Matrix metalloproteinase activated Multimodal 'Theranostic' Drug Delivery Imaging Agents for thrombosis

Stroke is a pathological outcome of occlusive thrombi in the cerebral artery. This occlusion leads to ischaemia, and, if not treated, brain damage, potentially leading to profound mental symptoms, paralysis, and death. The lifetime risk of stroke is 20-30%. Early treatment for stroke patients is critical since untreated thrombi have more polymerised fibrin, making them more resistant to degradation by thrombolytic drugs. Endovascular intra-arterial delivery (EID) of thrombolytics or mechanical clot disruption are highly effective therapies resulting in rapid revascularisation in a proportion of eligible patients with evidence of improved functional outcomes compared to medical therapy. However EID is complex, requiring a high degree of operator skill and sufficiently trained staff to provide a 24 hour a day service. EID is also currently only indicated for patients presenting in the first few hours of symptom onset. Therefore EID can only be delivered in highly specialised units necessitating patient travel to the unit. This in combination with a prespecified time-window for the delivery of EID and mechanical disruption limits the delivery of this treatment to patients. A common thrombolytic is Alteplase, a recombinant plasminogen activator. Unfortunately, a notable side-effect of thrombolytic drug therapy, is intracranial haemorrhage (ICH) which occurs in 2.4%-10% of patients. These patients have significantly worse prognosis, with an increased risk of death or disability.

This proposal sets out to develop a series of state of the art therapeutic imaging or 'theranostic' agents for advancing targeted and personalised therapy in stroke patients. It focuses on the development of Matrix metalloproteinase (MMP) activated 'smart theranostic' MR imaging agents targeting thrombi. The drug release system is 'smart' as it would only activate once present within the thrombus, due to release of MMPs by activated platelets, allowing for precise targeting of the drug to the thrombus. Furthermore, due to the MRI capability of the theranostic, it would also be possible to have the non-invasive visualization of thrombolytic drugs in-vivo.

As such the development of these 'smart theranostic' agents, will help to limit the unwanted side-effects of stroke therapy, whilst simultaneously increasing the ability to image thrombi in vivo effectively.
It is expected that the results of this work will significantly contribute to a better understanding of drug delivery with the aim to develop theranostic imaging agents for personalised treatments in ischemic stroke.

This proposal contains four aims;
(i) the development and validation of nanoparticles, non-toxic quantum dot (QD) based probes that will specifically bind platelets, whilst not affecting the platelet's activation or functional status. Involving the synthesis of InP/ZnS based QDs, synthesizing bifunctional chelates for gadolinium (MRI) and conjugation of antibodies specific for platelets.
(ii) Development of a MMP specific mechanism of drug release: including peptide conjugation chemistry between Altepase and QDs through cleaveable peptide sequences, responsive to MMP released from activated platelets. Also including physical studies using MMP enzymes.
(iii) Evaluation of the thrombolytic capability of the NP-drug delivery system: Washed platelets and whole blood will be incubated with NPs, with and without Alteplase, and we will use our panel of platelet functional assays to determine how our preparation affects platelet activation. Thrombolytic drugs need to be effective at removing preformed thrombi to re-establish blood flow. Therefore whole blood will be taken in corn trypsin, and thrombi will be formed on collagen in the presence of thrombin, to allow the formation of fibrin rich thrombi. These thrombi will then be post perfused with whole blood containing NPs, with and without Alteplase and thrombus height, surface area, dissolution and fibrin content will be analysed via realtime microscopy.
(iv): Evaluation of the NP-drug delivery system in vivo. Mice will go under general anaesthesia, left carotid arteries will be exposed and injury will be induced by applying 1x2 mm filter paper saturated with anhydrous 10% FeCl3. Real time thrombus progression will be recorded using MRI and backed up by intravital microscopy for up to 40 minutes. Once a thrombus is fully formed, NPs with and without Alteplase, will be injected via tail vein and real time thrombolysis (thrombus size and stability) will be monitored.

Research into Ischemic Stroke and Cardiovascular related disease (CVD) is highly critical as both the World Health Assembly, and World Health organization have action plans to reduce global mortality from non-communicable diseases such as CVD, by upto 25% by 2025, with the lifetime risk of stroke at 20-30%. Part of this will be the development of novel, specific therapies, which open new avenues for medical professions to target thrombosis.

The aim of the project is to establish an interdisciplinary platform of research for the development of novel theranostic agents for advancing targeted and personalised therapy in thrombosis. It features a unique mixture of basic science (synthetic and physical chemistry, cell biology, and in vivo imaging). The immediate impact of the research will thus be the fundamental scientific advances in molecular design, probe synthesis, drug delivery techniques and therapeutics, in stroke. In the medium term, this novel research will directly influence biomedical imaging and the treatment of thrombosis - and therefore have significant impact on both patients, medical staff, and also pharmaceutical companies. An unmet global challenge is the development of novel technologies and imaging modalities that will assist medics and clinicians to have a dramatic effect on stroke treatment.

This proposal should result in new techniques and scientific advances in imaging and thus impact the knowledge economy of both academic and industrial scientists concerned with the discovery and development of novel theranostic probes for the detection and therapy of stroke. These probes would then be highly amenable for collaborative development including scale-up synthesis and the entry into clinical trials with an industrial partner. From the outset of the project, we will seek collaborators such Novartis, GE Healthcare and Sanofi Avantis to ensure research focus and effective translation and development - and ultimately, effective translation, animal practice and maximum impact.

Ultimately this proposal is designed to produce a therapeutic tool, that has significant patient benefit. This will be due to its ability to deliver the drug to the relevant location in a specific manner. This will reduce the dose of drug required for a therapeutic benefit, but should also therefore cause less side-effects, benefiting the quality of life of stroke patients. In addition medical staff could additionally use this technology to image and localise thrombi, separate to its use as a therapy, meaning this is a multipotential probe. Furthermore, this proof of principal grant, once proven to work, will allow for this technology to be used to target other situations in which thrombosis is relevant, such as cancer metastasis, and therefore should further expand the impact of the proposal.

However, furthermore, at the scientific level, this proposal is focused on exploring new science of relevance to the medical domain by integrating chemistry, physics research groups at Hull University and the platelet biology research groups at Hull York Medical School, with imaging groups at the University of Leeds. All the expertise and equipment is present at Hull and Leeds, and as such the relevant research groups are in the correct place to produce novel and internationally relevant research.

Exploitation and application
Hull has in place arrangements for the exploitation of research, and any formal agreement to exploit for profit the results generated in this project would be negotiated through this office.