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, GreeceConsortium: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 isto work on the problem of delivering therapeutic agents, across the blood-brain barrier (BBB) at the efficacious doseto 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 OncoNanoBBBPPSUoBTEIALipid formulationLiposomes radiolabellingSPECT in vivo imagingLiposomes formulationLiposome characterizationAssessment of brain penetrabilityCNS drug discovery expertiseBiodistribution and safety studies
5 Research objectivesSynthesis a range of nanoparticles for in vitro and in vivo assessmentPhysicochemical characterisation (shape and size) of nanoparticles in different mediumsOptimise nanoparticles formulation using a variety of pharmaceutical excipientsStudy mechanism of action (MOA) using high resolution imaging using endothelial cells both in vitro and in vivoLabelling of nanoparticles with radioisotopes, without altering their biological propertiesAssess the ability of nanoparticles to enhance transport of a diverse set of existing cancer drugs in in vitro models of BBBEstablish the in vivo pharmacokinetics Structural Distribution Relationship (SDR) for these nanoparticles via different administration routesEstablish the in vivo neuropharmacokinetics (brain pharmacokinetics) Structural Distribution Relationship (SDR) for these nanoparticles utilised via optimised route identified from aboveEstablish and validate imaging protocols for screening of Cerense™-formulated chemotherapeutics in models of brain cancerAssess a number of CNS and non-CNS penetrating chemotherapeutics with and without the Cerense™ technology in in vivo model of brain cancerExplore a range of future applications for this technology in critical care medicine
6 Scientific methodology Year 1 and 2Synthesis of lipids and characterizationDesign of lipids to ensure radiolabellingSynthesis of liposomes and characterizationOptimize liposomes for drug loadingFunctionalization for targetingRadiolabelling of liposomes with Tc99mAssessment of different radiolabelling strategiesEvaluation of SPECT imaging for in vivo imagingOptimization of system geometryAssessment of imaging protocolsIn vivo studies in normal and tumor bearing miceComparison with ex vivo biodistribution dataYear 3 and 4Drug selectionliposomes loadingIntroduction of Cerense technologyComparison with reference liposomesIn vivo tests with functionalized liposomesComparison with Year 1 and 2 resultsLabelling with fluorophoresIn vivo optical imaging studiesDevelopment of intracranial tumorsIn vivo tests in those modelsAssessment of response to therapyUsing standard methodsWith PET imaging
7 Synthesis of lipidsBased 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 lipidsMethodology for synthesis of the lipids and liposomes.
9 Liposomes radiolabelling (I) Our aim is:to attach a radioisotope to liposomeswith high efficiencywith in vitro and in vivo stabilityuse the gamma photons, emitted from the isotope in order to study the biodistribution of the liposomesobtain successive imagesderive qualitative and semi-quantitative informationEncapsulation during manufacturing B. Reduction MethodD. Chelation Method C. “After-Loading” or Remote Labeling MethodLaverman 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-COOHSchematic representation of the surface chelation approach for preparing radioactive 99mTc-(CO)3-LP-PEC
11 Tomographic SPECT system SPECT imagingγ-camera headTomographic SPECT systemHeadLiverBladderInjection pointSpleen20052008Planar static bone imaging with Tc99m-MDP and dynamic study with a Tc99m-Bombesin derivativetailheadOur 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 imagingRecently 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 StudyspleenlivertailbladderheadDynamic image up to 70min p.iBiodistribution 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-PECScintigraphic 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 quantificationIn 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 targetingNovel glucose functionalized lipids are been formed in order to formulate liposomes with improved properties in terms of BBB targetingThe 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 imagingTEIA 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 imagingAccording our knowledge no such system is installed in GreeceIts technology is rather simple and interesting and complementary to the one that Greek researchers haveGreek secondees have started such activity in Athens and study SiPM detectors for optical light detectionTraining 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 coursesDuring the second year the project outcomes resulted to a goo number of dissemination activities in scientific and broader audienceThe project website contains all major project information and will now move from the teiath.gr domain to
22 Dissemination/Outreach Activities (II) PublicationsScientific JournalsEirini 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 ConferencesPreliminary 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, MayEirini 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, AthensEirini 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.