Development of a validated LC method for enantiomeric separation and determination of adrafinil and its related substances on a Chiralcel OJ-H column connected to PDA and polarimetric detectors in series.

A rapid and reliable high-performance liquid chromatographic method for resolution of enantiomers of adrafinil [(±)-ADL], a novel vigilance promoting agent, and its synthetic intermediates was developed. The separation was carried out on a Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase. The detection was carried out at 225 nm using a photodiode array (PDA) detector. The optical rotation and order of elution of enantiomers were assigned. The method is suitable not only for process development of ADL but also for quality assurance of bulk drugs and pharmaceuticals.

(-)-Dibromophakellin: an alpha2B adrenoceptor agonist isolated from the Australian marine sponge, Acanthella costata.

Bioassay-guided fractionation of the organic extract from the marine sponge Acanthella costata resulted in the isolation of the known natural product, (-)-dibromophakellin (1). Using a fluorescence imaging plate reader (FLIPR) based assay, compound 1 was identified as displaying agonist activity against the alpha(2B) adrenoceptor, with an EC(50) of 4.2muM. Debromination and Suzuki-Miyaura coupling reactions were undertaken in order to provide structure activity data about the pyrrole ring of this marine metabolite. These synthetic studies generated the known natural product analogues, (-)-phakellin (2), and (-)-monobromophakellin (3), along with the new synthetic derivatives (-)-4-bromo-5-phenylphakellin (5) and (-)-4,5-diphenylphakellin (6). Substitution of the C-5 Br of 1 with H (2 and 3) or phenyl (5 and 6) resulted in loss of activity indicating that Br at C-5 is required for agonist activity.

Screening for in vivo (anti)estrogenic activity of ephedrine and p-synephrine and their natural sources Ephedra sinica Stapf. (Ephedraceae) and Citrus aurantium L. (Rutaceae) in rats.

Formulations containing Ephedra sinica Stapf. (Ephedraceae) and Citrus aurantium L. (Rutaceae) are consumed worldwide for body weight control. Considering the related adverse effects and the risk potential, the aim of this study is to evaluate the effects of the thermogenic compounds ephedrine, p-sinephrine, E. sinica and C. aurantium in the female reproductive system through the uterotrophic assay in immature female rats. The animals (n = 6-7) received E. sinica 85.5 and 855.0 mg/kg/day, C. aurantium 25.0 and 50.0 mg/kg/day, ephedrine 5.0 mg/kg/day and p-synephrine 50.0 mg/kg/day for three consecutive days by oral gavage. For detection of antiestrogenicity, tamoxifen 20.0 mg/kg/day, E. sinica 855.0 mg/kg/day, C. aurantium 50.0 mg/kg/day, ephedrine 5.0 mg/kg/day and p-synephrine 50.0 mg/kg/day were administered to estrogen-treated females. Macroscopical alterations were evaluated in liver, kidneys, adrenals and uterus. All analyzed substances showed an antiestrogenic potential, but only ephedrine at 0.5 mg/kg/day presented a significative antiestrogenic effect (P < 0.01). Adrenals relative mass were reduced (P < 0.01) in all tested compounds when compared to the control, which seems to be related to the alfa-1-adrenoceptor agonist activity, which promote a vasoconstriction and reduction of the liquid in the organ. The endocrine system is highly complex and there are a number of ways in which a chemical may interfere with it, other in vivo and in vitro assays are being necessary to support this mechanism of action.

Study of retention parameters obtained in RP-TLC system and their application on QSAR/QSPR of some alpha adrenergic and imidazoline receptor ligands.

The retention constant (R(0)(m)) is determined for 11 selected adrenergic and imidazoline receptor ligands by reverse-phase-thin layer chromatography. It is established that the retention behavior of investigated compounds mostly depends on geometrical, electrostatic, and hydrogen bonding properties. Good correlations among hydrophobic parameters R(0)(m) versus log P for all eleven tested compounds are obtained. The satisfactory correlations are found between R(0)(m) versus apparent partition coefficient octanol-buffer pH 7.4 (log P') or apparent partition coefficient in four liposome systems (log K'(M)) and hypotensive activity (pC(25)) for five imidazolines. The results confirm the suitability of this parameter in quantitative structure-property and structure-activity relationships studies of these drugs.

Lipolysis induced by segment wall extract from Satsuma mandarin orange (Citrus unshu Mark).

The lipolysis induced by Satsuma mandarin orange (Citrus unshu Mark) was investigated using rat fat cells. Peel or segment wall extract from Satsuma mandarin orange induced the lipolysis in a concentration-dependent manner, whereas juice sac extract did not induce the lipolysis. High concentration of synephrine, which is an adrenergic amine, was detected in the peel or segment wall extract, whereas it was not detected in the juice sac extract. The segment wall extracts from Iyokan and orange had high lipolytic activity, whereas the extracts from grapefruit and lemon did not have lipolytic activity. The beta-antagonist inhibited the lipolysis elicited by the segment wall extract from Satsuma mandarin orange, whereas alpha-antagonist did not inhibit the lipolysis induced by the segment wall. The lipolysis induced by the segment wall was considerably higher in the visceral fat cells when compared to the subcutaneous fat cells. These results suggest that the segment wall, an edible fraction, from Satsuma mandarin orange might be useful as a functional food, especially as a fat-reducing material.

Determination of midodrine in human plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive fluorometric high-performance liquid chromatographic method was developed for the determination of midodrine in human plasma. After liquid-liquid extraction from plasma, the drug and 2-phenylglycinol (internal standard) were convened into the corresponding fluorescent derivatives by reaction with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives were separated within 30 min on a reversed-phase column using isocratic elution with acetonitrile-methanol-water (10:30:60, v/v) and were detected spectrofluorometrically at 485 nm with excitation at 400 nm. The detection limit for midodrine was 0.3 pmol (76 pg) per mL plasma at a signal-to-noise ratio of 3.

Isolation from beer and structural determination of a potent stimulant of gastrin release.

Beer was subjected to five successive chromatographic procedures to isolate the gastrin release-inducing activity, guided by bioassay of the fractions in anaesthetized Donryu rats. The procedures were: (1) hydrophobic interaction chromatography (aqueous effluent with an HP20 column); (2) weak cation-exchange chromatography (1 M acetic acid eluate with a CM Sephadex C-25 column); (3) gel filtration (methanol eluate with a Sephadex LH-20 column); (4) same as (2); (5) high-performance liquid chromatography (YMC-Pack ODS-AM with 7% acetonitrile-0.01 M HCl). The active component finally isolated had a specific activity approximately 10000 times higher than that of beer. It was identified by means of mass, 1H- and 13C-nuclear magnetic resonance spectral analyses as N-methyltyramine (NMT). The dose of NMT giving maximal gastrin-releasing activity was 25 microg/kg, and the 50% effective dose was approximately 10 microg/kg on oral administration to rats. NMT was isolated and identified as a gastrin release inducer in beer. Its concentration in beer is sufficient to account for most of the activity of beer.

Adrenergic influence of uterine muscle contractions stimulated by a glycoside from the root of Dalbergia saxatilis.

The mechanism of uterine muscle contraction stimulated by a triterpenoid glycoside (dalsaxin) isolated from the root of D. saxatilis was investigated by in vitro methods in the rat. Dalsaxin caused a dose-related increase in uterine muscle contraction. The contraction was single and transient and was abolished by moderate doses of isoprenaline (1.80 nmol-0.40 mumol) and salbutamol (0.13-25 mumol). Adrenaline (9.10 nmol) also caused a reversible decrease (92.6%; P < 0.01) in myometrial contraction stimulated by this glycoside (0.24 mg/ml). Uterine muscle responses to dalsaxin (0.24 mg/ml) were enhanced by the beta-adrenergic receptor antagonist, propranolol, in a dose related manner. Atipamezole (1.50 ng/ml) but not prazosin (7.72 nmol-15.60 nmol) substantially reduced (80%; P < 0.01) myometrial contractions induced by this uterine spasmogen. The results suggest that dalsaxin enhances uterine muscle contraction by stimulating post junctional alpha 2-adrenergic receptors, presumably by inhibiting plasma membrane adenylate cyclase system and its associated increase in intracellular cAMP content.

Preparation of mixed ligand immobilized artificial membranes for predicting drug binding to membranes.

Mixed ligand immobilized artificial membranes (IAMs) are surfaces that contain at least two immobilized membrane phospholipids which are designated as either the primary phospholipid or the secondary phospholipid. The primary immobilized phospholipid refers to the immobilized phospholipid that has the highest surface density. For this work, the primary immobilized phospholipid was a single-chain ether phosphatidylcholine (PC) analog. Four mixed-ligand IAMs were prepared by use of immobilized PC as the primary immobilized phospholipid. The secondary immobilized phospholipid ligand was either phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. All of these secondary phospholipids are bonded at approximately 6-10 mol % relative to the molar amount of immobilized PC. Each secondary phospholipid contains functional groups in the polar head group region that require protecting groups during the immobilization process. The four-step synthetic strategy to prepare mixed-ligand IAMs involves (i) immobilization of the PC analog at high density to silica propylamine (SPA), (ii) immobilization of the second phospholipid (PL) analog at low density, (iii) end capping residual amines with a long-chain anhydride followed by end capping with a short-chain anhydride, and (iv) deprotection of the polar head group protecting groups. The surface density of the mixed PLs bonded to the silica support was approximately 130 mumol of PLs/g of SPA. High-performance liquid chromatography using these mixed lipid IAMs can be exploited to rapidly predict the membrane binding properties of drugs.

[The separation of some racemic beta-blockers and alpha-sympathomimetics with HPLC following derivatization]. / Zur Trennung einiger racemischer beta-blocker und alpha-Sympathikomimetika durch HPLC nach Derivatisierung.

Five beta-adrenergic blocking agents and two alpha-sympathicomimetics were separated after derivatization with (R)-(-)-1-(Naphth-1-yl)-ethylisocyanate (NEIC). For two of these analytes the separation was successful also after reaction with (R)-(+)-1-phenylethylisocyanate (PEIC) by using HPLC with RP-18 phases and a water-methanolic eluent by UV-detection on 254 nm. After reaction with (1S)-(+)-campher-10-sulfonylchloride (CSC) an acceptable separation of norphenylephedrine also was possible. Analytes which can be derivated with NEIC and PEIC give no reaction with CSC (and vice versa).

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.