Joe Palandra

Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC-MS/MS.

A highly specific and sensitive immunoaffinity LC-MS/MS assay for quantification of human and cynomolgus monkey interleukin 21 (IL-21) was developed, qualified, and implemented. The workflow includes offline enrichment of IL-21 using an anti-IL-21 capture antibody, followed by isolation using magnetic beads, trypsin digestion, online enrichment of IL-21 derived tryptic peptides using antipeptide antibodies, and quantification using nanoflow LC-MS/MS. This assay was developed and qualified in human and cynomolgus monkey serum and tissues with a lower limit of quantitation of 0.78 pg/mL based on the intact cytokine. Both intra- and interbatch precision and accuracy, as well as stability and recovery, were found to be acceptable. IL-21 was not detected in serum from normal healthy volunteers or from autoimmune disease patients. However, IL-21 levels were quantified in cynomolgus monkey spleen and colon tissue and normal and inflammatory bowel disease (IBD) human colon tissue as well as hyperplasia human tonsils.

Enhancing the utility of alanine aminotransferase as a reference standard biomarker for drug-induced liver injury

Drug-induced liver injury (DILI) is the most frequent cause of discontinuation of new chemical entities during development. DILI can either be intrinsic/predictable or an idiosyncratic type. These two forms of DILI are contrasted in their manifestation and diagnosis. Even with regulatory guidance (FDA, 2009), there is still a gap in our ability to identify predictable DILI, both specifically and sensitively. Alanine aminotransferase (ALT) is the principal reference standard biomarker to diagnose DILI, yet its current application in preclinical to clinical translation for decision-making purposes has imperfections: (1) analytical ALT assay uniformity across industry would be aided by common analytical processes; (2) assessment of ALT toxicological performance in a large preclinical analysis would help to establish a true threshold of elevation for predictable DILI and improve translational use across various stages of pharmaceutical development and finally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to define and manage variations in clinical study subjects including rising body mass index (BMI) range and ALT upper limit of normal (ULN) in the broader population over time. The emergence of new hepatotoxicity biomarkers necessitates a parallel and equivalent assessment to the aminotransferases in a regulatory qualification model.

Endogenous ethanolamide analysis in human plasma using HPLC tandem MS with electrospray ionization

A sensitive and selective liquid chromatography tandem mass spectrometry (LC\MS\MS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50 microL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1 mm x 50 mm, 5 microm) at a flow rate of 0.30 mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5 mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M+H](+)-->m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05 ng/mL for AEA and LEA, 0.5 ng/mL for OEA and 1.0 ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.

Recommendations to qualify biomarker candidates of drug-induced liver injury

Certain compounds that induce liver injury clinically are not readily identified from earlier preclinical studies. Novel biomarkers are being sought to be applied across the pharmaceutical pipeline to fill this knowledge gap and to add increased specificity for detecting drug-induced liver injury in combination with aminotransferases (alanine and aspartate aminotransferase)--the current reference-standard biomarkers used in the clinic. The gaps in the qualification process for novel biomarkers of regulatory decision-making are assessed and compared with aminotransferase activities to guide the determination of safe compound margins for drug delivery to humans where monitoring for potential liver injury is a cause for concern. Histopathologic observations from preclinical studies are considered the principal reference standard to benchmark and assess subtle aminotransferase elevations. This approach correlates quite well for many developmental compounds, yet cases of discordance create dilemmas regarding which standard(s) indicates true injury. Concordance amongst a broader set of biomarker injury signals in a qualification paradigm will increase confidence, leading to accepted and integrated translational biomarker signals during safety assessment processes across the pharmaceutical industry, with academia, in government and in contractor laboratories.

High-performance liquid chromatography–tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.

Reduced serum myostatin concentrations associated with genetic muscle disease progression

Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC–MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.

Quantitative measurements of GDF-8 using immunoaffinity LC-MS/MS

Growth and differentiation factor 8 (GDF‐8) is a negative regulator of skeletal muscle mass and targeted by inhibitors to treat diseases associated with muscle loss. In order to enable clinical and translational investigations of GDF‐8 inhibitors, specific and sensitive measurements of GDF‐8 are necessary.