Medical device testing in vitro - pyrogenicity testing and ...DE, ES, FR, GB, IT, NL, SE) ......
Transcript of Medical device testing in vitro - pyrogenicity testing and ...DE, ES, FR, GB, IT, NL, SE) ......
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Thomas Hartung Doerenkamp-Zbinden Professor and Chair for Evidence-based Toxicology,
Director, Center for Alternatives to Animal Testing (CAAT) Joint appointment: Molecular Microbiology and Immunology
Johns Hopkins University, Baltimore, US
Professor of Pharmacology and Toxicology, University of Konstanz, Germany
Medical device testing in vitro - pyrogenicity testing and beyond -
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Promoting for 32 years alternatives to animal testing where they are not fit for purpose
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The Bernice Barbour Foundation
About 1/3 funding each from industry, philanthropy and research funding
…and individuals
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Examples for medical devices!
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Biocompatibility Testing - Material (solid, contact?) vs.
wash solution (diluted) vs. eluate (long-term, ill defined)
- Material vs. contaminant vs. degradation product vs. mixture
- In vivo: Confounder surgery - In vitro: Problem cell contact,
differentiation - In silico: Problem too few
positive materials, data avail.
- 3rd dimension: hazard – dose / duration – shape / surface
http://en.wikipedia.org/wiki/Biocompatibility
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Involved from about 2000 to 2004 (animal welfare and pyrogen group), then replaced by Albrecht Wendel for pyrogen testing
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Biocompatibility!• Incompatible: Release of substances in toxic
concentrations or of antigens
• Biotolerant: Release of substances in non-toxic concentrations
• Bioinert: No release of toxic substances
• Bioactive: positive interaction leading to reaction of tissue
Effect of Contaminants?!
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Medical device testing!• Material vs. contaminant!• Surgery vs. material!• Surface effects!• Material effects!• Long-term effects!• Immune reactions!• Shape-dependent!• Batch-dependent!
Pyrogen?
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cell associated immune receptors
TLRs
NODs
S.aureus
PAMPs
Pyrogen contamination
killing of bacteria release of inflammatory
mediators
web
page
s.sh
ephe
rd.e
du
ww
w. s
ange
r.ac.
uk
1013 own cells versus 1014 bacteria per person the gut of one person contains 1-2kg of bacteria = 50g of Gram-negative endotoxin = enough to kill one million people by injection = enough to induce fever in one billion people
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Pyrogen (bacterial and fungal contamination) testing still consumes more than 300.000 rabbits per year
Limulus assay is restricted to Gram-negative endotoxin, disturbed by many substances and does not reflect human pyrogen potency
1995 Whole blood pyrogen test 2003 and 2004 Validated 2006 Validity statement 2009 Accepted Eur. Pharmacopoeia and with limitations by ICCVAM / FDA PyroDetect (Biotest, now Merck/Millipore)
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ALTEX 2013, 30:169-208
Submitted to FDA / ICCVAM
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Endotoxin determination on PTFE filters
0.0 50.0 500.0 0
1
2
with filter
without filter
LPS (E. coli O-113) pg/ml
OD
(450
nm
)
TEFLON FILTER (5 µm)
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Lessons learned • alternative methods can outperform animal tests • importance of commercialization • 15 years from development to acceptance • Importance International harmonization • lack of enforcement of implementation • enabling technology • importance of public-private partnership
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Patient safety: non-endotoxin pyrogens New expression systems in gene technology (Gram-positive bacteria, insect and fungal cells) Medical devices Cellular therapies Environmental pyrogens
New challenges
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Risk of Pyrogenic Contamination
short-term
pyrogenic reactions • inflammatory reactions
long-term
example hemodialysis • immunodeficiency • amyloidosis • erythropoietin response ↓ • atherosclerosis
contact with tissue / blood consequences
Impairment of the patient reduced biocompatibility
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Pyrogen detection on biocompatible materials!
(contaminated)
Biocompatible Material
Rinsing solution
Limulus-Assay
Rabbit Assay
Rinsing solution/ Implant
Blood Cytokine, e.g. IL-1β
ELISA (MAT)
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Performance of the MAT for Devices
PERFUSION
STATIC CONTACT IL-1
IL-1
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Contamination of steel by manual handling
control A B C D ABCD pooled
1
10
100
1000
10000
donor
IL-1
ß (p
g/m
l)
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IL-1β
Static Contact
15-well steel block
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Endotoxin response on different materials tested in the 15well steel block
0 12,5 25 50 250 500 1
10
100
1000
10000
100000 polystyrol stainless steel elastomer PTFE
n.d. n.d. n.d.
LPS E.coli O 113 [pg/ml]
IL-1
β [p
g/m
l]
n.d.
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Elimination of pyrogenic residuals on titanium plates contaminated by manual handling
1
10
100
1000
10000
100000
IL-1
ß (p
g/m
l)
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Test of alginate microcapsules in the whole blood pyrogen test
+ LPS spike [50pg/ml] - LPS
EXTRACT
A l g i n a t e control
O D
[ 4 5 0 n
m ]
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
D E
MICROCAPSULE
A l g i n a t e control
O D
[ 4 5 0 n
m ]
0
1
2
3
D E
NaCl
NaCl
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Spider Silk
Spidersilk IL-1β
0
500
1000
1500
2000
2500
pg/ml O113
pg/m
l IL-
1 β
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forum Nr.2, August 1999, Greiner Labortechnik
Abb. 2 Abb. 3
32,4 nm 20,6 nm
Abb. 1
LPS is only partially removed
Atomic Force Microscopy of Polypropylen surface LPS Spike (500 ng/ml) and washing
Average peak hight
43,4 nm
Spike noSpike washed
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Neurosurgical aneurysm clips
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con 0,5 1 2 5 16 240
1000
2000
3000LPS [2 µg/well]LTA [2 µg/well]
coat-incubation time [h]solublestimuli
TNF-α
[pg/
ml]
Presentation of LTA on a surface increases 1000fold its inflammatory potency
Deininger, Immunobiol. 2008
Only 1% of LTA binds to surface
Surface bound LTA is more potent!
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Conclusions pyrogen testing!
• Current tests do not adequately control pyrogen contamination of medical devices
• Testing on surface is a paradigm shift
• Human relevant testing is possible
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Patents Method for assaying flowing media for microbial toxins EP 02 729 818.9 submitted 25.03.2002, under evaluation US 10/474 694 submitted 10.10.2003, under evaluation JP 2002-581 998 submitted 04.12.2003, granted 10.04.2009 Test procedure with biological system EP 0 741 294 B1 submitted 24.04.1996, granted 05.11.2003 (countries: AT, BE, CH/LI, DE, ES, FR, GB, IT, NL, SE) USP 5 891 728 submitted 02.05.1996, granted 06.04.1999 JPP 3 667 439 submitted 07.05.1996, granted 15.04.2005 Test for determining pyrogenic effect of a material EP 0 851 231 B1 granted 15.12.1997 (countries: AT, BE, CH/LI, DE, ES, FR, GB, IT, NL, SE) US CIP 10/761,237 submitted 23.12.1997 (under evaluation) JP 9-354572 submitted 24.12.1997, granted 04.04.2008
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Perspectives for other alternative methods
• Skin assays (mucosal models?)
• Genotoxicity • Sensitization • Cytotoxicity • Cell Transformation • Immunotoxicity
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Hartung T and Sabbioni E. Alternative in vitro assays in nanomaterial toxicology.
WIREs Nanomed. Nanobiotechnol. 2011, DOI: 10.1002/wnan.153
Nanoparticles testing – medical device testing on speed?
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New hazards from nano?!• No altered positive effects without
altered negative effects • Particulate matter is e.g. associated
with arteriosclerosis
• Inhalation toxicology • Reactive chemistry goes with surface • Changed kinetics
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Altered kinetics is enough to cause new effects!!!
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Cytotoxicity induced by Co-sol and Co-nano in Balb/3T3 cell line at 4h exposure
IC50
Cosol 41µM
Conano 9µM
Cytotoxicity 4h
0
20
40
60
80
100
1 10 100 1000
Concentration (µM)
CFE
(%)
Co-nanoparticlesCoCl2
Ponti et al., Mutagenesis 2009
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Co-nano in H2O (SEM
microphotograph)
We have to expect compared to parent compound: - Altered solubility - Altered hazard profile - Altered kinetics
Each and every NP is an individual !!!
These can affect in vitro and in vivo in different ways - Extrapolation to humans? - Classical risk factors? - In vivo extrapolation? - Low dose extrapolation?
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ZrO2-nano in H2O (SEM microphotograph)
Expected suitability of (validated) alternatives (none really evaluated) - Very useful:
refinement/reduction alternatives pyrogenicity
- Useful: skin irritation, corrosion, eye irritation, cytotoxicity, genotoxicity, cell transformation
- Possibly useful: skin absorption, embryotoxicity, myelotoxicity
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Workshop in Baltimore, October 2010 ALTEX 2011, 28, 236-241
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!
!
Conclusions for nanotox - The advent of nanotech amplifies
the problems of toxicology (amount of testing, new endpoints, complex kinetics…)
- We must take advantage of experiences from validation of in vitro tests
- Tox-21c and ITS approaches should be considered
- We need quality assurance beyond formal validation (e.g. EBT)
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Frontloading of toxicology
“fail early, fail cheap”
Anticipate human or regulatory problems?
“test early, develop clean”
Green Toxicology
InfoDay Green Toxicology Baltimore, 22 Nov 2013 Designing more biocompatible materials from start?
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Role Model REACH?!
Endpoint Informa.on: Annexes VI-‐XI
(Q)SARs Read Across
In-‐vitro
Waiving: • technical • exposure
Exis.ng informa.on
TESTING
Last resort
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model
modeling
model
model
HTS data genera.on
All models are wrong, some are useful. George Box
Validation
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Sure, animal tests are one-eye blind…
l Humans are not 70 kg-rats… l “One suit fits all”-models:
tests can only be either sensitive or specific
l Statistically underpowered l Too many endpoints without
statistical correction for multiple testing
l …
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Limitations of in vitro models l Cell identity l Mycoplasma l Dedifferentiation favored by
growth conditions and cell selection
l Cells are bored to death l Lack of oxygen l Tumor origin of many cells l Lack of metabolism and defense l Unknown fate of test
compounds in culture
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Tox-20c
Tox-21c EBT
Omics, high-content, HTS Bio-informatics & -engineering
Pathways of Tox (PoT) Human Toxome
Integrated Testing Strategies ITS
Organo-typic cultures Human-on- Chip
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http://en.wikipedia.org/wiki/Organ-on-a-chip
Human on Chip Approach • Prompted by DoD need for
medical countermeasure evaluation
• Could overcome many test shortcomings, especially using stem cells
• NAS panel • $200 million program • CAAT InfoDay 2012 • CAAT workshop 3D in 2012 • 10 May 2013 conference FDA /
NIH / DARPA / CAAT • GCCP for iPSC workshop to come
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• Interim decision points • Probabilistic / Bayesian approaches • Modeling and Machine Learning Commisioned white paper: Jaworska, J., and S. Hoffmann. 2010. Integrated Testing Strategy (ITS) - Opportunities to better use existing data and guide future testing in toxicology. ALTEX 27: 231–242.
The future of ITS
Bayesian network ITS for skin sensitization
CAAT workshop Jul 2013
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An atmosphere of departure in toxicology
New technologies from biotech and (bio-)informatics revolution
Mapping of pathways of toxicity (PoT)
NAS vision report Tox-21c
“We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments” F. Collins, NIH, 2008
“With an advanced field of regulatory science, new
tools, including functional genomics, proteomics, metabolomics, high- throughput screening, and
systems biology, we can replace current toxicology assays with tests that incorporate the mechanistic underpinnings of disease and of underlying toxic side effects.” M.A. Hamburg, FDA 2011
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Initiatives implementing Tox-21c
Organiza.on Approach Purpose Outcome
US EPA & Tox21 (ToxCast Program)
High-‐throughput tes.ng
Chemical priori.za.on (ini.ally)
“Biological signatures”
Hamner Ins.tute Case studies “Just do it” Proof-‐of-‐principle
NIH project (CAAT-‐US) Pathway mapping Pathway ID &
annota.on Human Toxome
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Mapping the Human Toxome by Systems Toxicology
Hewitt et al., 2005. Science, 307:1572-1573
Endocrine disruption • Use “omics” to map PoT for endocrine disruption
• Develop software tools
• Identify PoT
• Develop a process for PoT annotation, validation
• Establish public database on PoT.
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PoToMaC - The Pathways of Toxicty Mapping Center Transformative Research Grant: Mapping the Human Toxome by Systems Toxicology
7 companies, 3 stakeholders
European branch?
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Evidence-based Toxicology “Evidence-based medicine goes toxicology!”
Hoffmann and Hartung “Toward an evidence-based toxicology”,
Human Exp. Tox., 2006
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Mar 2011: US EBTC Oct 2011: Secretariat at CAAT Jan 2012: First conference hosted by
EPA Jun 2012: EU EBTC Diverse working groups Jul 2013: Symposium at IUTOX, Seoul,
Korea Sep 2013: Symposium at EuroTox,
Interlaken, Switzerland Systematic reviews increasingly embraced by IRIS and NTP
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EBT Collabora.on Steering CommiXees United States!
• Mel Andersen, Hamner*"• Rick Becker, ACC"• Kim Boekelheide, Brown"• Robert Chapin, Pfizer"• Rodger Curren, IIVS"• Suzanne Fitzpatrick, FDA"• Jack Fowle, EPA"• James Freeman, ExxonMobil"• Alan Goldberg, CAAT"• Thomas Hartung, CAAT"• Michael Holsapple, Battelle"• Richard Judson, EPA"• Francis Kruszewski, ACI"• Martin Stephens, CAAT"• William Stokes, NIEHS, NIH"• Raymond Tice, NTP"• Joanne Zurlo, CAAT"• + Sebastian Hoffmann (EU EBTC liaison)"
"
Europe!• Alan Boobis (Imperial College)"• Neil Carmichael (ECETOC)"• Thomas Hartung (CAAT)"• Jan Hengstler (Leibniz Research Centre)"• Sebastian Hoffmann (seh consulting + services)"• Philippe Hubert (INERIS)"• Joanna Jaworska (P&G)"• Ian Kimber (University of Manchester)"• Annette Kopp-Schneider (Cancer Research Centre)"• Marcel Leist (University of Konstanz)"• Jean-Roch Meunier (L’Oréal)"• Bennard van Ravenzwaay (BASF)"• Kai Savolainen (Institute of Occupational Health)"• Thomas Singer (Hoffmann-La Roche)"• Nigel Skinner (Agilent)"• Carl Westmoreland (Unilever)"• + Martin Stephens (US EBTC liaison)"
"* Affiliations for identification purposes only 56
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The difficulty lies, not in the new ideas,
but in escaping from the old ones.
John Maynard Keynes
(1883 - 1946)