Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Development and validation of a rapid LC-MS/MS method for simultaneous quantification of arecoline and its two active metabolites in rat plasma and its application to a pharmacokinetic study.

Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N-oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N-oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C18 analytical column. The mobile phase consisted of methanol and 2 mM ammonium acetate buffer solution containing 0.2% (v/v) formic acid (8:92, v/v) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m/z 156.2 → 53.2, m/z 142.2 → 44.2 and m/z 172.2 → 60.2 were selected for arecoline, arecaidine and arecoline N-oxide, respectively. The method was linear over the concentration range of 0.5-100 ng/mL for arecoline, 5-5000 ng/mL for arecaidine and arecoline N-oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N-oxide after oral treatment with 20 mg/kg arecoline to rats.

Liquid chromatography-tandem mass spectrometric assay for determination of unstable arecoline in rat plasma and its application.

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5v/v) at an isocratic flow rate of 300µL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1-1000ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45min in rats after oral treatment of 150mg/kg arecoline.

Pilot study of the pharmacokinetics of betel nut and betel quid biomarkers in saliva, urine, and hair of betel consumers.

Approximately 600 million people worldwide practise the carcinogenic habit of betel nut/quid chewing. Carcinogenic N-nitroso compounds have been identified in saliva or urine of betel chewers and the betel alkaloid arecoline in hair from habitual betel quid chewers. However, the pharmacokinetic parameters of these compounds have been little explored. Assessment of betel use by biomarkers is urgently needed to evaluate the effectiveness of cessation programmes aimed at reducing betel consumption to decrease the burden of cancers in regions of high betel consumption. In the search for biomarkers of betel consumption, we measured by liquid chromatography-mass spectrometry (LC-MS) the appearance and disappearance of betel alkaloids (characteristic for betel nuts), N-nitroso compounds, and chavibetol (characteristic for Piper Betle leaves) in saliva (n=4), hair (n=2), and urine (n=1) of occasional betel nut/quid chewers. The betel alkaloids arecoline, guvacoline, guvacine, and arecaidine were detected in saliva of all four participants and peaked within the first 2 h post-chewing before returning to baseline levels after 8 h. Salivary chavibetol was detected in participants consuming Piper Betle leaves in their quid and peaked ~1 h post-chewing. Urinary arecoline, guvacoline, and arecaidine excretion paralleled saliva almost exactly while chavibetol glucuronide excretion paralleled salivary chavibetol. No betel nut related compounds were detected in the tested hair samples using various extraction methods. From these preliminary results, we conclude that betel exposure can only be followed on a short-term basis (≤8 h post-chewing) using the applied biomarkers from urine and saliva while the feasibility of using hair has yet to be validated. Copyright © 2015 John Wiley & Sons, Ltd.

Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with ß-pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6×75mm, 3.5µm) using methanol: 5mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2-500ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC-MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration.

Comparative permeability of various chemical markers through human vaginal and buccal mucosa as well as porcine buccal and mouth floor mucosa.

A number of drugs undergo extensive first-pass metabolism after oral administration, necessitating large doses for effective therapeutic responses in the body. Buccal administration of drugs is becoming more popular because the drugs diffuse into the systemic circulation directly, circumventing the first-pass metabolism. Lower concentrations thus need to be administered and side effects may be minimized. In this study, one of the classic models for human buccal permeability, i.e. the porcine buccal mucosal model, is compared with the more recent human vaginal model and both these are in turn further compared to porcine mouth floor mucosa. To determine the permeability of the different markers (arecoline, 17beta-estradiol, water and vasopressin), a continuous flow-through perfusion system was used (20 degrees C, 24h). Mean steady state flux values were compared statistically using a t-test at a significance level of 5%. Porcine buccal mucosa showed a consistently lower permeability towards all the markers than the other mucosae tested. Porcine mouth floor mucosa was found to be more permeable than porcine buccal mucosa. From these studies we concluded that human vaginal and porcine mouth floor mucosae were superior models for human buccal mucosa than porcine buccal mucosa, using in vitro permeability studies with various chemical markers.

Comparative in vitro permeability of human vaginal, small intestinal and colonic mucosa.

Our previous experience with a continuous flow-through perfusion system has demonstrated its usefulness for studying diffusion kinetics of drugs across small intestinal mucosa for bioavailability/bioequivalence (BA/BE) studies. During the last decade, delivery of drugs to the colon for systemic absorption as well as for local delivery in certain colonic diseases, has been extensively investigated. For this reason, we sought to assess the in vitro comparative permeability of human vaginal, small intestinal and colonic mucosa using a flow-through perfusion method. It was clear from our studies that human colonic epithelium was statistically significantly (P<0.05) more permeable to water, 17beta-estradiol, arecoline and arecaidine than intestinal mucosa. However, both these mucosae were statistically significantly less permeable to the above four permeants than human vaginal mucosa. As previously shown for small intestinal mucosa, the low in vitro permeability of colonic mucosa to drugs with molecular weight >300Da may necessitate using other epithelial membranes, e.g. vaginal mucosa, as alternative barriers for in vitro BA/BE studies. We also concluded that the flow-through mucosal perfusion system used in our laboratory is therefore also potentially useful for determining the permeability of a therapeutic agent from the colon for registration purposes.

Areca nut extract lowers the permeability of vaginal mucosa to reduced arecoline and arecaidine.

Areca nut chewing has been implicated in the development of oral cancer and oral submucous fibrosis. Arecoline and arecaidine, which are alkaloids present in the areca nut, are thought to play a major role in the development of adverse effects resulting from this chewing habit. Because these alkaloids appear to be associated with the development of the above diseases, we determined their diffusion kinetics through human vaginal mucosa in the presence and absence of a 1% areca nut extract. Seven clinically healthy vaginal mucosa specimens (mean patient age+/-standard deviation, 52+/-13 years; age range, 38-74 years) were obtained during surgery. In vitro flux values of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. The flux values across vaginal mucosa of r-arecoline and r-arecaidine were decreased in the presence of 1% areca nut extract. For r-arecoline, these flux values were significantly lower statistically when compared to those obtained in the absence of areca nut extract. These findings concur with results previously obtained for water, where the astringent action of the tannins present in the areca nut extract was thought to alter the barrier properties of the epithelium, resulting in decreased permeability.

Diffusion of reduced arecoline and arecaidine through human vaginal and buccal mucosa.

Because alkaloids from areca nut, arecoline and arecaidine, have been implicated in the development of oral submucous fibrosis, we determined their diffusion kinetics through human buccal and vaginal mucosa. Four clinically healthy vaginal mucosa specimens (mean patient age +/- standard deviation: 47 +/- 15 years; age range: 31-60 years) and 4 buccal mucosa specimens from 2 male patients and 2 female patients (mean patient age +/- standard deviation: 31 +/- 9 years; age range: 17-53 years) were obtained during surgery. In vitro flux rates of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined by use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. Although statistically significant differences were observed between flux values for both alkaloids and tissues at certain time points, these were not considered to be of biological (clinical) significance. However, the flux rates across both mucosa of r-arecoline were significantly higher statistically than those of rarecaidine. The findings demonstrated the differences in the diffusion kinetics between r-arecoline and r-arecaidine across human buccal and vaginal mucosa, an observation that could be explained in terms of their ionisation characteristics. Additionally, the results obtained further support the hypothesis that human vaginal mucosa can be used as a model for buccal mucosa in studies of permeability to various chemical compounds.

Inhibition of brain choline uptake by isoarecolone and lobeline derivatives: implications for potential vector-mediated brain drug delivery.

Delivery of certain compounds to brain is restricted by the nature of the blood-brain barrier (BBB). Many valuable pharmaceuticals are excluded from the CNS due to hydrophilicity or charge. These limitations have been overcome by numerous methods. One method we use is to take advantage of saturable nutrient transporters located at the barrier. These systems transport hydrophilic and charged nutrients into brain such as choline, a quaternized neurotransmitter precursor. Using knowledge of their substrate specificity, it is possible to deliver agents into brain using these nutrient carriers. In this report, derivatives of lobeline and isoarecolone were evaluated to determine if they may gain access to brain by the blood-brain barrier basic amine transporter using the in situ brain perfusion technique. These compounds do bind the blood-brain barrier basic amine transporter and may enter brain by this transport system.

Clinical pharmacokinetics of arecoline in subjects with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetics and pharmacodynamics of intravenously administered arecoline in subjects with Alzheimer's disease. METHODS: Plasma arecoline concentrations were measured during and after high-dose (i.e., 5 mg intravenously over 30 minutes) and up to 2 weeks of continuous multiple-dose steady-state intravenous infusions of arecoline in 15 subjects with mild to moderate Alzheimer's disease. During multiple-dose infusions, the dose of arecoline was escalated from 0.5 to 40 mg/day. Psychometric tests were administered at baseline and every other dose to determine an "optimal dose" for each subject. This dose then was administered for 1 week using a randomized, placebo-controlled, double blind, crossover design. Plasma drug concentrations were measured by GC-MS. RESULTS: The optimal dose of arecoline varied fourfold across subjects (4 mg/day, n = 6; 16 mg/day, n = 3) with mean plasma half-lives of 0.95 +/- 0.54 and 9.3 +/- 4.5 (SD) minutes. Clearance and volume of distribution were 13.6 +/- 5.8 L/min and 205 +/- 170 (SD) L, respectively. At the dose that optimized memory, the mean plasma level was 0.31 +/- 0.14 (SD) ng/ml, and it predicted the optimal dose in all subjects. CONCLUSIONS: Because optimal dose variation is due to differing plasma kinetics, the plasma arecoline level measured at a single infusion rate can be used to choose the optimal dose for memory enhancement in patients with Alzheimer's disease.

Modification of the duration of action and pharmacodynamics of arecoline by tetraisopropylpyrophosphoramide.

Tetraisopropylpyrophosphoramide (ISO-OMPA) pretreatment in the mouse was examined for its ability to prolong the time course of arecoline (ARE) concentration using brain ARE concentration as an index. Brain ARE levels were also correlated with acetylcholine (ACh) and choline concentrations. Pretreatment with 40 mg kg-1 ISO-OMPA increased ARE brain concentrations from 3 nmoles g-1 to 76 nmoles g-1 15 min after i.p. administration of 25 mg kg-1 ARE. ARE (25 mg kg-1) alone significantly increased brain ACh and choline levels. In time course studies, administration of ARE (15 mg kg-1) produced a peak brain level of 7.9 nmoles g-1 at 3 min. ISO-OMPA pretreatment increased the peak level of ARE to 46.5 nmoles g-1 and the peak time to 7-15 min. The time of maximum brain elevation of ACh and choline produced by ARE alone lagged slightly behind the peak level time observed for ARE. ARE alone produced a maximal increase in ACh and choline levels to 34.1 and 57.1 nmoles g-1, respectively. In the presence of ISO-OMPA, ARE further increased ACh and choline levels to 48.2 and 103 nmoles g-1, respectively. After i.p. administration the highest concentration of ARE was found in the cortex, followed by the subcortex, and cerebellum. A significant elevation of ACh was observed in the cortex.

Intranasal absorption of physostigmine and arecoline.

Physostigmine, an acetyl cholinesterase inhibitor, and arecoline, a muscarinic agonist, have been shown to improve Alzheimer presenile dementia in some patients when administered parenterally. Both of these compounds are ineffective orally due to first-pass metabolism. The nasal route was examined as an alternative route of administration for both drugs. Nasal bioavailabilities and dispositions for both drugs were determined in rats. Physostigmine nasal bioavailability was 100% as compared with iv bioavailability, and that of arecoline was 85% when compared with bioavailability following im administration.

Regional brain metabolic responsivity to the muscarinic cholinergic agonist arecoline is similar in young and aged Fischer-344 rats.

In order to determine whether a functional deficit in brain cholinoception accompanies the reported loss of muscarinic receptors with age, local cerebral glucose utilization (LCGU) was measured in awake 3- and 24-month-old rats after the administration of the muscarinic cholinergic agonist arecoline. Awake, male Fischer-344 rats received isotonic saline or arecoline (0.05-50 mg/kg), i.p., and LCGU was measured in 95 regions with the [14C]2-deoxy-D-glucose technique. Plasma and brain pharmacokinetics of arecoline in rats of both ages were measured in a separate experiment and were found not to differ between the two age groups. Peak cerebral cortex arecoline concentrations, at 3 min after the administration of 5 mg/kg, i.p., were 1558 +/- 588 and 1830 +/- 317 ng/g in 3- and 24-month-old rats, respectively. LCGU effects were dose-dependent, with fewer regions activated by smaller doses. In 24-month-old rats, arecoline in one or more doses elevated LCGU significantly in 94% of the regions examined; no declines in LCGU were found. Increases in whole brain glucose utilization produced by all doses of arecoline, compared to control values, were similar in 3- and 24-month-old rats. After one or more doses, the rise in LCGU in 24-month-old animals, compared to that in 3-month rats, was not significantly different in 75 brain regions, greater in 13 and smaller in 7. These findings demonstrate that the functional responsivity of brain regions to a cholinergic agonist is, for the most part, age-invariant in the rat, implying that the function of muscarinic receptor and postreceptor mechanisms remains intact despite reported age-related losses of muscarinic receptors.