Validation of a method for the determination of AMG 579 in cerebrospinal fluid with a focus on sample collection procedures for clinical trials.

Analysis of pharmaceutical compounds in cerebrospinal fluid (CSF) may present challenges due to the combination of the low protein content in this matrix and relatively low drug concentrations, often corresponding to free drug concentrations in plasma, typically found in CSF. A 30% loss of AMG 579 was observed during preparation of quality control samples and further investigation determined that this loss was likely due to binding to collection tubes. This observation also highlighted the possibility of additional losses of AMG 579 that could occur during collection of clinical samples, such as binding to catheters used in the collection of CSF.

Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium.

Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumor cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (proheparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro.

Bioanalytical method validation: concepts, expectations and challenges in small molecule and macromolecule--a report of PITTCON 2013 symposium.

The concepts, importance, and implications of bioanalytical method validation has been discussed and debated for a long time. The recent high profile issues related to bioanalytical method validation at both Cetero Houston and former MDS Canada has brought this topic back in the limelight. Hence, a symposium on bioanalytical method validation with the aim of revisiting the building blocks as well as discussing the challenges and implications on the bioanalysis of both small molecules and macromolecules was featured at the PITTCON 2013 Conference and Expo.

Direct quantitative analysis of a 20 kDa PEGylated human calcitonin gene peptide antagonist in cynomolgus monkey serum using in-source CID and UPLC-MS/MS.

PEGylation is a successful strategy to improve the pharmacokinetic and pharmaceutical properties of therapeutic peptides. However, quantitative analysis of PEGylated peptides in biomatrix by LC-MS/MS poses significant analytical challenge due to the polydispersity of the polyethylene glycol (PEG), and the multiple charge states observed for both the peptide and PEG moieties. In this report, a novel LC-MS/MS method for direct quantitative analysis of 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkey serum is presented.

Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC-MS/MS.

An LC-MS/MS method using pre-column derivatization with phenylisothiocyanate (PITC) was developed to quantify glycine in human cerebrospinal fluid (CSF) and applied to the determination of glycine in human samples collected during clinical testing. The calibration curve range for the assay was 50-10,000 ng/mL and ¹³C₂¹⁵N-glycine was used as an internal standard. Artificial CSF was used as a surrogate matrix for standards due to the presence of endogenous glycine in human CSF and this approach was validated with additional experiments involving either standard addition, or stable labeled glycine as an alternate calibration standard for endogenous glycine.

Serum hepcidin but not prohepcidin may be an effective marker for anemia of inflammation (AI).

Anemia in cancer patients can result from a complex interaction of numerous factors including iron deficiency, inflammation, toxicity related to therapy and effect of cancer on the marrow. Determining effective anemia treatment can therefore be complex, requiring a combination of diagnostic tests. Research on iron metabolism has highlighted the importance of hepcidin and its potential role in development of anemia of inflammation (AI).

Pharmacokinetics of anti-hepcidin monoclonal antibody Ab 12B9m and hepcidin in cynomolgus monkeys.

Hepcidin is a key regulator responsible for systemic iron homeostasis. A semi-mechanistic PK model for hepcidin and a fully human anti-hepcidin monoclonal antibody (Ab 12B9m) was developed to describe their total (free + bound) serum concentration-time data after single and multiple weekly intravenous or subcutaneous doses of Ab 12B9m. The model was based on target mediated drug disposition and the IgG-FcRn interaction concepts published previously.

Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth.

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g.

Selective inhibitors of the mutant B-Raf pathway: discovery of a potent and orally bioavailable aminoisoquinoline.

The discovery and optimization of a novel series of aminoisoquinolines as potent, selective, and efficacious inhibitors of the mutant B-Raf pathway is presented. The N-linked pyridylpyrimidine benzamide 2 was identified as a potent, modestly selective inhibitor of the B-Raf enzyme. Replacement of the benzamide with an aminoisoquinoline core significantly improved kinase selectivity and imparted favorable pharmacokinetic properties, leading to the identification of 1 as a potent antitumor agent in xenograft models.

Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS.

Introduction: Hepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation.

Design and synthesis of conformationally constrained glucagon-like peptide-1 derivatives with increased plasma stability and prolonged in vivo activity.

A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated.

Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans.

The vanilloid receptor TRPV1 has been identified as a molecular target for the treatment of pain associated with inflammatory diseases and cancer. Hence, TRPV1 antagonists have been considered for therapeutic evaluation in such diseases. During Phase I clinical trials with AMG 517, a highly selective TRPV1 antagonist, we found that TRPV1 blockade elicited marked, but reversible, and generally plasma concentration-dependent hyperthermia.

Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays.

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS.

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW>4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution.

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine.

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20.

Glycosaminoglycans and the regulation of allergic inflammation.

Glycosaminoglycans (GAGs) are large, polyanionic molecules expressed throughout the body. The GAG heparin, co-released with histamine, is synthesised by and stored exclusively in mast cells, whereas the closely related molecule heparan sulphate is expressed, as part of a proteoglycan, on cell surfaces and throughout tissue matrices. These molecules are increasingly thought to play a role in regulation of the inflammatory response and heparin like molecules are now being seriously considered to hold potential in the treatment of inflammatory diseases such as asthma.

Evaluation of microdosing strategies for studies in preclinical drug development: demonstration of linear pharmacokinetics in dogs of a nucleoside analog over a 50-fold dose range.

The technique of accelerator mass spectrometry (AMS) was validated successfully and used to study the pharmacokinetics and disposition in dogs of a preclinical drug candidate (7-deaza-2'-C-methyl-adenosine; Compound A), after oral and intravenous administration. The primary objective of this study was to examine whether Compound A displayed linear kinetics across subpharmacological (microdose) and pharmacological dose ranges in an animal model, before initiation of a human microdose study. The AMS-derived disposition properties of Compound A were comparable to data obtained via conventional techniques such as liquid chromatography-tandem mass spectrometry and liquid scintillation counting analyses.

Pharmacokinetics of etoricoxib in patients with hepatic impairment.

The effect of hepatic insufficiency on the pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase-2, was investigated following administration of single and multiple oral doses to mild hepatic insufficiency patients (Child-Pugh score of 5 to 6), multiple oral doses to moderate hepatic insufficiency patients (Child-Pugh score of 7 to 9), and single intravenous doses to both mild and moderate hepatic insufficiency patients. A trend of decreasing systemic clearance with increasing hepatic impairment was observed. Absorption of etoricoxib was unaffected by hepatic impairment.

Platelets are essential for leukocyte recruitment in allergic inflammation.

Background: The role of platelets in inflammation is recognized but poorly characterized, and little is known of their interaction with leukocytes. However, platelet-leukocyte interactions have been demonstrated in cardiovascular disease, culminating in enhanced leukocyte recruitment.

Objectives: This study was undertaken to assess the possibility and potential role of similar phenomena occurring in asthmatic patients, a murine model of allergic inflammation, and in vitro adhesion studies.

Single- and multiple-dose pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase-2, in man.

The single- and multiple-dose pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase-2, were examined in two clinical studies. Single-dose pharmacokinetics--including dose proportionality, absolute bioavailability of the highest dose-strength (120-mg) tablet, and the effect of a high-fat meal on the bioavailability of that tablet--were investigated in a two-part, open, balanced crossover study in two panels of healthy subjects (12 per panel). Steady-state pharmacokinetics were investigated in an open-label study in which 24 healthy subjects were administered 120-mg single and multiple (once daily for 10 days) oral doses of etoricoxib tablets.

Amino acid and peptide analysis using derivatization with p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether and capillary electrophoresis with electrochemical detection.

The amine derivatization reagent p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether (NDTE) was used in conjunction with capillary electrophoresis (CE) and electrochemical detection (EC) for the pre-separation derivatization of primary amine analytes present in aqueous solution. Glycine, several dipeptides and angiotensin II were used as model analytes. A miniaturized EC detection cell was designed and fabricated, which featured a fractured-joint field decoupler with a fixed end-column carbon fiber electrode.