Method for individualized evaluation of antiemetic effect induced by 5-HT3 receptor antagonist.

5-HT3 receptor antagonists are widely used for prevention of chemotherapy-induced nausea and vomiting, though their antiemetic effects vary among patients. We investigated a method for evaluation of antiemetic effects in individual patients. We used the 5-HT3 receptor occupancy of serotonin for our evaluation, which was estimated based on the plasma concentration of granisetron and concentration of serotonin near the 5-HT3 receptor in the small intestine, obtained by measuring the urinary concentrations of granisetron and 5-hydroxyindoleacetic acid (5-HIAA)/creatinine (Cre). The mean cumulative percent for urinary excretion of granisetron at 24 h after administration and coefficient of variation were 16.19 ± 6.30% and 38.91%, respectively. The time course of urinary concentration of 5-HIAA/Cre also varied among the patients. The value for 5-HT3 receptor occupancy of serotonin without granisetron was higher than that prior to administration (blank), thus most treated patients had the possibility of induced emesis. In contrast, that with granisetron was lower than the blank value, indicating that those treated patients would not develop emesis. Furthermore, the estimated 5-HT3 receptor occupancy of serotonin in the small intestine and actual individual patient condition corresponded well, showing the validity of our method. Our results suggest that it is possible to evaluate individual antiemetic effects by estimating the 5-HT3 receptor occupancy of serotonin in the small intestine based on plasma concentrations of granisetron and serotonin near the 5-HT3 receptor in the small intestine using noninvasive urine samples. This method of individual evaluation is considered to be useful and effective.

Identification of novel metoclopramide metabolites in humans: in vitro and in vivo studies.

Metoclopramide (MCP) is frequently used to treat gastroparesis. Previous studies have documented MCP metabolism, but systematic structural identification of metabolites has not been performed. The aim of this study was to better understand MCP metabolism in humans. For examination of in vivo metabolism, a single oral 20-mg MCP dose was administered to eight healthy male volunteers, followed by complete urine collection over 24 h. In vitro incubations were performed in human liver microsomes (HLM) to characterize metabolism via cytochromes P450 and UDP-glucuronosyltransferases and in human liver cytosol for metabolism via sulfotransferases. Urine and subcellular incubations were analyzed for MCP metabolites on a mass spectrometer with accurate mass measurement capability. Five MCP metabolites were detected in vivo, and five additional metabolites were detected in vitro. The five metabolites of MCP identified both in vitro and in vivo were an N-O-glucuronide (M1), an N-sulfate (M2), a des-ethyl metabolite (M3), a hydroxylated metabolite (M4), and an oxidative deaminated metabolite (M5). To our knowledge, metabolites M1 and M4 have not been reported previously. M2 urinary levels varied 22-fold and M3 levels varied 16-fold among eight subjects. In vitro studies in HLM revealed the following additional metabolites: two ether glucuronides (M6 and M8), possibly on the phenyl ring after oxidation, an N-glucuronide (M7), a carbamic acid (M9), and a nitro metabolite (M10). Metabolites M6 to M10 have not been reported previously. In conclusion, this study describes the identification of MCP metabolites in vivo and in vitro in humans.

Disposition and metabolism of radiolabeled casopitant in humans.

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S)-(GW679769)] is a novel neurokinin-1 receptor antagonist being developed for the prevention of chemotherapy-induced and postoperative nausea and vomiting. The disposition of [(14)C]casopitant was determined in a single-sequence study in six healthy male subjects after single-dose 90-mg i.v. and 150-mg oral administration. Blood, urine, and feces were collected at frequent intervals after dosing. Plasma, urine, and fecal samples were analyzed by high-performance liquid chromatography/mass spectrometry coupled with off-line radiodetection for metabolite profiling. Moreover, urine was also analyzed with (1)H-NMR to further characterize metabolites. Plasma pharmacokinetic parameters for casopitant, a major metabolite (M13, coded as GSK525060), and total radioactivity were determined. Absorption of radioactivity after oral administration appeared to be nearly complete; elimination was principally via the feces both after oral and intravenous administration. Urinary elimination accounted for only <8% of total radioactivity. The main circulating metabolites were a hydroxylated derivative, M13 (coded as GSK525060), and, after oral administration, a deacetylated and oxidized metabolite, M12 (coded as GSK631832). In addition, many other metabolites were identified in plasma and excreta: the principal route of metabolism included multiple oxidations, loss of the N-acetyl group, modifications or loss of the piperazine group, and cleavage of the molecule. Casopitant was extensively metabolized, and only negligible amounts were excreted as unchanged compound. Some phase II metabolites were also observed, particularly in urine.

Square wave anodic stripping voltammetric determination of metoclopramide in tablet and urine at carbon paste electrode.

A simple, reliable and selective square wave anodic stripping (SWAS) voltammetric method at carbon paste electrode (CPE) of metoclopramide hydrochloride (MCP) in pharmaceutical dosage forms (tablet) and in biological fluids (spiked and real urine samples) has been developed and evaluated. Different parameters such as medium, supporting electrolyte, pH, accumulation potential, scan rate, accumulation time and ionic strength, were tested to optimize the conditions for the determination of MCP. The adsorbed form is oxidized irreversibly under optimal conditions, viz., 0.4M HCl-sodium acetate buffer (pH approximately 6.2), 0.2M KCl, a linear concentration ranges from 0.067 to 0.336, 0.067 to 0.269 and 0.067 to 0.269 ng/mL of MCP, at accumulation times 60, 120 and 180 s, respectively, can be determined successfully. The interferences of some common excipients and some metal ions were studied. The standard addition method was used to determine the MCP in pure solutions, tablets and in biological fluids with satisfactory results. The data obtained are compared with the standard official method.

Dose escalation pharmacokinetics of intranasal scopolamine gel formulation.

Astronauts experience Space Motion Sickness requiring treatment with an anti-motion sickness medication, scopolamine during space missions. Bioavailability after oral administration of scopolamine is low and variable, and absorption form transdermal patch is slow and prolonged. Intranasal administration achieves faster absorption and higher bioavailability of drugs that are subject to extrahepatic, first pass metabolism after oral dosing. We examined pharmacokinetics of 0.1, 0.2, and 0.4 mg doses of the Investigational New Drug formulation of intranasal scopolamine gel (INSCOP) in 12 healthy subjects using a randomized, double-blind cross-over study design. Subjects received one squirt of 0.1 g of gel containing either 0.1 mg or 0.2 mg/0.1 mL scopolamine or placebo in each nostril. Serial blood samples and total urine voids were collected after dosing and drug concentrations were determined using a modified LC-MS-MS method. Results indicate dose-linear pharmacokinetics of scopolamine with linear increases in Cmax and AUC within the dose range tested. Plasma drug concentrations were significantly lower in females than in males after administration of 0.4 dose. All three doses were well tolerated with no unexpected or serious adverse side effects reported. These results suggest that intranasal scopolamine gel formulation (INSCOP) offers a fast, reliable, and safe alternative for the treatment of motion sickness.

Application of packed capillary liquid chromatography/mass spectrometry with electrospray ionization to the study of the human biotransformation of the anti-emetic drug dolasetron.

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Pharmacokinetics of alizapride in children receiving chemotherapy for solid tumour.

The purpose of the present study was to determine the pharmacokinetics of alizapride to optimize its use in children aged 1 month to 15 years old who were receiving chemotherapy. Seventeen children were given a single 4 mg/kg alizapride infusion prior to the administration of cytostatic drugs. Blood and urine samples were collected within 10 h after onset of the infusion. Kinetic parameters were calculated and showed a decrease in plasma clearance expressed per unit of body weight with age. The current data suggest that dosage expressed per unit of body weight should be higher in children than in adults and higher in infants than in children.

N-dephenylated and N-phenyl urinary metabolites of mociprazine (CERM 3517) in beagle dogs after oral administration. A mass spectrometric determination.

CERM 3517 (mociprazine), a new anti-emetic compound, was administered orally to six beagle dogs at 10 mg/kg b.i.d. for four days. Unconjugated urinary metabolites were identified by GC-MS analysis against synthesized reference compounds, after solvent extraction, purification by TLC and concentration. Twenty one metabolites were identified indicating the following biotransformations: N-dephenylation followed by reactions on the exposed secondary amine such as methylation acetylation; and parahydroxylation on the phenyl ring, and monohyrdoxylation on the cyclohexyl ring in different positions. The parahydroxylation on the phenyl ring was confirmed by NMR analysis. Some reactions on the secondary amine were unexpected, such as N-formylation. N-dephenylation and N-formylation were confirmed not to be artifacts. The role of the para-hydroxyl intermediate was proved to be essential for the N-dephenylation after intravenous administration of meta- and para-hydroxylated derivatives of CERM 3517 to five beagle dogs.

On-line focusing of 5-hydroxy-tryptamine type 3 receptor antagonists via the combination of field-enhanced sample injection and dynamic pH junction in capillary electrophoresis with amperometric detection.

In the present work, an on-line dual focusing technique based on field-enhance sample injection (FASI) and dynamic pH junction (DypH) was developed for the analysis of two 5-hydroxy-tryptamine type 3 receptor (5-HT3) antagonists ondansetron (Ond) and tropisetron (Tro) by capillary electrophoresis with amperometric detection (CE-AD) system. By preparing the sample in a lower conductivity (FASI condition) and lower pH value (DypH condition) matrix relative to the background electrolyte (BGE) solution, a simple and effective dual focusing approach, FASI-DypH was achieved. In this stacking mode, a large amount of analytes could be electrokinetically injected into the capillary and stacked at the boundary of the sample and the BGE zone as a result of deprotonation and decrease in the electric field. Effects of separation, detection and FASI-DypH focusing conditions were investigated in detail. Under the optimum conditions, good separation for Ond and Tro was achieved within 8min. In comparison with the conventional CE-AD analysis method, the present dual focusing technique enabled the enhancement factors in terms of peak heights to reach 357-fold and 345-fold for Ond and Tro, respectively. The limits of detection (LODs) (S/N=3) for Ond and Tro were 2nM and 5nM, respectively. The intraday and interday repeatabilities (RSDs) were less than 4.5% and 2.9% for peak height and migration time, respectively. The proposed method was successfully applied for the analysis of Ond and Tro in human urine sample.

Metabolism, pharmacokinetics, and excretion of a nonpeptidic substance P receptor antagonist, ezlopitant, in normal healthy male volunteers: characterization of polar metabolites by chemical derivatization with dansyl chloride.

The excretion, biotransformation, and pharmacokinetics of ezlopitant [(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)-amine], a substance P receptor antagonist, were investigated in healthy male volunteers after oral administration of a single 200-mg (approximately 93 microCi/subject) dose of [(14)C]ezlopitant. The total recovery of administered radioactive dose was 82.8 +/- 5.1, with 32.0 +/- 4.2% in the urine and 50.8 +/- 1.4% in the feces. Mean observed maximal serum concentrations for ezlopitant and total radioactivity were achieved at approximately 2 h after oral administration; thus, ezlopitant was rapidly absorbed. Ezlopitant was extensively metabolized in humans, since no unchanged drug was detected in urine and feces. The major pathway of ezlopitant in humans was the result of the oxidation of the isopropyl side chain to form the omega-hydroxy and omega-1-hydroxy (M16) metabolites. M16 and omega,omega-1-dihydroxy (1,2-dihydroxy, M12) were identified as the major circulating metabolites accounting for 64.6 and 15.4% of total circulating radioactivity, respectively. In feces, the major metabolite M14 was characterized as the propionic acid metabolite and formed by further oxidation of the omega-hydroxy metabolite. The urinary metabolites were the result of cleaved metabolites caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. The metabolites (M1A, M1B, and M4), approximately 34% of the total radioactivity in urine, were identified as benzyl amine derivatives. These were polar metabolites that were further characterized using the reaction with dansyl chloride to derivatize the primary amines and phenol moieties to less polar analytes. The other metabolites were the result of O-demethylation, dehydrogenation of the isopropyl group, and oxidation on the quinuclidine moiety.

Quantitation of a novel antiemetic (ADR-851) in plasma and urine by reversed-phase high-performance liquid chromatography with fluorescence detection.

A sensitive and specific bioanalytical method for quantitation of a novel antiemetic (ADR-851) in plasma and urine has been developed and validated. The drug and internal standard (metoclopramide) are extracted from the plasma matrix by solid-phase extraction on cyanopropyl bonded-phase columns. After extraction, samples are separated by isocratic reversed-phase high-performance liquid chromatography. The parent drug, internal standard and a yet unidentified metabolite are detected by fluorescence. The method requires 1.0 ml of plasma or 0.1 ml of urine and has a lower limit of quantitation of 2 ng/ml with 10.9% relative standard deviation (R.S.D.). Method linearity has been established over a 2-800 ng/ml range when 1.0 ml of plasma is used. The intra- and inter-day imprecisions for the method are typically better than 6% and 11% R.S.D., respectively, in both plasma and urine over the entire dynamic range. The pooled estimate of bias is less than 5% and attests to the excellent accuracy.

Pharmacokinetics and bioavailability of the anti-emetic agent bromopride.

The pharmacokinetics of bromopride, an anti-emetic agent chemically related to metoclopramide, has been investigated in normal human subjects. After intravenous bolus doses of 10 mg, a one-compartment open model appeared adequate to describe the plasma drug concentration data. The systemic clearance of bromopride was 899 ml min-1 +/- 22 per cent CV, the volume of distribution was 2151 +/- 16 per cent CV, and the elimination half-life was 2.9 h +/- 21 per cent CV. Over a wide drug concentration range of up to 650 ng ml-1, bromopride was only 40 per cent bound to plasma proteins. The systemic availability of orally and intramuscularly administered solution doses of 20 mg of bromopride was 54 per cent and 78 per cent, respectively. Formulation of bromopride as the solid material in capsules delayed absorption but did not affect the extent of drug bioavailability. The pharmacokinetics of bromopride appeared similar to that of metoclopramide. No evidence for non-linear kinetics was found when bromopride was administered orally in the dose range 10-30 mg: after single oral doses of 10, 20, and 30 mg, peak mean plasma drug concentrations were 20 ng ml-1 +/- 32 per cent CV, 38 ng ml-1 +/- 16 per cent CV, and 64 ng ml-1 +/- 23 per cent CV, respectively.

Simultaneous measurement of the major metabolites of dolasetron mesilate in human urine using solid-phase extraction and high-performance liquid chromatography.

A method based on solid-phase extraction and high-performance liquid chromatography (HPLC) has been developed for the simultaneous quantitation of the principal active metabolites of dolasetron mesilate [i.e. MDL 74,156 (II), MDL 102,382 (III) and MDL 73,492 (IV)] in human urine. The method has been validated over the concentration range of 200-5000 pmol/ml for all three metabolites. Within-day and day-to-day coefficients of variation were less than 9 and 14%, respectively, for the three metabolites. The method allowed the simultaneous quantitation of III, IV and II and the evaluation of the urinary excretion of these metabolites in human urine following the administration of dolasetron mesilate.

The metabolic disposition of aprepitant, a substance P receptor antagonist, in rats and dogs.

The absorption, metabolism, and excretion of [14C]aprepitant, a potent and selective human substance P receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, was evaluated in rats and dogs. Aprepitant was metabolized extensively and no parent drug was detected in the urine of either species. The elimination of drug-related radioactivity, after i.v. or p.o. administration of [14C]aprepitant, was mainly via biliary excretion in rats and by way of both biliary and urinary excretion in dogs. Aprepitant was the major component in the plasma at the early time points (up to 8 h), and plasma metabolite profiles of aprepitant were qualitatively similar in rats and dogs. Several oxidative metabolites of aprepitant, derived from N-dealkylation, oxidation, and opening of the morpholine ring, were detected in the plasma. Glucuronidation represented an important pathway in the metabolism and excretion of aprepitant in rats and dogs. An acid-labile glucuronide of [14C]aprepitant accounted for approximately 18% of the oral dose in rat bile. The instability of this glucuronide, coupled with its presence in bile but absence in feces, suggested the potential for enterohepatic circulation of aprepitant via this conjugate. In dogs, the glucuronide of [14C]aprepitant, together with four glucuronides derived from phase I metabolites, were present as major metabolites in the bile, accounting collectively for approximately 14% of the radioactive dose over a 4- to 24-h period after i.v. dosing. Two very polar carboxylic acids, namely, 4-fluoro-alpha-hydroxybenzeneacetic acid and 4-fluoro-alpha-oxobenzeneacetic acid, were the predominant drug-related entities in rat and dog urine.