Royal jelly increases peripheral circulation by inducing vasorelaxation through nitric oxide production under healthy conditions.

AIMS: Royal jelly (RJ) has a variety of reported biological activities, including vasorelaxation and blood pressure-lowering effects. Although functional foods are positively used for health, the effects of RJ on the cardiovascular system in healthy individuals have not been well studied. Therefore, we investigated the mechanisms underlying the vasorelaxation effects of RJ in healthy control rats to evaluate whether the peripheral circulation was increased. MAIN METHODS: We used fresh RJ to examine the vasorelaxation effects and related mechanisms in Wistar rats using organ bath techniques. Furthermore, we measured changes in tail blood circulation, systolic blood pressure (sBP), and heart rate (HR) after the oral administration of RJ to control rats and nitro-l-arginine methyl ester (l-NAME)-treated rats (0.5 mg/ml dissolved in distilled drinking water for 1 week). Concentrations of acetylcholine (ACh) in the RJ were measured using a commercial kit. KEY FINDINGS: RJ caused vasorelaxation of isolated rat aortas and superior mesenteric arteries, and this effect was inhibited by atropine (10-5 M, 15 min) or L-NAME (10-4 M, 20 min) and endothelium-denuded arterial ring preparations. Oral RJ increased tail blood flow and mass in control rats 1 h after treatment without affecting velocity, sBP, or HR. These effects were not observed in L-NAME-treated rats. RJ contained approximately 1000 µg/g of ACh. SIGNIFICANCE: The present study demonstrated that RJ is composed of muscarinic receptor agonist(s), likely ACh, and induces vasorelaxation through nitric oxide (NO) production from the vascular endothelium of healthy rats, leading to increased tail blood circulation. Thus, fresh RJ may improve peripheral circulation in healthy individuals.

Structural characterization of cevimeline and its trans-impurity by single crystal XRD.

Cevimeline is muscarinic receptor agonist which increases secretion of exocrine glands. Cevimeline base is a liquid (m.p. 20-25 °C) at ambient conditions, therefore its pharmaceutical formulation as a solid hydrochloride hemihydrate has been developed. The synthesis of cevimeline yields its cis- and trans-isomers and only the cis-isomer is recognized as the API and used in the finished formulation. In this study structural and physicochemical investigations of hydrochloride hemihydrates of cis- and trans-cevimelines have been performed. Single crystal X-ray analyses of both cis- and trans-isomers of cevimeline are reported here for the first time. It was found that the cis-isomer, the API, has less dense crystal packing, lower melting point and higher solubility in comparison to the trans-isomer.

Evaluation of mechanism for antihypertensive action of Clerodendrum colebrookianum Walp., used by folklore healers in north-east India.

ETHNOPHARMACOLOGICAL RELEVANCE: The present investigation was aimed to justify the pharmacological basis in traditional use of Clerodendrum colebrookianum as antihypertensive agent in north-east India. MATERIALS AND METHODS: The aqueous extract (AECc), its aqueous, n-butanol (nBFCc), Ethyl-acetate (EtFCc) and Chloroform fractions of C. colebrookianum leaves were evaluated for antihypertensive potential by using fructose-induced hypertension model in rats and in isolated frog heart. The ex-vivo muscarinic action in isolated rat ileum, in-vitro assay for Rho-kinase (ROCK -II), phosphodiesterase-5 (PDE-5) and angiotension converting enzyme (ACE) were also carried out to establish the mechanism of action of samples. The total phenolic and flavonoied contents in test samples were estimated to establish phyto-pharmacological relationship. RESULTS: The 100µg/mL test samples were showed calcium antagonism in rat ileum and at 50µg/mL and 75µg/mL doses exhibited ROCK-II and PDE-5 inhibition respectively where, EtFCc was caused maximum 68.62% (ROCK-II) and 52.28% (PDE-5) inhibition, but none of the sample was exhibit effect in ACE at 100µg/mL. The test samples also showed negative inotropic and chronotropic effect on isolated frog heart and significant (P<0.001) reduction in systolic blood pressure and heart rate in hypertensive rats compared to control. The total phenolic content maximum 80µg gallic acid equivalents in nBFCc and flavonoids content maximum 69.57µg Quercetin equivalent in AECc were estimated. CONCLUSIONS: These observations established the traditional claim and thus C. colebrookianum could be a potent antihypertensive agent for use in future. The antihypertensive effect mediated by cholinergic action and following ROCK - II, PDE-5 inhibition of C. colebrookianum.

Development and validation of a reversed-phase HPLC method for determination of lesatropane and enantiomeric impurity.

Lesatropane is a novel muscarinic receptor agonist and is currently being under preclinical development in China as a single enantiomer drug for the treatment of primary glaucoma. A reversed-phase chiral HPLC method for determination of lesatropane and enantiomeric impurity was developed. Enantiomeric separation of lesatropane from its enantiomer (desatropane) was achieved in normal-phase mode with Chiralpak AD-H and in reversed-phase mode with Chiralpak AS-RH. The conditions using a Chiralpak AS-RH column and mobile phase of K(2) HPO(4) -KH(2) PO(4) (pH 7.0; 0.02 M)-acetonitrile (69:31, v/v) at a flow rate of 0.5 ml/min have been fully validated with satisfactory specificity, linearity, accuracy, and precision. The method was found to be suitable for the simultaneous quantitation of lesatropane and enantiomeric impurity desatropane.

The relationship between the drug concentration profiles in plasma and the drug doses in the colon.

After the dosing of an extended-release (ER) formulation, compounds may exist in solutions at various concentrations in the colon because the drugs are released at various speeds from the ER dosage form. The aim of this study was to investigate the relationship between the drug concentration profiles in plasma and the drug doses in the colon. Several drug solutions of different concentrations were directly administered into the ascending colon of dogs using a lubricated endoscope, and the effects of the drug dose on colonic absorption were estimated. As a result, dose-dependency of colonic absorption varied from compound to compound. Although the relative bioavailability of colonic administration of diclofenac, metformin and cevimeline compared to oral administration was similar regardless of the drug doses in the colon, colonic absorption of diltiazem varied according to the doses. From the results of the co-administration of verapamil and fexofenadine, it was clear that diltiazem underwent extensive hepatic and gastrointestinal first-pass metabolism, resulting in a low area under the curves (AUC) at a low drug dose. During the design of oral ER delivery systems, a colonic absorption study of candidate compounds should be carried out at several solutions of different drug concentrations and assessed carefully.

The Snake Venom Peptide Bj-PRO-7a Is a M1 Muscarinic Acetylcholine Receptor Agonist

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacologicalaction of these peptides resulted in the development of Captopril, one of thebest examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensinconvertingenzyme-independent mechanisms. Here, we show that Bj-PRO-7a (

Case report: acute unintentional carbachol intoxication.

INTRODUCTION: Intoxications with carbachol, a muscarinic cholinergic receptor agonist are rare. We report an interesting case investigating a (near) fatal poisoning. METHODS: The son of an 84-year-old male discovered a newspaper report stating clinical success with plant extracts in Alzheimer's disease. The mode of action was said to be comparable to that of the synthetic compound 'carbamylcholin'; that is, carbachol. He bought 25 g of carbachol as pure substance in a pharmacy, and the father was administered 400 to 500 mg. Carbachol concentrations in serum and urine on day 1 and 2 of hospital admission were analysed by HPLC-mass spectrometry. RESULTS: Minutes after oral administration, the patient developed nausea, sweating and hypotension, and finally collapsed. Bradycardia, cholinergic symptoms and asystole occurred. Initial cardiopulmonary resuscitation and immediate treatment with adrenaline (epinephrine), atropine and furosemide was successful. On hospital admission, blood pressure of the intubated, bradyarrhythmic patient was 100/65 mmHg. Further signs were hyperhidrosis, hypersalivation, bronchorrhoea, and severe miosis; the electrocardiographic finding was atrio-ventricular dissociation. High doses of atropine (up to 50 mg per 24 hours), adrenaline and dopamine were necessary. The patient was extubated 1 week later. However, increased dyspnoea and bronchospasm necessitated reintubation. Respiratory insufficiency was further worsened by Proteus mirabilis infection and severe bronchoconstriction. One week later, the patient was again extubated and 3 days later was transferred to a peripheral ward. On the next day he died, probably as a result of heart failure. Serum samples from the first and second days contained 3.6 and 1.9 mg/l carbachol, respectively. The corresponding urine concentrations amounted to 374 and 554 mg/l. CONCLUSION: This case started with a media report in a popular newspaper, initiated by published, peer-reviewed research on herbals, and involved human failure in a case history, medical examination and clinical treatment. For the first time, an analytical method for the determination of carbachol in plasma and urine has been developed. The analysed carbachol concentration exceeded the supposed serum level resulting from a therapeutic dose by a factor of 130 to 260. Especially in old patients, intensivists should consider intoxications (with cholinergics) as a cause of acute cardiovascular failure.

How much pilocarpine contaminates pilocarpine-induced tick saliva?

Pilocarpine is often applied or injected into ticks to induce salivation, and the resulting saliva used to test for various pharmacological, biochemical and immunological activities. To measure the amount of pilocarpine in pilocarpine-induced tick saliva, an HPLC-MS/MS method, based on capillary strong cation exchange chromatography online with an ion trap mass spectrometer, was used to measure pilocarpine in the pg to ng range. Results indicate large concentrations of pilocarpine in Ixodes scapularis Say and Amblyomma americanum (Linnaeus) (Acari: Ixodidae) saliva, ranging from 3 to 50 mm. Due to the known effects of pilocarpine on smooth muscle and immune cells, appropriate controls are proposed and discussed for proper interpretation of results using this saliva preparation.

A flow injection renewable surface technique for cell-based drug discovery functional assays.

A novel flow injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium-sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-à-vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed.

Quantitative colorimetric and gas chromatographic determination of arecaidine propargyl ester.

Arecaidine propargyl ester (APE) is a potent muscarinic agonist often used in pharmacological studies. To date, no sensitive quantitative analytical method for APE has been published. In this study, two methods for the quantitative determination of APE are compared: a colorimetric assay, based on the formation of the corresponding ferric(III)-hydroxamic acid complex, and a direct gas chromatographic method, using arecoline as the internal standard. The latter method was found to be more precise. The utility of the gas chromatographic assay was further demonstrated in a stability study of the drug in the biological fluid aqueous humor of rabbits.

The effects of a long-term powdered diet on the amounts of two principal neurotransmitters in the major salivary glands and on stimulated salivary secretion in mice.

The amounts of 2 principal neurotransmitters, acetylcholine (ACh) and norepinephrine (NE) in the 3 major salivary glands, and pilocarpine-, isoproterenol- and phenylephrine-induced salivation in male mice fed a powdered diet for 16 weeks were compared with those in mice fed a standard pellet diet (as control). There were no significant differences in the final body weights of mice fed the powdered diet and the control diet. The only salivary gland in the powdered diet fed mice to increase significantly in weight was the sublingual gland. Mice fed the powdered diet had significantly increased ACh concentrations and contents, but had decreased amounts of NE, in the submandibular and sublingual, but not the parotid glands. The salivation stimulated by pilocarpine was markedly decreased in mice fed the powdered diet, whereas the salivation stimulated by phenylephrine or isoproterenol was not. These findings indicate that reduced mastication affects not only the secretory function but also the amounts of these neurotransmitters in the salivary glands of mice.

The utility of salivary amylase as an evaluation of M3 muscarinic agonist activity in Alzheimer's disease.

1. In this double-blind, placebo-controlled phase I study of the safety/tolerance of two doses of xanomeline tartrate (100 mg and 115 mg tid) given to 12 AD patients, the authors measured serum amylase, fractionated into pancreatic and salivary isoenzymes, as a potential marker for M3 activity associated with maximally tolerated dose (MTD). 2. MTD of xanomeline was determined to be 100 mg tid based on intolerable adverse events at 115 mg tid. One patient at the 115 mg tid level presented with moderate hypersalivation and salivary amylase levels 400% of baseline. 3. Overall amylase results were not significant, however a trend in the results for salivary amylase in the 115 mg panel suggests that salivary amylase may be a useful marker for M3 activity.