French biotech company Trophos is seeking partners for its high-throughput image-based primary neuron drug-discovery program to fund its internal drug-discovery efforts, even as it eyes its third financing round and prepares to push its lead therapautic candidate into phase II clinical trials, a Trophos executive said last week.
The Marseilles-based company last week was also awarded its first US patent, number 6,949,354, "Methods for screening compounds active on neurons." The patent broadly covers screening applications on cultured primary neurons (see Patent Watch, this issue).
In addition, Trophos also has a US patent application pending (number 20040224315) for Flash Cytometer, an LED- and CCD -camera-based imaging instrument developed by Trophos scientists that serves as the core technology for the company's screening efforts.
To this point, Trophos has had some "rather small collaborations with other companies to use" the drug discovery platform, Rebecca Pruss, Trophos' chief scientific officer told CBA News in an interview. "That would be something we're interested in developing," Trophos said.
Trophos was founded in 2000 with the goal of developing therapeutics for motor neuron and neurodegenerative disease, in particular amyotrophic lateral sclerosis, spinal muscular atrophy, Huntington's disease, and Alzheimer's disease.
To do so, the company uses phenotypic screens using neuronal survival as an endpoint in order to better understand the mechanisms of neuronal death and specific targets involved in the process — a difficult prospect due to the challenges associated with culturing and assaying primary neurons.
"Neurons are an extremely delicate type of cell to grow, and are done so in very small amounts, because they don't divide once they are put into culture," Pruss said. "They're terminally differentiated cells, so the amount of material that you have to screen is quite limiting. That's the big challenge.
"So we need to design assays that use the minimal number of cells possible," she added. "If you use a very small number of cells, you need to have a way to count them with sensitivity that no other instrument — at least at the time that Tropos was started — had available.
To do this, the company developed the Flash Cytometer, which, according to Pruss, was the brainchild of Michel Delaage, a co-founder of the company and its chief scientific officer until his retirement in 2002. Delaage had a great deal of experience with flow cytometry, and he thought that the company could use a similar principle to analyze the number of neurons on a microplate using standard fluorescent probes.
"There are so few cells in a well — 100 perhaps — that you can't just count a 1-mm by 1-mm sample size because there might not be a single cell in that space. The idea was to be able to capture the whole well in a single image, and then apply fairly standard image-analysis techniques to identify the pixel clusters that contain the fluorescent object."
The Flash Cytometer illuminates plates with LEDs, which allows for multiplexed fluorescence, and captures images from each well with a CCD camera. Trophos says that the instrument can move across a 96-well plate and capture images in about two minutes.
"That means we can actually use live cells, and we don't have to fix them, wash them, stain them, all of which lead to variability," Pruss said. "That's the ideal thing about homogeneous assays. You can count the cells, put them back in the incubator, and count then again a second time, because you can count them so quickly. If you have to leave a plate out of the incubator for even 20 minutes, the neurons are very sensitive and start to die."
Trophos uses this approach to conduct "high-throughput" primary assays using small molecule libraries. Then, when possible hits are identified, Trophos reverts to more detailed interrogations, such as low-throughput but higher content sub-cellular imaging, to better understand the mechanism of action. Often times the company will use the same images obtained in a primary screen, but apply more sophisticated image-analysis software to them.
"We're using the machine only to count what we can see in whole cell bodies, but the images that are captured can actually be treated with other image-analysis programs that can perhaps optimize the image quality and bring up the details that we haven't exploited," she explained.
For instance, the company has used Molecular Devices' MetaMorph program to assay for parameters such as neurite outgrowth.
Pruss said the company can achieve a primary screening throughput of approximately 20,000 compounds per day. This throughput pales in comparison to most high-throughput screening efforts — biochemical or cell-based — but is relatively large for using primary neurons.
"Most companies have neuroscience departments that have people with the right technical knowledge for setting up and running these cultures, but usually on a benchtop, in a very low-throughput fashion, maybe for secondary screening or advanced assays," Pruss said.
"But massive screening of random compound libraries is usually done at specialized sites with automation and people who have specialized skills, but not necessarily the skill base to set up and run primary neuron cultures." Pruss claims that Trophos is the only company that uses primary neurons in a screening mode.
The company has yet to land a major screening collaboration with someone, so it has raised money primarily through financing rounds, which have netted Trophos approximately €11.4 million (around $13.8 million according to current conversion rates) since its inception. The company is also gearing up for a third round of financing, which Pruss said will support clinical trials of its Phase I compound.
"We're planning to develop that first for ALS, and then for SMA," she said. "Phase I should complete at the end of this year, so we're looking for money for the phase II round."
In addition, Trophos has secured some €7.6 million from patient advocacy organizations such as Association Française contre les Myopathies and the HighQ Foundation for Huntington's disease.
"They fund us because we look for general mediators of cell survival," Pruss said. "We don't take a target-based approach, but instead a whole systems approach."
— Ben Butkus ([email protected])