrine-induced contraction in isolated rat aortic strips have been compared. (+)-IP-66 and (+)-IP-30 were resp. 363 and 170 times more potent than respective (-) isomers in eliciting competitive alpha 1-adrenoceptor blockade. Similarly IP-66 (+) or (+/-) were extremely more effective than the (-) isomer in antagonizing norepinephrine-induced pressor responses in pithed rat.

Hymenin, a novel alpha-adrenoreceptor blocking agent from the Okinawan marine sponge Hymeniacidon sp.

A novel bromine-containing alkaloid, hymenin, has been isolated from the Okinawan marine sponge Hymeniacidon sp. as a potent alpha-adrenoceptor blocking agent and its structure determined to be 1 on the basis of the spectral data.

Identification of an alpha-adrenoceptor binding inhibitor: possible implications in diabetes mellitus.

An endogenous adrenergic antagonist extracted from porcine duodenal mucosa was tested for 3H-yohimbine (5 nM) binding inhibitory activity using whole brain membranes. Duodenal mucosa was scraped into liquid N2 and extracted in 2M acetic acid (HAc) + 1 mg/ml ascorbic acid. The supernatant was fractionated on SP-Sephadex C-25 with a stepwise pH gradient. The peak adrenoceptor binding inhibitory (ABI) activity eluted at pH 4.5 and fractionation of this material on Sephadex G-25 SF indicated a relative molecular mass (Mr) of 2000 to 3000. The ABI did not bind to a Pharmacia Pro RPC 5/5 column which resulted in further purification and indicated that the peptide is hydrophilic. ABI activity was specific for 3H-yohimbine and did not affect 3H-dihydroalprenolol binding to beta receptors. Incubation of pancreatic islets isolated from fasted rats with ABI completely reversed the effect of 2.5 X 10(-6)M norepinephrine (NE) on insulin release in the presence of 300 mg/dl glucose. I.v. injection of ABI in rats also prevented the increased serum glucose response to a glucose bolus caused by NE. Preliminary experiments indicate that ABI blocks the stimulation of platelet aggregation by epinephrine as well. The peptide identified in this study causes specific inhibition of binding to alpha-2 adrenergic receptors and exerts a potent glucose dependent effect on adrenergic inhibition of insulin release. Possible implications in diabetes mellitus are discussed.

The resolution of agonist alpha 2-adrenergic receptor complexes from unoccupied receptors or antagonist-alpha 2 receptor complexes using DEAE chromatography.

Alpha 2-adrenergic receptors linked to inhibition of adenylate cyclase activity in human platelet membranes can be isolated using DEAE chromatography following solubilization into digitonin-containing buffers. This procedure permits resolution of agonist-occupied receptors from antagonist-occupied receptors. Antagonist-occupied receptors co-elute with unoccupied receptors. Adenylate cyclase activity elutes independently of all alpha 2-receptor activities. A greater resolution of agonist-receptor complexes from antagonist-receptor complexes is obtained using DEAE chromatography than reported earlier using approaches that rely entirely on agonist-stabilized increases in apparent receptor size. Consequently, DEAE chromatography may be of considerable value in isolating these agonist-receptor complexes to permit identification of the membrane component(s) which are more stably associated with the receptor subsequent to agonist occupancy and thus might be involved in receptor-cyclase coupling.