Bioequivalence of an ezetimibe/simvastatin combination tablet and coadministration of ezetimibe and simvastatin as separate tablets in healthy subjects.

OBJECTIVE: To assess the bioequivalence of an ezetimibe/simvastatin (EZE/SIMVA) combination tablet compared to the coadministration of ezetimibe and simvastatin as separate tablets (EZE + SIMVA). METHODS: In this open-label, randomized, 2-part, 2-period crossover study, 96 healthy subjects were randomly assigned to participate in each part of the study (Part I or II), with each part consisting of 2 single-dose treatment periods separated by a 14-day washout. Part I consisted of Treatments A (EZE 10 mg + SIMVA 10 mg) and B (EZE/SIMVA 10/10 mg/mg) and Part II consisted of Treatments C (EZE 10 mg + SIMVA 80 mg) and D (EZE/SIMVA 10/80 mg/mg). Blood samples were collected up to 96 hours post-dose for determination of ezetimibe, total ezetimibe (ezetimibe + ezetimibe glucuronide), simvastatin and simvastatin acid (the most prevalent active metabolite of simvastatin) concentrations. Ezetimibe and simvastatin acid AUC(0-last) were predefined as primary endpoints and ezetimibe and simvastatin acid Cmax were secondary endpoints. Bioequivalence was achieved if 90% confidence intervals (CI) for the geometric mean ratios (GMR) (single tablet/coadministration) of AUC(0-last) and Cmax fell within prespecified bounds of (0.80, 1.25). RESULTS: The GMRs of the AUC(0-last) and Cmax for ezetimibe and simvastatin acid fell within the bioequivalence limits (0.80, 1.25). EZE/ SIMVA and EZE + SIMVA were generally well tolerated. CONCLUSIONS: The lowest and highest dosage strengths of EZE/SIMVA tablet were bioequivalent to the individual drug components administered together. Given the exact weight multiples of the EZE/SIMVA tablet and linear pharmacokinetics of simvastatin across the marketed dose range, bioequivalence of the intermediate tablet strengths (EZE/SIMVA 10/20 mg/mg and EZE/SIMVA 10/40 mg/mg) was inferred, although these dosages were not tested directly. These results indicate that the safety and efficacy profile of EZE + SIMVA coadministration therapy can be applied to treatment with the EZE/SIMVA tablet across the clinical dose range.

Characterization of the solubility of a poorly soluble hydroxylated metabolite in human urine and its implications for potential renal toxicity.

The solubility, in human urine, of the major hydroxylated metabolite (M1) of an experimental cognition enhancer was characterized through a series of in vitro experiments in an effort to estimate the probability of crystalluria occurring following oral administration of the parent compound. The aim of these experiments was to determine if a safety margin existed between clinically observed urine concentrations and the solubility of M1. The mean urine concentrations of M1 in young and elderly subjects following oral administration of the parent compound at the highest doses tested, were 4865 +/- 2368 ng/mL and 2764 +/- 791 ng/mL, respectively. In vitro solubility experiments with M1 were conducted in drug-free human urine (37 degrees C) from four male and four female healthy subjects under conditions of high and low urine osmolality. Mean concentrations (n = 16) of M1 in human urine to which solid M1 was added, were 3656 +/- 621 ng/mL, 4678 +/- 1169 ng/mL and 5378 +/- 2474 ng/mL after stirring for 24, 48 and 72 h, respectively, indicating that the ex vivo mean solubility of M1 in human urine is no greater then approximately 5 microg/mL. Addition of solid M1 to urine from human subjects dosed with the parent compound resulted in mean urine M1 concentrations 23.5% greater than those observed in vivo. The results from both experiments indicated a significant overlap between urine concentrations of M1 in vivo following the highest oral administration of the parent drug and M1 solubility measured in vitro, suggesting a high potential for in vivo saturation of urine with M1 with subsequent precipitation, crystalluria, and nephrotoxicity. Consequently, the results of these studies have placed restrictions on the dose that could be administered during clinical development of this compound.

Pharmacokinetics and bioavailability of Sinemet CR: a summary of human studies.

The pharmacokinetics of Sinemet CR, a controlled-release formulation containing carbidopa and levodopa, were investigated in healthy young and elderly volunteers and in patients with Parkinson's disease. Sinemet CR produced more sustained plasma levels of levodopa, carbidopa, and 3-O methyldopa than did conventional Sinemet. In elderly subjects, the corresponding steady-state plasma levels fluctuated in narrower ranges with Sinemet CR than those following the administration of Sinemet. Results indicate a levodopa bioavailability of 71% for Sinemet CR, in contrast to a bioavailability of 99% for Sinemet for these subjects. The carbidopa bioavailability of Sinemet CR was 58% relative to that of Sinemet. Systemic decarboxylase inhibition was comparable between the 2 regimens as indicated by the renal clearance of levodopa. The absorption of levodopa was slower and more protracted with Sinemet CR than with Sinemet. Food increased the levodopa bioavailability of Sinemet CR. This increase was attributed to an increased gastric retention time. No dose-dumping occurred with Sinemet CR in either the nonfasting or the fasting state. Levodopa bioavailability was lower in young volunteers than in elderly volunteers. This was attributed to an age-related decrease in gastric emptying and in 1st-pass metabolic decarboxylation in the gastrointestinal (GI) tract. In parkinsonian patients, as in healthy subjects, the Sinemet CR formulation produced more sustained levodopa plasma levels. These patients required a higher total daily dosage of Sinemet CR than of Sinemet for control of parkinsonian symptoms, but less frequent dosing was required during chronic therapy. Peak plasma levodopa levels increased proportionately with increasing Sinemet CR dosage. These observations were consistent with the pharmacokinetic characteristics of the formulation.

Synthetic and preliminary hemodynamic and whole animal toxicity studies on (R,S)-, (R)-, and (S)-2-methyl-3-(2,4,5-trihydroxyphenyl)alanine.

The synthesis, resolution, and absolute configuration assignment of 2-methyl-3-(2,4,5-trihydroxphenyl)alanine (6-OH-alpha-Me-Dopa) are reported. Hemodynamic studies in the rat have shown that this structural analogue and potential metabolite of the clinically useful drug (S)-alpha-Me-Dopa possesses weak hypotensive activity which resides in the R enantiomer. LD50 studies in mice have established that 6-OH-alpha-Me-Dopa is over four times more toxic than alpha-Me-Dopa. Chronic exposure to 6-OH-alpha-Me-Dopa leads to renal and hepatic lesions. The case of oxidation of this hydroquinone to the electrophilic quinone species may contribute to its enhanced toxicity compared to alpha-Me-Dopa.

Lack of pharmacokinetic and pharmacodynamic interaction between rizatriptan and paroxetine.

Rizatriptan is a potent, oral 5-HT(1B/1D) agonist with a rapid onset of action being investigated for the acute treatment of migraine. This study examined the clinical and pharmacolinetic interaction between rizatriptan and the selective serotonin reuptake inhibitor, paroxetine. In this two-period crossover study, 12 healthy young subjects (6 males and 6 females) received 1 mg rizatriptan following 14 days of treatment with placebo or paroxetine (20 mg once daily). Plasma was sampled for rizatriptan and N-monodesmethyl rizatriptan, a minor but active metabolite of rizatriptan. Safety evaluations included monitoring for adverse events, vital signs, and visual analog scale assessment of mood. Plasma levels of rizatriptan and N-monodesmethyl rizatriptan were not altered when rizatriptan was administered with paroxetine compared to the placebo. Clinically, coadministration of rizatriptan with paroxetine was well tolerated. Blood pressure, heart rate, and temperature changes during the observation period did not differ to a clinically significant degree when rizatriptan was administered with paroxetine compared to the placebo. No effects on mood occurred following treatment with the combination compared to rizatriptan alone. Adverse events following rizatriptan administration with paroxetine were similar to those reported when rizatriptan was given with the placebo.

High-throughput sample preparation procedures for the quantitation of a new bone integrin alpha(nu)beta(3) antagonist in human plasma and urine using liquid chromatography-tandem mass spectrometry.

High throughput LC-MS/MS assays to quantitate a new alpha(nu)beta(3) bone integrin antagonist (I) in human plasma and urine have been developed using instruments programmed to automate sample preparation procedures. Packard liquid handling system-MultiPROBE II EX was programmed for preparing calibration standards in control plasma and urine, acidifying all standards, quality control (QC), and clinical samples with necessary dilutions, and adding the internal standard to the acidified samples. TOMTEC Quadra 96 was programmed to perform the solid phase extraction (SPE) process on a 3M 96-well mixed phase cation standard density (MPC-SD) plate to isolate the analytes from the sample matrix. The extract collected from both types of matrices was directly injected into reversed-phase LC-MS/MS system with a Turbo Ion Spray (TIS) interface in the positive ionization mode. The plasma and urine assays have the calibration range of 0.5-1500 and 2-6000 ng/mL, respectively. Validation of the automated and the manual plasma assays showed that application of MultiPROBE II to sample preparation gave comparable accuracy and precision. Overall, the automated approaches with minimum manual intervention enhanced the throughput of sample preparation.

Analysis of nitrosamines in aqueous and biological fluids based on measurement of photochemically liberated nitrite.

A method is described for the analysis of nitrosamines in aqueous solution and in biological fluids (blood, plasma, and rat liver microsomal suspensions). The method is based on photochemical degradation of the nitrosamine in a controlled environment to yield the corresponding amine and nitrite ion, and the latter is subsequently used to form a chromophoric or fluorescent product. The analysis scheme is a modular three-component system consisting of a column to remove contaminating nitrite prior to photolysis, a photochemical reactor, and a chemical reactor. Additional modules are used to accommodate biological samples or large-volume (5--50 ml) aqueous samples. In this study, N-nitrosopyrrolidine, N-nitrosodimethylamine, and N,N-diethanolnitrosamine were utilized as substrates. Because of intersubstrate variability in the photochemical decomposition rate and overall nitrite yield, the structure (i.e., photochemical behavior) of the particular nitrosamine in the sample must be known prior to analysis. With a colorimetric readout, the sensitivity for analysis of N-nitrosopyrrolidine was 800 ng/ml for a 5-ml sample and the measurement precision was +/- 6% in the biological fluids. Fluorometric analysis improved sensitivity to 4 ng/ml with a precision of +/- 10% in biological media.

A sensitive method for assay of a novel tricyclic compound using coulometric electrochemical detection.

A reversed-phase high-performance liquid chromatographic method using coulometric electrochemical detection in the oxidative mode has been developed for the analysis of 3-(9-chloro-5,6-dihydro-11-H-pyrrolo[2,1-b][3]benzazepine-11-ylidene- N,N-dimethyl-1-propanamine(E)-Z-butenedioate hydrogen maleate (1) in plasma of patients dosed with 2-8 mg/kg/d of the drug. Concentrations as little as 0.1 ng/mL of 1 in plasma can be estimated with a mean coefficient of variation of 7.4 +/- 1.08%. The utility of the procedure was demonstrated by the analysis of 500 patient samples from a rising multiple-dose study.

Quantitation of norfloxacin, a new antibacterial agent in human plasma and urine by ion-pair reverse-phase chromatography.

A specific and sensitive high-performance liquid chromatographic method for the analysis of norfloxacin in human plasma and urine is described. Norfloxacin was extracted from the sample matrix using dichloromethane under neutral conditions, followed by back extraction into dilute phosphoric acid for chromatographic analysis on a reverse-phase column with a mobile phase consisting of methanol, phosphate buffer, and ion-pairing reagent (pH 3.0) at a flow rate of 2.0 mL/min. The ability of this method to distinguish intact norfloxacin from its metabolites was demonstrated. The method is linear, quantitative, and reproducible for both plasma analysis (0.05-2.5 microgram/mL) and urinalysis (1.0-500 micrograms/mL) using peak area ratios (norfloxacin-internal standard) for quantitation. The stability of norfloxacin and its metabolites in dilute phosphoric acid was studied. To assess the presence of norfloxacin conjugates in the urine of dosed individuals, the effects of urine hydrolysis on drug quantitation were examined. Urine and plasma levels of norfloxacin at selected time points following the administration of single drug doses are presented.

A sensitive analytical method for the determination of a novel lipoxygenase inhibitor, 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-one, in plasma using reductive electrochemical detection.

A reversed-phase high-performance liquid chromatographic assay using electrochemical detection in the reductive mode has been developed for the analysis of 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-one (1) in plasma to determine drug absorption. Free drug in plasma in concentrations as little as 0.25 ng/mL can be estimated with a mean coefficient of variation (CV) of 6.3 +/- 2.6%. Metabolites which can be converted to the parent drug by acid hydrolysis can be quantified in concentrations of 10 ng/mL or more, with a mean CV of 4.3 +/- 1.9%. To test the procedure, plasma was obtained from dogs receiving 14C-labeled 1. After acid hydrolysis of plasma, the electrochemical assay for parent drug showed good agreement with the radioactive equivalents in plasma, suggesting that parent drug and metabolites can be satisfactorily analyzed by this procedure.

Analytical methods for the determination of sulindac and metabolites in plasma, urine, bile, and gastric fluid by liquid chromatography using ultraviolet detection.

A high-performance liquid chromatographic method using a linear elution gradient has been developed for the analysis of sulindac, sulindac sulfone, and sulindac sulfide in plasma, urine, bile, and gastric fluid. The methodology uses reverse-phase, radial compression chromatography with gradient elution, and UV detection. Sulindac and its metabolites in plasma can be quantitated at 0.25 microgram/mL with a mean CV of 6.0 +/- 2.9%; urine, bile, and gastric fluid (0.5 microgram/mL) yield a mean CV of 5.5 +/- 1.9%.

Bioanalysis and disposition of alpha-fluoromethylhistidine, a new histidine decarboxylase inhibitor.

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of alpha-fluoromethylhistidine (alpha-FMH) in human biological samples. The plasma assay required isolation of the drug using a weak cation-exchange resin prior to HPLC analysis with UV detection. The urine assay employed postcolumn derivatization with o-phthalaldehyde (without a thiol) and fluorescence detection. The extent of metabolism of alpha-FMH in humans was studied in four healthy volunteers using tritium-labeled material. No significant differences in the plasma and urine concentrations of radioactivity and unchanged drug were detected. In addition, the radiochromatograms of selected urine samples revealed a single peak with a retention time corresponding to the unchanged drug. The evidence presented suggests negligible biotransformation of alpha-FMH in humans.

Pharmacokinetics of rizatriptan in healthy elderly subjects.

OBJECTIVE: Rizatriptan is a serotonin 5-HT1B/1D receptor agonist for acute treatment of migraine. Its pharmacokinetics were assessed in healthy elderly males and females receiving a single 10 mg tablet oral dose. The pharmacokinetic data (AUC(0-infinity) and Cmax) for the elderly in this study were compared with historical data from previous studies for healthy young adults (n = 65). METHODS: In a double-blind, parallel, placebo-controlled study, healthy elderly female and male subjects aged 65 or older (n = 8 each) received a single oral dose of 10 mg rizatriptan. Plasma and urine concentrations of drug were determined by HPLC with tandem mass spectrometry detection at several collection time points or intervals starting at predose and postdose over 24 h. RESULTS: In elderly subjects, the geometric mean values for AUC(0-infinity) and Cmax were 77.7 ng/h/ml and 21.9 ng/ml; the average values for tmax, half-life (t 1/2), renal clearance (Clr), and percent urinary excretion of dose (Ue) were 1.2 h, 1.8 h, 197 ml/min and 9.3%, respectively. The AUC(0-infinity) and Cmax of rizatriptan were similar in elderly and young subjects. The geometric mean AUC ratio of elderly to young was 0.96 with 90% confidence interval (0.83, 1.11), p > 0.25. The geometric mean Cmax ratio was 0.89 with 90% confidence interval (0.72, 109), p > 0.25. No significant pharmacokinetic differences were observed between elderly males and females. CONCLUSIONS: The plasma pharmacokinetics of rizatriptan appear to be similar in the elderly and young. In the elderly, the pharmacokinetics of rizatriptan do not appear to differ between male and female to a clinically significant extent.

LC-MS/MS determination of a farnesyl transferase inhibitor in human plasma and urine.

To support clinical pharmacokinetic studies in cancer patients, sensitive and specific methods for measuring 4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-1-(3-chlorophenyl) piperazinone (I), a farnesyl transferase inhibitor (FTI), in human plasma and urine were developed and validated. The methods are based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization (APCI) and tandem mass spectrometric (MS/MS) detection in the positive ion mode using a heated nebulizer interface. Drug and internal standard were isolated from plasma or basified urine using automated solid-phase extraction on cyano cartridges. The organic extracts were dried, reconstituted in aqueous acetonitrile and injected into the system. Chromatographic separation of I and internal standard (IS) was achieved using a BDS Hypersil C8 analytical column, with a mobile phase consisting of acetonitrile:methanol:water (50:4:46) and trifluoroacetic acid (0.05%) at a flow rate of 0.6 ml/min. MS/MS detection was performed on a PE-Sciex API 300 tandem mass spectrometer operated in selected reaction monitoring mode. The parent-->product ions monitored were m/z 406-->195 for analyte I and m/z 448-->195 for the internal standard. Unusual in this method is that quantitation is accomplished using a secondary product ion, m/z 195, of drug I and IS. The assays were validated over the concentration range of 0.5-1000 ng/ml (1.2 nM to 2.5 microM, respectively) in plasma, and 2.5-500 ng/ml (6.2 nM to 1.23 microM) in urine. Accuracy was within +/-10% of nominal concentration at all levels in urine, and all but the lowest standard in plasma (+/-14% at 0.5 ng/ml). Intraday precision (expressed as coefficients of variation, CVs) for standard replicates and interday precision for quality control (QC) samples were less than 8% at all concentrations in both matrices. Detailed descriptions of the extraction procedure and analytical methodology used in the assay of I in plasma and urine are presented. This procedure may have utility in the quantitation of other imidazole-based FTIs with cyanobenzyl substructures.

Liquid chromatographic-tandem mass spectrometric urine assay for a highly metabolized cyclic ureidobenzenesulfonamide: issues concerning assay specificity and quality control preparation.

An LC-MS-MS method was validated for the quantitation of a beta(3) agonist (A) in human urine to support Phase I studies. A was designed to accelerate metabolism for weight reduction. During assay development a significant loss of A was apparent from frozen urine quality control samples. The addition of 0.75% bovine serum albumin (BSA) in urine (v/v) was required to maximize the recovery of A from urine. Urine samples were basified and extracted into methyl t-butyl ether-isopropyl alcohol (90:10, v/v). The organic layer was washed, evaporated, reconstituted, and injected onto a 5 cm, C8 HPLC column prior to MS-MS analysis. The standard curve was linear from 5 to 500 ng/ml. Intraday precision for peak area ratios from BSA urine samples at seven separate concentrations over a range of 5-500 ng/ml (n=5) was <4.0% and calculated concentrations were within 91-115% of nominal concentrations. Interday precision for BSA urine quality control (QC) samples at four separate concentrations (n=10 of each) was <5.0% and individual calculated concentrations were within 90-111% of nominal concentrations. This work emphasizes that potential metabolites and quality control standards should be prepared and assayed as early as possible in method development, especially before the sample collection section of the clinical protocol is prepared. The methods described here have wide utility to other compounds containing basic benzene sulfonamides and to beta3 agonist candidates.

Determination of a beta(3)-agonist in human plasma by LC/MS/MS with semi-automated 48-well diatomaceous earth plate.

Methods for the determination of a beta(3)-agonist (A) in human plasma were developed and compared based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using a turbo ion spray (TIS) interface. Drug and internal standard were isolated from plasma by three sample preparation methods, liquid-liquid extraction, Chem Elut cartridges and 48-well diatomaceous earth plates, that successively improved sample throughput for LC/MS/MS. MS/MS detection was performed on a PE Sciex API 365 tandem mass spectrometer operated in positive ion mode and using multiple reaction monitoring (MRM). The precursor/product ion combinations of m/z 625/607 and 653/515 were used to quantify A and internal standard, respectively, after chromatographic separation of the analytes. Using liquid-liquid extraction and Chem Elut cartridges, the assay concentration range was 0.5-100 ng/ml. Using diatomaceous earth plates, the concentration range of the assay was extended to 0.5-200 ng/ml. For all three assays, the statistics for precision and accuracy is comparable. The assay accuracy ranged from 91-107% and intraday precision as measured by the coefficient of variation (CV) ranged 2-10%. The sample throughput was tripled when the diatomaceous earth plate method was compared with the original liquid-liquid extraction method.

Assay methodology for prednisolone, prednisolone acetate and prednisolone sodium phosphate in rabbit aqueous humor and ocular physiological solutions.

Prednisolone, prednisolone acetate and prednisolone sodium phosphate are glucocorticoids used for ocular, anti-inflammatory therapy. A reversed-phase high-performance liquid chromatographic assay using ultraviolet detection has been developed that affords baseline resolution of the above analytes in balanced salt solutions and rabbit aqueous humor. The drugs can be quantified at 0.025-0.05 micrograms/ml in the above matrices; 6 alpha-methylprednisolone is used as the internal standard. Both esters of prednisolone are vulnerable to chemical and enzymatic hydrolysis giving prednisolone. Analysis of aqueous humor samples shows prednisolone acetate penetrating/metabolizing primarily to prednisolone; prednisolone sodium phosphate penetrates the cornea giving the ester and alcohol.

Comparative corneal penetration of prednisolone sodium phosphate and prednisolone acetate in NZW rabbits.

Comparative corneal penetration studies in the literature with prednisolone sodium phosphate solution and prednisolone acetate suspension administered to rabbit eyes give conflicting results concerning the greater bioavailability of prednisolone acetate. A recent in vitro penetration study shows similar fluxes for both salt forms in terms of prednisolone, quantified by a specific HPLC assay. An in vivo comparison has been conducted in NZW rabbit eyes and shows similar bioavailability for both drugs. Prednisolone sodium phosphate, prednisolone acetate and the primary metabolite prednisolone are quantified by a specific HPLC assay in aqueous humor.

Determination of the antibiotic fludalanine in plasma and urine by high-performance liquid chromatography using a packed-bed, post-column reactor with o-phthalaldehyde and 2-mercaptoethanol.

Fludalanine is a novel anti-bacterial agent active against gram-negative and gram-positive bacteria. A high-performance liquid chromatographic assay has been developed using ion-pair chromatography to resolve fludalanine and the internal standard 3,3-difluoro-D-alanine from plasma and urine background. The mobile phase contains sodium dodecyl sulfonate and methanol in a phosphate buffer. Fludalanine is derivatized post-column with o-phthalaldehyde via a packed-bed chemical reactor. The adduct is detected fluorometrically. The plasma and urine assays are sensitive to 0.25 and 0.5 micrograms/ml, respectively.

An in vitro comparison of the permeability of prednisolone, prednisolone sodium phosphate, and prednisolone acetate across the NZW rabbit cornea.

Controversy and ambiguity in the literature concerning the corneal penetration of prednisolone acetate over Prednisolone Sodium phosphate in NZW rabbits has recently prompted comparative studies using specific chromatographic assays. In vitro, corneal penetration studies were performed using the Ussing Chambers to compare the permeability and flux of both esters and prednisolone at 0.5% using a reversed phase HPLC-UV assay. Chromatograms of samples from the receiver chambers show primarily the presence of prednisolone from both esters; only prednisolone phosphate penetrated the cornea intact. Flux measurements were similar for prednisolone and both salt forms in terms of the metabolite prednisolone. Permeability coefficient calculations give the relative comparison: prednisolone acetate greater than prednisolone greater than prednisolone sodium phosphate.