Molecular imaging as a tool to image drug delivery across the Blood Brain Barrier: OnconanoBBB project George Loudos Department of Biomedical Engineering, Technological Educational Institute of Athens, Greece Consortium: Technological Educational Institute of Athens (EL) Pharmidex (UK) University of Brighton (UK) Coordinator : Prof. George Loudos (EL) e-mail: gloudos@teiath.gr Website: http://www.bme.teiath.gr/ni/EnglishVersion/Project_files/2011_OnconNanoBBB/index.html FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Introduction: The problem • • • • • Brain cancer is an unmet medical need with few options for treatment and commonly associated with a poor prognosis in patients. Currently only up to 5% of small molecule drugs can cross the BBB; this presents a major hurdle in treating brain diseases, such as brain cancer. The BBB severely restricts the movement of hydrophilic solutes via the intercellular cleft. A safe, reliable and consistent method of delivering compounds across the BBB would therefore be highly desirable for the discovery and development of new therapeutics for disorders of the brain. The aim of the project is – to work on the problem of delivering therapeutic agents, across the blood-brain barrier (BBB) at the efficacious dose – to assess the application of in vivo imaging tools for drug delivery monitoring and assessment FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Introduction: Reasons for carrying out the project • • • • Traditional approaches to getting compounds into the brain are crude and include direct administration of therapeutic agents such as drugs or stem cells into the brain. Pharmidex has developed a drug delivery system (Cerense™) that transiently and reversibly opens the BBB to entry of molecules into the brain without inducing tissue injury. Cerense™ delivery technology unlocks the potential of CNS therapeutic opportunities by providing a novel technology that opens the blood–brain barrier. Cerense™ has been shown to increase substantially the brain penetration of a chemotherapeutic agent and markedly enhance its chemotherapeutic efficacy. FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Introduction: The consortium UoB TEIA •Lipid formulation •Liposomes radiolabelling •SPECT in vivo imaging PPS •Liposomes formulation •Liposome characterization •Assessment of brain penetrability •CNS drug discovery expertise •Biodistribution and safety studies OncoNanoBBB FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Research objectives • • • • • • • • • • • Synthesis a range of nanoparticles for in vitro and in vivo assessment Physicochemical characterisation (shape and size) of nanoparticles in different mediums Optimise nanoparticles formulation using a variety of pharmaceutical excipients Study mechanism of action (MOA) using high resolution imaging using endothelial cells both in vitro and in vivo Labelling of nanoparticles with radioisotopes, without altering their biological properties Assess the ability of nanoparticles to enhance transport of a diverse set of existing cancer drugs in in vitro models of BBB Establish the in vivo pharmacokinetics Structural Distribution Relationship (SDR) for these nanoparticles via different administration routes Establish the in vivo neuropharmacokinetics (brain pharmacokinetics) Structural Distribution Relationship (SDR) for these nanoparticles utilised via optimised route identified from above Establish and validate imaging protocols for screening of Cerense™-formulated chemotherapeutics in models of brain cancer Assess a number of CNS and non-CNS penetrating chemotherapeutics with and without the Cerense™ technology in in vivo model of brain cancer Explore a range of future applications for this technology in critical care medicine FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Scientific methodology Year 1 and 2 Year 3 and 4 • • Synthesis of lipids and characterization – Design of lipids to ensure radiolabelling • Synthesis of liposomes and characterization – Optimize liposomes for drug loading – Functionalization for targeting • • • – liposomes loading • • In vivo tests with functionalized liposomes – Comparison with Year 1 and 2 results – Assessment of different radiolabelling strategies • Evaluation of SPECT imaging for in vivo imaging • In vivo studies in normal and tumor bearing mice Introduction of Cerense technology – Comparison with reference liposomes Radiolabelling of liposomes with Tc99m – Optimization of system geometry – Assessment of imaging protocols Drug selection Labelling with fluorophores – In vivo optical imaging studies Development of intracranial tumors – In vivo tests in those models – Assessment of response to therapy – Using standard methods – With PET imaging – Comparison with ex vivo biodistribution data FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Synthesis of lipids • • Based on bibliography candidate lipids were selected. Taking into account the required radiolabelling steps 2 different lipids were initially formulated by TEIA and sent to UoB for liposome formulation. FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Synthesis of liposomes • DSPC, Chol, PEG-DSPE and DSPE-PEG2000-COOH (for LP-COOH) or DSPE-PEG2000PEC-2 (for LP-PEC) were dissolved in 2/1 chloroform/methanol in a molar ratio of 51.9: 44.9: 1.7: 1.5, resulting in >3mol% PEGylated lipids Methodology for synthesis of the lipids and liposomes. FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Liposomes radiolabelling (I) Our aim is: A. during manufacturing • Encapsulation to attach a radioisotope to liposomes B. Reduction Method – with high efficiency – with in vitro and in vivo stability • use the gamma photons, emitted from the isotope in order to study the biodistribution of the liposomes – obtain successive images – derive qualitative and semi-quantitative information D. Chelation Method C. “After-Loading” or Remote Labeling Method Laverman et al. Methods In Enzymology, Vol. 373, 2003 FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Liposomes radiolabelling (II) Schematic presentation of the direct or membrane labelling approach for preparing radioactive 99mTc-LP-COOH Schematic representation of the surface chelation approach for preparing radioactive 99mTc-(CO)3-LP-PEC FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 SPECT imaging Tomographic SPECT system γ-camera head Head Liver 2005 2008 Bladder Spleen Injection Planar static bone imaging with Tc99m-MDP and dynamic study with a Tc99m-Bombesin derivative point head tail FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 SPECT imaging (IV) 99mTc-NBRh1 - Dynamic Study spleen 80 liver 70 60 50 40 tail bladder head 30 Dynamic image up to 70min p.i 20 10 Pa ng re as Lu ng s Sp le en M us cl e s St om ac h In te rs ins Ki dn ey s He ar t Li ve r Bl oo d 0 Biodistribution data FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 12 Results: Biodistribution in Normal Mice Comparative study of organ uptake for the two 99mTc labelled liposomes in normal Swiss mice at time intervals of 5, 60 min and 24h. Biodistribution values represent the mean±st_dev of %ID/organ (3 animals per time point). FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Results: Planar normal mouse imaging A. LP-COOH B. LP-PEC Scintigraphic images (left) at 10min p.i. (one short 2min frame), (center) at ~1h p.i. (sum of all images from 10min to 52min for LP-COOH and up to 58 min for LP-PEC) and (right) at 24 p.i. of female normal Swiss mice intravenously injected with 3.7 MBq or 100μCi οf the radiolabelled LP-COOH (A) and 0.37-0.74 MBq or 10-20 μCi LP-PEC (B) FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Results: Planar U87MG mouse imaging Biodistribution study of organ uptake for (A) 99mTc LP-COOH (B) 99mTc(I)-(CO)3-LP-PEC in tumour bearing mice at 60 min p.i.. Biodistribution values represent the mean±st_dev of %ID/organ (3 animals were used per time point). Sum of all scintigraphic images from 10min to 56min for (left) 99mTc-LP-COOH and (right) 99mTc(I)-(CO) -LP-PEC of tumour bearing mice 3 intravenously injected with 3.7 MBq or 100μCi οf 99mTc-LP-COOH and 0.37MBq or 10 μCi of 99mTc(I)-(CO) -LP-PEC. 3 FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Results: Brain imaging & comparison with HMPAO Comparison of image contrast of 99mTc labelled liposomes (LP-PEC & GLP-PEC) and 99mTcHMPAO at 1h p.i. (as sum of all images from 10min to 60min) in female normal Swiss mice by a custom high resolution SPECT system (1.5mm spatial resolution). FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Scientific highlights so far • • • Two alternative radiolabelling methods have been established, with the chelator method providing more stable complexes, but requiring purification process, which decreases sensitivity. Planar SPECT imaging can provide spatiotemporal information on lipids biodistribution, which is comparable to ex vivo analysis; higher overall system resolution is expected to improve quantification In vivo imaging, as well as biodistribution data have confirmed passive targeting on U87MG tumors; those liposomes can be the reference systems for assessing drug delivery to BBB. FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Ongoing work (I) Liposome targeting • • Novel glucose functionalized lipids are been formed in order to formulate liposomes with improved properties in terms of BBB targeting The first lipid was synthesized in TEIA and sent to UoB for liposome formulation. Chemical formula and the characteristics of a glucose modified lipid FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Ongoing work (II) Drug selection • Taking into account the available drugs, as well as the techniques that are available to the consortium for their study, the consortium has decided to focus on Vincristine and Methotrexate, which were recently purchased and will now be tested (PPS) FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Scientific report: Ongoing work (III) Optical imaging • TEIA researchers, having a strong imaging background contributed in the installation of the system and initial evaluation tests. • TEIA and PPS work together on the full system evaluation, as well as on the design of in vivo studies, which will take place in terms of the project. • In Year 3 and 4 candidate liposomes will be functionalized with fluorescent agents and imaged in vivo. • Those results will be correlated with in vivo SPECT imaging • According our knowledge no such system is installed in Greece – Its technology is rather simple and interesting and complementary to the one that Greek researchers have – Greek secondees have started such activity in Athens and study SiPM detectors for optical light detection Training and Networking FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Dissemination/Outreach Activities (I) • • • During the first year of the project few results were available, thus the project was mainly mentioned in invited lectures and courses During the second year the project outcomes resulted to a goo number of dissemination activities in scientific and broader audience The project website contains all major project information and will now move from the teiath.gr domain to www.onconanobbb.eu FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Dissemination/Outreach Activities (II) Publications Scientific Journals 1. 2. Eirini Fragogeorgi, Irina N Savina, Theodoros Tsotakos, Eleni K Efthimiadou, Stavros Xanthopoulos, Lazaros Palamaris, Dimitris Psimadas, George Kordas, Sergey Mikhalovsky, Mohammad Alavijeh, George Loudos, "Comparative In vitro Stability and Scintigraphic Imaging for Trafficking and Tumour Targeting of a Directly and a Novel 99mTc(I)(CO)3 Labelled Liposome", submitted to International Journal of Pharmaceutics, 2013. Review article on the role of radiolabelled nanoparticles to assess drug delivery across BBB (submitted). International Conferences 1. 2. 3. 4. 5. Preliminary In Vitro and In Vivo Evaluation of Glucose Modified Stealth Liposomes labelled with technetium tricarbonyl core for Brain Targeting, European Molecular Imaging Meeting – EMIM, Antwerp, Belgium, 4-6 June 2014. Preliminary in vitro and in vivo evaluation of Liposomal nanoparticles for passive and active tumour targeting by scintigraphic and MRI imaging, Bianchi Patrick, Fragogeorgi Eirini, Efthymiadou Eleni, Xanthopoulos Stavros, Psimadas Dimitrios, Bouziotis Penelope, Kordas George, Loudos George, Silvio Aime, 3rd International Conference on PET/MR and SPECT/MR, Kos Island, May 19-21 2014. Eirini A. Fragogeorgi, Irina N. Savina, Theodoros Tsotakos, Elen Efthimiadou, Chris Tapeinos, Stavros Xanthopoulos, Lazaros Palamaris, George Kordas, Sergey Mikhalovsky, Mohammad Alavijeh, George Loudos, "Comparative in vitro and in vivo evaluation of nanosized liposome appropriately modified for being labelled with Tc-99m by two different radiolabelling approaches", Annual meeting of the COST Action TD1004, September 2013, Athens Eirini Fragogeorgi, "Non-Invasive Nanoparticles evaluation using in vivo imaging", 2nd Conference on Bio-Medical Instrumentation and related Engineering and Physical Sciences, BIOMEP, Saturday 22 June, 2013, Athens, Greece. Fragogeorgi I., I.N, Savina, T.Tsotakos, Varvarigou A.D., Mikhalovsky, S.V., Alavijeh M.S., Loudos G., "Radiolabelling optimization of new stealth liposomal nanoparticles with Tc-99m. Preliminary study with challenging promises for the imaging of lipid-based delivery systems across the BBB", EMIM 2013, 26-28 May 2013, Torino, Italy. FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Acknowledgements OnconanoBBB team • Andy Harris • Eirini Fragogeorgi • Eleftherios Fysikopoulos • Eleni Efthimiadou • George Loudos • Irina Savina • Mairead Stickings • Mansoor Chishty • Maria Georgiou • Matthew Illsley • Mo Alavijeh • Ray Whitby • Sergey Mikhalovsky • Theodora Christopoulou • Zeeshan Qaiser Other team members • Anil Mishra • Selina Teixeira • Dimitrios Psimadas • Theodoros Tsotakos • George Kordas • Lazaros Palamaris • Kostantinos Mikropoulos • Pavlos Papamichalis EU officers • Bronius Goosens • Julie Deacon • David Pina FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013 Thank you FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013