B.K. Matuszewski

Studies of the oral bioavailability of alendronate

Clinical studies were performed to examine the oral bioavailability of alendronate (4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonate monosodium). All studies, with the exception of one performed in men, involved postmenopausal women. Short‐term (24 to 36 hours) urinary recovery of alendronate after an intravenous dose of 125 to 250 μg averaged about 40% in both men and women. In women, oral bioavailability of alendronate was independent of dose (5 to 80 mg) and averaged (90% confidence interval) 0.76% (0.58, 0.98) when taken with water in the fasting state, followed by a meal 2 hours later. Bioavailability was similar in men [0.59%, (0.43, 0.81)]. Taking alendronate either 60 or 30 minutes before a standardized breakfast reduced bioavailability by 40% relative to the 2‐hour wait. Taking alendronate either concurrently with or 2 hours after breakfast drastically (>85%) impaired availability. Black coffee or orange juice alone, when taken with the drug, also reduced bioavailability (approximately 60%). Increasing gastric pH, by infusion of ranitidine, was associated with a doubling of alendronate bioavailability. A practical dosing recommendation, derived from these findings and reflective of the long‐term nature of therapy for a disease such as osteoporosis, is that patients take the drug with water after an overnight fast and at least 30 minutes before any other food or beverage.

Elimination and Biochemical Responses to Intravenous Alendronate in Postmenopausal Osteoporosis

Postmenopausal women with established vertebral osteoporosis were studied for 2 years to determine the terminal elimination half‐life and the duration of response to treatment with intravenous alendronate (30 mg) given over 4 days. The urinary excretion of alendronate followed a multiexponential decline. Approximately 50% of the total dose was excreted over the first 5 days, and a further 17% was excreted in the succeeding 6 months. Thereafter, there was a much slower elimination phase with an estimated mean terminal half‐life of greater than 10 years (n = 11). Urinary excretion of hydroxyproline and calcium decreased significantly from pretreatment values by day 3, reaching a nadir by 1 week (40% and 67% decrease, respectively). Thereafter, hydroxyproline remained suppressed for the following 2 years. In contrast, urinary calcium excretion returned gradually toward pretreatment values over the first year and during the second year was comparable to pretreatment values. Serum activity of alkaline phosphatase activity decreased over 3 months (23% reduction), increased gradually thereafter, and returned to pretreatment values at month 24. Bone mineral density measured at the spine increased by approximately 5% during the first year and remained significantly higher than pretreatment values at 2 years. We conclude that a short course of high doses of intravenous alendronate is associated with a prolonged skeletal retention of the agent. This open study also suggests that this regimen has a sustained effect on bone turnover persisting for at least 1 year.

Determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid in urine by automated pre-column derivatization with 2,3-naphthalene dicarboxyaldehyde and high-performance liquid chromatography with fluorescence detection

A sensitive (5 ng/ml) method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid in human urine is described. The procedure includes (1) the isolation of the drug from urine by co-precipitation of its calcium salt with endogenous phosphates in the presence of base, (2) a solid-phase anion-exchange sample clean-up and (3) automated pre-column derivatization of the primary amino group with 2,3-naphthalene dicarboxyaldehyde-cyanide reagent followed by fluorescence detection of the N-substituted cyanobenz[f]isoindole derivative. The derivative of the drug was synthesized and its spectral and fluorescence properties were evaluated. The fluorescence quantum efficiency was determined to be 0.82 in the mobile phase used for the assay. The derivative is also capable of accepting energy in an oxalate ester-hydrogen peroxide chemiluminescence system.

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection.

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 A), 150x4.6 mm C8 analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor-->product ion combinations of m/z 1093.7-->1033.6 and 1094.7-->1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-1000 ng/ml using ISP, and 2.5-500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of a novel 4-aza-steroidal inhibitor of 5 alpha-reductase in human plasma has been developed. The assay is based on a single solid-phase extraction and an efficient HPLC separation on two analytical columns in series. The assay has been fully validated and used to support Phase II and III clinical pharmacokinetic studies. The lowest limit of quantification was found to be at 1 ng/ml and allowed pharmacokinetic evaluation of the drug at doses down to 5 mg.

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid-liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50-2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.

Column-switching high-performance liquid chromatographic determination of a 2-pyridinone-based human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase inhibitor in human plasma

A method for the determination of a 2-pyridinone-based specific HIV-1 reverse transcriptase inhibitor in human plasma is described. Plasma samples are extracted using phenyl solid phase extraction columns. The extract is analyzed via HPLC using a column-switching system to remove interferences from late-eluting endogenous components. Detection is based on UV absorbance at 314 nm. The assay was linear in the concentration range of 10–500 ng/ml, when 1-ml aliquots of plasma were extracted. The mean precision of the assay, expressed as the coefficient of variation, was 3.8%. The assay has been validated and utilized to support human pharmacokinetic studies.

Determination of a peptide–doxorubicin, prostate-specific antigen activated prodrug, and its active metabolites in human plasma using high-performance liquid chromatography with fluorescence detection: Stabilization of the peptide prodrug with EDTA

A method for the determination of I, a peptide-doxorubicin conjugate that was evaluated for the treatment of prostate cancer, and two of its active metabolites, doxorubicin and leucine-doxorubicin is described. Blood samples were chilled immediately after being drawn in order to prevent ex vivo entry of the metabolites into red blood cells. EDTA (10 mg/ml final concentration) was used to prevent plasma-mediated degradation of the peptide portion of the prodrug. After the addition of internal standard, plasma was prepared for analysis using a C-8 solid-phase extraction column. In order to overcome secondary ionic interactions with the silica-based extraction column, the analytes were eluted with ammonium hydroxide in methanol. The extracts were evaporated to dryness, reconstituted, and assayed by step change, gradient, reverse phase HPLC with fluorescence detection. Two interfering metabolites found in post dose plasma were chromatographically separated by an adjustment of the mobile phase pH. The within-day reproducibility of the doxorubicin and leucine-doxorubicin chromatographic retention times was improved by a brief washing of the analytical column with 90% acetonitrile after each injection. The range of the standard curve was 12.5-1250 ng/ml for doxorubicin and 25-2500 ng/ml for I and leucine-doxorubicin.

Determination of a cyclic heptapeptide, a novel fibrinogen receptor antagonist, in human plasma by high-performance liquid chromatography with automated precolumn 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.

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor.

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.