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

Biomarkers Provide Answers to Critical Questions and Expedite Development of Therapeutics

Researchers from academia, pharma, and the biotech industry gathered at the National University Health System of Singapore last month for the “Translational Strategies for Therapeutics Discovery Through Dementia Biomarkers” conference.

Daniel Hutcheson, Ph.D., director of discovery research and head of neuroscience & CNS safety pharmacology at Maccine, and organizer of the conference, emphasized the important role various types of biomarkers are having and will have in the development of disease-modifying therapies that can intervene in the early stages of dementia.

In the future, clinicians will likely utilize an array of biomarkers that may include cerebrospinal fluid (CSF) sampling, imaging, cognitive testing, and measurement of changes in vasculature and blood flow to detect and monitor alterations in normal brain function.

Biomarkers can provide answers to critical questions, said Dr. Hutcheson. “Is my drug doing what I predict it will do? Is it impacting the target and can it modulate or prevent the progressive changes in the brain that underlie neurodegeneration?”

John Beaver, Ph.D., director of imaging at Maccine, talked about the company’s use of nonhuman primates to evaluate the effects of compounds intended to modulate CSF biomarkers believed to have a role in the pathophysiology of neurodegeneration. Placement of a cannula allows for serial sampling of relatively large volumes of CSF without causing stress to the animal, and supports the evaluation of pharmacokinetics and pharmacodynamics.

Eric Siemers, M.D., senior medical director of the Alzheimer global research team at Eli Lilly, identified three main roles for biomarkers in Alzheimer disease—more accurate diagnosis, which can also aid in patient selection for clinical trials; indicators of efficacy to inform decision making in Phase I and II trials; and, potentially, for use as surrogate markers to substitute for measures of, for example, cognition or performance of activities of daily living. Dr. Siemers noted a trend in the field toward the increasing consideration of putative surrogate biomarkers in Phase II trials.

We have “a toolbox of different biomarkers,” and the challenge is to select the best tool for a particular purpose,” observed Dr. Siemers. “Biomarkers allow you to be very much translational, to go back and forth between what you see in the laboratory and in the clinic.”

Despite the failure of Lilly’s gamma-secretase inhibitor semagacestat in Phase III testing last year, administration of the investigational drug was associated with a substantial change in plasma Aß levels, indicating that the drug was getting into the brain and “we knew we were hitting the target,” said Dr. Siemers.

Solanezumab, the Lilly monoclonal antibody drug in Phase III testing to assess its ability to delay progression of AD, selectively binds Aß. “After a single dose of solanezumab, data has shown that the concentration of Aß in the blood increases several hundred-fold.”

Maccine, a preclinical contract research organization, offers discovery support and regulatory safety assessment services. The company relies on naturalistic models, biomarkers, and clinical technology platforms to better represent the clinical disease state.

Targeting Early-Stage Disease

Alzheimer disease (AD) is the principal cause of dementia in an aging population and, as such, is the main focus of ongoing research into biomarker and drug development targeting the neurodegenerative disorders characterized as “dementias” due to their effects on cognition and memory.

A primary goal of this research is the detection of biomarkers associated with predementia—the pathophysiologic changes in the brain that precede clinical, symptomatic disease.

Professor Colin Masters, University of Melbourne, described amyloid plaque, a hallmark of Alzheimer disease, as a “signpost,” a preclinical biomarker of disease that “may occur well before the onset of clinical symptoms.” But even amyloid plaque is a relatively late marker of AD, and biomarker research is focusing on more upstream processes: the initial misfolding of amyloid protein—in particular, beta-amyloid (Aß), and more specifically Aß(1-42)—which leads to protein aggregation and dimer, trimer, and oligomer formation, triggering neuroinflammatory processes and having a toxic effect on neuronal synapses.

Professor Masters spoke about “dramatic progress” during the past several years toward the goal of validating biomarkers of predementia. This includes the use of labeled ligands for detecting Aß and associated advances in positron emission tomography (PET) imaging, as well as the development of strategies for measuring levels of Aß and tau protein in CSF.

The correlation of blood-based biomarkers to preclinical disease is an ongoing challenge, but “we’re closing in,” said Professor Masters, noting that the focus is not only on Aß and tau levels in the blood, but also other surrogate markers such as molecules associated with inflammation that are emerging from proteomics studies.

In addition, there is growing interest in phosphorylated tau protein (phospo-tau), as well as other proteins that may be more reactive and destructive than Aß or tau.

As amyloid accumulates in plaques in the brains of AD patients, Aß levels in the CSF decrease while phosphorylated tau protein levels increase, with resulting cell death and neuronal loss, explained Dr. Hutcheson. The company has used biomarkers to show in real time and in a dose-dependent fashion the ability of certain drugs to knock down Aß(1-42).

Researchers at the University of Melbourne are working to validate biomarkers for predementia: 11C-PIB in the differential diagnosis of AD. [Images created by Victor Villemagne]

“Transformation of Aß into soluble synaptotoxic oligomers is the main underlying neuropathology in AD,” said Mark Treherne, Ph.D., CEO of Senexis, and the therapeutic goal is to modulate Aß aggregation. In his talk, Dr. Treherne described the company’s work in developing orally bioavailable small molecule inhibitors that can penetrate the CNS and both block and reverse the toxic effects of Aß oligomerization.

SEN1500 is Senexis’ lead compound in development for AD. It binds directly to Aß monomers and oligomers and inhibits aggregation. At the conference, Dr. Treherne presented recent functional and physiological data that follow on previous behavioral findings for SEN1500 and a follow-up compound, SEN1576. He described the correlation between biochemical markers and the pharmacological effects of the compounds, which the company hopes will help translate these results into human subjects and support a future Phase II trial.

Senexis is also developing SEN1176, which suppresses Aß(1-42)-induced production of inflammatory compounds by macrophages, such as nitric oxide, TNF-α, IL-1β, and IL-6. It has shown good oral bioavailability and in vivo efficacy in a model of learning and memory.

We have demonstrated that our compounds are neuroprotective, and “preserving synaptic integrity is essential to having a drug that works,” added Dr. Treherne.

This amyloid aggregation process shows how Senexis’ compounds interact with soluble oligomers.

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

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.

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.

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