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Molecular imaging as a tool to image drug delivery across the Blood Brain Barrier: OnconanoBBB project George Loudos Department of Biomedical Engineering,

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Presentation on theme: "Molecular imaging as a tool to image drug delivery across the Blood Brain Barrier: OnconanoBBB project George Loudos Department of Biomedical Engineering,"— Presentation transcript:

1 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) Website:

2 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

3 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.

4 Introduction: The consortium
OncoNanoBBB PPS UoB TEIA Lipid formulation Liposomes radiolabelling SPECT in vivo imaging Liposomes formulation Liposome characterization Assessment of brain penetrability CNS drug discovery expertise Biodistribution and safety studies

5 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

6 Scientific methodology
Year 1 and 2 Synthesis of lipids and characterization Design of lipids to ensure radiolabelling Synthesis of liposomes and characterization Optimize liposomes for drug loading Functionalization for targeting Radiolabelling of liposomes with Tc99m Assessment of different radiolabelling strategies Evaluation of SPECT imaging for in vivo imaging Optimization of system geometry Assessment of imaging protocols In vivo studies in normal and tumor bearing mice Comparison with ex vivo biodistribution data Year 3 and 4 Drug selection liposomes loading Introduction of Cerense technology Comparison with reference liposomes In vivo tests with functionalized liposomes Comparison with Year 1 and 2 results 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

7 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.

8 Synthesis of liposomes
DSPC, Chol, PEG-DSPE and DSPE-PEG2000-COOH (for LP-COOH) or DSPE-PEG2000-PEC-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.

9 Liposomes radiolabelling (I)
Our aim is: to attach a radioisotope to liposomes 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 Encapsulation during manufacturing B. Reduction Method D. Chelation Method C. “After-Loading” or Remote Labeling Method Laverman et al. Methods In Enzymology, Vol. 373, 2003

10 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

11 Tomographic SPECT system
SPECT imaging γ-camera head Tomographic SPECT system Head Liver Bladder Injection point Spleen 2005 2008 Planar static bone imaging with Tc99m-MDP and dynamic study with a Tc99m-Bombesin derivative tail head Our group has unique imaging equipment in Greece that includes: - A high resolution SPECT systems that we use for dynamic planar studies, as well as SPECT imaging. A dual head PET system, which has been used for mouse FDG imaging Recently we developed a dedicated gamma camera for the university of Patras with a 10 x 10 FOV based on 4 PSPMTs, compact ADCs and FPGA electronics

12 99mTc-NBRh1 - Dynamic Study
SPECT imaging (IV) 99mTc-NBRh1 - Dynamic Study spleen liver tail bladder head Dynamic image up to 70min p.i Biodistribution data

13 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).

14 Results: Planar normal mouse imaging
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 MBq or μCi LP-PEC (B)

15 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)3-LP-PEC of tumour bearing mice intravenously injected with 3.7 MBq or 100μCi οf 99mTc-LP-COOH and 0.37MBq or 10 μCi of 99mTc(I)-(CO)3-LP-PEC.

16 Results: Brain imaging & comparison with HMPAO
Comparison of image contrast of 99mTc labelled liposomes (LP-PEC & G-LP-PEC) and 99mTc-HMPAO 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).

17 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.

18 Chemical formula and the characteristics of a glucose modified lipid
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

19 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)

20 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 

21 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 domain to

22 Dissemination/Outreach Activities (II)
Publications Scientific Journals 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 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 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, May 2013, Torino, Italy.

23 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

24 Thank you

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