June 15, 2006 (Vol. 26, No. 12)

Drug Discovery Based on Microfluidics, GPCR and Kinase Targets, and Cell-based Assays

With only 20 new drugs approved by the FDA in 2005, down from 36 in the previous year, there is still a serious gap between R&D and productivity in the pharmaceutical industry. Opening “MipTec”, recently held in Basel, Gianni Gromo, M.D., Ph.D., head of discovery research at Roche (www.roche.com), commented that there have been only four NCE and no biologics approvals by the FDA thus far in 2006 so, pharma’s woes continue. He went on to highlight some of the challenges and offer some potential ways in which the efficiency and effectiveness of drug research may be improved.

The industry cannot command nature to produce new drugs, he said. The process is complex, lengthy, and marked by unpredictability. There are unexpected successes, such as Nexium (esomeprazole), Lipitor (atorvastatin), and MabThera (rituximab). On the other hand, there are a number of compounds that have not done as well as expected, such as ximelagatran, the thrombin inhibitor. There have been surprise turnarounds too; Iressa was the leader in non-small-cell-lung cancer in 2004, and a year later, it was Tarceva.

Understanding Targets and Disease Process

In drug discovery, according to Dr. Gromo, much depends on having a proper understanding of targets and the disease process. Heart failure and obesity are two important therapeutic areas illustrating this. The four drugs now on the market for heart failure were not developed specifically for the condition, and more rational approaches have proved disappointing, explained Dr. Gromo. In obesity a plethora of targets have been discovered, yet it is not clear which one can best fool the stomach and the brain into safe and effective weight loss, he added. “We are not delivering enough because it is so hard to identify the right target.”

To meet the challenge of creating clinically differentiated medicines, either in a product class or as a clinical breakthrough, the first requirement is good people who understand drug hunting, are flexible, curious, and even foolish, said Dr. Gromo. As far as targets are concerned, these must be relevant to the diseasewith 563 kinases, it is a challenge to develop a truly selective kinase inhibitor, for instance. In fact, there are probably about 150 truly druggable targets for small molecules and maybe 1,000 for biologics.

As to the future, Dr. Gromo is looking to Asia for speedy drug development. Roche’s new 4,500-square-meter R&D center in Shanghai, focused on chemistry, has already got two patents deposited after just a year of operation. “While the number of chemistry graduates in Europe is going down, the scientific paper output from China is steadily increasing, and 70 percent of the publications are on chemistry,” he said.

Timothy Wells, Ph.D., head of research at Serono (www.serono.com), described the biotech view of drug discovery. “Patients want new drugs tomorrowtoday, if possible and doctors want these drugs to be safe, while governments want costs kept down, and shareholders want a return. It all amounts to a lot of pressure, but on the other hand, it is exciting because the human genome project means we now have a more complete picture. Despite all the hype, it has still changed the focal point of drug discovery.”

Although the cost of R&D is up and productivity down, the way to look at this gap is that current productivity is actually the result of R&D carried out 10 to 15 years ago. In biotech, the gap is narrower.

In the 1990s, there was an over investment in technology, said Dr. Wells, without thinking of the possible returns. “There is also a disconnect between what we do and actual human biology. But as we microminaturize the technology, it will help carry out more studies on human tissue.”

Serono has built its success upon recombinant human hormones and going forward is looking at new classes of hormones with as yet unknown functions. An example is osteopontin, which increases myelin formation and is being investigated as a new treatment for multiple sclerosis with interferon-beta, the current treatment. “We have already worked on inflammation, now we are looking at repair,” explained Dr. Wells.

Serono is also interested in developing new methods with current protein therapies using simple approaches like smaller needles and needle-less delivery. Additionally, the company is involved in antibody therapies, where it has many partnerships and in small molecules, for which the technical barriers to development have decreased.

The fact that two out of three compounds still fail in Phase II is unacceptable, commented Dr. Wells. “To improve, we need to have therapy based much more on human disease.” There are three aspects in doing this. First, is the target actually present in human disease and does the drug alter its course of action? Second, there needs to be more experimental medicine showing, as soon as possible, that the drug works in humans. And, finally, genetics can be used to help correlate the severity and response to treatment of a disease.

Methods to Speed Up R&D

Themes in drug discovery technologies presented at the meeting included microfluidics, new assays for GPCR and kinase targets, and an increased focus on the cell-based assay. Platforms based on automation, chips, and microfluidics are well suited to current trends toward miniaturization and multiplexing. For instance, the Sophion (www.sophion.dk) QPatch16 is an automated patch clamp system being developed to select drug candidates through their effect on the electrophysiological properties of ion-channel targets.

Whatman’s (www.whatman.com) soon to be launched CombiChip, a multiplex protein microarray for diagnosis of autoimmune diseases, recently received CE registration. CombiChip will allow researchers to discover patterns of autoantibodies from patients with autoimmune diseases, therefore, allowing the researcher to obtain a complete profile from a single sample.

Meanwhile, the IBM Zurich Research Lab wants to use its expertise in microfluidics, soft lithography, and surface chemistry to develop a powerful bioanalytic platform. Along with the University of Basel, they are now reportedly developing a micromosaic immunoassay that is 10 times faster than assays on a 96-well microtiter plate and uses 1,000 times less sample.

Changes in cell morphology, such as changes in neurite length and volume of cytoplasmic vacuoles, are an important indicator of cellular response to potential drugs that can be applied to in vitro toxicity screening. Timo Ylikomi, Ph.D., of the University Chip-Man Technologies (www.chipmantech.com), is using this idea in the company’s lead product, Cell-IQ, which uses machine-vision technology to drive a real-time and label-free system for monitoring the behavior of cells in the presence of potential drug molecules.

Machine-vision technology is the automated acquisition and analysis of images to obtain relevant data. The set-up can detect numerous endpoints, like cell death, cell division, and viability, in parallel and allows for the re-analysis of data without re-running the experiment. “This is a way of turning microscopy, which is usually descriptive, into data,” said Dr. Ylikomi. He and his team have been able to demonstrate several different patterns of cellular death dynamics, which can be used to profile the toxicity of potential drugs.

Chip-Man just entered into a collaboration with ArcDia(www.arcdia.com) to develop new fluorescence detection technology for the Cell-IQ system. This will allow extraction of even more detailed information on cellular response to drugs.

Researchers at ACEA Biosciences (www.aceabio.com), on the other hand, used changes in impedance to monitor cell growth, viability, and morphology to create the RT-CES assay platform. Without labels and in real time, the system identified cells undergoing mitotic arrest in response to exposure to a potential drug. Such antimitotic agents, including the taxanes and vinca alkaloids, play an important role in cancer therapy.

The system has a microelectrode array in the bottom of each well in a microtiter plate, and this can detect the changes in impedance. RT-CES offers real-time, dynamic monitoring. “It allows you to capture important information that could be missed by a standard end-point assay,” explained Yama Abassi, Ph.D., director of cell biology.

In development studies, the RT-CES has been used with a number of microtubule-disrupting drugs against human cancer cell lines. A novel antimitotic agent has also been identified, and signature activity profiles for DNA damaging, cytostatic, and actin-Axxam (www.axxam.com) developed an assay for monitoring transient changes in calcium concentration within the cell that is relevant to work on GPCR targets. Axxam works with photoproteins that emit light when calcium binds via a conformational change. A new photoprotein, Photina, on which the company has significant IP, is being used for cell-based assays in high-throughput screens.

The Bionas2500 analyzing system from Bionas (www.bionas.de) uses primary human hepatocytes as a model of human physiology, cultured on a silicon chip to monitor oxygen consumption, acidification, and adhesion via sensor electrodes, generating data online.

Another cell-based system comes from researchers at the University of Munich who are developing an Intelligent Microplate Reader, combining optical analysis with bio-electrical microsensor technology. Automatic fluidics supplies cells and tissue in a way that allows for ongoing cell culture with cellular and sub-cellular monitoring of metabolic and morphological responses to exposure to potential drug molecules. The system is modular, which means it can grow with the application.

Interest in GPCR and kinase targets continues to be intense. PerkinElmer (www.perkinelmer.com) recently launched two new technologies for kinase and GPCR target research, based on its AlphaScreen nonradiometric, homogeneous, bead-based system. The AlphaScreen PhosphoSensor kit is an antibody-free system that allows measurement of any kinase activity on any size protein substrates. This will allow work on kinases for which no antibody has yet been identified and will be especially relevant in kinase de-orphanization, where the substrate remains unknown.

Meanwhile, a new long-term licensing agreement will bring AlphaScreen together with TGR Biosciences’ (www.tgr-biosciences.com.au) SureFire ERK assay kits for the screening of GPCR activation in whole cells. “This will allow robust and sensitive screening of some of the toughest GPCR targets, which can’t be done by traditional methods,” said Achim von Leoprechting, Ph.D., European segment leader in drug discovery and life science research.

Roche Pharmaceuticals also developed some new kinase and GPCR assays using new fluorophores, involving a ruthenium complex based label from Roche Diagnostics.

Researchers at Promega (www.promega.com) are looking at novel bioluminescent assays for CYP450s, kinases, p-glycoprotein drug transporters, and monoamine oxidases in multiplex formats. Bioluminescence is very sensitive and may be easier to use in assays of these kinds than the standard fluorescent readout.

Evotec (www.evotec.com) is trying to bring forward high-content assays from lead optimization into primary high-throughput and secondary screening. A high-content assay is imaging based and can be multiplexed. It gives a lot of information on, for instance, the sub-cellular location of targets. These assays are valued for their physiological relevance and could be especially important in primary screening where there are no good alternatives.

In collaboration with the University of Toronto, Evotec is developing a high-throughput primary screening assay for orphan GPCRs (i.e., whose ligand has not been identified), which depends upon the target translocating to the nucleus when not bound to ligand and staying on the plasma membrane when it is bound. The assay allows quantification between the amount of GPCR in the nucleus and on the membrane.

Meanwhile, Imperacer is a new approach being developed at Chimera Biotec(www.chimera-biotec.com,/a>) using MorphoSys’ (www.morphosys.com) Antibodies by Design Technology. The system increases the sensitivity of immunoassay 1,000-fold. Imperacer involves substituting the conventional antibody-enzyme conjugate with an antibody-DNA conjugate, where the DNA part can undergo PCRin effect combining the advantages of PCR and ELISA.

Christopher Lipinski, Ph.D., senior research fellow at Pfizer (www.pfizer.com), emphasized the ongoing importance of medicinal chemistry to drug discovery. “The approved drug is a tool to probe the target,” he said. He believes that many new medicines could come from developing new clinical indications for these tools. Dr. Lipinski has been involved in setting up Melior Discovery (www.meliordiscovery.com), which is looking for new uses for old drugs using phenotypic screening in mice. “There is a lot of untapped value in existing compounds. Many have never been systematically explored after being rejected for efficacy in one application,” he explained.

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