estp pathology 2.0 april 17, 2018 magnetic resonance ... · magnetization some atomic nuclei spin,...

ESTP Pathology 2.0

April 17, 2018

Magnetic Resonance Imaging (MRI)

Applications in Non-clinical Pharmaceutical R&D

Peter Allegrini

Imaging and MRI expert

Consultant

Abraham Nyska

Toxicologic pathologist

Consultant

Enrico Vezzali


Idorsia Pharmaceuticals

Robert Maronpot


Maronpot Consulting

Thomas Lemarchand


TPL Path Labs

Physics, terminology and potential

Ischemic brain stroke

MRI associated to histology

Stem cells safety

Medical device safety

Drug local tolerance

Drug efficacy in brain primary tumor

Drug efficacy in lung tumor metastasis

Drug efficacy in chronic heart failure by ischemia

Drug efficacy in polycystic kidney disease

Heart hemodynamics

MR Spectroscopy in mitochondrial toxicity

Regulatory aspect

Summary

ESTP Pathology 2.0 MRI in R&D

Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Magnetic Resonance Imaging

Physics Terminology Potential

MRI in R&D #3

Magnetization

Some atomic nuclei spin, resulting in a magnetic moment and

thus align with an external magnetic field (like a compass).

“Magnetization” is the sum of all spins

Alignment either parallel or antiparallel to external field

low sensitivity of MRI

If excited, the spins precess (wobble) like a gyroscope

Precession frequency ω0 (≡ Larmor frequency) is linearly

dependent of external magnetic field B0

𝜔0 = γ𝐵0 The precession process induces electromagnetic radio wave with

Larmor frequency

Basic MR signal of MR images, spectra, etc.

From mri : Phyics by Evert J Blink

MRI in R&D #4

MRI friendly isotopes

From mri : Physics by Evert J Blink

Relevant Isotopes in Biomedical

Research(MRI: Magnetic Resonance Imaging;

MRS: Magnetic Resonance Spectroscopy)

MRI/MRS

MRI

MRS

MRS

MRS

Water

MRI in R&D #5

An MRI Acquisition (simplified)

From MRI made easy (… well almost) by

Prof HH Schild (Schering)

MRI in R&D #6

More physics: T1 relaxation (& it’s close relative T1ρ)

Excited magnetization recovers to its equilibrium state

Relaxation is defined by the T1 relaxation time (or rate)

From J Ridgway (2010). Cardiovascular

magnetic resonance physics for clinicians:

Part I. Journal of cardiovascular magnetic

resonance. 12

MRI in R&D #7

Even more physics: T2 relaxation (& it’s close relative T2*)

From J Ridgway (2010). Cardiovascular magnetic resonance

physics for clinicians: Part I. Journal of cardiovascular magnetic

resonance. 12

The individual spins of a magnetization lose phase coherence (synchronization)

Signal loss (T2 relaxation time or rate)

MRI in R&D #8

Relaxation Times

Important: T1 and T2 relaxation are two independent

processes, which happen simultaneously.

Furthermore: T1 and T2 relaxation are affected by the

close environment and the mobility of the

spins

MRI in R&D #9

Why are T1 resp. T2 important? MR image contrast!

T1, water/proton density, and T2 weighted images of human brain.

T1 weighted T2 weightedWater density weighted

Fat

Protein-rich fluid

Paramagnetic subst.

Tissue

Grey matter darker

than white matter

Water, CSF

Edema, tumor, infarction

Infl., hemorrhage

Bone, air

Water, CSF

Edema, tumor, infarction

Infl., hemorrhage

Tissue

White matter darker

than grey matter

Fat

Protein-rich fluid

deoxyHb

Bone, air

MRI in R&D #10

MR Image resolution

“Highly” resolved MR Images of a rat brain

Digital resolution 78 x 78 µm2; Slice thickness 1.2 mm

From PR Allegrini,

European

Pharmaceutical

Review (2012)

17;20-24

MRI in R&D #11

MR image contrast mechanisms

Relaxation times are the basis of image contrast in most instances

T1 relaxation time T1 weighted images

T2 relaxation time T2 weighted images

Contrast agents (T1 or T2 altering CA)

Inversion recovery (T1 contrast enhancement)

Proton/water density

Oxyhemoglobin to deoxyhemoglobin ratio

Flowing vs. stationary water

Water diffusion coefficient

Arterial spin labeling

Magnetization transfer

Many others ………..

MRI in R&D #12

Criteria for (in vivo) MR imaging approaches

When

Target validation (e.g. phenotyping of genetically modified animals)

PD / efficacy / toxicology/safety studies

What

Answer a biological question in a project (pharmacology, pathophysiology).

If biological question can be answered by various imaging approaches, choose the best suitable modality.

Imaging in competition to other (simpler, cheaper, faster, ...) techniques.

Read out characteristics

Non-invasive: Time course studies to capture dynamic changes, reduce animal experiments

Non-invasive: Integrated in multi-modality studies

Complex techniques: Functional read-outs (organ function in a broad sense)

Versatility: Multiple read-outs in a study

Results

Quantitative (including statistical analyses)

MRI in R&D #13




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Cerebral ischemia in the rat: Pathophysiological cascade

Symptoms

T2 weighted

MRI

Middle cerebral artery occlusion

Lack of perfusion

Hypoxia/ energy failure

Reversible lesion/

Cytotoxic edema

Permanent lesion /

Vasogenic edema (or

cyst, resp.)

Time-of-flight MR-Angiography

seconds

< 5min

minutes

- hours

> 6 hours

T2* weighted MRI

Arterial spin labelling perfusion imaging

Diffusion-weighted imaging

0

months

fMRIBrain function (lack of)

Brain plasticity

MRI in R&D #15

Quantification: Image segmentation and 3D rendering

From Inge A Mulder et al, Front.

Neuroinform.(31. Jan 2017)

MRI in R&D #16

Raw multi-slice MR image stack of cerebral ischemia in the rat 48h after onset of stroke

Segmented image stack of cerebral ischemia in the rat

Se

ge

me

nta

tio

n3

D R

en

de

rin

g

Quantification: MRI vs. Morphometry (gold standard)

MR image taken 2 days post

MCA occlusion: Edema

Matching histological frontal

section: Infarct From PR Allegrini & D Sauer, Magnetic

Resonance Imaging (1992) 773-778

MRI in R&D #17

Quantification: Predictive value

Untreated

Glutamate

receptor

antagonist

2 Days 15 Weeks

From E Weber, PR Allegrini

& D Sauer, in vivo (1993)

335-338

Edema (MRI) 2 days

post MCA occlusion vs.

total volume of atrophy

(morphometry) 2 to 3

months later: r = 0.83

Edema and cyst (MRI) 2 days

and 15 weeks post MCA

occlusion: Treatment effect

MRI in R&D #18




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

The EX-VIVO MRI (or “SMART HISTOLOGY) can be an excellent tool in preclinical

efficacy and safety evaluation, helping to:

Accurately localize the lesion;

Count the lesions;

Measure their volume;

Information about the homogeneity of the lesions.

For EX – VIVO MRI, the fixed samples were transferred from the fixative (like the 10%

buffered formalin) to Fluorinert (a proton-free and biologically inert perfluorocarbon) and,

following the scanning, the tissue were returned to formalin, for further histology

processing.

MRI-based histology – “Smart sections”

MRI in R&D #20

Comparison Classical MRI versus Compact MRI

=

Tempel Brami C, Schiffenbauer Y, Nyska A, Abramovitch R, Ezov N, Spector I, Maronpot RR (2015):

Practical Applications of in vivo and ex vivo MRI in toxicologic pathology using a novel high-performance compact MRI system.

Toxicologic Pathology 43(5):633-50. MRI in R&D #21




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

MRI in Preclinical Safety Evaluation of Stem Cells

Objective

Using compact MRI technology for the assessment of tumorigenicity following

intrathecal transplantation of human Embryonic Stem Cells (hESC) in mice

Protocol

Two mice injected with the vehicle and 3 injected with hESC

Intrathecal Injection within the inter-vertebral (L5 to L6) groove.

Daily clinical evaluation

In vivo MRI on 2 occasions (days 25 & 48)

Sacrifice on day 55, followed by formalin fixation, ex vivo MRI, and histopathology

A Safety Evaluation of Stem-Cells in a Mouse Model

MRI in R&D #23

In-Vivo (A-C) MRI of

Teratoma in the Spinal

Cord of a Mouse 25 (A)

and 48 (B-C) Days, and Ex

Vivo (D) MRI on Day 55,

After Intrathecal Injection

of hESCs

Teratoma in the Spinal cord after Intrathecal

Injection of hESC

Expanding lumbar

lesions (bright areas,

arrows)from day 25 to

48

High magnification of

the spinal teratoma

In Vivo 3D Rendering and

Segmentation of the Spinal

Cord Teratoma on Day 48MRI in R&D #24

In Vivo 3D Rendering

of the Brain Showing

Extent of the

Teratoma

In vivo MRI day 48 (A), ex vivo MRI day 55 (B-C) and

histopathology (D) of 2 different areas of the brain of a NOD-

SCID mouse after intrathecal injection of hESCs, showing

distinctive lesions

Teratoma in the Brain after Intrathecal Injection of

hESC

MRI in R&D #25

Paresis developed in mice injected with the hESC

Both in vivo and ex vivo MRI located abnormal areas in the spinal

column and brain

Histopathology confirmed malignant teratoma

The MRI technique can be used for time course observations for

testing the carcinogenic potential of novel stem cells intended for

clinical use.

The MRI (in vivo and ex vivo) were effective in accurately localizing

the teratomas in the brain and spinal cord.

Results and Conclusions

MRI in R&D #26




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Objective

Evaluation of in vivo MRI as a tool for assessment of degradation of a bio-

degradable implanted device

Protocol

A double layer of a 5x5 mm2 device was implanted in the right paralumbar

muscle of Sprague Dawley rats. A plastic bead was implanted

subcutaneously just over the device to enable accurate localization and

follow-up of the implantation site

MRI in Evaluation of Biodegradable Implanted

Device

MRI in R&D #28

In Vivo Follow-up of Implanted DeviceDay 5 Day 30 Day 60

T1

T2 Inflammation

bead

implant

MRI in R&D #29

Ex Vivo MRI of Implanted Device

implant

MRI in R&D #30

Ex Vivo MRI of Implanted Device - 3D Rendering & Segmentation

Volume of device : 32.2 mm3

MRI in R&D #31

Histopathology of the Implantation Site 60 Days

Post Implantation

Note the Cavities (red arrows) Lined by Mature Connective Tissue Capsule (blue arrow).

No Inflammatory Reaction is Present

MRI in R&D #32

In vivo MRI could detect inflammation related to the implantation process

and follow its progressive reduction. In addition, in vivo MRI was able to

demonstrate the presence of the test device in the para-lumbar muscles up to

16 weeks post implantation.

Accurate quantification of the device volume and shape was possible using

ex vivo MRI.

It can be concluded that the combination of in vivo and ex vivo MRI with

histopathology can help in assessing the tolerability and bio-degradation of biodegradable devices.

Summary and Conclusion

MRI in R&D #33




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Objective

A feasibility study for testing the utility of ex-vivo MRI in evaluating local

subcutaneous toxic (i.e., irritantancy) effects induced by injection of test

compounds

Protocol

The study was performed in pig. Surgically removed infusion sites were analyzed

by EX-MRI, 2 weeks after a 24-hour continuous infusion of different locally irritant

formulations

MRI in Tolerability (Irritancy) Testing of Drugs:

Local Safety of Subcutaneous Formulations in a

Minipig Model

MRI in R&D #35

MRI (T1) Histology

Subcutaneous Drug Injection into Pig Skin MRI vs.

Histology

MRI in R&D #36

Affected Volume 2200 mm3

3-D Rendering and Segmentation (Quantification) of

Affected Volume – Ex Vivo MRI

MRI in R&D #37

Ex-vivo MRI was effective in identifying the location and quantifying the extent

of subcutaneous necrosis and inflammation caused by different formulations

Applying ex-vivo MRI on fixed infusion site samples from different dose

formulations provides a quantitative determination of relative irritancy


MRI in R&D #38




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Objective

Validate the In vivo and ex vivo compact MRI as a method for the assessment of

anti-cancer efficacy testing in a mouse brain model.

Protocol

Murine GL-261 glioma cells were stereotactically injected into the right brain

hemisphere of CB6F1 mice

MRI in Efficacy Testing of Anti-Cancer Drugs:

Application of Compact MRI in a Mouse Brain

Tumor Model

MRI in R&D #40

Longitudinal Evaluation of Tumor Growth

In Vivo MRI (T2)

Day 15 Day 17 Day 20

axial

coronal

0

5

10

15

15 17 19 21

Exponential

tumor growth

Time (days)

MRI in R&D #41

Tumor volume 6.6 mm3

Day 17 – axial

Tumor Segmentation – Ex Vivo MRI (T2)

MRI in R&D #42

Day 15

Day 20

Correlation of In Vivo MRI vs. Histology

MRI in R&D #43

In vivo and ex vivo MRI evaluation provided a way to follow the time-related

growth, and when applied to an anticancer study, could show time-related

shrinkage of tumors

This model demonstrates the utility of using MRI for longitudinal studies and

would be useful for testing the efficacy of anti-cancer drugs


MRI in R&D #44




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Objective

Validate the compact MRI as an additional more accurate and high-throughput

method for the assessment of anti-cancer efficacy testing in a mouse lung

metastatic model.

Protocol

Intravenous injection of breast cancer cell line into the tail of 8-10 weeks-old

BALB/c mice. This Tumor is an animal model for Stage IV human breast cancer.

Animals were terminated 18 days post tumor injection and Ex-Vivo MRI scanning

was accomplished on the lungs

MRI in Efficacy Testing of Anti-Cancer Drugs:

Application of Compact MRI in a Mouse Lung

Metastasis Model

MRI in R&D #46

Macroscopic View of the Lungs (Before MRI

Scanning)

Arrows Indicate Nodules of Metastatic Mammary

Cancer

MRI in R&D #47

Ex-Vivo MRI 3D Rendering and Quantification of

Lesions

ROI Modality Color Voxels Volume mm³

lesions MR red 8255 62.9806

MRI in R&D #48

Ex vivo MRI evaluation provided a way to accurately determine the number and volume of

the tumors within the lungs. This method is superior to the currently used macroscopic

counting of the cancer nodule

This model demonstrated the utility of ex-vivo MRI for comparative testing of the

efficacy of anti-cancer drugs


MRI in R&D #49




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Sham operation Coronary ligation

Effects of vasodilators (Enalapril) in chronic heart

failure induced by coronary ligation in rats

Masson’s

trichrome

sections

Morphometry

gross

detection

Image

analysis fine

detection

MRI in R&D #51

SHAM CHF



MRI in R&D #52



MRI in R&D #53

Microscopic measurement by morphology and image analysis

Heart + Infarct

Volume

mm3

Sham Vehicle Enalapril

Collagen

Volume

mm3

Sham Vehicle EnalaprilCollagen

%Sham Vehicle Enalapril

Mean 540 475 542 11 56 74 2.0 11.8 13.2

% Sham / Vehicle 88 114 513 132 603 111

Macroscopic measurement by caliperInfarct length

mmSham Vehicle Enalapril

Mean 0.0 10.5 12.1

% Sham / Vehicle 115

MRIHeart

Volume

mm3

Sham Vehicle Enalapril

Infarct

Volume

mm3

Sham Vehicle EnalaprilInfarct

%Sham Vehicle Enalapril

Mean 849 886 932 0 68 80 0.00 6.87 7.24

% Vehicle 105 117 105



MRI in R&D #54




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Polycystic Kidney Disease:

• Structure

• Cysts volume

Other possible applications

MRI in R&D #56




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Heart hemodynamics:

• stroke volume

• ejection fraction

• wall thickness

• chambers volume

Other possible applications

MRI in R&D #58




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

Resting state 31P spectrum of calf muscles Dynamic 31P spectra of calf muscle with

simultaneous muscle stimulation

(temporal resolution 30 s)

Recovery period

pH

Other possible applicationsMitochondrial toxicity: assessment of mitochondrial capacity by MR

Spectroscopy (MRS)

Phosphorous spectroscopy (skeletal muscle)

MRI in R&D #60

Energy metabolism: Mitochondrial Toxicity

,0.00

,0.05

,0.10

,0.15

,0.20

,0.25

,0.30

,0.35

,0.40

,0.45

Day -1 Day 9

k PCr[m

in-1]

Vehicle

DPI

$$$*-38%

PCr recovery time rate

signifivantly reduced by

Complex I inhibition

Time [min]

-10 -5 0 5 10 15 20

PC

r [%

of

ba

se

lin

e]

40

50

60

70

80

90

100

110

Animal B2 @ day 9 (Vehicle)

Animal C1 @ day 9 (DPI)

Phosphocreatine recovery

Muscle stimulation Recovery

Statistics

Two Way Repeated Measure ANOVA

Post hoc Fisher LSD Method

Diphenyleneiodonium (DPI): Complex I inhibitor Metformin

(for type 2 diabetes)

Metformin inhibits Complex I

From Wessels B, et al. (2014) Metformin Impairs

mitochondrial Function in Skeletal Muscle of Both

Lean and Diabetic Rats in a Dose-Dependent

Manner. PLoS ONE 9(6) MRI in R&D #61




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali

MRI in preclinical development is currently not GLP-compliant *

GLP exceptions to the validation of non-routine endpoints, like MRI, is accepted *

Imaging complementary to histopathology enhances targeted sampling and assessment *

Imaging complementary to histopathology is a desirable complement of a regulatory submission *

Regulatory authorities accept imaging reports, provided sufficient controls **

Only images used for data generation must be authenticated and archived ***

The Scientific and Regulatory Policy Committee (SRPC) of the STP is filing a position paper about

investigative tools in toxicologic pathology and related regulatory approach

* Maronpot RR, Nyska A, Troth SP, Gabrielson K, Sysa-Shah P, Kalchenko V, Kuznetsov Y, Harmelin A, Schiffenbauer YS, Bonnel D,

Stauber J, Ramot Y (2017):Regulatory Forum Opinion Piece: Imaging Applications in Toxicologic Pathology-Recommendations for Use in

Regulated Nonclinical Toxicity Studies. Toxicol Pathol 45(4):444-471

** Long RE, Smith A, Machotka SV, Chlipala E, Cann J, Knight B, Kawano Y, Ellin J, Lowe A (2013)

Scientific and Regulatory Policy Committee (SRPC) paper: validation of digital pathology systems in the regulated nonclinical environment.

Toxicol Pathol 41(1):115-122

*** Tuomari DL, Kemp RK, Sellers R, Yarrington JT, Geoly FJ, Fouillet XL, Dybdal N, Perry R (2007)

Society of Toxicologic Pathology position paper on pathology image data: compliance with 21 CFR Parts 58 and 11.

Toxicol Pathol 35(3): 450-455

Regulatory aspect

MRI in R&D #63




Stem cells safety







Heart hemodynamics


Regulatory aspect

Summary


Abraham Nyska

Enrico Vezzali

Peter Allegrini

Peter Allegrini

Enrico Vezzali


Strengths

• Quantitative data (measurements, counts)

• 3D and deep assessment

• Wide range of contrast (especially for soft tissues)

• Homogeneity/heterogeneity of whole organs (e.g.: uterus or brain)

• Smart histopathology (identification, location, size and correlation)

• Longitudinal and not destructive evaluation 3Rs compliant

• Dual purpose (in vivo & ex vivo)

Weaknesses

• Resolution(giving precision and reproducibility)

=acquisition time

(not always compatible with in-vivo requirements)+

magnetic induction(permanent magnets limited to 1 Tesla magnetic fields)

• Project-specific set-up resources (working hours)

Opportunities

• MRI complementary to histopathology without overlapping

• Granular grading based on measurements (%, number of lesions)

• Accurate (no detectable lesion missed, e.g.: low tumor burden/load)

Challenges

• Large organs

• Physiological movements (peristalsis, respiration, heartbeat)

estp pathology 2.0 april 17, 2018 magnetic resonance ... · magnetization some atomic nuclei spin,...

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