November 15, 2005 (Vol. 25, No. 20)
Eliminating Toxic and Ineffective Compounds at an Early Stage
R&D costs for each new drug on the market are estimated to be $800 million. Traditional molecular screening, although high-throughput, does not provide information about the effects of test compounds on cellular functions. Also, 2-D cell cultures do not accurately reflect in vivo drug responses.
Therefore, as demonstrated in presentations at Cambridge Healthtech’s “Tissue Models for Therapeutics” conference in Boston, many companies are developing techniques to create tissue constructs that provide more accurate compound screening and to eliminate toxic and ineffective compounds at an early stage.
InvivoSciences (www.invivosciences.com) developed a screening method based on cellular responses that produce mechanical changes in biological tissue constructs cultivated in 96-well plates. This is used to validate targets and optimize leads in one step. These can be assembled to provide models for specific disease conditions.
“With phenotypic screening, you can start without a specific target in mind, but find the target that causes disease, and later on, optimize the chemical structure of the target,” explains Tetsuro Wakatsuki, Ph.D., company co-founder and chief scientist. The tissue contains cells of specific types and defined extracellular matrix components.
“We first developed the cardiac tissue model. The main application now is to study mechanical and structural responses to active compounds on fibrotic tissue after a heart attack,” he says. Other potential applications for fibrotic tissue include kidney, liver, and skin. Another available tissue model is vascular smooth muscle cells that is being used to identify potential hypertensive drugs.
The company says it is still developing this screening method, and the next phase will be commercialization. Other applications being explored include testing chemicals for cosmetics and potentially, specific human tissue models for personalized medicine. Dr. Wakatsuki says they are currently seeking collaborators for other screening projects.
“Most people are looking at what’s going on inside the cell. But, there’s a lot going on outside the cell too, for example, protease for the cell matrix. We can address that using our tissue models. We have a collaboration now where we are looking at collagenase activity. The collagenase inhibitor creates different tissue mechanical properties,” states Dr. Wakatsuki.
Generating Motor Neurons from Mouse ES Cells
Curis (www.curis.com) is another company that developed a unique method to screen potential drug candidates. They use a proprietary small molecule Hedgehog agonist to promote the differentiation of motor neurons from mouse embryonic stem (ES) cells.
These neurons are found in the spinal cord and brain stem, and control movement by connecting to other tissues, usually muscles. Damage to these motor neurons can occur by a spinal cord injury or disease, such as amyotrophic lateral sclerosis or spinal muscular atrophy (SMA).
The company received a $5.4 million, three-year grant from the Spinal Muscular Atrophy Foundation to identify compounds to treat this disease, which is the second leading genetic cause of infant death. It is currently developing motor neuron assays to further SMA drug candidates.
“We’re using mouse embryonic stem cells that have GFP driven by the HB9 promoter, a post-mitotic motor neuron marker. This allows you to visualize the differentiation of the mouse ES into motor neurons because they will appear green, GFP+, under the microscope. What makes us unique is that we have a particular Hedgehog agonist that enables us to get a high percentage of the ES cells to differentiate into motor neurons,” explains Amy Sinor, Ph.D., company scientist. “In the past, people used fibroblasts, but we’re able to study the cell type that’s actually affected by SMA,” she adds.
Dr. Sinor says her group has set up a screen using wild-type (normal) motor neurons to look for ways to promote their survival. The ES cells used for this are not deficient in any gene. The point is to show they could set up this particular screen and look for small molecules.
“Soon we will be using motor neurons that are differentiated from mouse ES cells featuring the SMA genotype. These ES cells will have the human transgene for SMN2 and the mouse SMN gene will be knocked out. Then, we will use our survival assay to validate previously identified small molecule candidates that can rescue the motor neurons and promote axonal outgrowth.
“The nice thing about our cells is you can use them for screening and they are flexible. You can manipulate them, which you can’t do easily with primary motor neurons. We hope to look at other motor neurons and neurodegenerative diseases and apply the same approach,” states Dr. Sinor.
3-D Prostate Tissue Models Enhance Drug Discovery Efforts
Researchers at the Yorkshire Cancer Research Lab at the University of York, U.K., have developed a way to model the prostate in 3-D. Headed by Norman Maitland, Ph.D., the 15-year effort is now being commercialized via Pro-Cure Therapeutics (www.pro-curetherapeutics.com). The company says it is focused on improving drug development via cell models for testing new compounds and rediscovery of older failed drugs.
“We found if you take prostate cells and add extracellular matrix, then seed it with prostate mesenchymal cells, you can reconstruct a working prostate gland; not the full tissue, but individual small glands that secrete prostatic proteins,” says Dr. Maitland. The same thing happens with prostate cancer cells, he adds, but the cells in the 3-D model are a “very different type of cell than the ones that grow on plastic. It tries to differentiate a normal prostate, but it fails. So, we thought this was an excellent model to study hormone response.”
His group has been working with companies’ proprietary drugs to see whether there is a differential response between 2-D and 3-D models. Screening is done in 96-well plates, and drugs are monitored in real-time.
“When we look at a new generation of drugs, especially ones that attack enzymes or intracellular structures, there is a very distinct difference between 2-D and 3-D. Drug dosages and susceptibility of cell types might be different.” In addition, studies found that 2-D screens indicated that a compound was killing 100% of the cancer, but when the same compound was tested in the 3-D model, a portion of the cells were resistant to the agent. Dr. Maitland says they will be looking at this phenomenum more closely.
“What we want to do is change the paradigm of how pharmaceutical companies are testing their new drugs. We’d like to convince them that the 3-D models really represent a closer approximation of real tumors, and they will be able to reduce the number of mice studies in the pre-tox stage,” states Dr. Maitland.
Primary Cell-Based Assays Reflect Disease Physiology
Since it is known that various cell types are present in diseased tissue, BioSeek (www.bioseekinc. com) developed in vitro systems that expose cells to a disease environment. “Tissue models are generally healthy tissue, but when you have disease, there are multiple cells that infiltrate the disease site,” says Ivan Plavec, Ph.D., senior director of technology development. “So if you want to study a drug effect, or use your system to study or develop drugs, you need to create a disease-like environment,” explains Dr. Plavec.
BioMAP systems are cell-based assays that use human primary cells, or combinations of primary cells, in a single well to capture cell-cell interactions. These are simultaneously activated to replicate pathway interactions found in disease physiology.
“We’ll combine cells to come up with something that’s most relevant for that particular tissue. Then we take the panel with several BioMAP systems, profile compounds in the panel, and use this as a tool to develop therapeutics,” states Dr. Plavec.
The in vitro environments are optimized such that the cell response represents what is occurring in vivo. Then test compounds are added to the cell cultures. “We want to see that the response to drug treatment is in agreement with what’s known about the effect of that drug in vivo. Once we see these two being in close approximation, then we say we’re modeling the right biology,” says Dr. Plavec.
Optimized sets of protein readouts that represent the biology of disease areas are measured. Depending on the mechanism of action, compounds induce specific changes in the expression of protein readouts, providing a specific BioMAP profile. These profiles are stored in the company’s database and analyzed using proprietary statistical and modeling algorithms. Newly identified leads are compared to the BioMAP profile of known therapeutics to identify compounds that exhibit desired activity.
Current tissue models include skin, vasculature, lung, and joint. Dr. Plavec says although the company initially started with inflammation, they are interested in expanding into cardiovascular disease, metabolism, heart disease, and fibrosis. In addition, with companies more interested in biology, Dr. Plavec says their in vitro disease models should be better predictors of drug activity than animal models.
Miniature Primary Cell Assays
VitraBioscience (www.vitrabio. com) has a proprietary cell assay platform that has enabled them to reduce the number of primary cells required to generate data. The CellCard platform contains carriers made of biocompatible materials with encoded microparticles that allow multiple cells to be tested in a single assay well.
“We were able to miniaturize the assays, using primary cells, from 10,000 cells for an assay to about 100 cells,” says Simon Goldbard, Ph.D., company founder and vp of R&D. This allows for early predictive toxicity screens like apoptosis and in vitro patient stratification.
Dr. Goldbard presented a case study using adipocytes from 10 randomized patients. The cells were distributed into a 96-well plate, and different Glitazones were added. These drugs act by reducing peripheral insulin resistance by activating the PPAR (peroxisome proliferator-activated receptor-gamma) pathway.
One drug showed that 60% of patients responded well and 40% didn’t. “It turns out, in the general population, these are the same percentages of responders versus non-responders,” says Dr. Goldbard, adding, “This may just be a coincidence since we only looked at 10 patients, but it’s a nice coincidence.”
He says the company is proposing that this is the right way to screen drugs, using primary cells in the disease model, i.e., adipocytes for diabetes, osteoblasts for bone disease, etc. “We are proposing that everything should be done this way. Even target validation should be done within the disease model.”
The technology has been validated for GPCR, kinases, nuclear receptors, and phenotypesproliferation, apoptosis and cytotoxicity. Besides drug screening, the assays can also be used to prioritize drug leads and for in vitro toxicology. The additional primary cells the company is interested in using for these assays, include osteoblasts and HUVec cells (endothelial cells involved in vascular processes, a valuable model for angiogenesis research).