John Y.-K. Hsieh

Safety, Tolerability, and Pharmacokinetics of Escalating High Doses of Ivermectin in Healthy Adult Subjects

Safety and pharmacokinetics (PK) of the antiparasitic drug ivermectin, administered in higher and/or more frequent doses than currently approved for human use, were evaluated in a double‐blind, placebo‐controlled, dose escalation study. Subjects (n = 68) were assigned to one of four panels (3:1, ivermectin/placebo): 30 or 60 mg (three times a week) or 90 or 120 mg (single dose). The 30 mg panel (range: 347–594 μg/kg) also received a single dose with food after a 1‐week washout. Safety assessments addressed both known ivermectin CNS effects and general toxicity. The primary safety endpoint was mydriasis, accurately quantitated by pupillometry. Ivermectin was generally well tolerated, with no indication of associated CNS toxicity for doses up to 10 times the highest FDA‐approved dose of 200 μg/kg. All dose regimens had a mydriatic effect similar to placebo. Adverse experiences were similar between ivermectin and placebo and did not increase with dose. Following single doses of 30 to 120 mg, AUC and Cmax were generally dose proportional, with tmax ∼4 hours and t1/2 ∼18 hours. The geometric mean AUC of 30 mg ivermectin was 2.6 times higher when administered with food. Geometric mean AUC ratios (day 7/day 1) were 1.24 and 1.40 for the 30 and 60 mg doses, respectively, indicating that the accumulation of ivermectin given every fourth day is minimal. This study demonstrated that ivermectin is generally well tolerated at these higher doses and more frequent regimens.

Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human plasma

Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree((R)) centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree((R)) device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 microgram/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 microgram/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 microgram/ml and stored at -70 degrees C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).

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.

High-throughput simultaneous determination of the HIV protease inhibitors indinavir and L-756423 in human plasma using semi-automated 96-well solid phase extraction and LC-MS/MS

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.

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.

Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and automation

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE II EX robotic liquid handling system.

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.

Development and Application of HPLC Methods with Tandem Mass Spectrometric Detection for the Determination of Hydrochlorothiazide in Human Plasma and Urine Using 96‐Well Liquid‐Liquid Extraction

Abstract Sensitive and selective methods for the determination of hydrochlorothiazide (HCTZ) in human plasma and urine were developed. Both methods were based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection. HCTZ and hydroflumethiazide (HFTZ), used as an internal standard, were isolated from human plasma and urine by liquid‐liquid extraction in the 96‐well plate format using a Tomtec Quadra 96 workstation. The MS/MS detection was performed on an Applied Biosystems‐Sciex API 3000 tandem mass spectrometer interfaced with a heated nebulizer probe and operated in the negative ionization mode. The developed methods were validated in the concentration ranges of 1–100 ng/mL and 0.05–20 µg/mL in human plasma and urine, respectively. The precision of both methods, as expressed by the coefficients of variation (C.V.), was less than 5.6% at all concentrations within the standard curves with acceptable accuracy. The plasma and urine methods provided sufficient sensitivity and high sample throughput to map out the pharmacokinetics of HCTZ in human subjects following an oral dose of HCTZ/Losartan, in support of a human clinical trial designed to evaluate the bioequivalence of HCTZ between two formulations.

High-throughput liquid chromatographic determination of rofecoxib in human plasma using a fully automated on-line solid-phase extraction system

A fully automated on-line solid-phase extraction method combined with high-performance liquid chromatography-fluorescence detection for the determination of rofecoxib in human plasma is described. A Spark Holland PROSPEKT system was used to perform all the automated on-line sample extraction and quantitation tasks, including sample loading, cartridge conditioning and washing, analyte elution, and start of a data acquisition system. Under postcolumn ultraviolet–irradiation conditions, the analytes were photochemically converted to highly fluorescent products and then monitored by a fluorescence detector. The assay was validated in the concentration range of 0.5–100 ng/mL using 0.25 mL of human plasma. The utility of this method was demonstrated by analysis of plasma samples from human subjects receiving oral doses of rofecoxib. ‡ This work was presented at the Fifteenth Annual International Symposium on Laboratory Automation and Robotics, October 1997, Boston, MA, USA.

Determination of a Selective GABA‐A α5 Receptor Inverse Agonist in Human Plasma by High‐Performance Liquid Chromatography with Tandem Mass Spectrometric Detection

Abstract A sensitive and selective method for the determination of a compound with activity at the benzodiazepine site of GABA‐A receptors (I, 3‐(5‐methyl‐isoxazol‐3‐yl)‐6‐(1‐methyl‐1H‐[1,2,3]triazol‐4‐yl‐methoxy)‐[1,2,4]‐triazolo[3,4‐A] phthalazine) and its active 5‐hydroxymethyl‐isoxazole metabolite (II) has been developed. The method is based on high performance liquid chromatography (HPLC) with tandem mass spectrometeric (MS/MS) detection. The analytes (I and II) and internal standard (III) were isolated from human plasma by a 96‐well solid phase extraction technique using a Tomtec Quadra 96 system. The MS/MS detection was performed on a PE Sciex API 3000 tandem mass spectrometer interfaced with a heated nebulizer probe. This semi‐automated HPLC‐MS/MS method was validated in the concentration range of 0.1–25 ng/mL, with the lower limit of quantification (LLOQ) of 0.1 ng/mL for both analytes. The precision of the assay, as expressed by the coefficients of variation (CV), was less than 8% at all concentrations within the standard curve for both I and II, with acceptable accuracy. This method provided sufficient sensitivity and high sample throughput to completely map out the pharmacokinetic time‐course in human subjects following oral doses of I.