Loudos George Molecular imaging as a tool to image drug delivery

Report
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: [email protected]
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
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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
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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
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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
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Synthesis of lipids and characterization
– Design of lipids to ensure radiolabelling
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Synthesis of liposomes and characterization
– Optimize liposomes for drug loading
– Functionalization for targeting
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– liposomes loading
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In vivo tests with functionalized liposomes
– Comparison with Year 1 and 2 results
– Assessment of different radiolabelling
strategies
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Evaluation of SPECT imaging for in vivo
imaging
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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
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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
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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
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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
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liver
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60
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tail
bladder
head
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Dynamic image up to 70min p.i
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Pa
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Biodistribution data
FP7-PEOPLE-2011-IAPP OnconanoBBB, BIOMEP 2014, Athens, 13 September 2013
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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
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intravenously injected with 3.7 MBq or 100μCi
οf 99mTc-LP-COOH and 0.37MBq or 10 μCi of
99mTc(I)-(CO) -LP-PEC.
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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
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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
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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
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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)
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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
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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
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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

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