Determination of an endothelin receptor antagonist in rat plasma by radioimmunoassay.

A quantitative method based on radioimmunoassay for the determination of an endothelin receptor antagonist (C(31)H(33)NO(7), I) has been developed and validated. The immunogen was prepared by coupling I to the bovine serum albumin via the N-hydroxysuccinimide ester of I from which the radioligand was also prepared by the reaction with [125I]-iodotyrosine. The method was specific and no immunoactive material other than the parent drug was detectable in mammalian plasma. This direct assay, using 50 microl of rat plasma is sensitive (0.4 ng/ml), without matrix interference, and has sufficient sensitivity, specificity, accuracy and precision for the analysis of dosed rat plasma samples.

Development and implementation of an electrochemiluminescence immunoassay for the determination of an angiogenic polypeptide in dog and rat plasma.

A quantitative method based on electrochemiluminescence immunoassay for the determination of the angiogenic agent aFGF-S117 has been developed and validated. Two polyclonal antibodies specific to aFGF-S117 and a wild-type aFGF antibody were selected for the analysis. The assay was based on the non-competitive sandwich immunoassay principle in which the drug is trapped with a biotinylated antibody that is immobilized on a streptavidin magnetic particle. The drug is then sandwiched with a ruthenium chelated second antibody. The assay demonstrates good accuracy and reproducibility at plasma concentration of 0.5 ng/ml.

Determination of an echinocandin, MK-0991, in mammalian plasma by radioimmunoassay.

A quantitative method based on radioimmunoassay for the determination of the antifungal agent, CANCIDAS (MK-0991) has been developed and validated. The immunogen was prepared by coupling MK-0991 to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to MK-0991 was selected for RIA. The assay was based on the competitive immunoassay principle in which the drug competes with iodinated drug for a limited quantity of specific antibody. The bound tracer was separated via goat anti-rabbit globulin. The assay demonstrates good accuracy and reproducibility at plasma concentration down to 10 ng/ml. The specificity of the RIA method was confirmed by cross-validating against an established HPLC method.

Ketoconazole induces G0/G1 arrest in human colorectal and hepatocellular carcinoma cell lines.

Ketoconazole is an oral-antifungal agent that has been used worldwide in the treatment of some hormone-dependent human cancer. In this study, we demonstrated that ketoconazole (20 microM) induced various types of human cancer cell growth arrest in the G0/G1 phase. Our results revealed that ketoconazole-induced growth arrest was more profound in COLO 205 and Hep G2 (with wild-type p53) than in HT 29 (p53 His(273) mutant) and Hep 3B (with deleted p53) cells. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated by ketoconazole (10 microM) treatment in COLO 205 but not in HT 29 cells. The ketoconazole-induced G0/G1 phase arrest in COLO 205 cells was attenuated by p53-specific antisense oligodeoxynucleotides (20 microM) treatment. These results suggested that the p53-associated signaling pathway is involved in the regulation of ketoconazole-induced cancer cell growth arrest. By Western blot analysis, we demonstrated that cyclin D3 and CDK4 protein but not other G0/G1 phase regulatory protein levels were decreased by ketoconazole-treatment in both COLO 205 and HT 29 cells. Our study provides the basis of molecular mechanisms for ketoconazole in growth inhibition of human cancer cells and such results may have significant applications for cancer chemotherapy.

Determination of pilocarpic acid in human plasma by capillary gas chromatography with mass-selective detection.

A novel, highly sensitive method for the determination of pilocarpic acid (PA) in human plasma is described. In addition, the method provides for the conversion of the lactone, pilocarpine (P), to PA so that a total drug presence can be determined. Using novel high-performance liquid chromatographic conditions capable of separating P, isopilocarpine (I-P), PA and isopilocarpic acid (I-PA) from each other and from endogenous plasma impurities, it was confirmed that P exclusively and quantitatively converts to PA in heparinized human plasma during storage. For the determination of PA, the selective extraction of PA from protein-free plasma was accomplished using two different solid-phase extraction (SPE) cartridges in two consecutive SPE steps. After extraction, PA was lactonized with trifluoroacetic acid back to P, and both P and an internal standard were acylated using heptafluorobutyric anhydride (HFBA). The trifluoroacetylated derivatives were monitored using gas chromatography (GC) with mass spectrometric (MS) detection. This procedure allowed the sensitive and reliable determination of PA with a limit of quantification (LOQ) of 1 ng/ml, which could not be achieved using previously described methods. The assay was validated in the concentration range of 1 to 10 ng/ml with an intra-day precision (expressed as the coefficient of variation, C.V.) ranging from 9.9 to 0.5%. Inter-day precision for the quality control standard at 2.5 ng/ml showed a C.V. of 10.2%. Accuracy ranged from 94 to 102%. The assay was used to monitor the maximum systemic exposure to P, administered by the ocular route, in terms of total plasma PA (P and PA).

An automated sample preparation and high performance liquid chromatographic method for the determination of MK-591, a novel leukotriene biosynthesis inhibitor, in human plasma.

A high performance liquid chromatography assay utilizing an automated sample preparation procedure for the determination of a novel leukotriene biosynthesis inhibitor, (MK-591), in human plasma has been developed. After aliquoting plasma samples and adding internal standard manually, the BenchMate Workstation executed protein precipitation and solid-phase extraction. Following evaporation to dryness, the residue was reconstituted and chromatographed isocratically on a cyano-phase analytical column. MK-591 and the internal standard were separated from each other and from endogenous plasma substances and detected with an absorbance detector. The assay has been validated in the concentration range 10-1000 ng ml-1 and has the sensitivity and specificity necessary to quantify plasma concentrations from several clinical studies.

Absence of a pharmacokinetic interaction between losartan and hydrochlorothiazide.

To support the use of a combination of losartan, a highly specific and selective AT1 angiotensin II receptor antagonist, and hydrochlorothiazide for treatment of hypertension, a pharmacokinetic drug interaction study was conducted. In this open-label, randomized, three-period, crossover study, patients with mild to moderate hypertension received a 12.5-mg tablet of hydrochlorothiazide, a 50-mg losartan tablet, or a combination tablet of 12.5 mg of hydrochlorothiazide and 50 mg of losartan for 7 days. Twelve patients (age range, 35-55 years; mean age, 44 years) were allocated to treatment. Drug interactions were evaluated by comparing the 24-hour area under the concentration-time curve (AUC24) for losartan and its active metabolite, E-3174, when losartan (50 mg) was given alone or in combination with 12.5 mg hydrochlorothiazide. The urinary recovery over the 24-hour period of hydrochlorothiazide was compared for hydrochlorothiazide (12.5 mg) given alone or in combination with 50 mg losartan. A clinically significant interaction was defined as a treatment difference of more than 35%. There was no evidence of a clinically significant effect of hydrochlorothiazide on the pharmacokinetics of losartan or E-3174, as the geometric mean AUC24 ratio (90% confidence interval [CI]) was 1.02 (0.95, 1.09) for losartan and 1.02 (0.96, 1.09) for E-3174. Based on urinary recovery over a 24-hour period of hydrochlorothiazide, losartan did not affect the pharmacokinetics of hydrochlorothiazide, as the geometric mean ratio of urinary hydrochlorothiazide recovery (90% CI) was 0.898 (0.79, 1.20). There was a minor (17%) decrease in the AUC24 of hydrochlorothiazide after administration of the combination tablet. Coadministration of hydrochlorothiazide and losartan was well tolerated.

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection.

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(-)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.

Fully automated methods for the determination of hydrochlorothiazide in human plasma and urine.

LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology Robot and BenchMate Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid-liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate performed buffer and organic solvent addition, liquid-liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2-100 ng ml-1 in plasma and 0.1-20 micrograms ml-1 in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.

Robotic sample preparation and high-performance liquid chromatographic analysis of verlukast in human plasma.

A fully automated HPLC assay has been developed and validated for the quantitation of verlukast, a leukotriene D4 antagonist, in human plasma. An upgraded Zymate I robotic system was utilized to perform protein precipitation and on-line injection followed by reversed-phase HPLC with fluorescence detection. Inter-day accuracy and precision were 100.8 and 4.6%, respectively, for the low quality control standards (0.125 microgram/ml). The automated robotic method was shown to be more efficient and accurate than the manual method.

Determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

Determination of tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol) in human plasma and urine has been developed and validated. The method utilizes reversed-phase high-performance liquid chromatographic separation and fluorescence detection of a pre-column derivative. The assay was linear in the ranges of 1-200 micrograms/ml plasma and 5-500 micrograms/ml urine with accuracies (mean percent recovery) all within 10% of 100% and precisions (coefficient of variation) all less than 10%.

Oral pharmacokinetics and food interaction of the leukotriene D4 receptor antagonist verlukast.

The influence of dose and food on the pharmacokinetic profile of orally administered verlukast, a leukotriene D4 receptor antagonist, was investigated in 12 healthy male volunteers. This was an open, four-period, single dose, randomised, crossover design including the following doses: one 75 mg tablet, one 250 mg tablet, 500 mg (2 x 250 mg) and 500 mg immediately following a standard meal. There were dose-related increases in the AUC, although after 500 mg verlukast this was disproportionately greater than with 75 mg (P = 0.04). Similarly, there were dose-related increases in C(max). No differences were observed in the t(max) between treatments. With respect to food, there was a 22% decrease (P = 0.02) in C(max) after 500 mg, and the AUC was 13% less (P = 0.052). The differences in the plasma concentration profiles betweeen fasted and fed states are not considered to be of clinical importance.

Determination of the HMG-CoA reductase inhibitors simvastatin, lovastatin, and pravastatin in plasma by gas chromatography/chemical ionization mass spectrometry.

A general method for the assay of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors lovastatin, pravastatin, and simvastatin in plasma has been developed and validated. The analytes are isolated from plasma by a solid-phase extraction procedure which separates the lactone and acid forms of the drugs. The lactone is converted to the acid form, which is subsequently derivatized by pentafluorobenzylation of the carboxyl group, and trimethylsilylation of the hydroxyl functions. Derivatized samples of intrinsic and converted acid are assayed by gas chromatography/mass spectrometry using negative chemical ionization mass spectrometry. The method has sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples containing the drugs administered at therapeutic doses. The method thus permits determination of both the lactone and hydroxy acid forms of lovastatin and simvastatin, and is also applicable to the assay of pravastatin.

Determination of a cyclic heptapeptide, a novel fibrinogen receptor antagonist, in human plasma by high-performance liquid chromatography with automated pre-column derivatization, column switching and fluorescence detection.

A sensitive high-performance liquid chromatographic (HPLC) assay for the determination of the cyclic heptapeptide Ac-Cs-Asn-Dtc-Amf-Gly-Asp-Cys-OH (Dtc = beta,beta-dimethylthioproline, Amf = p-aminomethylphenylalanine) in human plasma has been developed. The key steps in the assay include: solid-phase extraction of the drug from plasma, chemical derivatization of the primary amino group with naphthalene-2,3-dicarboxyaldehyde in the presence of N-acetyl-D-penicillamine as a nucleophile to form a fluorescent benzo[f]isoindole derivative, and HPLC with column switching to provide the necessary chromatographic separation of the derivative from endogenous plasma components. The assay has been validated in the concentration range 1-10 ng/ml of plasma.

Plasma drug profiles and tolerability of MK-571 (L-660,711), a leukotriene D4 receptor antagonist, in man.

We have studied the tolerability and plasma drug profiles of a leukotriene D4 receptor antagonist, MK-571, given intravenously and as an oral solution in two separate trials. Study I (i.v.) involved 2 panels of 6 healthy men in a double-blind, alternating, incrementally increasing dose study with single doses up to 1500 mg. There was good tolerability at all doses. Plasma was assayed stereospecifically by HPLC for the S(+) and R(-) enantiomers of MK-571. For each enantiomer AUC values increased more than proportionately with increasing dose, suggesting nonlinear kinetics. The S(+) enantiomer was cleared more rapidly than the R(-) enantiomer. The apparent initial volume of distribution was less than 101 for both enantiomers. Study II (oral) involved 18 healthy subjects in 3 parallel groups who took multiple oral doses of 100, 300, and 600 mg t.i.d. for 31 doses. MK-571 administration was well tolerated, with only mild to moderate gastrointestinal discomfort at the highest dose. Total MK-571 (plasma samples assayed nonstereoselectively) was rapidly absorbed after oral administration, reaching peak concentrations at 1-2 h. Mean 8 h AUC increased from dose 1 to dose 31 in all subjects at all doses, suggesting a modest extent of accumulation (about 50%) of total MK-571 in plasma with a t.i.d. dosage regimen.

Stereoselective determination of R(-)- and S(+)-MK-571, a leukotriene D4 antagonist, in human plasma by chiral high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(-)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D4 antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine-acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine-pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r2 greater than 0.999) for concentrations of enantiomers of 1 from 0.05 micrograms/ml, the lowest quantitation limit, up to 2.5 micrograms/ml. Intra-day coefficients of variation at 0.05 microgram/ml were 2.4% for the R(-)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(-)- and S(+)-1 were 2.6 and 3.6%, respectively. The utility of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.

High-performance liquid chromatographic determination of cilastatin and its major metabolite N-acetylcilastatin in rat plasma, urine and bile.

A new high-performance liquid chromatographic method coupled with solid-phase (C8) sample extraction has been developed for the simultaneous quantification of cilastatin and its major metabolite N-acetylcilastatin in rat plasma, urine and bile. The method is linear, reproducible and reliable with a detection limit of 1 microgram/ml in all three fluids. Plasma concentrations of cilastatin and N-acetylcilastatin at selected time intervals and biliary and urinary recoveries of cilastatin and N-acetylcilastatin following an intravenous dose of 10 mg/kg cilastatin are presented.

Disposition and metabolism of sodium 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma -oxobenzenebutanoate in rats and dogs.

The disposition of sodium 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma-o xo benzenebutanoate (L-648,051) was determined in rats and dogs. L-648,051 is a potent receptor antagonist for leukotriene D4 and is potentially useful in the treatment of asthma and other allergic disorders. After a dosage of 10 mg/kg iv, L-648,051 declined rapidly with a half-life of approximately 2 min in rat and dog plasma. Although the compound was well absorbed, it exhibited poor bioavailability due to efficient first-pass metabolism. In rats receiving 25, 50, and 150 mg/kg po, bioavailabilities were 0.5, 4.8, and 38.7%, respectively. In dogs, bioavailability of 10 and 50 mg/kg po was 0 and 23%, respectively. Two metabolites were identified, 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl-gamma- hydroxybenzenebutanoic acid (metabolite I), formed by ketoreduction, and 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl] benzeneacetic acid (metabolite II) formed by catabolic oxidation of the butanoic acid moiety of L-648,051. Ketoreduction resulted in the production of a chiral center and two enantiomers of metabolite I. In vitro studies suggest that rat erythrocytes formed the (+)-enantiomer exclusively. When L-648,051 was administered orally or iv to rats, both the (+)- and (-)-enantiomers were observed in the plasma. The data suggest that either two L-648,051 ketoreductases were present or that inversion of the hydroxyl stereocenter of metabolite I occurred.

Enantioselective disposition and protein binding of [(5,6-dichloro-9a-propyl-3-oxo-2,3,9,9a-tetrahydro-1-H-fluoren-7 -yl)-oxy] acetic acid, a new cerebral antiedemic agent, in rats.

[(5,6-Dichloro-9a-propyl-3-oxo-2,3,9,9a-tetrahydro-1-H-fluoren-7-y l)-oxy] acetic acid (DPOFA) is an agent capable of reducing the swelling of astroglial cells in brain tissues. In vitro studies have demonstrated that the (R)-(+)-form of DPOFA is more effective than its (S)-(-)-form in inhibiting tissue swelling. The purpose of this study is to compare the elimination kinetics of the enantiomers. A new stereoselective HPLC procedure was developed for the simultaneous quantitation of (R)-(+)- and (S)-(-)-enantiomers in plasma and bile samples. After iv administration of the racemic mixture (40 mg/kg), rats cleared the (R)-(+)-enantiomer more rapidly than the (S)-(-)-isomer; time-averaged total plasma clearances were 8.88 +/- 0.55 and 4.20 +/- 0.70 ml/min/kg (mean +/- SD), respectively. Similar results were observed when the individual isomers were administered (20 mg/kg iv). Both (R)-(+)- and (S)-(-)-enantiomers were highly bound to plasma protein. The (R)-(+)-isomer had a higher unbound fraction (2%) than did the (S)-(-)-enantiomer (0.8%). The intrinsic clearance of unbound drug for (R)-(+)- and (S)-(-)-enantiomers were 434 +/- 27 and 490 +/- 84 ml/min/kg, respectively, suggesting that the differences in the elimination of the enantiomers in rats were attributable to stereoselectivity in plasma protein binding rather than to enzyme activity. In vitro studies with isolated hepatocytes supported the hypothesis that there was no stereoselectivity in metabolism of the enantiomers.