Final Report Summary - NANODIARA (Development of novel nanotechnology based diagnostic systems for Rheumatoid Arthritis and Osteoarthritis)
Executive Summary:
The NanoDiaRA project on the development of modified superparamagnetic iron oxide nanoparticles (SPION) as a diagnostic tool for the detection of early stages of rheumatoid arthritis (RA) and osteoarthritis (OA) is a four years research project in the field of “nanotechnology-materials-processes” of the FP7 program. It started in February 2010 having a budget of European funding of 8,917,307.00 EUR and ended in January 2014. Building up on a large knowledge in nanoparticle research and in research in connective tissue pathology and new biomarker technologies and on the clinical expertise of RA and OA, this project aimed to translate know-how in nanomaterials and processing into pre-clinical application, hoping that a next step of clinical investigation could follow by another project.
In this regard, a variety of SPION were developed bearing polyvinyl alcohol (PVA) as the principal surface component. An important step on the way towards scaling-up SPION production was reached by using a special magnetic reactor and partially automated processes for synthesis and functionalization with high batch-to-batch conformity documented by Standard Operation Procedures (SOPs). This allowed the transfer of knowledge and the production even at other production sites and in much larger amounts (up to several mg). A large step forward was made in combining SPION with antibodies (biomarkers) and the use of this in the diagnostics of a particular disease state in the human body. For in vitro diagnostics, a modular device was used in combination with the multiplexing IncaArray system. SPION constructs for targeted biomarker capturing, concentration and following release were functionalized using neutravidin via biotin bridging. They were shown to be stable in physiological solutions and compatible for biomarker capturing with a biomarker-specific antibody to enable tissue and molecular targeting. For new biomarkers, SPION were tested for in vitro applications and assessment of biomarkers in biofluids. It could be shown that SPION have a very good potential to increase the specificity and the output for small protein quantities.
These results made SPION also interesting for tissue-targeted contrast agent for magnetic resonance imaging (MRI). Safe and functionalized SPION with different biomarkers were required for use in RA and OA. The toxicity tests undertaken with PVA-SPION showed very promising results, e.g. that they were well tolerated in rats and did not change hematological and clinico-chemical parameters, that they did not activate the generation of pro-inflammatory cytokines and did not cause changes in macrophages and immune cells at in vivo concentrations. By using antibodies raised towards new epitopes identified by mass spectrometry as proteolytic cleavages in specific tissue proteins, a range of new biomarker detection tools were identified. Cleavage neo-epitopes were identified using synovial fluid and cartilage tissue samples. It could be demonstrated that biodistribution for ferumoxytol and PVA-SPION after intra-articular injection in the knee was similar in the number of SPION and their location in the knee joint; the same uptake by macrophages lining the joint cavity was also noted. In comparison with gadolinium chelate that showed a general uptake in the oedematous tissue, a persistent line of SPION iron oxide uptake was seen that localized over time to the lining of the inflamed synovial membrane. SPION were also functionalized with antibodies for MRI imaging. First appearance was that the particles were more localized to the articulation and around the meniscus, rather than just in the synovium (compared to not functionalized SPION). Further cryosections of arthritic knees were Prussian blue stained to visualize the SPION clusters and only with P2D3 SPIONs the articular cartilage surface was stained.
By using dexamethasone treatment, it is also possible to follow the therapeutic response by SPION-enhanced MRI. This imaging method is sensitive enough to detect small changes in the inflammatory response after treatment in the AIA model, which could not be observed with the non-specific gadolinium chelate experiments. In parallel to the SPION and biomarker development, other methods and patient cohorts were studied to diagnose and detect early osteoarthritis.
Summarized:
• SPION were developed that are not toxic and even do not activate cytokines (PVA crosslinked SPION /-25mV)
• SPION synthesis, coating and functionalization has been developed in a semi-automated process with a good batch-to-batch consistency and SOP's to be translated for GMP production
• SPION were developed that show higher and other specificity than Gd contrast agents (PVA & crosslinked SPION /-25mV)
• Specific antibodies have been developed which can be used in the Arrayon platform
• Arrayon platform has been developed for 3 antibodies in parallel and demonstrated their usefulness
• SPION could be functionalized with antibodies for in vitro and in vivo application
• SPION functionalized with antibodies for in vivo applications showed a different specificity that SPION alone
• MRI images with SPION are shown in the same contrast (white on black) as Gd
• MRI images with and without SPION contrast enhancer can be segmented by a computer program and by this improve the diagnostic outcome
• Novel molecular biomarkers for joint disease have been identified
The complex structure of a research and development project with about 50 permanent collaborators in seven countries required special electronic tools to exchange the SOPs, samples and sample descriptions and results. For this, an electronic sample book (ESB) was developed, used and further exploited. Internal education courses, summer schools and external seminars for consortium members and trainees and separate events for lay people were held every year.
Project Context and Objectives:
The NanoDiaRA Project dealing with the development of modified superparamagnetic iron oxide nanoparticles (SPION) as a diagnostic tool for the detection of early stages of rheumatoid arthritis (RA) and osteoarthritis (OA) has as its main objectives the development of nanotechnology-based novel diagnostic tools for easy and early detection of biomarkers in inflammatory and degenerative diseases, especially RA and OA. RA, which develops in about 1% of the population, is a chronic inflammatory disorder that involves acute and chronic synovial inflammation causing the destruction of cartilage and subchondral bone. Significant pain, morbidity and mortality and reduced capacity to work result from this disease; organs like the heart, the lungs and the vascular system are often also affected. There is no cure for RA, but novel agents such as inhibitors of cytokines (e.g. TNF alpha) as well as immunomodulatory drugs (e.g. methotrexate) have drastically changed disease outcome in many of the patients. By using modified superparamagnetic iron oxide nanoparticles (SPION) for in vitro application, such as bioassays of molecular and cellular biomarkers of arthritis pathology in tissues and body fluids, and for in vivo diagnostics of disease activity and response to therapy e.g. by using magnetic resonance imaging (MRI), disease onset and progression can be detected and determined. In contrast, established diagnostic methods like ELISA for in vitro analyses of patient’s body fluids and MRI without contrast enhancers or computer tomography (CT) and ultrasound (US) do not provide sufficient data for an early definite diagnosis or a clear estimation of the prognosis.
Figure 1: Overview of the NanoDiaRA Project.
FIGURE SEE ATTACHMENT
As shown in Figure 1, the starting position was to focus on the two strategies of diagnosis: on one hand on development of new ultrasensitive immunoassays including use of a benchtop multi-assay body fluid analytical system for widespread use in clinical centers, and on the other hand diagnostic SPION-based in vivo imaging at the molecular level to detect disease-related molecular changes prior to those seen with existing imaging. For both strategies, the development of a platform technology based on SPION was performed to increase the sensitivity and specificity of bioassays and imaging. In both cases it was necessary to search for new RA and OA biomarkers. Specific pathologically cleaved tissue molecules were identified for detection using ultrasensitive immunoassays to measure and identify early events in arthritis tissue destruction. The in vivo application aimed at detecting early inflammation and articular cartilage destruction in joints of RA and OA patients. For this, early Adjuvant-Induced Arthritis (AIA) in rat stifle joints was studied with new MR imaging algorithms. The final goal was to create tissue and molecule-targeted SPIONS by decorating SPION with antibodies raised against the degradation site of specific joint tissue structures, such as articular cartilage, so to detect joint damage and changes occurring in relation to the early onset, progression and treatment of RA and OA at a molecular level. In parallel to this research, it was important to have also a clinical focus on the functional status of RA and OA patient cohorts at the various partner hospitals (Charité, UTartu and ULund) and to combine this with biomarker and MR imaging analysis using specific segmentation techniques, which were also used in the AIA rat studies. The exchange of samples and results was done by an electronic sample book (ESB) and a document management system (DMS) developed and installed for this purpose.
Figure 2: Workpackages in NanoDiaRA.
FIGURE SEE ATTACHMENT
Figure 2 shows the structure of the project. The research and clinical workpackages No 2, 3a and 3b, 4 and 7 are mainly depending on WP1 which is responsible for the SPION development and up-scaling. WP9 and 5 are responsible for coordination, while WP6 and 8 are related to the societal challenges and the dissemination and exploitation of the results.
Based on various research activities proposed with coated SPION at EPFL, it was decided to further develop SPION specifically coated with variations of polyvinyl alcohol (PVA), with silica and a mixture of both and to functionalize them for the various applications proposed in the document of work (DoW). All these developments were documented and processes described in SOPs which were archived in the ESB developed in WP5.
Two different activities were planned: at EPFL for the in vivo application with G1 and G4 and the in vitro application with G2, and at UniFR with G3 (see Figure 3). G2 and G3 nanoparticles were prepared as beads of 50 to 200 nm with a TEOS-APTES coating which allowed a biotin-streptavidin functionalization of antibodies developed by ULund and AnaMar and provided for further investigations as well as additional antibodies from external company IBEX (Canada). The G2/G3 SPION were developed for increasing the sensitivity of the bioassay developed in WP4 with Arrayon and benchmarked with various biofluid samples of UTartu, ULund and Charité.
In fall 2012, after a set of preliminary toxicity tests at Charité and Merck Serono, it was decided not to continue with G1, as individual tests showed possible negative side effects of pure PVA on blood platelets and cytokine activation. Thus, further development was concentrated on a cross-linked PVA/TEOS SPION version called G4.1 which should be further functionalized to achieve a SPION-antibody nanoparticles (G4.2) useful in targeted molecular imaging. The approach was similar to G2/G3, but without using the biotin-neutravidin linker which leads to large particles not useful in MRI and which may also activate the immune system. Multiplex COMP, C2C and CILP biomarker analytics was approved in synthetic surrogate matrices: synthetic serum, synthetic urine and synthetic synovial fluid.
Figure 3: SPION development for in vitro and in vivo application.
Figure 4: Quality assessment by Standard Operation Procedures (SOPs) regularly updated and transferred to ESB.
BOTH FIGURES SEE ATTACHMENT
The G4.1 particles were successfully developed by January 2013, and the SOPs were then transferred to the industrial partner Merck Estapor. An important step for achieving clean and not agglomerated nanoparticles is to wash the final solution again by a magnetic washing step which eliminates the particles and the suspension from any still free floating PVA or other additional contamination and finally to avoid any contamination by endotoxins. Both conditions could be achieved only in the late stage of the project by using an industrial reactor normally used for water cleaning with magnetic particles. The requested concentration was prepared by a newly built magnetic concentrator at EPFL. The SPION were studied at Charité to deliver a comparison/benchmarking to other batches. These G4.1 particles were then functionalized with antibodies and tested by MRI.
G1 and G4 SPION particles were provided to the various partners in WP2, WP3a and WP3b, WP4 and WP7. For the in vivo application by MRI (WP2), the SPION generation G1 and G4.1 were used and benchmarked to gadolinium and ferumtoxyl. Safety aspects were a primary concern, and potential toxicity was investigated in cells from healthy and RA/OA donors (WP3b), and also in animal studies (WP2, WP7) to characterize their behaviour in vivo. Pharmacokinetic (PK) studies were performed to measure the effects of these nanoparticles on the body and to determine the biodistribution and uptake by various organs (WP7).
In parallel to the research activities, clinical investigation in view of disease and functional parameters was performed (WP3b). At Charité, serum and urine samples from patient cohorts that are at an early stage of joint damage due to OA or RA were valuated using the diagnostic tool developed by Arrayon called IncaArray. In parallel, the KANON trial, a randomized control trial comparing a surgical and a non-surgical treatment strategy in young, active adults with an acute ACL tear, was analyzed using a segmentation tool for MRI images at PMU and incorporated into the NanoDiaRA project data-pool. As a third clinical group, UTartu examined middle-aged patients with chronic knee problems on the prevalence and progression of radiographic knee osteoarthritis (KOA) over 3 and 9 years. Collected sera and urine samples from UTartu were used by partners, amongst others to test the IncaArray system at Arrayon. Measurements were done for all three biomarkers (urinary C2C, serum CILP and COMP) following the main aim to test whether biomarkers are able to differentiate OA patients with and without progressive structural changes in their knee joints. This knowledge is crucial for using specific biomarkers in combination with SPION.
Four of nine work packages dealt with the coordination of the project (WP 5 and 9), the ethical, legal and social aspects (WP6), the education of young investigators and the dissemination and exploitation of results at conferences, by publications and by exchanges with industry (WP8).
While WP9 was coordinating the project between partners and the EU Commission regarding legal and financial issues, WP5 was concerned with scientific coordination and the development and implementation of a web-based document system for sample, results and protocol exchange. The system was applied successfully and could be further developed for the application in other projects in United Kingdom and in Switzerland, latter starting in 2014. The project underwent an amendment in WP6 (ELSI) and WP7 (Toxicology) and a new WP leader in WP3a (Biomarker) had to found as Prof. Dick Heingård passed away in May 2013. His collaborator PhD Patrik Önnerfjord took over, however Dick Heingård‘s considerable knowledge and many of his ideas and results that were not documented were lost.
The ethical, legal and social implications (ELSI) were dealt with by WP6. The main aim to inform and educate lay people, consumers and patients was done by conferences and several seminars organized on these issues and presented by reports. Papers were published, and as the initially planned book was abandoned, a larger reports will be published on the webpage of NanoDiaRA in the form of an electronic book.
Dissemination and exploitation of results was the objective of WP8 and undertaken by all partners by presenting the results at international, European and national conferences, seminars and workshops and by publishing them in peer-reviewed journals. Further to this, exploitation of the novel imaging results was undertaken through a workshop in Zurich with companies outside the consortium. Education of young investigators was a continuous task, and four “summer” and “winter” schools were organized by consortium members and held in Lausanne and Kandersteg, Switzerland, Lund, Sweden, and Berlin, Germany.
In summary, it can be stated that the translational approach to bring nanoparticle and biomarker knowledge together for use in clinical application worked very well and that most of the objectives of this project could be achieved. Such large consortia with high and very detailed expertise in the various fields however need a certain time to come to a level of understanding which is necessary to easily exchange the information. The various project and workpackage meetings, the summer schools and trainings of young investigators which accompanied the research were very helpful in this respect, and the objectives of these work packages were fully achieved. The research itself faced several challenges in view of high safety requirements for in vivo applications, high specificity of antibodies, reliability of processes in nanoparticle synthesis, coating and antibody functionalization and the chronology of the various research activities. Looking back from the knowledge and experience gained in these four years, the Consortium would propose to put the objectives for a next complex project into a five-years-term to better handle unexpected events.
The objectives to develop SPION for early diagnostics in RA and OA could be achieved in a first aspect. It was successfully demonstrated that SPION technology with fixed coatings is safe and specific for depicting inflammatory events in joints by using magnetic resonance imaging. The benchmarking with Gd-based contrast agents showed other kind of specificity over time to the lining of the inflamed synovial membrane instead of uptake in the oedematous tissue. A first demonstration of functionalized SPION with cartilage antibodies showed increased localization around the cartilage rather than in the synovium, however these early investigations need more careful control studies to further understand the mechanism of the attached antibody. SPION technology showed also to be a successful tool in the in vitro diagnostics in multiple arrays and for unknown biomarker detection in biofluids. In the assay technology, SPION can be used in a combined approach with the assay to up-concentrate the amount of biomarkers in larger quantities of biofluids and by this increase the specificity in biofluids of e.g. rare diseases or those with unknown biomarkers. However it can be stated that a fifth year would have increased the opportunities to be nearer to clinical translation especially in respect to antibody functionalized SPION.
The various investigations of RA and OA progression in view of functional data as well as the computation of quantitative parameters of cartilage and immunohistochemical studies of various patient cohorts helped to better understand the progression of RA and OA and to later combine the early diagnostics with SPION with other diagnostic tools.
Project Results:
In this section there are many images and graphics included. Please see attachment for the complete description of the main S&T results/foregrounds.
Potential Impact:
The Document of Work discussed the early diagnosis of rheumatoid arthritis and osteoarthritis in view of improving a European health problem and by this an improvement of European social and economic cohesion. The aim was to achieve this goal by introducing new in vitro and in vivo diagnostic tools to detect the disease events earlier and more specifically than by x-ray radiology which can detect only already established degradation and by immune assays normally used for detecting biomarkers in biofluids.
A new article of the Center for Surveillance, Epidemiology, and Laboratory Services, Centers for Disease Control and Prevention (CDC), U.S. Department of Health and Human Services, Atlanta, USA reported that during 2010 to 2012 about 10% of adults in USA had arthritis-attributable activity limitations (AAAL). Approximately half of all adults with heart disease or diabetes had arthritis, and one fourth of adults with either condition and arthritis had AAAL. Approximately one third of adults who were obese also had arthritis, and 15% of those adults had AAAL. A current U.S. Department of Health and Human Service initiative addresses the burden of multiple chronic condi¬tions, which now affect one in four adults and are increasingly common with the aging of the population. The literature outcomes mentioned in the DoW in 2009 have not much changed and research in drug development did not show real break trough for treating both diseases. On the contrary, RA and OA will lead to an substantially increase in health care costs, limits severely the patients in their workforce, reduce the ability to perform normal activities and therefore reduce drastically the quality of life. A report on the limitations of activities of people with musculoskeletal disorders from 2012 still highlights that these diseases amongst them RA are the “major cause of morbidity throughout the world, having a substantial influence on quality of life (QOL)”. The discussion summarized that even developed countries have to face a larger burden of such disorders due to an aging population, and that for these diseases – although associated with great deal of disability, impairment, and handicap – the research remains unrecognized, under-appreciated, and under-ressourced. We will discuss this topic later on.
The list of treatments including all kinds of medications like non-steroidal anti-inflammatory agents (NSAIDs), corticosteroids, and disease modifying anti-rheumatic drugs (DMARDs), but also the reduction of joint stress, physical and occupational therapy, and surgical intervention, proposed today by highly recommended clinics has not much changed. DMARDs therapy is shown to cost between 6’500 and 10’000 EUR per patient per annum and TNF-a therapy costs up to 25'000 EUR per patient per year. However, comparing the costs of treatment with costs for the society if patients are not treated effectively, the treatment of RA can lead to a reduced cost to society as the cost for loss of productivity to society can be same or even higher than the costs of a treatment. The development of potential therapies is driving the testing market in Europe as such diseases are expected to expand rapidly. Many global key players are active on the market of molecular diagnostics being aware that the varying symptoms of patients especially in the early phase of RA and OA are considered difficult to diagnose. Multiple tests have to be conducted before making a diagnosis and therefore novel in vitro diagnostics are needed. The trend towards integrated healthcare and a gradual shift to ‘proactive’ testing rather ‘reactive’ diagnosis is likely to benefit this market in long term and companies have to align their positioning strategies to benefit from the changing markets and obtain maximum return on investment. Therefore we are convinced that the early diagnostic measures to find possible biomolecular indications for the diseases are important ways to better handle the treatment. The increasingly important tools in the development of new targeted therapies are related with new biomarkers and the main requirements for quantitative imaging so to connect cellular processes with therapy. One of the main aims of the NanoDiaRA project was to qualify imaging biomarkers of cartilage tissue degradation and an important step forward was done in understanding which biomarkers are in particular important, and to find ways to combine them with the imaging technology.
Developing new technologies and trying to introduce them into a market also means to educate and inform experts and lay people as well. A Frost & Sullivan publication of 2013 mentioned that for autoimmune diseases like rheumatoid arthritis diagnostic tests are available, however that the underlying key mechanism which are related to such a disease still seems to be vague and unclear and that research to better understand such key regulatory processes are only of moderate interests across Europe. Although the associations in Europe which primarily govern autoimmune research have educational programs for lay people and the scientific community, we think that education and information regarding both the disease itself and the new developments in disease diagnostics and therapies would be wise to be improved. Having Robin Poole, Professor emeritus of McGill University and former Director of the Canadian Arthritis Network as Scientific and Clinical Advisor attending most of NanoDiaRA Meetings we learned that consumer groups demand more choice in diagnosis, pain and disease management for their members, and are pressuring for better early diagnosis and treatment. In Europe the European Union pays particular attention to the needs of diseased people and to enable them to fulfil the roles and responsibilities of citizenship and have the same individual choices and control over their life as non disabled people. For RA patients this would mean to reduce the heavy burden of disease, to improve of the physical and psychological dependence and to minimise their dependence on nursing staff and family and to increase their workforce and the quality of life.
NanoDiaRA tried to educate and inform in different ways, a) by training the young investigators of the project through summer schools and by exchanging them between the different laboratories to learn new and other methods, b) by informing and discussing with experts the value chain of nanotechnological products in medicine and by this especially the risks and legal sides of such products, c) by exchanging with lay people about diagnostic and therapeutic approaches and to inform them about ethical, legal and societal implications, and d) by informing the wider European and international scientific and technological community about the project. In addition, further contacts with other European projects in this field were established, companies were contacted and the results discussed with them for further exploitation.
1.4.2 The potential impact
The value of iron oxide nanoparticles (SPION) as contrast agents were accepted by both FDA and EMA for imaging enhancement in liver and lymph nodes. Having in mind this and various studies already undertaken with a novel iron oxide nanoparticle formulation based on amino polyvinyl-alcohol (PVA), it was subsequently the next step to develop and use these nanoparticles for the RA and OA diagnostic market.
SPION are characterized by their paramagnetic properties that only occur on the nanoscale (below 100 nm). This makes them distinguishable from other “nano” products in nanomedicine like the liposome technology, which is nanoscale (below or around 100 nm) but without providing “fundamentally new properties and functions”. Applications of nanoparticles in medicine and diagnostics have to meet stringent safety requirements, notably no toxicity in the application, reliable and safe production, knowledge about bio-distribution and clearance of such particles when administered to the body and of course a higher performance in diagnostics than competing materials. The research project was therefore created to develop such systems along the whole value chain up to pre-clinical investigations; further development to clinics is beyond the scope of this project. Three main objectives describe the integrated interdisciplinary research efforts which are being made: (i) application of SPION technology as a contrast agent in targeted molecular imaging by MRI, (ii) development of specific multimodal array technologies using SPION for concentration of biomarkers in biological fluids to increase sensitivity and specificity of the array and (iii) biomarker development based on proteomic investigations and ELISA assays and the search for possible new biomarkers in biofluids using the SPION technology (biomarker-fishing). For all these tasks reliable and industrially effective transferable processes were developed, creating meaningful SOPs to evaluate the safety of these particles for human application and improving our understanding of arthritic disease, disease progression and its biologic answer through corresponding biomarkers and their assay technology.
The most profitable nanomedical products today are anticancer delivery systems based on liposomes, showing a steadily increasing market, as these products answer an urgent demand of new therapies able to better fight this highly aggressive and increasingly frequent disease. Although not much is published so far or available as nanotherapeutical approach, RA is mentioned as an important field e.g. in the review of Christine T.N. Pham on “Nanotherapeutic approaches for the treatment of rheumatoid arthritis.“ Current strategies and recommendations for RA are centered on early, intensive treatment with disease-modifying anti-rheumatic drug (DMARD) in monotherapy or in combination with non-steroidal anti-inflammatory drugs (NSAIDs) or by using biologics like cytokine antagonists (TNF, IL-1 and IL-6 inhibitors or receptor antagonists), B cell depleting agents, and T cell co-stimulation modulator. Some combinations may even reach to achieve positive resonance for up to 70% of the patients. However, there are several drawbacks especially to current biologic therapy as it is cost intensive and accompanied in some cases by infectious complications, and the loss or failure to maintain response overtime. The treatments using e.g. glucocorticoids and given systemically or in some cases intra-articularly (ia) may also be associated with a number of unwanted side effects, like e.g. increased risks of cardiovascular diseases, osteoporosis, infections.
Moreover, the optimal and effective therapeutic approach to an individual cannot be predicted at this point due to the heterogeneity of the patients, thus necessitating a “trial and error” approach that increases costs and delays clinical response. NanoDiaRA-developed SPION have the potential to early diagnose inflammatory events by either macrophage in vivo magnetic resonance imaging or even more specific with specific tissue binding antibodies. These SPION formulation which have shown to monitor therapies can be a very helpful tool in the pre-clinical and clinical development phase of future drugs either by decorating the SPION with biological or other drugs or by monitoring them. The next step on this development will be the demonstration of GMP production; the SOPs for translation of processes established.
A breakthrough for a marketable product cannot longer be only in the hands of researchers and scientists but companies have now to take over the responsibility for the next development step towards clinics to have an impact on innovation in Europe and the creation of further working places. This is especially true for projects like NanoDiaRA, where inorganic nanoparticles for medical applications are developed. Such particles are treated from the regulatory side as medication regardless of their use only for diagnostic. This leads to relative high barrier to transfer the results from academia to industries and finally to clinics. Because NanoDiaRA was aware of this problem from the beginning, parameters like reliability, reproducibility, toxicity, biocompatibility, scalability, acceptability by clinical doctors and patients, social impact and acceptance has guided the project development beside the also important scientific questions and constraints to reach finally the project goal. We are convinced that with this approach, which leads maybe to a lower number of scientific publications during the project period, but to very useful, scalable and very well documented processes ready for scale-up, we would have on a long term a substantial impact regarding the clinical use of nanoparticle for diagnosis and therapy.
Figure 13: Historical and projected markets for nanotherapeutics (Tx) and nanodiagnostics (Dx).
FIGURE SEE ATTACHMENT
Imaging diagnosis has gained importance in recent years as shown in Figure 11. The main benefit of molecular imaging for in vivo diagnosis lies in the early detection of disease and the monitoring of disease activity and response to treatment, „supporting the development of individualized medicine and the real-time assessment of therapeutic and surgical efficacy“. Comparing different methods, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) offers greater sensitivity but a lack of anatomical context. In contrast, MRI provides accurate anatomical detail but no data on disease activity. At present it is the preferred option for cellular tracking. To enhance the image quality, contrast agents are used. This market for MRI products is reported to be more than $1 billion annually and is currently dominated by gadolinium chelate-based agents, which are the subject of class-action lawsuit concerns due to the potential release of gadolinium into the body. The global contrast agent market is expected to strongly increase if the targeted MRI imaging, which is in the center of NanoDiaRA project, becomes successful to specifically localize the molecular target. Advances in MRI technology especially in the spatial resolution and the fact that MRI is a non-invasive, irradiation-free technology will push this development. NanoDiaRA has alos further improved the imaging quality by developing new imaging procedures like the positive contrast MRI technique (dUTE – difference Ultrashort echo time). The development should also focus on reduced concentrations of the agents at the targeted side, as until now high quantities for cell recognition and high affinity are necessary to balance MRI’s inherently low sensitivity. Increased market penetration of medical imaging equipment is expected to lead to significant opportunities for the use of contrast agents. Lodhia mentioned that „for molecular and cellular imaging with MRI, the current research sets a platform for the further development of SPIONS“ and sees the „next step in SPION development [...] towards molecular imaging“ as a „useful complementary role“ to nuclear medicine and PET. If the development of specific targeting, iron oxide nanoparticles could be successful, the market-potential may be large enough to successfully reintroduce this type of nanoparticles for molecular imaging. This type of particles is not restricted to MRI, MPI (magnetic particle imaging, a method developed by Philips) , showing a higher resolution is also based on this type of SPION. The fact that SPION-antibody specific MRI could be demonstrated in NanoDiaRA for the first time with RA biomarker raises hope that molecular imaging will also be used for early RA diagnostics.
1.4.3 Further Education and Training to improve the chances in careers
In total, 200 people were partially or fully funded by the FP7 project; 66 working places could be established specifically for the project. Two third of the collaborators were woman, whereby 5 of the around 50 permanent female collaborators were pregnant during the project time (having 6 pregnancies). Further education and training was one of the important tasks of NanoDiaRA to increase the future career opportunities of the young investigators, especially also women. The project NanoDiaRA has shown a high diversity in activity and in research fields covering the whole value chain of a product from basic research (necessary to understand biomarker and particle characteristics) towards nanoparticle development and pre-clinical investigations as some clinical investigations on the functional status of patients as well. The project therefore promoted inter-university and university-industry contacts and cooperation between young investigators, trainees and technicians. This was done by organising four summer schools of at EPF Lausanne, University of Lund, Charité University Hospital Berlin and a fourth as “winter school” in Kandersteg, Switzerland together with the Competence Center for Materials Science and Engineering, CCMX. The aim was to provide one week of lectures, specific break-out sessions, lab work, practical training and insight into the work processes of the hosting partner institution for young researchers/clinicians active in the Consortium as well as the presentation of current science & technology from external experts to increase the internal overall knowledge in the different fields and to allow all collaborators to understand the typical language of the other professional and scientific sector. We could recognize that each scientific or industrial domain had its own conceptual metaphors which were not always understood by the other research community. In addition, we stated that engineers present their ideas and results mainly in a way that formulas play a role while biologists and clinicians are using pictograms for their presentations. In addition to the many expressions and abbreviations it was necessary to achieve a communication basis so that everybody could understand the main activities, and many of the collaborators learned additional skills and knowledge. The first summer school took place at EPFL; two more followed at ULund and Charité to cover all aspects from technology to clinic by courses, practical work and visits. Lab work and student seminars accomplished the summer school. The summer schools were based on three different main topics:
- Nanotechnology: synthesis, coating, functionalization of nanoparticals, physical and chemical understanding of the properties and processes, understanding about legal and ethical standards
- Cell and molecular biology in RA and OA: Introduction to rheumatic diseases, tissue organization: Cells and Matrix, turnover of matrix, cell biology in practice, inflammation, diagnostic oportunities in rheumatic diseases, practical work
- Clinical aspects of RA and OA: RA therapy - from gold standards to new approaches, materials & methods in the development of nanomedicine, diagnostics in RA and OA, practical lab course, nanotherapy, data banking and legal aspects
Figure 14: Participants at the summer school in Lund, May/June 2012
Figure 15: Participants at the summer school at Charité Berlin, July 2013.
BOTH FIGURES SEE ATTACHMENT
The project delivered also short reports from young investigators to learn more about the impact about such training exchanges as seen in some examples of the exchange practice in NanoDiaRA:
- Training visit from a collaborator of University Geneva, Switzerland at University of Nijmegen, Netherlands: learning more about the AIA rat model and erosion and inflammation histological scoring, AIA model induction (intra-articular injection), subcutaneous injection, emulsion preparation, introduction to the rat knee joint anatomy in absence and presence of AIA, followed by a training on the assessment of the degree of inflammation (infiltration scoring) and extent of joint destruction (erosion scoring) under the supervision of a senior scientist
- Training of a collaborator of the Parcelsus Private University in Salzburg, Austria at University of Geneva: to get insight into the image acquisition procedures at the University of Geneva, to discuss further image analysis methods to be employed in the workpackage, and to participate in an MR imaging session to provide insights into the animal work, the histological evaluation performed, and the MR imaging methodology, which is the basis for the analyses done at PMU, so to allow to know more about the requirements for the next software application to be developed at PMU.
- Training of collaborators at Charité University Hospital, Berlin Germany by a collaborator from EPF Lausanne, Switzerland for demonstration of protein separation from serum using the SPION technology, discussion about the sample types and the nanoparticles. All separation devices, equipment and materials (e.g. a reactor, valve, pump, connectors, tubes, Ni-Fe wire) needed for a full demonstration were transported to Charité.
- Training of collaborators of EPFL and University of Fribourg, Switzerland at company Arrayon, Neuchâtel, Switzerland to exchange about the antibody functionalization of SPION for in vitro application.
- Training of collaborators of EPFL and University of Lund, Sweden to exchange about the antibody functionalization of SPION for in vivo application.
Such exchanges between laboratories in different countries and between industry and academia enabled the young investigators to learn to use and improve so-called “soft” and “hard” skills in another environment – engineering in clinics, research at a company and more – and to exchange further personal information also in the environment of another culture.
These experiences are highly valuable when changing the employer and even when changing from university to industry. Young investigators received some form of information advice and guidance not only from the direct contact but also had always the opportunities to further discuss with principal investigators or other experienced collaborators, which clearly opened their horizon.
1.4.4 Dissemination activities and exploitation of results
The NanoDiaRA Consortium has presented results of the project more than 160 times at various conferences, workshops and seminars to an audience of mostly experts but also to lay people or patient organisations. Presentations at such events were given either orally or by posters while presentations at fairs like in Aarhus in 2012 were also provided interactively by electronic media. The following two diagrams show the activity of NanoDiaRA to present their results by oral or poster presentations whereby for one event the various partners were counted, who were presenting the project. Various workshops and scientific meetings were organised within NanoDiaRA (at least twice a year) and external speakers were invited (like Prof. Christoph Garnier from Inselspital Bern and Kenneth A. Dawson, University College Dublin), but also conferences and workshops for external people with invited speakers were organised. Two conferences were organised in Switzerland by MatSearch and EPFL (Lausanne 2012 and Bern 2013), one in Berlin Germany by EUAK (2012) and one in Portugal by MatSearch in Collaboration with the EU project NANOFOL.
Figure 16: Overview of the countries in which collaborators held presentations about NanoDiaRA work (in total 77).
Figure 17: Overview of the countries in which collaborators presented posters about NanoDiaRA work (in total 44).
Figure 18 shows the partner allocations of the poster authors of NanoDiaRA (in total 113 – multiple authors counted).
Figure 19 shows the partner allocation of the oral presentation authors of NanoDiaRA (in total 124 – multiple authors counted).
ALL FIGURES SEE ATTACHMENT
These diagrams depict the many activities of NanoDiaRA regarding dissemination of the results. Beside these presentations at national and international conferences, the results were also presented in peer reviewed publications. 33 publications were prepared during the project and up to now 25 have been published, the other 8 are under review. Further 13 papers are in progress. One article could be published in Chemical review having an impact factor of > 40, 12 of the documents were published in journals with impact factors between 4.6 and 10, the rest was published in journals with impact factors below 4.5. Another 21 publications are in preparation and partially submitted and further 8 were published in journals which are not reviewed.
Exploitation of the results was organised by MatSearch in contact with a company specialized in diagnostic imaging, a company specialized in SPION products and a university having a GMP facility. These contacts are foreseen to be used for follow-up projects. MatSearch and some partners organised a further application for a new project with a similar approach but was not successful.
1.4.5 Conclusions
In the four years of the NanoDiaRA project, it was possible to generate besides the scientific results a high positive social impact. 66 new jobs were provided, and many, especially young scientists could receive financing for their research. The quota of women working for the project was clearly above average. Interdisciplinary training and work exchange lead to well-educated young researchers in our project with insight into other disciplines and scientific cultures.
RA and OA are diseases for which better treatment and earlier diagnosis are a strong need, and developing SPION for targeted MRI-imaging for this market is a novel approach. The project tried to prepare for a further exploitation of our results from the beginning by creating standards, doing thorough and broad research on possible toxicity in each phase of the development, and by communicating our research to the scientific community as well as to the public. The results and developments of our project were presented on many international conferences by talks, posters and other media, by materials for the public like flyers, press releases, public discussions and the NanoDiaRA homepage, and of course by scientific publications; a task which is still ongoing.
Further exploitation towards new national and European projects occurred by various contacts with external companies and research groups for SPION technology and for biomarker development. Negotiations are ongoing under Non Disclosure Agreements.
List of Websites:
The NanoDiaRA public website is updated on a regular basis by EUAK and MatSearch. The website is available at http://www.nanodiara.eu/
Administrative Coordination
Priv.-Doz. Dr. med. Felix Thiele, M. Sc.
Europäische Akademie zur Erforschung von Folgen wissenschaftlich-technischer Entwicklungen Bad Neuenahr-Ahrweiler GmbH
Wilhelmstr. 56
53474 Bad Neuenahr-Ahrweiler
Germany
felix.thiele@ea-aw.de
www.ea-aw.org
The NanoDiaRA project on the development of modified superparamagnetic iron oxide nanoparticles (SPION) as a diagnostic tool for the detection of early stages of rheumatoid arthritis (RA) and osteoarthritis (OA) is a four years research project in the field of “nanotechnology-materials-processes” of the FP7 program. It started in February 2010 having a budget of European funding of 8,917,307.00 EUR and ended in January 2014. Building up on a large knowledge in nanoparticle research and in research in connective tissue pathology and new biomarker technologies and on the clinical expertise of RA and OA, this project aimed to translate know-how in nanomaterials and processing into pre-clinical application, hoping that a next step of clinical investigation could follow by another project.
In this regard, a variety of SPION were developed bearing polyvinyl alcohol (PVA) as the principal surface component. An important step on the way towards scaling-up SPION production was reached by using a special magnetic reactor and partially automated processes for synthesis and functionalization with high batch-to-batch conformity documented by Standard Operation Procedures (SOPs). This allowed the transfer of knowledge and the production even at other production sites and in much larger amounts (up to several mg). A large step forward was made in combining SPION with antibodies (biomarkers) and the use of this in the diagnostics of a particular disease state in the human body. For in vitro diagnostics, a modular device was used in combination with the multiplexing IncaArray system. SPION constructs for targeted biomarker capturing, concentration and following release were functionalized using neutravidin via biotin bridging. They were shown to be stable in physiological solutions and compatible for biomarker capturing with a biomarker-specific antibody to enable tissue and molecular targeting. For new biomarkers, SPION were tested for in vitro applications and assessment of biomarkers in biofluids. It could be shown that SPION have a very good potential to increase the specificity and the output for small protein quantities.
These results made SPION also interesting for tissue-targeted contrast agent for magnetic resonance imaging (MRI). Safe and functionalized SPION with different biomarkers were required for use in RA and OA. The toxicity tests undertaken with PVA-SPION showed very promising results, e.g. that they were well tolerated in rats and did not change hematological and clinico-chemical parameters, that they did not activate the generation of pro-inflammatory cytokines and did not cause changes in macrophages and immune cells at in vivo concentrations. By using antibodies raised towards new epitopes identified by mass spectrometry as proteolytic cleavages in specific tissue proteins, a range of new biomarker detection tools were identified. Cleavage neo-epitopes were identified using synovial fluid and cartilage tissue samples. It could be demonstrated that biodistribution for ferumoxytol and PVA-SPION after intra-articular injection in the knee was similar in the number of SPION and their location in the knee joint; the same uptake by macrophages lining the joint cavity was also noted. In comparison with gadolinium chelate that showed a general uptake in the oedematous tissue, a persistent line of SPION iron oxide uptake was seen that localized over time to the lining of the inflamed synovial membrane. SPION were also functionalized with antibodies for MRI imaging. First appearance was that the particles were more localized to the articulation and around the meniscus, rather than just in the synovium (compared to not functionalized SPION). Further cryosections of arthritic knees were Prussian blue stained to visualize the SPION clusters and only with P2D3 SPIONs the articular cartilage surface was stained.
By using dexamethasone treatment, it is also possible to follow the therapeutic response by SPION-enhanced MRI. This imaging method is sensitive enough to detect small changes in the inflammatory response after treatment in the AIA model, which could not be observed with the non-specific gadolinium chelate experiments. In parallel to the SPION and biomarker development, other methods and patient cohorts were studied to diagnose and detect early osteoarthritis.
Summarized:
• SPION were developed that are not toxic and even do not activate cytokines (PVA crosslinked SPION /-25mV)
• SPION synthesis, coating and functionalization has been developed in a semi-automated process with a good batch-to-batch consistency and SOP's to be translated for GMP production
• SPION were developed that show higher and other specificity than Gd contrast agents (PVA & crosslinked SPION /-25mV)
• Specific antibodies have been developed which can be used in the Arrayon platform
• Arrayon platform has been developed for 3 antibodies in parallel and demonstrated their usefulness
• SPION could be functionalized with antibodies for in vitro and in vivo application
• SPION functionalized with antibodies for in vivo applications showed a different specificity that SPION alone
• MRI images with SPION are shown in the same contrast (white on black) as Gd
• MRI images with and without SPION contrast enhancer can be segmented by a computer program and by this improve the diagnostic outcome
• Novel molecular biomarkers for joint disease have been identified
The complex structure of a research and development project with about 50 permanent collaborators in seven countries required special electronic tools to exchange the SOPs, samples and sample descriptions and results. For this, an electronic sample book (ESB) was developed, used and further exploited. Internal education courses, summer schools and external seminars for consortium members and trainees and separate events for lay people were held every year.
Project Context and Objectives:
The NanoDiaRA Project dealing with the development of modified superparamagnetic iron oxide nanoparticles (SPION) as a diagnostic tool for the detection of early stages of rheumatoid arthritis (RA) and osteoarthritis (OA) has as its main objectives the development of nanotechnology-based novel diagnostic tools for easy and early detection of biomarkers in inflammatory and degenerative diseases, especially RA and OA. RA, which develops in about 1% of the population, is a chronic inflammatory disorder that involves acute and chronic synovial inflammation causing the destruction of cartilage and subchondral bone. Significant pain, morbidity and mortality and reduced capacity to work result from this disease; organs like the heart, the lungs and the vascular system are often also affected. There is no cure for RA, but novel agents such as inhibitors of cytokines (e.g. TNF alpha) as well as immunomodulatory drugs (e.g. methotrexate) have drastically changed disease outcome in many of the patients. By using modified superparamagnetic iron oxide nanoparticles (SPION) for in vitro application, such as bioassays of molecular and cellular biomarkers of arthritis pathology in tissues and body fluids, and for in vivo diagnostics of disease activity and response to therapy e.g. by using magnetic resonance imaging (MRI), disease onset and progression can be detected and determined. In contrast, established diagnostic methods like ELISA for in vitro analyses of patient’s body fluids and MRI without contrast enhancers or computer tomography (CT) and ultrasound (US) do not provide sufficient data for an early definite diagnosis or a clear estimation of the prognosis.
Figure 1: Overview of the NanoDiaRA Project.
FIGURE SEE ATTACHMENT
As shown in Figure 1, the starting position was to focus on the two strategies of diagnosis: on one hand on development of new ultrasensitive immunoassays including use of a benchtop multi-assay body fluid analytical system for widespread use in clinical centers, and on the other hand diagnostic SPION-based in vivo imaging at the molecular level to detect disease-related molecular changes prior to those seen with existing imaging. For both strategies, the development of a platform technology based on SPION was performed to increase the sensitivity and specificity of bioassays and imaging. In both cases it was necessary to search for new RA and OA biomarkers. Specific pathologically cleaved tissue molecules were identified for detection using ultrasensitive immunoassays to measure and identify early events in arthritis tissue destruction. The in vivo application aimed at detecting early inflammation and articular cartilage destruction in joints of RA and OA patients. For this, early Adjuvant-Induced Arthritis (AIA) in rat stifle joints was studied with new MR imaging algorithms. The final goal was to create tissue and molecule-targeted SPIONS by decorating SPION with antibodies raised against the degradation site of specific joint tissue structures, such as articular cartilage, so to detect joint damage and changes occurring in relation to the early onset, progression and treatment of RA and OA at a molecular level. In parallel to this research, it was important to have also a clinical focus on the functional status of RA and OA patient cohorts at the various partner hospitals (Charité, UTartu and ULund) and to combine this with biomarker and MR imaging analysis using specific segmentation techniques, which were also used in the AIA rat studies. The exchange of samples and results was done by an electronic sample book (ESB) and a document management system (DMS) developed and installed for this purpose.
Figure 2: Workpackages in NanoDiaRA.
FIGURE SEE ATTACHMENT
Figure 2 shows the structure of the project. The research and clinical workpackages No 2, 3a and 3b, 4 and 7 are mainly depending on WP1 which is responsible for the SPION development and up-scaling. WP9 and 5 are responsible for coordination, while WP6 and 8 are related to the societal challenges and the dissemination and exploitation of the results.
Based on various research activities proposed with coated SPION at EPFL, it was decided to further develop SPION specifically coated with variations of polyvinyl alcohol (PVA), with silica and a mixture of both and to functionalize them for the various applications proposed in the document of work (DoW). All these developments were documented and processes described in SOPs which were archived in the ESB developed in WP5.
Two different activities were planned: at EPFL for the in vivo application with G1 and G4 and the in vitro application with G2, and at UniFR with G3 (see Figure 3). G2 and G3 nanoparticles were prepared as beads of 50 to 200 nm with a TEOS-APTES coating which allowed a biotin-streptavidin functionalization of antibodies developed by ULund and AnaMar and provided for further investigations as well as additional antibodies from external company IBEX (Canada). The G2/G3 SPION were developed for increasing the sensitivity of the bioassay developed in WP4 with Arrayon and benchmarked with various biofluid samples of UTartu, ULund and Charité.
In fall 2012, after a set of preliminary toxicity tests at Charité and Merck Serono, it was decided not to continue with G1, as individual tests showed possible negative side effects of pure PVA on blood platelets and cytokine activation. Thus, further development was concentrated on a cross-linked PVA/TEOS SPION version called G4.1 which should be further functionalized to achieve a SPION-antibody nanoparticles (G4.2) useful in targeted molecular imaging. The approach was similar to G2/G3, but without using the biotin-neutravidin linker which leads to large particles not useful in MRI and which may also activate the immune system. Multiplex COMP, C2C and CILP biomarker analytics was approved in synthetic surrogate matrices: synthetic serum, synthetic urine and synthetic synovial fluid.
Figure 3: SPION development for in vitro and in vivo application.
Figure 4: Quality assessment by Standard Operation Procedures (SOPs) regularly updated and transferred to ESB.
BOTH FIGURES SEE ATTACHMENT
The G4.1 particles were successfully developed by January 2013, and the SOPs were then transferred to the industrial partner Merck Estapor. An important step for achieving clean and not agglomerated nanoparticles is to wash the final solution again by a magnetic washing step which eliminates the particles and the suspension from any still free floating PVA or other additional contamination and finally to avoid any contamination by endotoxins. Both conditions could be achieved only in the late stage of the project by using an industrial reactor normally used for water cleaning with magnetic particles. The requested concentration was prepared by a newly built magnetic concentrator at EPFL. The SPION were studied at Charité to deliver a comparison/benchmarking to other batches. These G4.1 particles were then functionalized with antibodies and tested by MRI.
G1 and G4 SPION particles were provided to the various partners in WP2, WP3a and WP3b, WP4 and WP7. For the in vivo application by MRI (WP2), the SPION generation G1 and G4.1 were used and benchmarked to gadolinium and ferumtoxyl. Safety aspects were a primary concern, and potential toxicity was investigated in cells from healthy and RA/OA donors (WP3b), and also in animal studies (WP2, WP7) to characterize their behaviour in vivo. Pharmacokinetic (PK) studies were performed to measure the effects of these nanoparticles on the body and to determine the biodistribution and uptake by various organs (WP7).
In parallel to the research activities, clinical investigation in view of disease and functional parameters was performed (WP3b). At Charité, serum and urine samples from patient cohorts that are at an early stage of joint damage due to OA or RA were valuated using the diagnostic tool developed by Arrayon called IncaArray. In parallel, the KANON trial, a randomized control trial comparing a surgical and a non-surgical treatment strategy in young, active adults with an acute ACL tear, was analyzed using a segmentation tool for MRI images at PMU and incorporated into the NanoDiaRA project data-pool. As a third clinical group, UTartu examined middle-aged patients with chronic knee problems on the prevalence and progression of radiographic knee osteoarthritis (KOA) over 3 and 9 years. Collected sera and urine samples from UTartu were used by partners, amongst others to test the IncaArray system at Arrayon. Measurements were done for all three biomarkers (urinary C2C, serum CILP and COMP) following the main aim to test whether biomarkers are able to differentiate OA patients with and without progressive structural changes in their knee joints. This knowledge is crucial for using specific biomarkers in combination with SPION.
Four of nine work packages dealt with the coordination of the project (WP 5 and 9), the ethical, legal and social aspects (WP6), the education of young investigators and the dissemination and exploitation of results at conferences, by publications and by exchanges with industry (WP8).
While WP9 was coordinating the project between partners and the EU Commission regarding legal and financial issues, WP5 was concerned with scientific coordination and the development and implementation of a web-based document system for sample, results and protocol exchange. The system was applied successfully and could be further developed for the application in other projects in United Kingdom and in Switzerland, latter starting in 2014. The project underwent an amendment in WP6 (ELSI) and WP7 (Toxicology) and a new WP leader in WP3a (Biomarker) had to found as Prof. Dick Heingård passed away in May 2013. His collaborator PhD Patrik Önnerfjord took over, however Dick Heingård‘s considerable knowledge and many of his ideas and results that were not documented were lost.
The ethical, legal and social implications (ELSI) were dealt with by WP6. The main aim to inform and educate lay people, consumers and patients was done by conferences and several seminars organized on these issues and presented by reports. Papers were published, and as the initially planned book was abandoned, a larger reports will be published on the webpage of NanoDiaRA in the form of an electronic book.
Dissemination and exploitation of results was the objective of WP8 and undertaken by all partners by presenting the results at international, European and national conferences, seminars and workshops and by publishing them in peer-reviewed journals. Further to this, exploitation of the novel imaging results was undertaken through a workshop in Zurich with companies outside the consortium. Education of young investigators was a continuous task, and four “summer” and “winter” schools were organized by consortium members and held in Lausanne and Kandersteg, Switzerland, Lund, Sweden, and Berlin, Germany.
In summary, it can be stated that the translational approach to bring nanoparticle and biomarker knowledge together for use in clinical application worked very well and that most of the objectives of this project could be achieved. Such large consortia with high and very detailed expertise in the various fields however need a certain time to come to a level of understanding which is necessary to easily exchange the information. The various project and workpackage meetings, the summer schools and trainings of young investigators which accompanied the research were very helpful in this respect, and the objectives of these work packages were fully achieved. The research itself faced several challenges in view of high safety requirements for in vivo applications, high specificity of antibodies, reliability of processes in nanoparticle synthesis, coating and antibody functionalization and the chronology of the various research activities. Looking back from the knowledge and experience gained in these four years, the Consortium would propose to put the objectives for a next complex project into a five-years-term to better handle unexpected events.
The objectives to develop SPION for early diagnostics in RA and OA could be achieved in a first aspect. It was successfully demonstrated that SPION technology with fixed coatings is safe and specific for depicting inflammatory events in joints by using magnetic resonance imaging. The benchmarking with Gd-based contrast agents showed other kind of specificity over time to the lining of the inflamed synovial membrane instead of uptake in the oedematous tissue. A first demonstration of functionalized SPION with cartilage antibodies showed increased localization around the cartilage rather than in the synovium, however these early investigations need more careful control studies to further understand the mechanism of the attached antibody. SPION technology showed also to be a successful tool in the in vitro diagnostics in multiple arrays and for unknown biomarker detection in biofluids. In the assay technology, SPION can be used in a combined approach with the assay to up-concentrate the amount of biomarkers in larger quantities of biofluids and by this increase the specificity in biofluids of e.g. rare diseases or those with unknown biomarkers. However it can be stated that a fifth year would have increased the opportunities to be nearer to clinical translation especially in respect to antibody functionalized SPION.
The various investigations of RA and OA progression in view of functional data as well as the computation of quantitative parameters of cartilage and immunohistochemical studies of various patient cohorts helped to better understand the progression of RA and OA and to later combine the early diagnostics with SPION with other diagnostic tools.
Project Results:
In this section there are many images and graphics included. Please see attachment for the complete description of the main S&T results/foregrounds.
Potential Impact:
The Document of Work discussed the early diagnosis of rheumatoid arthritis and osteoarthritis in view of improving a European health problem and by this an improvement of European social and economic cohesion. The aim was to achieve this goal by introducing new in vitro and in vivo diagnostic tools to detect the disease events earlier and more specifically than by x-ray radiology which can detect only already established degradation and by immune assays normally used for detecting biomarkers in biofluids.
A new article of the Center for Surveillance, Epidemiology, and Laboratory Services, Centers for Disease Control and Prevention (CDC), U.S. Department of Health and Human Services, Atlanta, USA reported that during 2010 to 2012 about 10% of adults in USA had arthritis-attributable activity limitations (AAAL). Approximately half of all adults with heart disease or diabetes had arthritis, and one fourth of adults with either condition and arthritis had AAAL. Approximately one third of adults who were obese also had arthritis, and 15% of those adults had AAAL. A current U.S. Department of Health and Human Service initiative addresses the burden of multiple chronic condi¬tions, which now affect one in four adults and are increasingly common with the aging of the population. The literature outcomes mentioned in the DoW in 2009 have not much changed and research in drug development did not show real break trough for treating both diseases. On the contrary, RA and OA will lead to an substantially increase in health care costs, limits severely the patients in their workforce, reduce the ability to perform normal activities and therefore reduce drastically the quality of life. A report on the limitations of activities of people with musculoskeletal disorders from 2012 still highlights that these diseases amongst them RA are the “major cause of morbidity throughout the world, having a substantial influence on quality of life (QOL)”. The discussion summarized that even developed countries have to face a larger burden of such disorders due to an aging population, and that for these diseases – although associated with great deal of disability, impairment, and handicap – the research remains unrecognized, under-appreciated, and under-ressourced. We will discuss this topic later on.
The list of treatments including all kinds of medications like non-steroidal anti-inflammatory agents (NSAIDs), corticosteroids, and disease modifying anti-rheumatic drugs (DMARDs), but also the reduction of joint stress, physical and occupational therapy, and surgical intervention, proposed today by highly recommended clinics has not much changed. DMARDs therapy is shown to cost between 6’500 and 10’000 EUR per patient per annum and TNF-a therapy costs up to 25'000 EUR per patient per year. However, comparing the costs of treatment with costs for the society if patients are not treated effectively, the treatment of RA can lead to a reduced cost to society as the cost for loss of productivity to society can be same or even higher than the costs of a treatment. The development of potential therapies is driving the testing market in Europe as such diseases are expected to expand rapidly. Many global key players are active on the market of molecular diagnostics being aware that the varying symptoms of patients especially in the early phase of RA and OA are considered difficult to diagnose. Multiple tests have to be conducted before making a diagnosis and therefore novel in vitro diagnostics are needed. The trend towards integrated healthcare and a gradual shift to ‘proactive’ testing rather ‘reactive’ diagnosis is likely to benefit this market in long term and companies have to align their positioning strategies to benefit from the changing markets and obtain maximum return on investment. Therefore we are convinced that the early diagnostic measures to find possible biomolecular indications for the diseases are important ways to better handle the treatment. The increasingly important tools in the development of new targeted therapies are related with new biomarkers and the main requirements for quantitative imaging so to connect cellular processes with therapy. One of the main aims of the NanoDiaRA project was to qualify imaging biomarkers of cartilage tissue degradation and an important step forward was done in understanding which biomarkers are in particular important, and to find ways to combine them with the imaging technology.
Developing new technologies and trying to introduce them into a market also means to educate and inform experts and lay people as well. A Frost & Sullivan publication of 2013 mentioned that for autoimmune diseases like rheumatoid arthritis diagnostic tests are available, however that the underlying key mechanism which are related to such a disease still seems to be vague and unclear and that research to better understand such key regulatory processes are only of moderate interests across Europe. Although the associations in Europe which primarily govern autoimmune research have educational programs for lay people and the scientific community, we think that education and information regarding both the disease itself and the new developments in disease diagnostics and therapies would be wise to be improved. Having Robin Poole, Professor emeritus of McGill University and former Director of the Canadian Arthritis Network as Scientific and Clinical Advisor attending most of NanoDiaRA Meetings we learned that consumer groups demand more choice in diagnosis, pain and disease management for their members, and are pressuring for better early diagnosis and treatment. In Europe the European Union pays particular attention to the needs of diseased people and to enable them to fulfil the roles and responsibilities of citizenship and have the same individual choices and control over their life as non disabled people. For RA patients this would mean to reduce the heavy burden of disease, to improve of the physical and psychological dependence and to minimise their dependence on nursing staff and family and to increase their workforce and the quality of life.
NanoDiaRA tried to educate and inform in different ways, a) by training the young investigators of the project through summer schools and by exchanging them between the different laboratories to learn new and other methods, b) by informing and discussing with experts the value chain of nanotechnological products in medicine and by this especially the risks and legal sides of such products, c) by exchanging with lay people about diagnostic and therapeutic approaches and to inform them about ethical, legal and societal implications, and d) by informing the wider European and international scientific and technological community about the project. In addition, further contacts with other European projects in this field were established, companies were contacted and the results discussed with them for further exploitation.
1.4.2 The potential impact
The value of iron oxide nanoparticles (SPION) as contrast agents were accepted by both FDA and EMA for imaging enhancement in liver and lymph nodes. Having in mind this and various studies already undertaken with a novel iron oxide nanoparticle formulation based on amino polyvinyl-alcohol (PVA), it was subsequently the next step to develop and use these nanoparticles for the RA and OA diagnostic market.
SPION are characterized by their paramagnetic properties that only occur on the nanoscale (below 100 nm). This makes them distinguishable from other “nano” products in nanomedicine like the liposome technology, which is nanoscale (below or around 100 nm) but without providing “fundamentally new properties and functions”. Applications of nanoparticles in medicine and diagnostics have to meet stringent safety requirements, notably no toxicity in the application, reliable and safe production, knowledge about bio-distribution and clearance of such particles when administered to the body and of course a higher performance in diagnostics than competing materials. The research project was therefore created to develop such systems along the whole value chain up to pre-clinical investigations; further development to clinics is beyond the scope of this project. Three main objectives describe the integrated interdisciplinary research efforts which are being made: (i) application of SPION technology as a contrast agent in targeted molecular imaging by MRI, (ii) development of specific multimodal array technologies using SPION for concentration of biomarkers in biological fluids to increase sensitivity and specificity of the array and (iii) biomarker development based on proteomic investigations and ELISA assays and the search for possible new biomarkers in biofluids using the SPION technology (biomarker-fishing). For all these tasks reliable and industrially effective transferable processes were developed, creating meaningful SOPs to evaluate the safety of these particles for human application and improving our understanding of arthritic disease, disease progression and its biologic answer through corresponding biomarkers and their assay technology.
The most profitable nanomedical products today are anticancer delivery systems based on liposomes, showing a steadily increasing market, as these products answer an urgent demand of new therapies able to better fight this highly aggressive and increasingly frequent disease. Although not much is published so far or available as nanotherapeutical approach, RA is mentioned as an important field e.g. in the review of Christine T.N. Pham on “Nanotherapeutic approaches for the treatment of rheumatoid arthritis.“ Current strategies and recommendations for RA are centered on early, intensive treatment with disease-modifying anti-rheumatic drug (DMARD) in monotherapy or in combination with non-steroidal anti-inflammatory drugs (NSAIDs) or by using biologics like cytokine antagonists (TNF, IL-1 and IL-6 inhibitors or receptor antagonists), B cell depleting agents, and T cell co-stimulation modulator. Some combinations may even reach to achieve positive resonance for up to 70% of the patients. However, there are several drawbacks especially to current biologic therapy as it is cost intensive and accompanied in some cases by infectious complications, and the loss or failure to maintain response overtime. The treatments using e.g. glucocorticoids and given systemically or in some cases intra-articularly (ia) may also be associated with a number of unwanted side effects, like e.g. increased risks of cardiovascular diseases, osteoporosis, infections.
Moreover, the optimal and effective therapeutic approach to an individual cannot be predicted at this point due to the heterogeneity of the patients, thus necessitating a “trial and error” approach that increases costs and delays clinical response. NanoDiaRA-developed SPION have the potential to early diagnose inflammatory events by either macrophage in vivo magnetic resonance imaging or even more specific with specific tissue binding antibodies. These SPION formulation which have shown to monitor therapies can be a very helpful tool in the pre-clinical and clinical development phase of future drugs either by decorating the SPION with biological or other drugs or by monitoring them. The next step on this development will be the demonstration of GMP production; the SOPs for translation of processes established.
A breakthrough for a marketable product cannot longer be only in the hands of researchers and scientists but companies have now to take over the responsibility for the next development step towards clinics to have an impact on innovation in Europe and the creation of further working places. This is especially true for projects like NanoDiaRA, where inorganic nanoparticles for medical applications are developed. Such particles are treated from the regulatory side as medication regardless of their use only for diagnostic. This leads to relative high barrier to transfer the results from academia to industries and finally to clinics. Because NanoDiaRA was aware of this problem from the beginning, parameters like reliability, reproducibility, toxicity, biocompatibility, scalability, acceptability by clinical doctors and patients, social impact and acceptance has guided the project development beside the also important scientific questions and constraints to reach finally the project goal. We are convinced that with this approach, which leads maybe to a lower number of scientific publications during the project period, but to very useful, scalable and very well documented processes ready for scale-up, we would have on a long term a substantial impact regarding the clinical use of nanoparticle for diagnosis and therapy.
Figure 13: Historical and projected markets for nanotherapeutics (Tx) and nanodiagnostics (Dx).
FIGURE SEE ATTACHMENT
Imaging diagnosis has gained importance in recent years as shown in Figure 11. The main benefit of molecular imaging for in vivo diagnosis lies in the early detection of disease and the monitoring of disease activity and response to treatment, „supporting the development of individualized medicine and the real-time assessment of therapeutic and surgical efficacy“. Comparing different methods, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) offers greater sensitivity but a lack of anatomical context. In contrast, MRI provides accurate anatomical detail but no data on disease activity. At present it is the preferred option for cellular tracking. To enhance the image quality, contrast agents are used. This market for MRI products is reported to be more than $1 billion annually and is currently dominated by gadolinium chelate-based agents, which are the subject of class-action lawsuit concerns due to the potential release of gadolinium into the body. The global contrast agent market is expected to strongly increase if the targeted MRI imaging, which is in the center of NanoDiaRA project, becomes successful to specifically localize the molecular target. Advances in MRI technology especially in the spatial resolution and the fact that MRI is a non-invasive, irradiation-free technology will push this development. NanoDiaRA has alos further improved the imaging quality by developing new imaging procedures like the positive contrast MRI technique (dUTE – difference Ultrashort echo time). The development should also focus on reduced concentrations of the agents at the targeted side, as until now high quantities for cell recognition and high affinity are necessary to balance MRI’s inherently low sensitivity. Increased market penetration of medical imaging equipment is expected to lead to significant opportunities for the use of contrast agents. Lodhia mentioned that „for molecular and cellular imaging with MRI, the current research sets a platform for the further development of SPIONS“ and sees the „next step in SPION development [...] towards molecular imaging“ as a „useful complementary role“ to nuclear medicine and PET. If the development of specific targeting, iron oxide nanoparticles could be successful, the market-potential may be large enough to successfully reintroduce this type of nanoparticles for molecular imaging. This type of particles is not restricted to MRI, MPI (magnetic particle imaging, a method developed by Philips) , showing a higher resolution is also based on this type of SPION. The fact that SPION-antibody specific MRI could be demonstrated in NanoDiaRA for the first time with RA biomarker raises hope that molecular imaging will also be used for early RA diagnostics.
1.4.3 Further Education and Training to improve the chances in careers
In total, 200 people were partially or fully funded by the FP7 project; 66 working places could be established specifically for the project. Two third of the collaborators were woman, whereby 5 of the around 50 permanent female collaborators were pregnant during the project time (having 6 pregnancies). Further education and training was one of the important tasks of NanoDiaRA to increase the future career opportunities of the young investigators, especially also women. The project NanoDiaRA has shown a high diversity in activity and in research fields covering the whole value chain of a product from basic research (necessary to understand biomarker and particle characteristics) towards nanoparticle development and pre-clinical investigations as some clinical investigations on the functional status of patients as well. The project therefore promoted inter-university and university-industry contacts and cooperation between young investigators, trainees and technicians. This was done by organising four summer schools of at EPF Lausanne, University of Lund, Charité University Hospital Berlin and a fourth as “winter school” in Kandersteg, Switzerland together with the Competence Center for Materials Science and Engineering, CCMX. The aim was to provide one week of lectures, specific break-out sessions, lab work, practical training and insight into the work processes of the hosting partner institution for young researchers/clinicians active in the Consortium as well as the presentation of current science & technology from external experts to increase the internal overall knowledge in the different fields and to allow all collaborators to understand the typical language of the other professional and scientific sector. We could recognize that each scientific or industrial domain had its own conceptual metaphors which were not always understood by the other research community. In addition, we stated that engineers present their ideas and results mainly in a way that formulas play a role while biologists and clinicians are using pictograms for their presentations. In addition to the many expressions and abbreviations it was necessary to achieve a communication basis so that everybody could understand the main activities, and many of the collaborators learned additional skills and knowledge. The first summer school took place at EPFL; two more followed at ULund and Charité to cover all aspects from technology to clinic by courses, practical work and visits. Lab work and student seminars accomplished the summer school. The summer schools were based on three different main topics:
- Nanotechnology: synthesis, coating, functionalization of nanoparticals, physical and chemical understanding of the properties and processes, understanding about legal and ethical standards
- Cell and molecular biology in RA and OA: Introduction to rheumatic diseases, tissue organization: Cells and Matrix, turnover of matrix, cell biology in practice, inflammation, diagnostic oportunities in rheumatic diseases, practical work
- Clinical aspects of RA and OA: RA therapy - from gold standards to new approaches, materials & methods in the development of nanomedicine, diagnostics in RA and OA, practical lab course, nanotherapy, data banking and legal aspects
Figure 14: Participants at the summer school in Lund, May/June 2012
Figure 15: Participants at the summer school at Charité Berlin, July 2013.
BOTH FIGURES SEE ATTACHMENT
The project delivered also short reports from young investigators to learn more about the impact about such training exchanges as seen in some examples of the exchange practice in NanoDiaRA:
- Training visit from a collaborator of University Geneva, Switzerland at University of Nijmegen, Netherlands: learning more about the AIA rat model and erosion and inflammation histological scoring, AIA model induction (intra-articular injection), subcutaneous injection, emulsion preparation, introduction to the rat knee joint anatomy in absence and presence of AIA, followed by a training on the assessment of the degree of inflammation (infiltration scoring) and extent of joint destruction (erosion scoring) under the supervision of a senior scientist
- Training of a collaborator of the Parcelsus Private University in Salzburg, Austria at University of Geneva: to get insight into the image acquisition procedures at the University of Geneva, to discuss further image analysis methods to be employed in the workpackage, and to participate in an MR imaging session to provide insights into the animal work, the histological evaluation performed, and the MR imaging methodology, which is the basis for the analyses done at PMU, so to allow to know more about the requirements for the next software application to be developed at PMU.
- Training of collaborators at Charité University Hospital, Berlin Germany by a collaborator from EPF Lausanne, Switzerland for demonstration of protein separation from serum using the SPION technology, discussion about the sample types and the nanoparticles. All separation devices, equipment and materials (e.g. a reactor, valve, pump, connectors, tubes, Ni-Fe wire) needed for a full demonstration were transported to Charité.
- Training of collaborators of EPFL and University of Fribourg, Switzerland at company Arrayon, Neuchâtel, Switzerland to exchange about the antibody functionalization of SPION for in vitro application.
- Training of collaborators of EPFL and University of Lund, Sweden to exchange about the antibody functionalization of SPION for in vivo application.
Such exchanges between laboratories in different countries and between industry and academia enabled the young investigators to learn to use and improve so-called “soft” and “hard” skills in another environment – engineering in clinics, research at a company and more – and to exchange further personal information also in the environment of another culture.
These experiences are highly valuable when changing the employer and even when changing from university to industry. Young investigators received some form of information advice and guidance not only from the direct contact but also had always the opportunities to further discuss with principal investigators or other experienced collaborators, which clearly opened their horizon.
1.4.4 Dissemination activities and exploitation of results
The NanoDiaRA Consortium has presented results of the project more than 160 times at various conferences, workshops and seminars to an audience of mostly experts but also to lay people or patient organisations. Presentations at such events were given either orally or by posters while presentations at fairs like in Aarhus in 2012 were also provided interactively by electronic media. The following two diagrams show the activity of NanoDiaRA to present their results by oral or poster presentations whereby for one event the various partners were counted, who were presenting the project. Various workshops and scientific meetings were organised within NanoDiaRA (at least twice a year) and external speakers were invited (like Prof. Christoph Garnier from Inselspital Bern and Kenneth A. Dawson, University College Dublin), but also conferences and workshops for external people with invited speakers were organised. Two conferences were organised in Switzerland by MatSearch and EPFL (Lausanne 2012 and Bern 2013), one in Berlin Germany by EUAK (2012) and one in Portugal by MatSearch in Collaboration with the EU project NANOFOL.
Figure 16: Overview of the countries in which collaborators held presentations about NanoDiaRA work (in total 77).
Figure 17: Overview of the countries in which collaborators presented posters about NanoDiaRA work (in total 44).
Figure 18 shows the partner allocations of the poster authors of NanoDiaRA (in total 113 – multiple authors counted).
Figure 19 shows the partner allocation of the oral presentation authors of NanoDiaRA (in total 124 – multiple authors counted).
ALL FIGURES SEE ATTACHMENT
These diagrams depict the many activities of NanoDiaRA regarding dissemination of the results. Beside these presentations at national and international conferences, the results were also presented in peer reviewed publications. 33 publications were prepared during the project and up to now 25 have been published, the other 8 are under review. Further 13 papers are in progress. One article could be published in Chemical review having an impact factor of > 40, 12 of the documents were published in journals with impact factors between 4.6 and 10, the rest was published in journals with impact factors below 4.5. Another 21 publications are in preparation and partially submitted and further 8 were published in journals which are not reviewed.
Exploitation of the results was organised by MatSearch in contact with a company specialized in diagnostic imaging, a company specialized in SPION products and a university having a GMP facility. These contacts are foreseen to be used for follow-up projects. MatSearch and some partners organised a further application for a new project with a similar approach but was not successful.
1.4.5 Conclusions
In the four years of the NanoDiaRA project, it was possible to generate besides the scientific results a high positive social impact. 66 new jobs were provided, and many, especially young scientists could receive financing for their research. The quota of women working for the project was clearly above average. Interdisciplinary training and work exchange lead to well-educated young researchers in our project with insight into other disciplines and scientific cultures.
RA and OA are diseases for which better treatment and earlier diagnosis are a strong need, and developing SPION for targeted MRI-imaging for this market is a novel approach. The project tried to prepare for a further exploitation of our results from the beginning by creating standards, doing thorough and broad research on possible toxicity in each phase of the development, and by communicating our research to the scientific community as well as to the public. The results and developments of our project were presented on many international conferences by talks, posters and other media, by materials for the public like flyers, press releases, public discussions and the NanoDiaRA homepage, and of course by scientific publications; a task which is still ongoing.
Further exploitation towards new national and European projects occurred by various contacts with external companies and research groups for SPION technology and for biomarker development. Negotiations are ongoing under Non Disclosure Agreements.
List of Websites:
The NanoDiaRA public website is updated on a regular basis by EUAK and MatSearch. The website is available at http://www.nanodiara.eu/
Administrative Coordination
Priv.-Doz. Dr. med. Felix Thiele, M. Sc.
Europäische Akademie zur Erforschung von Folgen wissenschaftlich-technischer Entwicklungen Bad Neuenahr-Ahrweiler GmbH
Wilhelmstr. 56
53474 Bad Neuenahr-Ahrweiler
Germany
felix.thiele@ea-aw.de
www.ea-aw.org