Final Report Summary - PHARMEA (The PHAMEA platform: multi-electrode array technology based platform for industrial pharmacology and toxicology drug screening) Executive summary: The PHARMEA project aimed to develop novel tools intended to improve the drug discovery process in industry, especially in the field of Central nervous system (CNS) diseases, where the current rate of success to bring a new drug to the market is very low. The PHARMEA platform is based on the technology platform of Micro-electrode array (MEA)s, which have been widely used for electrophysiological experiments on neuronal and cardiac tissues over the past decades. Some of the key advantages of MEA technology include ease of use, non-invasive measurements and simultaneous multi-site recording and stimulation capability. However, the MEA tools currently available in the market have several shortcomings, which include lack of adequate throughput that is required in the drug discovery industry, data analysis tools to efficiently process the high volumes of data generated, and user friendly and flexible interface that also automates standard CNS protocols. The PHARMEA project addressed these shortcomings by developing a novel MEA-based platform and applications that will significantly increase throughput of MEA experiments, facilitate MEA experiments on various culture models, as well as associated applications tailored for the drug discovery industry. Furthermore, the PHARMEA project aimed at developing and automating biological assay protocols that are common used in ion-channel based drug discovery activities, which adds significant value to and brings out the benefit of the proposed tools for drug screening applications. The novel PHARMEA drug-screening platform includes the following key features: - Application specific integrated circuit (ASIC)-based integrated monitoring and stimulation hardware; - real-time data processing for the extraction of relevant biological signals and reduction of the amount of data to handle, which allows to work with higher number of electrodes; - novel 256-electrodes multiwell MEA biochips based on glass and porous polymer substrates allowing experimentation with dissociated cell cultures, acute tissue slices and organotypic tissue slice cultures; - novel high-level data analysis software, including graphical user interface and extraction of biological signal parameters, allowing faster readout from the large amount of generated raw data. Working prototypes of the PHARMEA instrument have been realised and its key features of real-time data analysis, data reduction, and use of multiwell format MEAs have been validated using heart culture preparations and hippocampal brain slices. Altogether, we believe that this new PHARMEA platform has a good potential to accelerate the uptake of MEA tools in the drug discovery industry, thereby significantly impacting on the overall drug discovery process. It is hoped that this will lead to better and safer drugs, and at the same time increase the market opportunity and competitive edge of the various sponsoring Small and medium-sized enterprise (SME)s in the lucrative CNS drug discovery industry. Project context and objectives: The context and the main objectives are to address problems in drug discovery experienced by the pharmaceutical industry. The main problem addressed by the PHARMEA project in drug discovery is the following: Ion channels represent highly attractive targets for drug discovery and are implicated in a diverse range of disorders, in particular in the central nervous and cardiovascular systems, but only account about 5-7 % of all pharmaceutical targets in modern medicine. Moreover, the assessment of cardiac ion-channel activity on new chemical entities, i.e. assessment of potential cardiovascular effects, has now become an integral component of drug discovery programmes. Thus despite their attractiveness as drug discovery targets, ion channels remain an under-exploited target class, which is in large part was due to the labour-intensive and low-throughput nature of patch-clamp electrophysiology, which made it also an expensive drug development technique. About 75 % of drugs for the CNS present undesirable secondary effects, a problem that is largely attributed to the high complexity of the CNS. In order to avoid these undesirable secondary effects, it is considered important that the compounds be properly tested for their impact on the cellular function. Besides drug safety issues, the drug discovery process is considered to be highly inefficient, especially the screening/lead optimisation segment, typically yielding plenty of false readouts that become costly downstream. In general, drugs and chemicals are first screened in cell free in vitro systems to profile initial biological effects. As the compounds are selected for further development, more sophisticated systems are employed to more closely mimic conditions in man. Prior to testing in humans (clinical trials), a number of animal experiments are conducted for both efficacy and toxicity. However large numbers of molecules continue to be abandoned both at this animal stage and later in human trials, as it is difficult to predict ultimate effects. Therefore, two developments are needed. Firstly, a more sensitive / accurate screen should be employed to screen out compounds before they are introduced into animal systems thereby increasing the chance of ultimate success. Secondly, a more technically sophisticated system, which more closely mimics physiological conditions, is considered more useful in profiling compounds. Currently, primary drug screening is done using high throughput systems that are capable of remarkable throughput in terms of the numbers of compounds that can be screened. However, only a few of these can be tested subsequently in animal models as the secondary screening methods are generally slow and labour intensive. There are currently electrophysiological monitoring systems available commercially, but these systems are suited only for basic research and low throughput compound testing, as experiments cannot be done in parallel. Towards addressing this problem, there is an increasing trend to develop cell based high content assays aimed at resolving bottlenecks between discovery and lead optimisation. In the CNS most lead optimisation still involves the use of animal models, as simple in vitro systems do not reflect the complex characteristics of brain pathophysiology. In vivo CNS models are highly labour intensive, expensive and are particularly unable to cope with the numbers of lead candidates from High throughput screening (HTS). One of the project partners has developed a series of tissue culture based organotypic models for high content screening applications in neurodegenerative diseases. These models are based on ex-vivo brain tissues, which can be used to determine structure-biological activity relationships and predict in vivo efficacy and safety. This new technology has the potential to act as an effective filter between lead candidates and preclinical development helping to significantly reduce attrition at an early stage. One of the promising approaches is the electrophysiological recording of neuronal activity in complex cell culture systems. Since electrical activity is the functional output of the processes in the CNS, it could provide a sensitive endpoint to detect the early effects of pharmacological agents. Novel technologies, such as MEAS, provide a simpler and less time-consuming alternative to conventional techniques. However, the available MEA tools in the market have several shortcomings which include: - lack of adequate throughput required in drug discovery industry; - data analysis tools to efficiently process the high volumes of data generated; and - user friendly and flexible interface that also automates standard CNS protocols. The PHARMEA project addressed these shortcomings by developing novel MEA tools and applications that will significantly increase throughput of MEA experiments, facilitate MEA experiments on various culture models, as well as associated applications tailored for the drug discovery industry. Specifically, the MEA tools developed will increase the number of channels or measurement sites for simultaneous recording and stimulation, from about 60- or 256-channels today to up to 1024 channels that are constructed from several modules of 256-channel each, along with the corresponding intelligent data handling and processing strategies. Furthermore, the PHARMEA project aimed at developing and automating biological assay protocols that are common in ion-channel based drug discovery activities, which adds significant value to and brings out the benefit of the proposed tools for drug screening applications. The novel PHARMEA drug-screening platform includes the following key features: - ASIC-based integrated monitoring and stimulation hardware; - real-time data processing for the extraction of pertinent biological signals and reduction of the amount of data to handle, which allows to work with higher number of electrodes; - novel high-level data analysis software including graphical user interface and extraction of biological signal parameters allowing faster readout from the large amount of raw biological data generated; - novel 256 electrodes multiwell MEA biochips based on glass and porous polymer substrates allowing experimentation with dissociated cell cultures, acute tissue slices and organotypic tissue slice cultures. Project results: The main result obtained during the PHARMEA project is the development of working prototypes of the PHARMEA platform that include the following key components: 1. Hardware: The PHARMEA platform is built around a commercial PXI chassis, which allows connecting several 256-channel amplifier interfaces within the same instrument. Two special plug-in modules, one for the power supply and another for data acquisition that controls the operation of the amplifiers, are used for each 256-channel MEA interface. The amplification and stimulation electronics are based on a 64-channel ASIC. Four ASICs are used in each MEA interface in order to obtain a 256-channel MEA interface. On-board electronics allowing real-time data analysis of the biological signals as well as data reduction, i.e. elimination of noise signal without biological content, have been developed. Novel 256-electrode MEA biochips based on wellplate format (4, 8, 32 and 60 wells) were developed on solid substrates as well as on porous polymeric substrates. These MEA biochips are intended for dissociated cell cultures as well as for acute and organotypic tissue slice preparations. The main advantage of the multiwell MEA formats is that they enable multiple experiments in parallel on the same device, which permits to generate data from control cell cultures, as well as from various experimental conditions, e.g. to study effects of different compounds or one compound at different concentrations versus control. A novel manufacturing technology of MEA biochips based on porous materials has been developed. It is based on microcontact printing technology that allows printing conductive materials onto a polymer substrate. This technique allows fast and low-cost manufacturing of MEA biochips. 2. Software: New software for controlling the PHARMEA instrument has been developed. It includes the possibility to select electrodes to be monitored and the desired amplifier gain via a graphical map corresponding to the MEA used. It also enables real-time filtering of signals. Spike detection, signal parameters extraction, and extraction of complex features, such as QT prolongation can also be achieved in off-line mode. The software allows to easily compare and to plot the extracted information from the biological raw data, providing to users a valuable readout from the experiments. PHARMEA platform validation was achieved with biological experiments addressing drug safety and drug screening applications. Embryonic chicken heart preparations were used for QT prolongation experimentation and were tested against Ivabradine, known to induce QT prolongation. The results obtained with the PHARMEA platform confirm the QT prolongation of Ivabradine. Organotypic and acute hippocampal tissue slices were used for drug screening validation, where spontaneous activity, as well as stimulation induced burst activity could be recorded and analysed. Working prototypes of the PHARMEA instrument have been realised and its key features of real-time data analysis, data reduction, and use of multiwell format MEAs have been validated using heart culture preparations and hippocampal brain slices. Potential impact: The main project result is a novel drug-screening platform, the PHARMEA platform, based on MEA technology that addresses the main drug discovery problems described above, as well as the current main shortcomings of existing MEA technology tools. This platform provides the following key benefits for drug screening applications: - It monitors multiple cellular sites simultaneously, while also providing global systems activity arising from complex inter-dependent cellular interactions in real-time, e.g. synaptic interactions. - It is non-invasive and allows repeatable long-term experiments of several weeks, useful for long-term evaluation of drug action in vitro. - It can be implemented with real neurons, as well as with brain tissue slices. - It is a label free cell-based assay. Therefore, the PHARMEA platform provides an innovative solution that employs the inherent synaptic connectivity of a live in vitro neuronal network, in order to monitor and interpret intracellular and trans-cellular molecular events. It is expected that the results obtained using this technology approach will have a beneficial impact on the overall drug discovery process and will hopefully result in better and safer drugs, especially in the field of CNS diseases where the current rate of success to bring a new drug to the market has been very low. Another expected impact of this technology will be the potential to reduce the number of animals used for drug testing applications, as the MEA technology would already provide more information about functionality and toxicity of new compounds more upstream of the drug discovery process, avoiding further testing of molecules that have already shown side effects on cell cultures and/or tissue preparations. Main dissemination activities The main project dissemination activities involved attending several international conferences, where new technology and project results were presented. It includes: - Swiss e-print conference - The first Swiss conference on printed electronics and functional materials, 1-2 December 2011, Basel Switzerland. This conference had international attendance and was focusing on printed electronics. Partner EIG presented a poster on its novel microcontact printing technology for MEA biochip manufacturing. - MEA meeting 2012 - The 8th International meeting on substrate-integrated microelectrode arrays, 10-13 July 2012, Reutlingen, Germany. This bi-annual international conference is the most relevant conference about MEA technology and applications. Many MEA technology users from all over the world attend this meeting. Partner Ayanda made a poster presentation of the main PHARMEA project results. - ICANN 2012 - The international conference on artificial neural networks, 11-14 September 2012, Lausanne, Switzerland. This annual conference is the annual flagship conference of the European Neural Network Society. Partner EIG-ENG DEPT made a poster presentation of the novel data analysis tools that were developed during the PHARMEA project. A paper on biological results including biological recordings data that had been generated during the PHARMEA project has been recently published. The paper reference is the following: Tomás J. Ryan et al., Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behaviour, Nature Neuroscience, Volume 16, number 1, pages 25-32, 2013. Companies involved in results exploitation Ayanda Biosystems had previously two main areas of activity: - Cancer diagnostics, where Ayanda was developing a blood test for detection of ovarian and breast cancer based on proprietary biomarker technology. - MEAs, where Ayanda manufactured and sold MEA biochips to customers worldwide. In spring 2011, Ayanda Biosystems SA brought in a strategic investor to help with the development of its cancer diagnostics program. As the investor's interest was solely the cancer program and not MEA-related activities, the board took the decision to divest all MEA-related activities and to continue only the cancer diagnostics activity within Ayanda. The people related to MEA activities took this opportunity to start a new company named Qwane Biosciences SA ('Qwane'), which took over the MEA-related know-how, IP, and all the inventory, and continues operations including associated EU research and development (R&D) projects. The new company was created in July 2011 and its operations started in September 2011. Qwane is focussing its activities on the further development, manufacturing and sales of MEA technology related products, i.e. MEA biochips, data acquisition hardware and data analysis tools. It should be noted though, that Ayanda Biosystems SA and Qwane Biosciences SA are completely unrelated entities, except for a few common shareholders between the two companies. At the end of the PHARMEA project (end of August 2011), Dr Marc Heuschkel, who had become the CEO of the new company, continued the management of the PHARMEA project during the remaining 3 months of the project extension until the end of November 2011. The project dissemination activities after the end of the project will be done under the name of Ayanda. However, the project results exploitation will be done by Qwane as Ayanda stopped its MEA related activities. Qwane will also further develop the ProMEA software in order to address the market need of efficient data analysis tools to get a fast readout from raw biological data. Bio-logic is a scientific instrument manufacturer that provides instruments in the fields of electrochemistry and rapid-kinetics and spectroscopy. Bio-Logic started the commercialisation of its MEA-based electrophysiology data acquisition systems named BioMEA in spring 2009. Unfortunately, it was only in a later stage that a major limitation was discovered in the main data acquisition component of the instrument, the ASIC called AGNES. The AGNES ASIC presents an offset problem, which generates large artefacts when data acquisition is combined with electrical stimulation. It could be partially solved by software filtering of the signals but this solution reduced the frequency bandwidth of recorded signals, which resulted in loss of one of the unique selling propositions of the BioMEA and PHARMEA instruments compared to competition in the market. After several BioMEA instrument customers complained about this limitation, Bio-Logic decided to stop the commercialisation of the BioMEA instrument beginning of 2011. In the meantime, in order to overcome this limitation, partner CEA-LETI has been able to get project external funding to overcome the problem, validate the updated ASIC, and adapt the PHARMEA electronics for integration of this important component of the PHARMEA platform. Even though there was this major drawback with the BioMEA instrument, Bio-Logic is still interested to commercially exploit the PHARMEA project results. Capsant, due to unanticipated changes in its financial position, had to reduce drastically its activities in the beginning of 2011. However the company still exists and is generating revenues from IP licenses, the company's operational activities have been stopped and the company's site in Romsey (UK) has been closed. Capsant believes in the capabilities of the MEA-based technologies and the high market potential it can achieve in the field of safety pharmacology applications. Capsant is currently providing services via others and would be happy to promote the PHARMEA platform to its customers helping the other SMEs in their effort of PHARMEA instrument exploitation. Further steps to be done before exploitation The obtained PHARMEA platform at the end of the project corresponds to an instrument prototype, where the key features have been validated to be functional. Thus the current instrument does not yet correspond to an instrument that can be directly commercialised as is. What we learned over the project is that the consortium had underestimated the amount of work and the necessary time to achieve all the project goals when preparing the project proposal. This was mainly the case for the development of the instrument hardware, where due to a new architecture the hardware had to be redesigned from scratch with exception of the ASIC. The complexity of the software to be developed made its development very challenging. The programming and debugging of the software during its integration with the PHARMEA hardware were very time consuming, which didn't allow achieving all functionalities completely within the time frame of the project. Further steps of development and industrialisation are necessary in order to achieve a commercially exploitable instrument. It includes the following tasks: - complete and validate the updated ASIC AGNES for solving artifact issues on stimulation functionalities; - redesign of casing and improve temperature control of the electronics hardware; - further programming to improve the ProMEA software to obtain a complete stand-alone solution: a module for controlling the electrical stimulation is currently missing, the user interface has still to be improved, and the data analysis and biological parameter output have to be enriched; - the introduction into a liquid-handling robot in a later step is envisaged. These steps will require time and supplementary funding from the involved SMEs and partner CEA before the PHARMEA platform instrument can be commercialised. Avenue for project results exploitation Without the update on the AGNES ASIC done by CEA (the new ASIC is property of CEA as done outside the PHARMEA project), the PHARMEA platform cannot be commercialised due to large artefacts when combining the recording and stimulation functionalities. This problem lead to the withdrawal of the BioMEA instrument in 2011. It is thus necessary to discuss between the SME partners and the CEA about the potential exploitation of the PHARMEA instrument after termination of the project. The most probable scenario for exploitation of the PHARMEA instrument once completed is still that pharmaceutical and biotechnology companies are expected to either acquire and implement the technology in-house and/or to access it through service providers. Thus the route to market will be either via sale of licenses for the technology or by the provision of specialist assay services. It is likely that specific partnerships may emerge for specialised applications. Qwane may exploit the resulting PHARMEA platform instrument as well as the wellplate format 256 electrodes MEA biochips once completed. Conditions of the instrument exploitation will have to be negotiated with Bio-Logic and CEA. Bio-Logic might then commercialise the PHARMEA platform instrument acting as the system hardware manufacturer. Capsant will take advantage of their network and contacts in the pharmaceutical industry and help to promote the new products generated through the PHARMEA project once ready for commercialisation. Qwane intends to exploit the software developed by EIG-ENG DEPT and completed by Qwane (after ensuring copyrights) with the PHARMEA platform, but also as a standalone MEA-based data analysis tool for analysis of data generated by other data acquisition systems. The biological protocols developed by Synome will provide a commercial advantage for the PHARMEA platform over other data acquisition systems in the market. Project website: http://www.PHARMEA.net Note that this website will be available under this address until end of September 2013. Afterwards, the internet pages will be made available in the website of Qwane Biosicences at following internet address: http://www.qwane.com