New Point of Care Diagnostics for Clinical Enzyme Biomarkers

For both point of care diagnostic and drug development applications, there is a critical need to develop assays that measure biomolecular interactions. Immense importance is given to assays that enable rapid, high-sensitivity monitoring of enzyme activity. Historically, the detection of enzymes has required time-intensive, laborious separation methods to monitor alterations in the physical structure of a substrate, or the use of radiolabelled substrates. Less cumbersome optical methods have been developed that utilise fluorescent detection technology. Although very popular, these methods usually involve the derivatisation of the biological substrate with a fluorophore, which can alter the molecular interactions. Herein, we describe a powerful assay developed by our group for the detection of a specific class of enzymes, phospholipases
(PLs), and we will optimise it in view of a pre-commercial demonstration and a pre-clinical validation of the product in selected patient groups to assess performance and end-user feedback and refine the demonstration product performance ahead of full commercial development.

This project will be a collaboration between Imperial College London (ICL), Imperial Innovations, Hammersmith Hospital and Mologic Ltd. Previously, the group of Prof. Stevens has developed a powerful platform for the detection of biomedical enzymes (PLs) based on the functionalisation of gold nanoparticles (NPs) with synthetic peptides. Dysregulation of PLs is a feature of several life-threatening diseases such as pancreatitis, atherosclerosis, acute sepsis, arthritis and some cancers. Since pancreatitis is very difficult to detect, and often requires expensive imaging tests, we will focus on this disease as a key initial market goal. The prototype we wish to develop for revealing PL activity is a colorimetric assay based on the aggregation of gold NPs. Gold NPs have the interesting property that when they are dispersed they form a bright red solution and when they aggregate become deep blue. The PL activity is determined via the degradation of liposomes
formed from the natural substrate lipids and filled with a NP crosslinking agent (synthetic peptide). Release of the crosslinker aggregates the gold NPs (functionalised with a complimentary peptide), inducing colour change that can be correlated to the activity of PL in a rapid, quantitative manner.

The potential for commercialisation of our assay resides in some clear selling points. Firstly, this method uses substrates that are entirely unmodified from the natural state. This capability is unlike any other current assay and makes our system considerably more flexible and more biologically relevant. Secondly, this assay measures enzyme activity rather than just concentration, meaning that it is appropriate for drug-screening applications. Finally, the components used in our assay are stable in storage for more than four months.

The goals of the present project are to scale up the technology developed and patented by us into a lateral flow device (LFD) and to study the performance of the LFD with both simulated laboratory and actual physiological samples. The device will be designed and manufactured by Mologic at their facility and the performance/optimisation study will be carried out at ICL. These data will subsequently be used to re-optimise the performance of the device to meet the end-user requirements in a pre-registration clinical evaluation using patient samples.

Since there is currently no LFD capable of detecting enzymes, the project will involve the early stage design of a new type of device. There also exists a significant business opportunity to use these technologies in the field of drug development and drug screening.

1) Acute pancreatitis is a relatively common disease with over 60,000 hospital admissions per year in the UK with about 12% fatality. There is no simple diagnostic kit for this disease, and as such it is often detected at a late stage, increasing the burden on the patient. As stated in the Summary, the key initial market as proof of commercial concept will be the
development of a LFD for the diagnosis of acute and chronic pancreatitis. To this end the major beneficiaries will be patients and clinicians. The development of a simple and rapid test for diagnosis of pancreatitis that could be used in Accident and Emergency departments (A&E), in GP surgeries or at the hospital bedside, and will enable early disease
treatments and better patient outcomes.

2) In addition to the diagnostic device application, there exists the potential to use this technology in the field of drug development and drug screening. The past five years have seen a decline in the number of new drugs approved. One reason is that many drugs fail to show the same behaviour in the body that they do in initial chemical tests. Often,
enormous human and economic capital is devoted to developing compounds that later prove to be ineffective. This is a particular problem in the development of drugs that target enzymes. Since enzymes perform subtle actions on large, complicated molecules they can be difficult to monitor. But without such precise measurements, companies will continue to
waste time and money. Clearly, by using the test, drug companies will benefit from being able to reduce the number of false leads and thus the total cost of development. Preliminary work carried out in collaboration with the Drug Development Centre at ICL and from a number of pharmaceutical companies confirms strong interest in this approach.

3) The UK has a pressing need to innovate in order to drive growth. A 'smart growth' strategy includes innovators and businesses working together to provide a product that will benefit soeirty as a whole. In this regard, the goal of fabricating an innovative diagnostic device, combined with its commercialization and the final goal of improving healthcare embodies exactly the type of project that will impact directly on the UK's smart growth. There is also growing interest in the applications of nanotechnology for healthcare, which is emerging as a strong candidate as a catalyst for growth in the healthcare sector. Nanotechnology offers the opportunity to interact with biology at the molecular level, offering many
possibilities and powerful tools to improve diagnosis and therapies, making medicine more effective in the fight against lifethreatening diseases. It is crucial for the improvement of UK's competitiveness and the quality of life of its citizens, due to its potential to lead to applications that benefit both society and the economy.