Cerebral open flow microperfusion (cOFM) is a minimally invasive, in vivo sampling technology that allows continuous long-term sampling of cerebral fluid in living animals. The decisive advantage of cOFM is that the cOFM probe is membrane–free and comprises macroscopic openings which offer the possibility for a multitude of applications without restriction regarding size, lipophilicity or protein binding effects of the collected substances. The cOFM probe is designed to elicit minimal tissue reactions and allows for reconstitution of the blood-brain barrier (BBB). Thus, cOFM can sample cerebral fluids in living and freely moving animals with intact BBB. During this webinar, Dr. Joanna Hummer introduces cOFM and presents how cOFM is used as an in vivo sampling technology in neuroscience for drug development. Dr. Florie Le Prieult, presents data her team collected using cOFM during a pharmacokinetic studies of therapeutic antibodies. Her study includes head-to-head comparison of cOFM and microdialysis.

1. Cerebral Open Flow Microperfusion (cOFM) for In-Vivo
Cerebral Fluid Sampling – Comparison of cOFM and
Microdialysis
Joanna Hummer, PhD
Deputy Head of Biomedical
Tissue Monitoring
JOANNEUM RESEARCH HEALTH
Florie Le Prieult, PhD
Lab Head
In vivo Neuroscience
Pharmacokinetics
AbbVie

2. Cerebral Open Flow Microperfusion (cOFM) for In-Vivo
Cerebral Fluid Sampling – Comparison of cOFM and
Microdialysis
An application and technical
expert present an overview of
cOFM and its use

3. Cerebral Open Flow
Microperfusion (cOFM)
for In-Vivo Cerebral
Fluid Sampling –
Comparison of cOFM
and Microdialysis
October 14th, 2021
Webinar

4. Who We Are
Boutique CRO for research projects & drug development programs
4
We support your clinical and preclinical activities
by providing:
(Pre)clinical PK/PD/BE studies at the target tissue level
In-vitro release testing (IVRT)
Customized bioanalyses (PK, PD) – GLP certified lab
Metabolomics
Data management
Biostatistics
Medical writing

5. Dermal Tissue Adipose Tissue Brain Tissue
Open Flow Microperfusion
✓ provides unfiltered interstitial fluid
✓ PK/PD at target tissue
✓ CE-certified for clinical use (aOFM/dOFM)
5

6. Source: JOANNEUM RESEARCH
Biomedical
Tissue
Monitoring
in the Brain

7. How to Measure Substances in the Brain In-Vivo?
7
Biomedical Tissue Monitoring
Blood Brain Barrier Microdialysis Cerebral Open Flow Microperfusion
Credit: https://www.researchgate.net/figure/Brain-
vasculature-separates-the-circulating-blood-from-the-CNS-
tissue-The-vessels-are_fig1_320026611
Credit: JOANNEUM RESEARCH
Membrane
-free
no size
exclusion
unfiltered cISF
intact BBB
long sampling
times

8. Source: JOANNEUM RESEARCH
Cerebral
Open Flow
Microperfusion

9. Concept
9
Cerebral Open Flow Microperfusion
Credit: JOANNEUM RESEARCH
cOFM is a unique
tool to investigate
pharmacokinetics
(PK) and
pharmacodynamics
(PD) in the brain
with an intact BBB

10. Implantation & Sampling
10
Credit: JOANNEUM RESEARCH
Implantation and sampling are 14 days apart
-> intact Blood Brain Barrier (BBB)
14 days
Push Pull
Cerebral Open Flow Microperfusion
Ø 0.5 mm

11. Intact Blood Brain Barrier
11
Cerebral Open Flow Microperfusion
Birngruber T, Ghosh A, Perez-Yarza V, Kroath T, Ratzer M, Pieber TR, et al. Cerebral open flow microperfusion: a new in vivo technique for continuous measurement of
substance transport across the intact blood-brain barrier. Clin Exp Pharmacol Physiol 2013;40:864–71. https://doi.org/10.1111/1440-1681.12174.
Evans Blue BBB permeability BBB marker – Sodium fluorescein (Naf)

12. Time-resolved Substance Concentration Monitoring
12
Cerebral Open Flow Microperfusion
✓ No tissue scaring
✓ No biofouling
→ cOFM allows
continuous and
long-term
sampling
Sampling
Implantation
Sampling 3
0 1w 2w 3w 4w 5w 6w 7w
Sampling Sampling
Healin
g
dumm
y
Healin
g
dummy
Concentration
Time

13. Minimal Tissue Reaction
13
Cerebral Open Flow Microperfusion
Birngruber T. Long-term implanted cOFM probe causes minimal tissue reaction in the brain. PLoS One 2014;9:e90221–e90221.
https://doi.org/10.1371/journal.pone.0090221.
Quantification of Astrocytes Quantification of Macrophages
✓ cOFM probe
material elicits
minimal tissue
reaction
✓ No glial scar
✓ 15 days and
30 days after
cOFM
implantation

14. Different Brain Regions
14
Cerebral Open Flow Microperfusion
ISF sampling in
Striatum
Cortex
Hippocampus
Hypothalamus
..and others
CSF sampling in
Lateral
ventricle
Third
ventricle
Cisterna
Magna
Credit: JOANNEUM RESEARCH

15. Cerebral Open Flow Microperfusion
Monitoring of Substances without Limitation
Lipophilic substances
Amitriptyline (logP: 4.9), fluoxetine (logP:
4.6), …
Big molecules and compounds
Antibodies (Trastuzumab, anti-BACE1, ...)
Proteins (amyloyid ß, tau, albumin, IGGs, …)
Nanoparticles (Doxorubicin, liposomes, …)
Hormones
Leptin (16 kDa), GLP-1 (3297 Da)
Biomarkers
Interleukins, eicosanoids, growth factors,
glucose, …
15

16. Applications
16
Cerebral Open Flow Microperfusion
Monitoring of substance transport across the intact BBB
& time-resolved concentration profile in the brain
BBB
Pharmacokinetics
Pharmacodynamics
Monitoring of BBB function and permeability changes
Monitoring of biomarkers in the brain
Established:
Rodents (mice, rats)
Ongoing Development:
Pigs
Non-human primates
Humans

17. Amitrityline Sampling with cOFM and Microdialysis
17
Case Study
Small (277 Da) lipophilic
(logP 4.92)
CMA 12 (PAES, 20 kDA MWCO)
Highly protein bound (95%)
Dosing: 25 mg/kg i.p.
Altendorfer-Kroath T, Schimek D, Eberl A, Rauter G, Ratzer M, Raml R, et al. Comparison of cerebral Open Flow Microperfusion and Microdialysis when sampling
small lipophilic and small hydrophilic substances. J Neurosci Methods 2019;311:394–401. https://doi.org/10.1016/j.jneumeth.2018.09.024.

18. Other Applications
18
Neurodegenerative Diseases
Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow
microperfusion. Le Prieult et al. (2021), mAbs, 13:1, 1918819, DOI: 10.1080/19420862.2021.1918819
Obesity/Diabetes
Time-resolved hypothalamic open flow micro-perfusion reveals normal leptin transport across the blood–brain barrier in leptin resistant mice,
Maximilian Kleinert (2018). Molecular Metabolism, Volume 13, Pages 77-82. DOI: 10.1016/j.molmet.2018.04.008
Inflammation/BBB integrity
Assessment of blood-brain barrier function and the neuroinflammatory response in the rat brain by using cerebral open flow microperfusion
(cOFM). Ghosh et al., PLoS One. 2014 May 22;9(5). DOI: 10.1371/journal.pone.0098143.
Nanoparticles
Enhanced doxorubicin delivery to the brain administered through glutathione PEGylated liposomal doxorubicin (2B3-101) as compared with
generic Caelyx,(®) / Doxil (® ) -- cerebral open flow microperfusion pilot study. Birngruber et al. (2014) J Pharm Sci. 2014 Jul;103(7):1945-1948.
DOI: 10.1002/jps.23994
Glioblastoma (work in progress)

19. 19
Cerebral Open Flow Microperfusion
cOFM setup is combined with
Raturn® and Culex®
Simultaneous dosing & sampling
(cISF, CSF & blood)
Awake freely moving animals
cOFM service @ JR HEALTH
Set Up

20. Contact us
Key Learnings
cOFM samples cerebral fluids with an intact blood brain
barrier.
cOFM is membrane-free and samples all substances from
the interstitial fluid independent of size, lipophilicity, or
protein-binding.
cOFM enables continuous sampling for up to several days
and a long implantation period for up to several weeks.
cOFM studies provide data for drug development as well as
unique insights into brain metabolism and signaling.
joanna.hummer@joanneum.at
cofm-services.com
Icons in this presentation are made by Smashicons
and Freepick from www.flaticon.com & edited by
JOANNEUM RESEARCH HEALTH
openflowmicroperfusion.com
croservices.joanneum.at
Visit our websites
Follow us

21. Florie Le Prieult
Team Leader PK/Neuro PK
Sr. Scientist II, DMPK-BA, AbbVie
cOFM online Webinar
Oct. 14, 2021 | Virtual Event
A WINDOW TO THE BRAIN: on-line measurement of
biologics concentrations and target engagement using
microdialysis
Measuring Target Exposure to Assess
Drug Delivery Method Success

22. 22
Disclosure statement
Florie Le Prieult is employee of AbbVie and may own AbbVie stock. AbbVie sponsored and funded the study;
contributed to the design; participated in the collection, analysis, and interpretation of data, and in writing, reviewing,
and approval of the final publication.

23. Pitfalls to be
considered
Objectives to
reach
Brain penetration
can be fast or slow
Brain homogenate
contains brain
capillary
endothelium,
residual blood,
CSF, ISF and all cell
types
Understand brain
distribution and
“real”
concentration in
relevant brain
compartments
Understand
relevance of active
transport
processes
Understand time-
dependence of
brain uptake
Route of
application and
dose have an
influence on brain
disposition
Biologics brain penetration: ~0.1% of blood concentration
Usual methodology: Single dose injection and collection of
brain homogenates at 24h
The brain to plasma/serum ratio is part of the data required to understand antibody brain disposition
Establishing a quantitative
brain microdialysis setup
Assessing the kinetics of
antibody brain disposition
Investigating pharmacokinetics
and target engagement

24. 24
Microdialysis and cerebral Open-Flow Microperfusion
are potential methods to sample the brain interstitium
Potential
Read-Outs
• Antibody conc in interstitium (ISF) and brain (PK and terminal)
• Target concentration in interstitium and brain (PK and terminal)
• Target engagement in interstitium - Microdialysis
• Target engagement in CSF – PK (PK and terminal)
• Translational model mouse/human – PK/TE Modeling
Le Prieult et al., mAbs, accepted Apr. 2021, DOI 10.1080/19420862.2021.1918819

25. 25
Workflow for the micordialysis or cOFM Studies
ISF antibody characterization: Innovative
methods to allow high-sensitive and parallel
measures antibodies and target
Hippocampus
Day 0
Day 14
Day 18
Continuous ISF sampling
(variable fraction time possible)
Strict aseptic surgery
Tissue collection
for analysis
Possibility of chronic
microdialysis
or
Disconnection of microdialysis setup
Connection to microdialysis setup
Histology, brain and serum concentrations

26. 26
In order to interpret the results correctly, quality
checks are essential

27. 27
Dosing of non-target binding antibody
and comparison to a „classical“ brain PK study
in vitro recovery back-calculation required for
microdiaysis
Kinetic effects of the dosing route can
be identified easily in ISF
Antibody concentrations recovered in
ISF by microdialysis overlap with the
pharmacokinetic profile of the
antibody: after 10h, the concentrations
reach a plateau at about 0.5-1% of the
serum concentration.
IP dosing; Dose: 43 mpk IP and IV dosing; Dose: 43 mpk
Le Prieult et al., mAbs, accepted Apr. 2021, DOI 10.1080/19420862.2021.1918819

28. 28
Absolute quantification for cOFM
Absolute quantification is performed at steady state (24h-30h p.a.) using the Zero-Flow-Rate method : step-wise
changes of flow rates in order to extrapolate (non-linear regression) the theoritical concentration at 0 µL/min
Le Prieult et al., mAbs, accepted Apr. 2021, DOI 10.1080/19420862.2021.1918819

29. 29
cOFM allows an intermittent
measurement over long time periods
▪ ISF sampled directly after injection and 1 week after injection in the same animals
▪ New probes were used for both sessions
▪ Different ISF disposition can be observed between mAbs
Antibodies concentrations in ISF
0 10 20 30 40 50
10
100
1000
10000
100000
1000000
160 165 170 175 180 185
Time post injection (h)
mAb
(pg/mL)
mAb3
mAb4
mAb3 mAb4
mAb3 mAb4
Time (h)
* *
No terminal time point
*

30. 30
Case example of Target Engagement (TE) using
microdialysis:
Quantification of extracellular Tau in brain interstitium
Tau intracellular >> Tau extracellular => standard methods, e.g. brain
homogenate, cannot capture TE
anti-Tau mAbs are targeted against extracellular Tau in interstial fluid (ISF) to bind seeding-competent Tau species and
prevent spreading of Tau pathology

31. 31
Target engagement can be directly measured in the ISF
Degree of affinity to monomeric tau could directly influence the dose-response curves of anti-tau antibodies. If so,
levels of TE for mAb1 and mAb2 should be different based on their differential affinities for tau.
Measurement of free
tau in ISF
Measurement of
antibodies in ISF
Anti-target mAb1 with low affinity Anti-target mAb2 with high affinity

32. 32
Summary
• Microdialysis and cOFM allow antibody and biomarker/target quantification in brain ISF
• High time resolution possible and allow kinetic measurements in the same animal
• Target engagement and biomarker results can be coupled to the measure of the test article with highly sensitive and
parallel analytical methods
• Modeling approaches have been established to link ISF and CSF concentrations in mice, providing input on the
expected concentrations in the ISF of humans from CSF samples

33. 33
Acknowledgements
Florie Le Prieult, Ina Mairhofer, Aysel Güler, Mario Mezler, Sonja Julier, Erica
Barini, Gudrun Plotzky, Kerstin Schlegel, Karen Bodié, Linda Nolan, Volker
Berweck, Franziska Hanke, Klaus Diry, Klaus Magin, Christina Boch, Kerstin
Kaygisiz, Axel Meyer, Diana Claußnitzer, Lynne Rueter, Loic Laplanche
Thank you very much for your attention
Reference: Le Prieult et al., mAbs, accepted for publication Apr. 2021, DOI 10.1080/19420862.2021.1918819

35. Joanna Hummer, PhD
Deputy Head of Biomedical
Tissue Monitoring
JOANNEUM RESEARCH HEALTH
Florie Le Prieult, PhD
Lab Head
In vivo Neuroscience
Pharmacokinetics
AbbVie
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