Anti-repulsive Guidance Molecule C (RGMc) Antibodies Increases Serum Iron in Rats and Cynomolgus Monkeys by Hepcidin Downregulation.

High levels of hepcidin, the main regulator of systemic iron metabolism, lead to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies ABT-207 and h5F9-AM8 which inhibit hemojuvelin/repulsive guidance molecule C (RGMc) and downregulate hepcidin. We conducted single-application and dose response studies to understand the antibodies' mechanism and subchronic toxicology studies to exclude safety-related concerns. Investigation was carried out at different biological levels through qPCR, Affymetrix, liquid chromatography coupled with mass spectrometry (LC-MS/MS), histopathology, serum iron, unsaturated iron binding capacity (UIBC), and drug concentration measurements. After a single application of these antibodies, hepcidin expression in liver and its serum protein levels were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of approximately 80-fold higher than ABT-207. When hepcidin levels were downregulated, iron deposition in the liver was visible histologically 1 week post application. These antibody-mediated iron depositions were not associated with any adverse toxicologically relevant effect at the doses and time points evaluated. Iron depositions seen after 14 weekly treatments with ABT-207 were reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease.

Implementing DBS methodology for the determination of Compound A in monkey blood: GLP method validation and investigation of the impact of blood spreading on performance.

BACKGROUND: This article describes validation work for analysis of an Abbott investigational drug (Compound A) in monkey whole blood with dried blood spots (DBS). The impact of DBS spotting volume on analyte concentration was investigated. RESULTS: The quantitation range was between 30.5 and 10,200 ng/ml. Accuracy and precision of quality controls, linearity of calibration curves, matrix effect, selectivity, dilution, recovery and multiple stabilities were evaluated in the validation, and all demonstrated acceptable results. Incurred sample reanalysis was performed with 57 out of 58 samples having a percentage difference (versus the mean value) less than 20%. A linear relationship between the spotting volume and the spot area was drawn. The influence of spotting volume on concentration was discussed. CONCLUSION: All validation results met good laboratory practice acceptance requirements. Radial spreading of blood on DBS cards can be a factor in DBS concentrations at smaller spotting volumes.

High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography.

Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC-MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC-MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC-MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 degrees C) temperature and the low temperature (4 degrees C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2-4 degrees C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS-->SSA to < or = 0.10% and the conversion of SSA-->SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL(-1) for both SS and SSA and a sample volume of 100 microL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100+/-20% of the original analysis results.

HPLC-MS/MS determination of a hardly soluble drug in human urine through drug-albumin binding assisted dissolution.

ABT-263 is under development for treatment of cancer. In order to support clinical trials, an analytical method for ABT-263 quantification in human urine became necessary. Due to the extremely poor solubility of ABT-263 in aqueous and most common organic solvents, a critical step was to dissolve the drug into urine matrix. Although other potential approaches could be used, addition of powder albumin was found to be the most advantageous. Albumin powder does not significantly alter urine sample volume (< or = 2.8%) and a range of albumin to urine sample volume ratios can be allowed for full recovery of drug and thus accurate quantification. The procedure is fairly simple and can potentially be a universal approach for compounds with low solubility in urine, but strong protein binding. The method has been validated to support clinical trials.

Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS.

Liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) has played an important role in pharmacokinetics and metabolism studies at various drug development stages since its introduction to the pharmaceutical industry. This article reviews the most recent advances in sample preparation, separation, and the mass spectrometric aspects of high-throughput quantitative bioanalysis of drug and metabolites in biological matrices. Newly introduced techniques such as ultra-performance liquid chromatography with small particles (sub-2 microm) and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC-MS/MS. Sample preparation formatted to 96-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly impacting throughput. On-line solid-phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both time and labor required to produce bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media including large particles for turbulent flow chromatography, restricted access materials (RAM), monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland systems. In the end, this paper also discusses recent studies of matrix effect in LC-MS/MS analysis and how to reduce/eliminate matrix effect in method development and validation.

A monolithic-phase based on-line extraction approach for determination of pharmaceutical components in human plasma by HPLC-MS/MS and a comparison with liquid-liquid extraction.

An automated procedure using monolithic-phase based on-line extraction is described for pharmaceutical component analysis in plasma by LC-MS/MS. In this approach, a short monolithic C(18) 4.6 mm x 10 mm cartridge is used for high flow extraction at 4 mL/min. Plasma samples were subjected to protein precipitation first with acetonitrile, and the supernatant was diluted and loaded onto a monolithic cartridge. Sample elution was accomplished with narrow-bore LC-MS/MS system. A method for determination of Amprenavir (APV) and Atazanavir (AZV) in human plasma was developed with this approach. After 0.1 mL of plasma was transferred into each well of a 96-well plate by a liquid handler, the rest of sample preparation time typically only takes about 20 min. A Phenomenex Luna C18(2) 2.0 mm x 150 mm analytical column was used for the separation at a flow rate of 0.3 mL/min. The run time for each sample was 4 min. The standard curve range was 2.77-1520 ng/mL for Atazanavir, and 4.50-2560 ng/mL for Amprenavir. The accuracy (%bias) at the lower limit of quantitation (LLOQ) for Atazanavir was 2.7% and the precision (%CV) at the LLOQ was 7.9%, while the accuracy at LLOQ for Amprenavir was -1.3% and the precision at LLOQ was 7.8%. The inter-day %bias and %CV of the quality control samples of Atazanavir were < or = 4.5% and < or = 6.5%, respectively. The inter-day %bias and %CV of the quality control samples of Amprenavir were < or = 1.1% and < or = 7.2%, respectively. Coefficients of determination, a measure of linearity, ranged from 0.993 to 0.999. Very low carry-over (0.006%) even after high standard sample was demonstrated in the monolithic-phase based method. Other characteristics of such method include high recovery and good tolerance to matrix effect, which was demonstrated by 12 lots of plasma. The back pressure of the monolithic extraction cartridge remained the same after 450 samples injected. The performance of the monolithic-phased on-line extraction method was compared with that done by an automated 96-well liquid-liquid extraction procedure, which was carried out using hexane:ethyl acetate as the extraction solvent. The results showed that similar precision and accuracy were achieved by both methods.

Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4 receptor agonist and radioligand.

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.