The in vitro liposaccharide stimulant (LPS) whole blood assay is the major assay for this biomarker hunting, Dr. Qi noted. Two of the big challenges of biomarker discovery are a high degree of interindividual variability and sensitivity that is responsiveness to low concentration of inhibitor (compound).
“The statistics play an important role here,” he said. “We have to do iterative experiments with a certain number of donors. The statistical analysis guides us toward optimal LPS concentration for in vitro stimulation, that is the optimal inhibition window for an IKKbeta inhibitor.”
Two genes were identified as high potential biomarkers—IL6 and IL1b as mRNA biomarkers—then subsequently as protein biomarkers. In both cases, responses were statistically significant at their EC50 concentrations in the gene (Taqman) and protein (ELISA) expression experiments with a full dose range of inhibitor. These two genes will serve as ex vivo predictive MOA biomarkers in the Phase I trial.
The Phase I trial ex vivo study protocol will: 1) Dose volunteers with compound/ inhibitors, 2) take blood at estimated Cmax and Cmin, and 3) stimulate cells from blood with LPS and look for downstream readouts—ELISAs for IL-6, IL1b.
Jin Sam You, Ph.D., of Monarch LifeSciences noted that cardiovascular disease (CVD) and atherosclerosis is the leading cause of death in the U.S.
CVD is greatly exacerbated by metabolic syndrome which is characterized by a group of risk factors including obesity, dyslipidemia, glucose intolerance, insulin resistance, and hypertension. Many studies have linked high saturated fat and fructose diets to insulin resistance and diabetes, Dr. You said. Ossabaw swine fed an excess calorie, high trans-fat/cholesterol diet develop all components of MetS.
Pigs provide a good research model in translational research because they have anatomy, physiology, and pathophysiology comparable to humans, You continued. They develop similar vascular disease and have similar lipid profiles. Additionally, their vessel size is similar to that of humans, which allows for clinically relevant interventions to be assessed. The Ossabaw miniature swine is unique among pig animal models in that the Ossabaw pig mimics humans by developing metabolic syndrome and atherosclerosis when maintained sedentary on an excess calorie atherogenic diet.
Differential Expression Analysis
In the discovery phase, differential expression proteomic analysis was obtained using an LC/MS-based label-free protein quantification method to profile the global protein expression.
Subsequent statistical modeling was utilized to select proteins for a predictive or diagnostic biomarker panel, Dr. You added. Candidate biomarkers will be validated by developing and using targeted MRM/SRM mass spec assays (LifeMarker™ assays) and testing the candidate biomarkers in new, larger sample sets. This panel of biomarkers will then be used in the development of clinically useful diagnostic tools that are more predictive than those currently available.
Atherosclerosis in Ossabaw swine was demonstrated to increase with severity of MetS as confirmed grossly and by angiography and intravascular ultrasound. The label-free LC/MS-based quantitative proteomic analysis method of protein expression profiles was validated in this model by reproducing known correlations to atherosclerosis and metabolic syndrome, Dr. You concluded. The many proteins identified provide a great opportunity to find biomarkers and predictive panels of biomarkers for cardiovascular disease.
Mass Spec Platforms
Critical to using mass spectrometry platforms for biomarker development is the need to progress from the general profiling of proteins to the targeting of specific protein panels, Randall W. Nelson, Ph.D., research professor and director of the molecular biosignatures analysis unit at The Biodesign Institute, Arizona State University.
The value of such a targeted approach, Dr. Nelson observed, lies in the ability to differentiate microheterogeneity in the proteins under investigation—variation in the chemical structure of the amino acid sequence of a protein that does not produce a major change in its properties such as gene, translational, and posttranslational modifications—and to generate data on only the specific molecular determinants relevant to disease. His talk focused on using targeted mass spectrometric immunoassays to investigate human plasma and urinary proteins in healthy and disease cohorts.
Dr. Nelson’s group uses mass spectrometry to identify biomarkers by looking for specific variances in proteins, characterizing them one by one. Plasma protein is increasingly their area of interest.
While Dr. Nelson served as the president, CEO, and founder at Intrinsic Bioprobes, the company adopted a strategy by looking at routine proteins in more detail and identified post-translational modifications, point mutations, variants, and truncations that defined the proteome. These variants, in turn, provide information that can be used to design targeted diagnostic assays, stratify patients in clinical trials, monitor patients more effectively, and facilitate pharmacokinetic studies by following in vivo changes of therapeutic proteins.
Dr. Nelson shared results with the conference illustrating the ability to detect low level protein variants relevant to type 2 diabetes, and how these findings are subsequently used to develop advanced assays for disease diagnosis and monitoring. Currently, these assays are out of R&D and being used in CLIA labs. Dr. Nelson forsees diagnostic use within the next 5–10 years.