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Oct 15, 2011 (Vol. 31, No. 18)

Dementia Studies Making Strides

Biomarkers Provide Answers to Critical Questions and Expedite Development of Therapeutics

  • Consortia-Driven Research

    Trevor Twose, Ph.D., is CEO of Mithridion, which is developing M1/M4 subtype-selective muscarinic receptor agonists that result in decreased levels of Aß and restoration of cognitive deficits.

    The M1 subtype, in particular, acts on cognition and memory and has disease-modifying potential, explained Dr. Twose. While proof of concept has been achieved for the ability of M1/M4 agonists to improve cognition, their ability to modify the course of AD has not yet been demonstrated.

    Muscarinic receptors work through G-protein coupled receptors (GPCRs) to trigger the inositol phosphate (IP) pathway. Mithridion used xanomeline, a known muscarinic agonist, as the basis for a primary in vivo nonisotopic, time-resolved FRET assay designed to measure selective muscarinic M1 activity.

    Subsequent lead optimization has led to the development of MI-10-022, a fourth-generation lead candidate that is selective for M1/M4 activity, is about seven times more potent than xanomeline, according to Mithridion data, efficiently crosses the blood-brain barrier, and is resistant to metabolism.

    MI-10-022 is also a potential first-in-class monotherapy for schizophrenia, according to Dr. Twose. The compound has completed preclinical development and is ready to enter IND-enabling studies, including toxicology, analytical methods development, and optimization of production and large-scale manufacturing.

    Biomarkers have been critical to the development of MI-10-022. “They let us answer the question of how well our experimental compounds engage the muscarinic M1 receptor in the hippocampus,” said Dr. Twose. They have also allowed the company to perform studies in the salivary glands of animals to measure simultaneously in the same animal the compound’s activity against the M1 and M3 subtypes to assess its comparative selectivity.

    “Research is exploding in this area,” concluded Dr. Twose, with the emergence of AD-focused consortia including the Alzheimer’s Disease Neuroimaging Initiative (ADNI) based at the University of California, San Francisco, and the more recently formed Dominantly Inherited Alzheimer Network (DIAN) consortium out of Wash.ington University in St. Louis.

    By following individuals with the inherited form of AD who carry dominant mutations in the presenilin-1 gene, which are 100% penetrant for early-onset AD, it is possible to correlate CSF and imaging biomarkers with the onset of preclinical disease.

    Professor Masters is a member of the core research team of AIBL, the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing. Launched in 2006, AIBL is a prospective longitudinal study that includes patients with AD, mild cognitive impairment (MCI, a precursor to AD), and healthy volunteers.

    At the Singapore conference, Professor Masters described several goals for AIBL: to develop and confirm diagnostic biomarkers and psychometrics for objective monitoring of disease progression; to understand the role of lifestyle factors; and to inform the development of preventive and therapeutic strategies. For biomarker research overall, Professor Masters concluded that “PET scans are proving enormously valuable, and we are getting close to being able to see registration at the FDA level for ligands to detect amyloid.”

    When that occurs, “testing for preclinical disease will be a reality.” In the future, PET scanning might be used more broadly as a screening tool downstream of a suspicious CSF or blood test.

    The aim of AIBL is “to get a handle on the natural history of AD, including preclinical progression and [variability in the] rates of progression. The single major problem we have is confounding diseases—mainly vascular disease and small strokes in the brain,” with the effects of ischemia caused by stroke being difficult to distinguish from preclinical AD.

  • New Approach to Protein Detection and Profiling

    Click Image To Enlarge +
    In ProteoMiner technology each bead features a different hexapeptide ligand with affinity for specific proteins in a sample. Samples are applied to the beads, allowing proteins to bind their specific ligands. Proteins in excess are washed away, and those proteins bound to the beads are eventually eluted, allowing furth downstream analysis.

    The wide dynamic range and complexity of biological samples and bodily fluids has hampered biomarker studies aimed at identifying disease-specific proteins and their post-translationally modified (PTM) variants. Even when highly sensitive mass spectrometers are used, the presence of high-abundance proteins obscures the detection of lower-abundance proteins and PTMs often associated with a disease or physiological state.

    To address this problem, immunodepletion columns targeting the most abundant proteins within plasma or serum have been used. However, owing to differences in the most abundant proteins among various samples, their applicability to other samples is limited

    An ideal solution would be universally applicable across samples: it would boost differential analyses (i.e., identify changes inherent to a specific disease or biological states) and protein/PTM detection studies. The commercially available hexapeptide libraries, including ProteoMiner from Bio-Rad, partially deplete high-abundance proteins and simultaneously concentrate low-abundance proteins in a variety of samples.

    In two recent publications, researchers at the University of Minnesota have investigated if hexapeptide libraries would boost differential analyses and PTM detection in human saliva. Saliva promises a noninvasive alternative to serum, long the gold standard, for biomarker discovery.

    Sri Bandhakavi et al. applied hexapeptide libraries to successfully identify differences in low-abundance proteins in human saliva putatively associated with breast cancer. They demonstrated a novel workflow that enables delineation of low-abundance proteins and retains quantitative information subsequent to treatment with hexapeptide libraries. Importantly, the workflow is readily transferable to other samples/studies and offers a new approach for boosting sensitivity of biomarker investigations by researchers.

    For analysis of PTMs, Bandhakavi et al. and Matthew Stone et al. coupled hexapeptide library treatment of saliva with N-glycopeptide and phosphopeptide enrichment followed by mass spec. Samples treated with hexapeptide libraries identified twice as many N-glycoproteins (and their sites of modification) and phosphoproteins (and their sites of modification).

    Although these studies were restricted to human saliva, hexapeptide libraries have been applied to boost overall protein detection in a variety of biological samples and bodily fluids.

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