Catecholamine inputs to expiratory laryngeal motoneurons in rats.

Many respiration-related interneurons and motoneurons receive a catecholaminergic input, but the extent and distribution of this input to recurrent laryngeal motoneurons that innervate intrinsic muscles of the larynx are not clear. In the present study, we examined the catecholaminergic input to expiratory laryngeal motoneurons in the caudal nucleus ambiguus by combining intracellular labeling of single identified motoneurons, with immunohistochemistry to reveal tyrosine hydroxylase immunoreactive (catecholaminergic) terminal varicosities. Close appositions were found between the two structures, with 18 ± 5 close appositions per motoneuron (n = 7). Close appositions were more frequently observed on distal rather than proximal dendrites. Axosomatic appositions were not seen. In order to determine the source of this input, microinjections of cholera toxin B subunit (1%, 20 nl) were made into the caudal nucleus ambiguus. Retrogradely labeled neurons, located in the ipsilateral nucleus tractus solitarius and the area postrema, were tyrosine hydroxylase-positive. Our results not only demonstrate details of the extent and distribution of potential catecholamine inputs to the expiratory laryngeal motoneuron, but further indicate that the inputs, at least in part, originate from the dorsomedial medulla, providing a potential anatomical basis for previously reported catecholaminergic effects on the laryngeal adductor reflex.

Effects of allicin on hyperhomocysteinemia-induced experimental vascular endothelial dysfunction.

This study was designed to investigate the effect and mechanism of allicin on hyperhomocysteinemia-induced experimental vascular endothelial dysfunction in rats. Fifty male Wistar rats were randomly divided into five groups: the normal control rats (NC), the high-methionine-diet rats (Met), the high-methionine-diet rats treated with folic acid, vitaminB6 and vitaminB12 (Met+F), or with low-dose allicin (Met+L), or with high-dose allicin (Met+H). After 6 weeks, we collected blood samples of all groups to determine plasma endothelin (ET), serum homocysteine (Hcy), nitric oxide (NO), superoxide dismutase (SOD), malondialdehyde (MDA), and detected the expression of basic fibroblast growth factors (bFGF), transforming growth factor beta (TGF-ß), tumor necrosis factor-alpha (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) in the aorta. The Hcy and the expression of TGF-ß in both the Met+L and Met+H groups were significantly lower than the Met and Met+F groups. The ET, ET/NO ratio and the MDA levels of the Met+L and Met+H groups were significantly lower than the Met group. The SOD and NO levels and the expression of bFGF, TNF-α and ICAM-1 of the Met+L and Met+H groups were significantly higher than the Met group. Our data indicate that allicin inhibits lipid peroxidation induced by hyperhomocysteinemia and regulates the excretion and equilibrium of ET and NO, and suggest that allicin might be useful in the prevention of endothelial dysfunction caused by hyperhomocysteinemia.

Substance P, tyrosine hydroxylase and serotonin terminals in the rat caudal nucleus ambiguus.

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.

Ongoing pain in the MIA model of osteoarthritis.

Osteoarthritis (OA) is a chronic pain condition characterized by pain during joint use as well as pain at rest (i.e., ongoing pain). Although injection of monosodium iodoacetate (MIA) into the intra-articular space of the rodent knee is a well established model of OA pain that is characterized by changes in weight bearing and hypersensitivity to tactile and thermal stimuli, it is not known if this procedure elicits ongoing pain. Further, the time-course and possible underlying mechanisms of these components of pain remain poorly understood. In these studies, we demonstrated the presence of ongoing pain in addition to changes in weight bearing and evoked hypersensitivity. Twenty-eight days following MIA injection, spinal clonidine blocked changes in weight bearing and thermal hypersensitivity and produced place preference indicating that MIA induces ongoing and evoked pain. These findings demonstrate the presence of ongoing pain in this model that is present at a late-time point after MIA allowing for mechanistic investigation.

Determinations of mifepristone and its metabolites and their pharmacokinetics in healthy female Chinese subjects.

The aim of this study is to establish an HPLC method for simultaneous determinations of mifepristone and its metabolites, mono-demethylated mifepristone, di-demethylated mifepristone and C-hydroxylated mifepristone in plasma and to evaluate the pharmacokinetic characteristics of mifepristone tablet. Twenty healthy female Chinese subjects were recruited and a series of blood samples were collected before and after 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 8.0, 12.0, 24.0, 48.0, 72.0 and 96.0 hours administration by a single oral dose of 75 mg mifepristone tablet. Mifepristone and its three metabolites were extracted from plasma using ethyl acetate and determined by high performance liquid chromatography. The main pharmacokinetic parameters of mifepristone and its metabolites, including Cmax, tmax, MRT, t(1/2), V, CL, AUC(0-96 h) and AUC(0-infinity), were calculated by Drug and Statistical Software Version 2.0. The simple, accurate and stable method allows the sensitive determinations of mifepristone and its metabolites in human plasma up to 4 days after oral administration of 75 mg mifepristone tablet and the clinical applications of their pharmacokinetic studies.

Bioequivalence of nifedipine softgel and capsule in healthy Chinese volunteers by liquid chromatography-mass spectrometry.

The study aimed to compare and evaluate the bioequivalence of Calcigard-10 softgel and Adalat 10 capsule in healthy Chinese volunteers in a randomized, two-way cross over study design with a washout period of 7 days. A sensitive and reproducible electro-spray ionization liquid chromatography-mass spectrometry (ESI-LCMS) assay was developed and validated to determine nifedipine in human plasma using nitrendipine as internal standard. Nifedipine and nitrendipine were extracted from plasma using liquid-liquid extraction with methylene chloride as extraction solvent. The separation was performed by a Diamonsil ODS column (150 x 4.6 mm, 5 microm). The mobile phase was consisted of acetonitrile-5 mM ammonium acetate (52:48, v/v), delivered at flow rate of 1 mL/min. The 90% confidence intervals for the ratio values of logarithmic transformed Cmax and AUC were calculated to evaluate the bioequivalence of two preparations. The values of Cmax (92.3-112.7%), AUC0-t (84.5-95.1%) and AUC0-inf (84.4-95.5%) are within the interval criterion of 70-143% for Cmax and 80-125% for AUC. The Calcigard-10 softgel and Adalat 10 capsule are bioequivalent.

Comparison of different pharmacodynamic models for pharmacokinetic-pharmacodynamic (PK-PD) modeling of carvedilol.

The paper is aimed to investigate the pharmacokinetic (PK) and the pharmacodynamic (PD) properties of carvedilol using indirect response and effect-compartment link models, and compare the fitness of PK-PD models. Twenty male healthy Chinese volunteers received a single oral dose of 20 mg of carvedilol. The plasma concentrations of carvedilol were determined by reversed-phase HPLC method with fluorescence detection, and the pharmacokinetic parameters were calculated by DAS2.0. The mean arterial blood pressure was measured and the pharmacodynamics of carvedilol was characterized by tail-cuff manometry. The main pharmacokinetic parameters of carvedilol were as follows, t1/2 (4.56 +/- 2.56) h, Cmax (46.29 +/- 21.07) ng x mL(-1), AUC(0-infinity) (173.76 +/- 87.36) ng x mL(-1) x h. The estimated Kin was (0.41 +/- 0.31)% h(-1), Kout was (0.40 +/- 0.26) h(-1), the IC50 value was (24.40 +/- 21.10) ng x mL(-1) and the area under the effect curve (AUE) was (3.82 +/- 1.46)% h for the indirect response PD model. The Ke0 was (0.35 +/- 0.27) h(-1), the EC50 was (24.30 +/- 24.30) ng x mL(-1), and the AUE was (5.65 +/- 2.54)% h for the effect-compartment model. The HPLC method can be used for the pharmacokinetic study of carvedilol. The proposed effect-compartment link model provided more appropriate and better-fitting PK/PD characteristics than the indirect response model in Chinese healthy volunteers according to Akaike's information criterion values.

Determination of 2,3-dimercaptosuccinic acid in mice blood and tissues by HPLC with fluorescence detection.

2,3-Dimercaptosuccinic acid (DMSA) is an orally effective chelating agent for the treatment of heavy metal poisoning. The increasing therapeutic use of DMSA has stimulated the need for sensitive and selective methods for its determination in biological samples, as well as study on pharmacokinetics and tissue distribution. According to the previously reported method, an improved method was established for the determination of DMSA in mice blood and tissues, in which oxidized DMSA was reduced by the disulfide-reducing agent, dithiothreitol (DTT), and DMSA was converted to a highly fluorescent and stable derivative by reaction with monobromobimane (mBBr) in alkaline solution. Acetonitrile was used for deproteinization and dichloromethane was used for condensation and purification, which significantly shortened the amount of time used to process the sample. Meanwhile isocratical elution was performed and excellent separation of the DMSA derivative was obtained, this enabled a run finish within 20 min. The limits of quantitation were 0.025 microg/ml in brain and 0.1 microg/ml in blood, lung, heart, intestine, liver, spleen and kidney, respectively. The calibration curves were linear in all samples (r(2)>0.992) with a range of 0.025-1.6 microg/ml for brain homogenate and 0.1-6.4 microg/ml for blood and homogenates of lung, heart, intestine, liver, spleen and kidney, respectively. Therefore, the method is simple, rapid and sensitive, and it could be applicable to the studies in an animal model to evaluate the distribution of DMSA in blood and tissues.

Development and validation of a sensitive LC-MS method for the determination of promethazine hydrochloride in human plasma and urine.

We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for determination of Promethazine hydrochloride in human plasma (0.5 ml) or urine (0.1 ml). The lower limit of quantification in human plasma and urine was 1.00 ng/ml. The inter- and intra-day precisions (CV %) in both plasma and urine were lower than 10%, the mean method accuracies and recoveries from spiked plasma samples at three concentrations were more than 97%. The developed method was successfully applied to determine Promethazine hydrochloride in human plasma and urine, and proved suitable to clinical pharmacokinetic study.

Development and validation of a sensitive LC-MS method for the determination of tramadol in human plasma and urine.

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS) method was developed and validated for the determination of Tramadol in human plasma and urine. The analyte was separated on a Diamonsil C18 column with ammonium acetate (5 mmol x L(-1))-methanol (50:50,v:v) adjusted PH by caustic soda at a flow rate of 0.8 ml min(-1), and analyzed by mass spectrometry is in positive ion mode. The ion mass spectrum of m/z were 264.1 for Tramadol and 248.0 for Tinidazole (I.S.), respectively. The weighted (1/x2) calibration curve was linear over plasma concentration range 1.00-400.00 ng/ml and urine concentration range 0.01-16.00 microg/ml, with a correlation coefficient (r) of 0.9995 and 0.9997, respectively. The lower limit of quantification in human plasma was 1.00 ng/ml. The inter-and intra-day precisions (CV%) in both plasma and urine were lower than 10%, the mean method accuracies and recoveries from spiked plasma samples at three concentrations ranged from 98.2 to 100.1% and 61.6 to 62.9%, respectively. The developed method was successfully applied to determine Tramadol in human plasma and urine, and provided suitable profiles for clinical pharmacokinetic study of Tramadol.

Pharmacokinetics, safety and tolerance of single- and multiple-dose of a novel compound tramadol hydrochloride injection (35 mg tramadol hydrochloride, 45 mgl promethazine hydrochloride) in Chinese healthy subjects.

The pharmacokinetics of Tramadol and Promethazine after a single dose of 40, 80 and 120 mg intramuscular injecting CTHI was evaluated in healthy volunteers. Physical exam, vital signs, clinical laboratory tests and electrocardiogram measurements were monitored to assess the safety and tolerance of the drug. The plasma levels of Tramadol and Promethazine in serial samples were measured by a validated HPLC-MS method. No subject showed any drug-related clinically significant changes on physical examination, vital signs or laboratory tests.

Construction of expression system of rabbit aldehyde oxidase cDNA for the clarification of species differences.

A remarkably large species difference in cinchonidine oxidation activity catalyzed by aldehyde oxidase (AO) has been known, in particular between rabbit and monkey. As the first step in clarifying the phenomenon from the view point of structures of the active site, we attempted to construct an expression system of rabbit AO cDNA. The nucleotide sequences of cloned full-length rabbit AO cDNA were determined and confirmed to agree completely with those of genome DNA. The expression system in Escherichia coli was constructed in reference to the previously established method for monkey AO. Both expressed rabbit and monkey AO proteins correctly reproduced the remarkable species differences observed in their liver cytosols towards cinchonidine and methotrexate. Namely, the expressed rabbit AO protein showed extremely high activities than did that of monkey AO. A difference in the structure of the active site might be responsible for the substrate-dependent species difference towards the relatively bulky molecules of cinchonidine and methotrexate. The use of molecular biology techniques will be very useful to verify the hypothesis.

[Advances in the study of regulation of novel organic cation transporter-2].

Novel organic cation transporter-2 (OCTN2), a member of the organic cation transporter family, may transport carnitine and multiple organic cationic drugs. Thus OCTN2 possesses substantial roles in physiology and pharmacology. A number of researches have proven that many factors can regulate the expression and/or function of OCTN2 via different pathways, and then may affect the homeostasis and disposition of drugs. This paper reviews recent progresses in this field.

Determination of bergenin in human plasma after oral administration by HPLC-MS/MS method and its pharmacokinetic study.

A highly sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and applied to the determination of bergenin concentration in human plasma. Bergenin and the internal standard (IS) thiamphenicol in plasma were extracted with ethyl acetate, separated on a C(18 )reversed-phase column, eluted with mobile phase of acetonitrile-water, ionized by negative ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor --> product ions of m/z 327.1 --> 192 for bergenin and 354 --> 185.1 for the IS, respectively. The linear range of the calibration curve for bergenin was 0.25-60 ng mL(-1), with the lowest limit of quantification of 0.25 ng mL(-1), and the intra/inter-day relative standard deviation (RSD) was less than 10%. The method is suitable for the determination of low bergenin concentration in human plasma after therapeutic oral doses, and has been first and successfully used for its pharmacokinetic studies in healthy Chinese volunteers.

[HPLC-MS/MS method for determination of sodium cromoglycate concentration in human plasma and its pharmacokinetics].

The study established an HPLC-MS/MS method for determining the concentrations of sodium cromoglycate in human plasma and evaluated the pharmacokinetics of nasal drops and nasal spray. A C18 column was used to separate sodium cromoglycate in plasma with a mobile phase of a mixture of ammonium-methanol (involves 50% acetonitrile) (15:85) at a flow rate of 0.4 mL x min(-1). Electronic spray ionization (ESI) and multiple-reaction monitoring (MRM) were used for the determination of sodium cromoglycate in human plasma. The linear range of the standard curve of sodium cromoglycate was from 0.3 to 20 ng x mL(-1), and the minimum concentration of detection was 0.3 ng x mL(-1). The extraction recovery was more than 94.1%, intra-day and inter-day RSD were less than 14.3%. After a single dose of sodium cromoglycate, the main pharmacokinetic parameters of nasal spray and nasal drops were as follows, T(1/2)(1.82 +/- 0.54) h, (1.59 +/- 0.52) h; Tmax (0.47 +/- 0.12) h, (0.44 +/- 0.15) h; Cmax, (9.79 +/- 4.66) ng x mL(-1), (10.88 +/- 4.05) ng x mL(-1); AUC(0-5 h)(11.52 +/- 3.46) ng x mL(-1) x h x h, (12.63 +/- 4.23) ng x mL(-1) x h, Fr(93.6 +/- 13.8)%. The method is sensitive, rapid and accurate. It is suitable for therapeutic drug monitoring and human pharmacokinetic study of sodium cromoglycate.

Determination of betamethasone in human plasma by liquid chromatography with tandem mass.

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of betamethasone in human plasma. The analyte was isocratically eluted on a Venusil XBP C8 column (200 mm x 3.9 mm ID, 5 microm) with methanol-water mol x L(-1) ammonium formate) (80:20) at a flow rate of 0.4 mL x min(-1), and detected (containing 5 mmol x L(-1) ammonium formate) (80:20) at a flow rate of 0.4 mL x min(-1), and detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 393.3-->355.2 for betamethasone and m/z 361.3-->343.2 for prednisolone (IS). Betamethasone was extracted from 0.5 mL human plasma with ethyl acetate. The average recovery is 88.24% and the low limit of quantitation (LLOQ) was 0.5 ng x mL(-1). The 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.5-80.0 ng x mL(-1). The method was successfully applied to the pharmacokinetic study of compound betamethason injection in healthy Chinese volunteers.

Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass.

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers.

Determination and pharmacokinetic study of norcantharidin in human serum by HPLC-MS/MS method.

A sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry method was developed and applied to the determination of norcantharidin concentration in human serum. Norcantharidin (NCTD) and cyclophosphamide (IS) in serum were extracted with acetone, separated on a C18 reversed-phase column, gradiently eluted with a mobile phase of acetonitrile-water containing 2 mm ammonium acetate and 0.1% formic acid (pH 3), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor-->product ions of m/z 169.3-->123.1 for NCTD and 261.2-->140.2 for IS, respectively. The linear range of the calibration curve for NCTD was 2.5-50 ng/mL, with a lowest limit of quantification of 2.5 ng/mL, and the intra/inter-day RSD was less than 10%. The method was suitable for determination of low NCTD concentration in human serum after therapeutic oral doses, and has been successfully used for pharmacokinetic studies in healthy Chinese volunteers.

[Development and evaluation of terpene penetration enhancers].

Major advantages of transdermal drug delivery system (TDDS) include avoiding of drug degradation in hepatic and gastrointestinal, predetermining release rate and blood drug level, reducing administration frequency and adverse reactions, and increasing patient compliance. But its application was limited by stratum corneum barrier and low skin permeability of drugs. Terpene penetration enhancers are low toxic and can improve the skin permeability and enhance the permeating veloc both of hydrophilic and lipophilic drugs. Terpenes can also significantly enhance the cumulative release amount of drug at its low concentration comparing with other synthetic penetration enhancers, and play an important role in the TDDS. This review presented the source, classification, mechanism and applications of terpenes, combined use with other enhancers and methods. Optimization, evaluation and prospective applications of terpene penetration enhancers were also discussed.

[Determination of curcumol in plasma by HPLC-MS/MS method and its pharmacokinetics in Beagle dogs].

To establish a high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry quantitative detection method for the determination of curcumol, the main ingredient of zedoary turmeric oil fat emulsion, and investigate its pharmacokinetics in Beagle dogs, nine healthy Beagle dogs were divided into three groups, and blood samples were collected at scheduled time points after intravenous injection of 7.5, 10 and 12.5 mg x kg(-1) zedoary turmeric oil fat emulsion. The concentrations of curcumol were determined and pharmacokinetics was calculated. A good linearity was obtained from 0.25 to 100 ng x mL(-1) in plasma. The relative recoveries were from 91.33% to 103.17%, and the absolute recoveries were from 31.61% to 37.20%. The intra-day and inter-day variances (RSD) were < 15%. The main pharmacokinetic parameters of curcumol after intravenous injection of 7.5, 10 and 12.5 mg x kg(-1) zedoary turmeric oil fat emulsion were as follows, T1/2 : (2.0 +/- 0.4), (1.7 +/- 0.2) and (2.3 +/- 0.8) h, AUC(0-infinity): (15.1 +/- 2.7), (18.3 +/- 2.0) and (29.5 +/- 4.0) ng x mL(-1) x h; MRT: (0.9 +/- 0.1), (0.8 +/- 0.2) and (0.8 +/- 0.1) h, CL: (21.9 +/- 4.0), (24.9 +/- 6.0) and (18.4 +/- 1.2) L x h(-1) x kg; Vd : (65.4 +/- 26.5), (62.0 +/- 13.4) and (61.2 +/- 19.8) L x kg(-1), respectively. The developed method was rapid, highly sensitive and specific and could be used in curcumol pharmacokinetic studies in vivo. A three-compartment model was best fit to the plasma concentration--time curves obtained in Beagle dogs and the plasma AUC was increased proportionally with doses.