The signals from cell surface receptors to downstream molecules within the cell make for a complex system, one that is only now beginning to yield a mere fraction of its workings. One molecule can elicit multiple activities that branch outward through numerous pathways, many of which intersect synergistically in ways that are difficult to predict. This lack of prediction leads to the fundamental problem associated with any pharmaceutical—the unintended consequences of treatment, also known as side effects.
High-throughput technology platforms and computing power act together in ways that are greater than their sum, especially when dealing with the millions of data points needed to understand these pathways. As in other areas of biology that are exploding thanks to these technologies, pathway research is beginning to identify the physiologic matrix underlying disease processes in a more selective manner, raising the probability of discovering ever-more selective agents.
In addition, these efforts are uncovering potential targets for treating refractory disease and are providing a more detailed look at the basic receptor mechanisms that drive cell signaling. At the recent Cambridge Healthtech Drug Discovery Conference in Boston, researchers converged to discuss some of these results.
Alexis Borisy, president and CEO at CombinatoRx (www.combinatorx.com), said that “the network is all-important” to elucidate new combinations to identify the selective activity of new agents. CombinatoRx uses a high-throughput multidimensional discovery platform focused on searching all possible combinations. “By screening through tens of millions of combinations in wet biological assays, cHTS finds pairs of molecules that are interacting synergistically, unmasking previously silent effects.”
Essentially, “the CombinatoRx drug discovery engine, cHTS™, redefines a target. Instead of a single molecule, the platform identifies the correct nodes in a network, which can then be collectively treated as a target.” One candidate that has emerged from this approach is a dissociated steroid, which has “the power and effectiveness of a glucocorticoid, without the safety issues and side effects,” claimed Borisy.
A combination of dipyridamole and prednisone, CRx-102, is in Phase IIb trials for the treatment of both rheumatoid arthritis and osteoarthritis. Another agent in Phase II trials, CRx-191, is a combined steroid/antidepressant (mometasone and nortryptiline) for the treatment of skin inflammation, including psoriasis. A third candidate is in Phase II trials; CRx-401 combines bezafibrate and diflunisal to treat type 2 diabetes patients with elevated triglycerides and low HDL levels.
Finding Beneficial Pathways
Joel E. Tocker, Ph.D., director of inflammation research at Amgen (www.amgen.com), is pursuing early work on the activity of IL-17 and the IL-17 receptor complex as inflammatory mediators in asthma, inflammatory bowel disease, rheumatoid arthritis, and psoriasis.
Dr. Tocker reported that Amgen is making progress in understanding the IL-17 pathway, especially its pathologic role in autoimmune disease and inflammation, adding that “quite a lot of work is being done with effector T cells that specifically produce large amounts of IL-17.
“While there may be a role of IL-17 in immune surveillance against viral or bacterial pathogens, there is also data indicating that too much IL-17 is pathogenic and can elicit a proinflammatory response. It can induce the accumulation of leukocytes as well as have particularly interesting effects on cell populations within joints such as osteocytes, synoviocytes, chondrocytes, and osteoblasts. The key is going to be how much IL-17 to inhibit, so as to avoid making the host vulnerable to infection.”
Currently Amgen is characterizing various large molecules using in vitro assays and proof-of-biologic concept studies in animal models to test the hypothesis that interceding in this pathway is beneficial.
In addition to joint disorders, the IL-17 family of ligands is also involved in signaling within a complex interplay of other interleukins such as IL-17E, which is associated with eosinophilia and populations of TH-2 lymphocytes that are correlated with asthma.
“Other data shows that IL-17 is elevated in colitis and selectively elevated in psoriatic plaque compared to adjacent nonaffected skin,” said Dr. Tocker. He however added that the “greatest body of evidence for IL-17 is associated with rheumatoid arthritis (RA). The anti-TNFs are quite effective in controlling and managing RA, but we hope we can capture patients who are responding incompletely to available biologics.” Amgen is also pursuing an agent targeting allergic asthma with an antibody to the IL-4 receptor.
Matthew Peters, Ph.D., principle scientist at AstraZeneca (www.astrazeneca.com), discussed the utility of cellular dielectric spectroscopy (CDS) in distinguishing Gs, Gi/o, and Gq signaling, “enabling one to monitor activation pathways by different ligands within a single assay.”
CDS is particularly sensitive to inverse agonists for GPCR. “Most people in the past have not looked for these.” Quoting data from Terry Kenakin, author of A Pharmacology Primer, Dr. Peters revealed that “if you look at drugs on the market now, almost all (about 85%) are inverse agonists.”
“The real advantage of this technique is that it can see a whole cell assay in real time, in a label-free way. You don’t have to clone in a new receptor with a tag, or use a small molecule with radio labeling. Previously, it’s been difficult to tell which of the three pathways’ receptors were activated.”
With CDS, cells are grown in standard tissue cultures in a 96-well format, each of which contain gold-film electrodes. “When a weak AC current is applied to the cells, the membranes act like resistors. If a receptor is coupling through Gs, there is a rapid decrease in impedance, if through Gi/o, there is a rapid increase, and if through Gq, there is a dip in impedance followed by an increase,” stated Dr. Peters.
CDS is a fairly new technology; AstraZeneca purchased its first instrument in early 2006. Few papers have been published thus far, but Dr. Peters feels CDS is being adopted by a lot of drug companies. “Its ability to demonstrate signal pathway deconvolution is an unproven opportunity that will likely have clear benefits to drug discovery.” Its only drawback in its current state, said Dr. Peters, is its modest throughput, “thus CDS is probably positioned to be a secondary or tertiary functionality for in vitro pharmacology.”
Brian Wong, M.D., Ph.D., director of research discovery in rheumatology at Roche Pharmaceuticals (www.roche.com), is focused on targeting key disease-critical pathways with small molecules at the preclinical stage. “We have learned a lot about RA in the last ten years, and this knowledge provides, in part, the underpinning of our discovery strategy.”
Roche has received recent approval in both the U.S. and Europe for using MabThera (rituximab) as a B-cell-selective treatment for RA. Originally developed as a treatment for B-cell lymphoma, said Dr. Wong, “MabThera targets CD20, which is specifically expressed on most B-cell populations,” and hence dampens B-cell accumulation in the RA-affected joint.
Actemra (tocilizumab) is another new product that inhibits the IL-6 receptor, which is responsible for systemic inflammatory responses mediated by both T and B cells. It is in Phase III trials for RA patients in both the U.S. and Europe.
“How these drugs work can tell you a lot about the pathophysiology of disease, and this has led Roche to target other genes in those pathways to find small molecules that will be cheaper to produce and orally available,” continued Dr. Wong.
A well known target in this effort is p38, “a kinase that is expressed fairly ubiquitously and broadly regulates cytokine signaling, including that mediated by TNF, IL-6, IL-1, and numerous others. Therefore by targeting p38, one may be able to replicate the anticytokine activity targeted by the biologics.”
T-5224, a AP-1 inhibitor in Phase I studies, was in-licensed by Roche from Toyama Chemical (www.toyama-chemical.co.jp). AP-1 is key in regulating inflammation and immune-cell function. “T-5224 blocks the ability of AP-1 to bind to DNA and induce the transcription of key inflammation genes such as cytokines.” Currently Roche is developing plans to test the agent in RA in its own studies.
Finally, Dr. Wong commented that companies continue to look for ways to enhance their drug discovery and development process. In the future, “monitoring the activity of key pathways in patients will help clinicians make better decisions during drug development.
“For example, one can monitor p38 activity in patients through directly measuring phosphorylation of p38 substrates in immune cells in blood.” In this way, the dosing of agents that interact with p38 can be much more fine-tuned to optimize safety and efficacy. Technologies such as phosphoflow cytometry enable quantification of multiple pathways in specific cell-types.
OSI Pharmaceuticals (www.osip.com) has accumulated a large body of evidence derived in large part from evaluating patient response data to Tarceva® (erlotinib), an anti-EGFR tyrosine kinase inhibitor that is used to treat non-small-cell-lung cancer and pancreatic cancer. Patient response data and evidence from other lines of investigation support the emerging value of targeting the signaling pathways that govern the epithelial-mesenchymal transition (EMT) involved in tumor progression. “EMT is an environmentally driven event that takes place at the invasive edge of tumors,” according to David M. Epstein, Ph.D., vp of oncology research.
“OSI has decided that now is an opportune time to center on EMT research for developing rational combinations of molecular targeted agents for non-chemotherapeutic intervention in cancer.”
The EMT story unfolded through initial observations that patients whose tumor cells expressed E-cadherin responded well to Tarceva, while those whose tumors expressed little or no E-cadherin appeared to derive less benefit when Tarceva was combined with chemotherapy.
Subsequent investigations have uncovered the role of numerous molecules in the signaling cascades that govern the transition from the epithelial phenotype to a more mesenchymal morphology, an event considered critical to tumor progression and metastasis. Equally important is understanding the reverse pathway, the mesenchymal-to-epithelial transition.
Currently, OSI is showing that epithelial cell lines can be driven to the mesenchymal phenotype by incubating them with certain growth factors such as hepatocyte growth factor and TGF-ß.
Dr. Epstein is enthusiastic about OSI-906, an orally active, potent, and selective inhibitor of the insulin-like growth factor-1 receptor inhibitor that may also influence blockade of EGFR signaling through observed cross-talk between the two receptors.