Non Toxicology of Nicotine and Aerosol from a Heated Tobacco · Methods ‐design OECD TG 412/413...
Transcript of Non Toxicology of Nicotine and Aerosol from a Heated Tobacco · Methods ‐design OECD TG 412/413...
Patrick Vanscheeuwijck, PhD
Director Pre-Clinical Toxicology
Non‐Clinical Toxicology of Nicotine and Aerosol from a Heated Tobacco Product
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Offering adult smokers satisfying products that reduce risk
• Smoking is addictive and causes a number of serious diseases
• Worldwide it is estimated that more than one billion people will continue to smoke in the foreseeable future*
• Successful harm reduction requires that current adult smokers be offered a range of Reduced Risk Products (RRPs) so that consumer acceptance can be best fulfilled
• Our Heated Tobacco Product (HTP) is a RRP.*The Tobacco Atlas 3rd Edition. American Cancer Society, 2009Figure adapted from Clive Bates presentation to E-Cigarette Summit (19 Nov 2013)
HARM REDUCTIONPRODUCT
ACCEPTANCE AND USAGE
REDUCED-RISK PRODUCT =X
PMI’s Goal for Harm Reduction
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Reduced-Risk Products (“RRPs”) is the term the company uses to refer to products with the potentialto reduce individual risk and population harm in comparison to smoking cigarettes. PMI’s RRPs are invarious stages of development and commercialization, and we are conducting extensive and rigorousscientific studies to determine whether we can support claims for such products of reduced exposureto harmful and potentially harmful constituents in smoke, and ultimately claims of reduced diseaserisk, when compared to smoking cigarettes.
Before making any such claims, we will rigorously evaluate the full set of data from the relevantscientific studies to determine whether they substantiate reduced exposure or risk. Any such claimsmay also be subject to government review and authorization as is the case in the US today.
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Outline & Scope
Short description of design and methods used in a series of in vivo inhalation studies assessing theeffects of nicotine aerosol, conventional cigarette smoke and aerosols from potentially Risk-Reduced Product (pRRP) [or candidate Modified Risk Tobacco Product] – Heated Tobacco Product
Description of effects of nicotine (in combination with pyruvic acid) using pre-clinical and systemstoxicology endpoints
Toxicity of cigarette smoke exposure and the effect of a HTP – interpretation of findings usingnicotine-exposure knowledge
Application of a mouse disease model for cardiovascular and respiratory disease – effects ofcigarette smoke exposure, switching to HTP aerosol exposure and cessation
HTP: Heated Tobacco Product
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Methods – in vivo studies
Regulatory rat inhalation studies (OECD protocols; 28-day, 90-day*, OECD GLP)• Aerosol generation: using appropriate standards
– Smoking machines: conventional cigarettes, reduced risk products
– Collison nebulizers: nicotine/salt solution, nicotine containing liquids
• Aerosol characterization: demonstrate that aerosol is representative and follows guidelines– Determination of aerosol markers (e.g. TPM, CO, carbonyls), breathing zone
– Particle size distribution
• Bio-monitoring: demonstrate that exposure is representative– Respiratory physiology
– Urinary particulate and gas phase biomarkers of exposure (e.g. nicotine metabolites, mercapturic acid metabolites from acrolein, benzene, acrylonitrile, NNK) blood carboxyhemoglobin)
*OECD Guidelines for the Testing of Chemicals, Section 4.Test No. 413: Subchronic Inhalation Toxicity: 90-day Study, OECD 2009Test No. 412: Subacute Inhalation Toxicity: 28-Day Study, OECD 2009
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Methods ‐ design
OECD TG 412/413 Endpoints –Standard Toxicology1. In-life observations2. Hematology3. Clinical chemistry4. Organ weights5. Inflammatory cells in BALF6. Histopathology
OECD ‘Plus’-Systems Toxicology1. Transcriptomics2. Genomics3. Lipidomics4. Proteomics
Subset of dataEg Histopathology or Inflammatory Data
Computational analysisComputational Network BiologyBiological Impact Factor Analysis
Systemic toxicity
• Test atmospheres presented in low/medium/high concentration: up to MTD: 50 µg Nicotine/l* for nicotine-containing aerosols – 23 µg Nicotine/l for cigarette smoke; concentration-response
• Endpoints:
*50 µg/l nic corresponds to 13.6 mg/kg, daily dose; way above acute toxicity levels- or the nicotine amount from approx. 130 cig/day for a 60 kg human
Executed according to OECD GLPs
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MethodsExposure – nebulized nicotine –nicotine‐salt solutions
Fres
h
Aero
sol
E xha
led
A ir
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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Exposure and Bio‐monitoringUrinary nicotine metabolites demonstrate appropriate exposure to ‘respirable’ aerosol
Test atmosphere concentrations Particle size determination Urinary nicotine metabolites
Means ± SEM; ***p ≤ 0.001
Means ± SD
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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Systemic effects
Means ±SEM; *p≤0.05, r**p ≤ 0.01, ***p ≤ 0.001 relative to control
Nicotine concentration-dependent changes in:– Body weight development (approx. 5% body weight gain reduction in ‘high’ concentration group)
– Organ weights (relative to body weight): Liver, higher than controls; Spleen, lower
– Clinical chemistry: Alkaline phosphatase and alanine aminotransferase: higher than controls; cholesterol and glucose: lower than controls – all in normal ranges
– Hematology: neutrophils, higher than controls; lymphocytes, lower – all in normal ranges
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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Histopathology of non‐respiratory tract organsLiver effects: nicotine concentration-dependent cytoplasmic vacuolation – vacuoles contain PAS-positive material, likely to consist of glycogen granules
Means ±SEM; *p≤0.05, r**p ≤ 0.01, ***p ≤ 0.001 relative to control
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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Summary & Conclusion
• Exposure to nicotine aerosols, alone or in combination with pyruvic acid, results in mild changes in toxicity in a 28-day inhalation toxicology study with respect to:– Systemic effects: increased liver weight, blood neutrophil counts, activity of liver enzymes and decreased
blood levels of cholesterol and glucose
– Histopathological effects: increased vacuolation of and glycogen content in hepatocytes
Changes in liver gene expression and lipidomics provide mechanistic explanation for the changes observed, i.e. alterations in xenobioitic and lipid metabolism
Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague–Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology.
Blaine Phillips, Marco Esposito, Jan Verbeeck, Stephanie Boue, Anita Iskandar, Gregory Vuillaume, Patrice Leroy, Subash Krishnan, Ulrike Kogel, Aneli Utan, Walter K. Schlage, Monali Bera, Emilija Veljkovic, Julia Hoeng, Manuel C. Peitsch, and Patrick Vanscheeuwijck.
Inhalation toxicology 2015 Aug;27(9):405-31, PMID 26295358
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Methods – Conventional cigarette smoke and aerosol from a HTP 90‐day inhalation study (OECD TG 413)
BV1"
WA
ST
E
Approximately 3.9m
App
roxi
mat
ely
1.2m
Control room
Clean corridor
Exposure room Fres
h
Aero
sol
E xha
led
A ir
Potentially Reduced-Risk product: Aerosol from Heatsticks and Tobacco Heating System, HTP, commercialized as iQOS(also designated as P1 or THS 2.2)
Conventional cigarettes:Smoke from University of Kentucky Standard Reference Cigarette 3R4F
Assessment of smoke/aerosol – Health Canada Intense smoke protocol
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Smoke chemistry – determination of 58 constituents in conventional cigarette smoke and HTP (THS2.2) aerosol
• Smoke chemistry data shows a strong reduction of HPHC* in THS2.2 relative to 3R4F (data per mg nicotine, 100 % is data from 3R4F)
*HPHC: Harmful or Potentially Harmful Constituents
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• Test atmosphere characterization – gas phase and particulate matter markers indicate that:‒ Target nicotine concentrations met‒ HPHCs in test atmospheres reflect aerosol chemistry data
Sham
3R4F
_L_8
3R4F
_M_1
5
3R4F
_H_2
3
THS2
.2_L
_15
THS2
.2_M
_23
THS2
.2_H
_500
15
30
45
60
mea
n co
ncen
tratio
n of
nico
tine
( g/
L +/
- SD
)
0 8 15 23 500
100
200
300
400Sham3R4FTHS2.2
test atmosphere concentration of nicotine (g/L)
mea
n co
ncen
tratio
n of
CO
(ppm
+/-
SD)
8 15 23 500.0
0.5
1.0
1.5
2.03R4FTHS2.2
test atmosphere concentration of nicotine (g/L)m
ean
conc
entra
tion
ofac
rolei
n (
g/L
+/- S
D)
Nicotine concentrations in test atmospheres
Acrolein concentrations in test atmospheres
CO concentrations in test atmospheres
Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Exposure
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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413) ‐Biomarkers of exposure
• Bio-monitoring parameters indicate that:– Urinary biomarkers of nicotine correlate with nicotine levels in test atmosphere– Urinary biomarkers of HPHC correlate with chemical composition of aerosols – The HTP causes much less upper respiratory tract irritation resulting in higher uptake of aerosol
0 8 15 23 500
100
200
300
400
500Sham3R4FTHS2.2
test atmosphere concentration of nicotine (g/L)
conc
entra
tion
of S
PMA
(ng/
ml,
mea
n +/
- SD
)0 8 15 23 50
0
5000
10000
15000
20000Sham3R4FTHS2.2
test atmosphere concentration of nicotine (g/L)
conc
entra
tion
of H
PMA
(ng/
ml,
mea
n +/
- SD
)
Urinary benzene metabolite
Urinary acrolein metabolite
Data for male rats only, after 4 weeks of exposure. The data for female rats and different collection time points are very similar.
0 8 15 23 500
2000
4000
6000
8000Sham3R4FTHS2.2
test atmosphere concentration of nicotine (g/L)
tota
l nic
otin
e m
etab
olite
s(n
mol
, mea
n +/
- SD
)
Urinary nicotine metabolites
0 8 15 23 5050
100
150
200
250Sham3R4FTHS2.2
test atmosphere concentration of nicotine (g/L)
resp
irato
ry m
inute
volum
e( m
l/ m
in, m
ean
+/- S
EM)
Respiratory Minute Volume
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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413) ‐Systemic toxicity
• No remarkable toxic effects in THS 2.2
• Body weight effect in males only
• Nicotine concentration-related increases in– Neutrophil count in blood
– Relative weight of liver
– Liver enzyme activity (normal range)
• Mild liver effects
Effects similar to those of nicotine exposure
-20 0 20 40 60 80 100 120 140200
300
400
500
600Sham3R4F_L_83R4F_M_153R4F_H_23THS2.2_L_15THS2.2_M_23THS2.2_H_50
90d exposure<-----------------------------><-------------->
90d exposure+ 42d recovery
Study Days
mea
n bo
dy w
eight
(g)
Body weight development, male rats
Alkaline phosphatase activity, female rats
* p < 0.05 as compared with SHAM at a given time point, # p < 0.05 as compared with 3R4F at target nicotine 15 g/l at a given time point, § p < 0.05 as compared with 3R4F at target nicotine 23 g/l at a given time point, and + p < 0.05 as compared with the same group at the 90-day exposure time point
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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413) ‐Lung inflammation
• Very low numbers of inflammatory cells are recruited in the lungs (BALF) of THS2.2 aerosol-exposed rats
Remarks: •Data for male rats; the data for female rats are very similar.•* p < 0.05 as compared with SHAM at a given time point, # p < 0.05 as compared with 3R4F at target nicotine 15 g/l at a given time point, § p < 0.05 as compared with 3R4F at target nicotine 23 g/l at a given time point, and + p < 0.05 as compared with the same group at the 90-day exposure time point
Means ± SEM
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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Histopathology of respiratory tract organs
Rel
ativ
eS
ever
ity
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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Histopathology of respiratory tract organs ‐ Nose
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Cigarette smoke and HTP aerosol– 90‐day inhalation study (OECD TG 413)Histopathology of respiratory tract organs – Bronchi and lungs
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Methods ‐ design
OECD TG 412/413 Endpoints –Standard Toxicology1. In-life observations2. Hematology3. Clinical chemistry4. Organ weights5. Inflammatory cells in BALF6. Histopathology
OECD ‘Plus’-Systems Toxicology1. Transcriptomics2. Genomics3. Lipidomics4. Proteomics
Subset of dataEg Histopathology or Inflammatory Data
Computational analysisComputational Network BiologyBiological Impact Factor Analysis
Systemic toxicity
• Test atmospheres presented in low/medium/high concentration: up to MTD: 50 µg Nicotine/l* for nicotine-containing aerosols – 23 µg Nicotine/l for cigarette smoke; concentration-response
• Endpoints:
*50 µg/l nic corresponds to 13.6 mg/kg, daily dose; way above acute toxicity levels- or the nicotine amount from approx. 130 cig/day for a 60 kg human
Executed according to OECD GLPs
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System Toxicology ResearchIdentify and Represent Disease Mechanisms
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Understanding of disease onset and progression
Chronic Cigarette Smoke
Exposure
Molecular changes
Disruption ofBiological
Mechanism
Cell / Tissue
Changes
Physiological changes
Disease (CVD, COPD, Lung cancer)
Biological Networks – Systems Biology/ToxicologyAnalytical Chemistry Medicine
Chronic exposure to cigarette smoke affects a number of biological networks associated with smoking-related diseases in a causal chain of Key Events known as Adverse Outcome
Pathways
Gh
β-catenin signaling
NFKB signaling
Cytokine signaling
Inflammation
Cigarette smoke
DNA damage
gtpof(Kras) gtpof(Hras)
taof(Myc)
taof(Mycn)
kaof(PI3K)
PDGF family
Fgf2
SMC differentiation regulation
β-Adrenergicsignaling
taof(Snai1)
Fhit
Response to oxidative
stress
catof(Sod1)
taof(Nrf2)
Hypoxia-induced angiogenesis
ROS
Apoptotic signaling
Oxidative phosphorylationLiterature-derived
knowledge
Data-derivedknowledge Identify & Build
Biological Networkmodels
Hoeng et al., Case Study: The Role of Mechanistic Network Models in Systems Toxicology. Drug DiscoveryToday, 2013, 19:183-192. (PMID: 23933191)
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Methods ‐ Systems Toxicology AssessmentUse Disease Mechanism Understanding for Product Assessment
23Hoeng J et al. A network-based approach to quantifying the impact of biologically active substances. Drug Discovery Today, 2012, 17:413-418 (PMID: 22155224)
Multi-OmicsData
Systems Response Profile
Inflammation
Cell Proliferation
Cell Stress
Apoptosis
DNA Damage
Necroptosis
Senescense
Autophagy
Previously BuiltBiological Network
models
Network PerturbationAmplitudes
Exposure
Sham
3R4F
pRRP
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MethodsFrom Gene Expression Profiles to Biological Impact Factor
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Effects of nicotine – 28‐day inhalation study (OECD TG 412)Application of systems toxicology on liver changes
System response profile of liver transcriptomics reveal up- and down-regulated gene expression
Up-regulated:
• Xenobiotic metabolism
• Oxidative phosphorylation
• Fatty acid/cholesterol metabolism
• Cell cycle
Down-regulated:
• Intracellular signaling
Lipidomics confirm downregulated glycerolipids and sterols
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Cigarette smoke and HTP aerosol– 90‐day inhalation studySystems biology evaluation – Systems Response Profile analysis of lung (90 days)
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Cigarette smoke and HTP aerosol– 90‐day inhalation studySystems biology evaluation – Network Perturbation Analysis of lung (90‐days)
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• Pre-clinical toxicology: Exposure of rats to aerosol from THS2.2 – even at a multiple of the aerosol concentration - results in a dramatically lower biological effects as compared to exposure to 3R4F in– Systemic toxicity; where effects were observed, they are related to nicotine exposure
– Lung inflammation
– Histopathology of respiratory tract organs
• Systems toxicology: Exposure of rats to aerosol from THS2.2 results in a dramatically lower biological network perturbations in the transcriptomes of the nasal epithelium and lung tissue
Cigarette smoke and HTP aerosol– 90‐day inhalation studySummary and conclusions
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ApoE-P1 Switching Study Design
[Time]
[Dis
ease
Ris
k]
Point ofintervention
Comparing switching to the HTP P1* with ongoing smoking and benchmarking against cessation
MethodsSwitching Study in an Animal Model of Disease – cardiovascular disease/emphysema
Mice:- ApoE-/- mice: animal model for aspects of CVD and COPDExposure:- 8 months with dissections at 1,2,3,6 and 8 months- Test atmosphere nicotine concentrations: approx. 30 µg/l- Whole body, 3 x 1 h/day
3R4FCombustible Cigarette: 3R4F
Fresh Air3R4FCESSATIONHTP3R4FSWITCHING
HTPHTPFresh AirControl: Sham/Air
Month 2 Month 8Start
Groups InhalationPoint of intervention
Non-clinical study
*P1: HTTP:THS2.2 or iQOS
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Switching Study in an Animal Model of DiseaseResult Summary: Disease Endpoint ‐ Lung function and Lung Volume
Lung volume (absolute, measured)
0 40.0
0.5
1.0
1.5Sham3R4F
ZRHCessation
Switch
1 2 3 6 8Month
Dis
plac
ed li
quid
wei
ght
(g)
3R4FCessationSwitchingHTPAir
Groups
Month 1 Month 2 Month 3
Month 6 Month 8
Means ± SEM
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Switching Study in an Animal Model of DiseaseResult Summary: Tissue changes – Lung Emphysema
*: Statistically significant compared to sham*: Statistically significant compared to 3R4F:CONT at DT2
Histopathology Score
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Total cells absolute
0 1 2 3 4 5 6 7 8
0
10
20
30Sham3R4F
THS 2.2Cessation
Switch
Tota
l Cel
ls (x
105 )
+/- S
EM
Switching Study in an Animal Model of DiseaseResult Summary: Disease Mechanism ‐ Lung Inflammation
Study monthStudy month
Differential counts
Alveolar macrophages absolute
0 2 4 6 8
0
5
10
15Sham3R4F
THS 2.2Cessation
Switch
Alv
eola
r mac
roph
ages
(x 1
05 ) +/
- SEM
Neutrophils absolute
0 2 4 6 8
0
2
4
6
8Sham3R4F
THS 2.2Cessation
SwitchN
eutr
ophi
ls (x
105 )
+/- S
EM
Alveolar dendritic cells absolute
0 2 4 6 8
0.0
0.5
1.0
1.5
2.0Sham3R4F
THS 2.2Cessation
Switch
Alv
eola
r den
driti
c ce
lls (x
105 )
+/- S
EM
Lymphocytes absolute
0 2 4 6 8
0
1
2
3
4
5Sham3R4F
THS 2.2
Cessation
Switch
Lym
phoc
ytes
(x 1
05 ) +/
- SEM
3R4FCessationSwitchingHTPAir
Groups
Free lung cells in Broncho-alveolar lavage fluid (BALF)
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Switching Study in an Animal Model of DiseaseResult Summary: Disease Mechanism ‐ Lung Inflammation
3R4F P1 Cess. Switch.
0 2 4 6 80
1
2
3
4
MMP activity
Sham
3R4FTHS 2.2Cessation
Switch
Month
MM
P ac
tivity
(m
U/m
l) +/
- SD
3R4FCessationSwitchingP1Air
Groups
Multiple analyte profiling in Broncho-alveolar Lavage Fluid
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3R4F
Cessation
Switch
P1
Switching Study in an Animal Model of Disease Result Summary: Differential Gene Expression ‐ Lung
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Switching Study in an Animal Model of DiseaseResult Summary: Disease Mechanisms – Network Perturbations ‐ Lung
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*
**
## #### ##
### # ##
Different from sham:* : p<0.05**: p<0.01
Different from 3R4F:# : p<0.05##: p<0.01
Sha
m
25.0 %
3R4F
38.5 %
HTT
P: P
1
27.5 %
Ces
satio
n
28.1 %
Sw
itch
29.9 %
Aortic Arches at month 8
Air 3R4F P1 Cess. Swit. Air 3R4F P1 Cess. Swit.
Switching Study in an Animal Model of Disease Result Summary: Disease Endpoint ‐ Aortic Plaque Growth
Image analysis of stained atherosclerotic plaques
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Means ± SEM
0
5
10
15
20
25
30
Means ± SEM
0
1
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Means ± SEM
Plaq
ue V
olum
e (m
m3 )
Plaq
ue S
urfa
ce (m
m2 )
Mea
n O
cclu
sion
(%)
Air 3R4F P1 Cess. Swit. Air 3R4F P1 Cess. Swit. Air 3R4F P1 Cess. Swit.
Plaque Volume Aortic Arch Occlusion Plaque Surface
3D-Reconstructions
Switching Study in an Animal Model of Disease Result Summary: Disease Endpoint ‐ Aortic Plaque size at Month 83D Reconstruction from High Resolution micro-Computed Tomography
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Switching Study in an Animal Model of Disease Summary and Conclusions
• The ApoE-/- mouse model is suitable for studying smoke-related aspects of cardiovascular disease and COPD
• Continuous exposure to aerosol from a pRRP for up to 8 months does not increase cardiovascular disease, inflammation and emphysema
• Switching from cigarette smoke exposure after 2 months to fresh air exposure reverses the onset of disease as measured in apical, functional, and molecular endpoints
• Switching from cigarette smoke exposure to pRRP aerosol exposure reverses the onset of disease in a similar manner as cessation
Use of Apoe−/− Mice in an 8-Month Systems Toxicology Inhalation/Cessation Study to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared with Conventional Cigarettes Phillips B, Veljkovic E , Boué S , Schlage WK, Vuillaume G, Martin F, Titz B , Leroy P, Buettner A, Elamin A, Oviedo A, Cabanski M, Guedj E, Schneider T, Talikka M, Ivanov NV , Vanscheeuwijck P, Peitsch MC, Hoeng J.Toxicological Sciences (2016) 149, 411-32 (PMID 26609137)
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Overall conclusion
• Exposure of rodents to cigarette smoke results in clear toxicological effects and development of known diseases
• Nicotine, when administered via inhalation exposure, causes limited toxicological effects after 28 or 90 days
• Aerosol from a pRRP, HTP elicits much lower toxicity (overall more than 80%) than cigarette smoke, even at a multiple of the nicotine exposure concentration. Most effects can be attribute to nicotine
• Aerosol from a pRRP, HTP does not lead to smoke-related disease and switching from smoke to pRRP aerosol exposure is almost identical to cessation.
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