Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies.

Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5-15 ng protein (per 5 µg total protein per lane), precision of 3.3 to 25.5%, and accuracy of - 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at - 70°C. Furthermore, our WB method was compared to the results from our recently published LC-MS method. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissues (created in Biorender.com). Step 1 involves protein extraction from skeletal muscle tissue lysates from control, DMD, or BMD biospecimen. Step 2 measures total protein concentrations. Step 3 involves running gel electrophoresis with wild-type dystrophin (wt-DYS) from muscle tissue extracts alongside mini-dystrophin STD curve and mini-DYS and protein normalization with housekeeping GAPDH.

Ultra-Sensitive Measurement of IL-17A and IL-17F in Psoriasis Patient Serum and Skin.

Interleukin 17 is a family of cytokines that play a central role in many autoimmune and inflammatory diseases. IL-17A has been implicated as a key driver of psoriasis, mediating a chronic cycle of T-cell activation, keratinocyte proliferation and angiogenesis. It has been hypothesized that expression of IL-17A and the related cytokine IL-17F could be used as predictive biomarkers for therapeutic response, though they have been difficult to measure locally or in circulation because of their low abundance. We developed ultrasensitive methods for measuring IL-17A and IL-17F in human serum samples and found that serum from psoriasis patients had higher and a broader range of concentrations of both IL-17 proteins compared to healthy volunteers. We also adapted these methods for tissue biopsies and saw higher concentrations of both IL-17 proteins in psoriatic lesions, but they were undetectable in non-lesional skin from the same patients.

When close is not close enough: a comparison of endogenous and recombinant biomarker stability samples.

Dr Stephanie Fraser is an Associate Research Fellow in the Pharmocokinetics, Dynamics and Metabolism department at Pfizer, Groton, Connecticut. Since 2010 she has led a small but ambitious group of scientists that provide ligand-binding and immunoassay-based support to clinical biomarker programs across multiple therapeutic areas. Prior to joining Pfizer, Stephanie spent 5 years in preclinical toxicology at Charles River Laboratories where she managed a flow cytometry laboratory. She received her PhD in cellular and molecular biology from the University of Nevada, Reno in 1999 and has since focused on biomarker development and fit-for-purpose bioanalytical assays. Stability for biomarkerassays should be established during method validation using actual samples. Due to contradictory reference papers and a near absence of biomarker guidance documents actual samples are commonly replaced with spiked validation samples. This practice often fails to identify the stability of the endogenous biomarker. Spiked QC and endogenous biomarker sample data were collected for two immunoassays, TGF- ß1 and IL-13. Following one freeze/thaw cycle purified TGF-ß1 recovery ranged between 87-110% whereas endogenous TGF-ß1 was 5-96%. Spiked recombinant IL-13 validation samples were stable for 4 months, whereas placebo samples were stable for 15 months. In these two cases stability established with purified and recombinant protein did not reflect the endogenous protein stability.

Due diligence in the characterization of matrix effects in a total IL-13 Singulex™ method.

After obtaining her PhD in Cellular and Molecular biology from the University of Nevada, Reno, Stephanie has spent the last 15 years in the field of bioanalysis. She has held positions in academia, biotech, contract research and large pharma where she has managed ligand binding assay (discovery to Phase IIb clinical) and flow cytometry (preclinical) laboratories as well as taken the lead on implementing new/emergent technologies. Currently Stephanie leads Pfizer's Regulated Bioanalysis Ligand Binding Assay group, focusing on early clinical biomarker support. Interleukin (IL)-13, a Th2 cytokine, drives a range of physiological responses associated with the induction of allergic airway diseases and inflammatory bowel diseases. Analysis of IL-13 as a biomarker has provided insight into its role in disease mechanisms and progression. Serum IL-13 concentrations are often too low to be measured by standard enzyme-linked immunosorbent assay techniques, necessitating the implementation of a highly sensitive assay. Previously, the validation of a Singulex™ Erenna(®) assay for the quantitation of IL-13 was reported. Herein we describe refinement of this validation; defining the impact of matrix interference on the lower limit of quantification, adding spiked matrix QC samples, and extending endogenous IL-13 stability. A fit-for-purpose validation was conducted and the assay was used to support a Phase II clinical trial.

Active glucagon-like peptide 1 quantitation in human plasma: a comparison of multiple ligand binding assay platforms.

There are a wide variety of ligand binding assay platforms available for implementation in present day bioanalytical laboratories. Selecting the platform that best suits a particular project's needs is highly dependent upon multiple assay characteristics. The active form of glucagon-like protein (GLP-1) is a biomarker of interest for type 2 diabetes (T2DM), and therefore a common target for quantitation. Previous projects requiring active GLP-1 measurements involved the use of a labor intensive ELISA, spurring an investigation towards other potential assay platforms. To that end, four separate ligand binding assay formats (standard ELISA, electrochemiluminescence, Gyrolab, and Singulex) were evaluated. The platforms were compared for numerous assay parameters including dynamic range, sample volume requirements, throughput, and cost. Additionally, thirty individual donor plasmas were run with each assay as representative study samples. Although our evaluation did not show any platform that was better than others in all assay characteristics, there was one that was best in sensitivity (Singulex) and one that was best in throughput and sample volume requirements (Gyrolab). The lack of a technology that was best in all categories underscores the importance of due diligence when selecting an assay platform; there are no silver bullets, and one must take into account what is necessary for project needs and the intended use of the data.

Prandial ghrelin attenuation provides evidence that des-acyl ghrelin may be an artifact of sample handling in human plasma.

BACKGROUND: Plasma acyl and des-acyl ghrelin are thought of as components of total ghrelin, but this has never been validated using ex vivo spiking experiments, human sample collection comparisons and fit-for-purpose translatable assays. RESULTS: Acyl ghrelin plasma stability was analyzed by LC-MS/MS and it revealed that acyl ghrelin is enzymatically and chemically converted to des-acyl ghrelin in the presence of active serine proteases and HCl. ELISAs with less than 30% total error were used to assess acyl ghrelin behavior in matched authentic human samples. Acyl and total ghrelin were not statistically different in 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride samples and acyl ghrelin losses in K(2)EDTA plasma were accounted for in des-acyl ghrelin formation. CONCLUSION: Acyl ghrelin is total ghrelin and des-acyl ghrelin should not be detectible in healthy human plasma under optimal sample handling and assaying conditions.

Comparison of four distinct detection platforms using multiple ligand binding assay formats.

Several detection platforms are available for ligand binding assays (LBA), each claiming superiority in sensitivity and dynamic range. However, little information exists in the literature directly comparing the various LBA platforms for quantitation. We have tested four common platforms to evaluate and compare the interchangeability of detection platforms by comparing sensitivity and dynamic range to a colorimetric LBA. The detection platforms compared are: colorimetric, chemiluminescence, time-resolved fluorescence (TRF) and electrochemiluminescence (ECL). Five different LBA protocols were tested with each of the detection endpoints. The assay protocols include the following ligand binding assay formats: direct binding, sandwich ELISA, competitive and cell based ELISA. We found that no detection platform consistently performed better than all the others and it was not possible to predict which platform would perform best for a given assay protocol. We also found surprising differences in assays (plate coating efficiency, low signal) which add to difficulty in choosing the best platform ad hoc. We propose here that in developing new assay protocols for detection of biotherapeutic agents, multiple detection platforms should be tested in order to forward the best assays possible and for the right reasons.

Comparison of an antibody capture and a cell capture ligand-binding assay to quantify a monoclonal therapeutic in serum.

BACKGROUND: Ligand-binding assays are a tool used for the quantification of antibody therapies. When assay format changes are required during the drug development process it is advisable to assess these formats ensuring the resulting data can be compared. In this article, we outline the method and results obtained comparing an anti-idiotype capture and a cell-capture ligand-binding assay. RESULTS: Comparison of results for all quality controls between assays were within acceptance limits, with the exception of the low quality control. Statistical analysis of the results demonstrated 95% power to detect a 20% difference between data sets. Subsequent analysis of unknown samples further confirmed 98% power to detect a 20% difference between data sets. CONCLUSION: Results obtained using two assay formats are statistically comparable to each other.

A practical guide for the stabilization of acylghrelin in human blood collections.

OBJECTIVE AND METHODS: To better understand acylghrelin plasma stability, human synthetic acylghrelin was spiked into plasma and tracked by liquid chromatography tandem mass spectrometry. To investigate the best method for quantifying clinical plasma acylghrelin levels, pre- and postprandial human blood was collected from healthy volunteers (n=6) using various sample collections and treatments. Plasma ghrelin levels from human blood collections were analysed by enzyme-linked immunosorbant assay (ELISA). RESULTS: Acylghrelin's half-life in plasma was approximately 45 min with the formation of des-acylghrelin approaching 50% before the end of the 60-min incubation. Loss of acylghrelin inversely correlated with an increase in des-acylghrelin (P<0.008; r(2) =0.870). Plasma pretreated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or protease inhibitor cocktail without acidification resulted in no detectible acylghrelin losses. Acylghrelin measurements with AEBSF-treated blood were minimally 40% higher than sodium citrate/citric acid, K(2) EDTA, aprotinin/HCl and P800 collections. HCl addition to AEBSF-treated plasma did not provide enhanced acylghrelin stability and induced deacylation at and above the 100 mM final concentration. Pre- and postprandial ghrelin attenuation was investigated using aprotinin/HCl, AEBSF, protease inhibitor cocktail and no treatment for blood and plasma preparations. Fasting samples treated with AEBSF and protease inhibitor cocktail were approximately threefold higher than aprotinin/HCl and control treatments (P<0.03). Pre- and postprandial ghrelin attenuation was approximately twofold different (P<0.04) with significant counterintuitive trends in aprotinin/HCl and K(2) EDTA groups. CONCLUSIONS: Our data suggest that AEBSF addition to K(2) EDTA blood immediately after collection without plasma acidification, processing on ice and 14-day 70 °C storage is the best treatment for accurately quantifying acylghrelin in human plasma.

Combination therapy enhances the inhibition of tumor growth with the fully human anti-type 1 insulin-like growth factor receptor monoclonal antibody CP-751,871.

PURPOSE: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor. EXPERIMENTAL DESIGN: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (K(d) = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties. RESULTS: This antibody blocks binding of IGF-1 to its receptor (IC(50) 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC(50) 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. CONCLUSION: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.

Design and SAR of thienopyrimidine and thienopyridine inhibitors of VEGFR-2 kinase activity.

Novel classes of thienopyrimidines and thienopyridines have been identified as potent inhibitors of VEGFR-2 kinase. The synthesis and SAR of these compounds is presented, along with successful efforts to diminish EGFR activity present in the lead series.