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Examining Drug Candidates for Pulmonary Arterial Hypertension: Ups and
Downs of Mul=ple Animal Models
Ups and Downs of Mul=ple Animal Models
Von Romberg (1891) – ‘pulmonary vascular stenosis’ increase in pulmonary blood pressure
-‐pulmonary artery, pulmonary vein, or pulmonary capillaries -‐shortness of breath, dizziness, fainCng -‐exacerbated by exerCon -‐heart failure
Normal pulmonary arterial pressure = 12–16 mm Hg PAH = mean pulmonary artery pressure > 25 mm Hg
Pulmonary Hypertension
This is PAH
USA -‐ ~200,000 hospitalizaCons/yr, ~15,000 deaths/yr 1980s -‐untreated median survival of 2–3 years from Cme of diagnosis
-‐cause of death -‐-‐ cor pulmonale Recent -‐outcome study show 89% survival at 2 yrs with Rx -‐future expectaCon is median survival of 10+ years -‐pregnancy is contraindicated in PAH
Pulmonary Hypertension
PATHWAYS OF PAH Schermuly et al – Nature Reviews Cardiology August 2011
Animal Models of PAH
MONOCROTALINE RAT Monocrotaline (MCT) is a 11-‐membered macrocyclic pyrrolizidine plant alkaloid A single SQ injecCon into rats results in hepaCc generaCon of toxic metabolite – MCT pyrrole Phase II metabolism of MCT is through glutathione conjugaCon ReacCve metabolite is transported to lungs, injuring pulmonary vasculature
Monocrotaline Rat
Three weeks a^er MCT (60 mg/kg, SQ)
5HT2B antagonism in MCT rat
Platelets take up 5HT in blood, deliver to sites of microvascular injury and coagulaCon 5-‐HT is a mitogen for pulmonary endothelial cells, SMC and myofibroblasts PaCents that ingested 5HT2B agonists develop PAH (fenfluramine) 5HT2B knockout mice resist development of hypoxic vasoconstricCon C-‐122 is novel antagonist for 5HT2B receptors
5HT2B antagonism in MCT rat
Serotonin plays a role in proliferaCve and funcConal components of PAH
Antagonism of 5HT2B helps to prevent the development of PAH in rats treated with MCT Pros of MCT model -‐well characterized -‐reproducible -‐similar to human condiCon
-‐muscularizaCon of PAs -‐increases PAP -‐RV hypertrophy
-‐generally works across species -‐easy to interrogate prevenCon Cons of MCT model -‐dis-‐similar to human condiCon
-‐lacks EC proliferaCon, lumen -‐addiConal organ toxicity -‐more of challenge to interrogate reversal
Hypoxia – Cofactor model
Hypoxia + Co-‐admin Induced PAH Model -‐exposure to hypoxia alone results in vasoconstricCon of the pulmonary arterial tree in mammalian research species – (HPV)
Hypoxia alone
Hypoxia PAH Model -‐exposure to hypoxia alone results in vasoconstricCon of the pulmonary arterial tree in mammalian research species – (HPV) -‐chronic hypoxia exposure results in moderate PAH
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Mouse RVSP (mmHg)
Vehicle Normoxia 21 Days
Vehicle 10% Hypoxia 21 Days
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Rat SPAP (mm Hg)
Normoxia Vehicle 28 Days
Hypoxia Vehicle 28 Days
Hypoxia – Cofactor model
Hypoxia + Cofactor Induced PAH Model exposure to chronic robust hypoxia results in pathophysiology similar to effect of monocrotaline
-‐thickening and muscularizaCon of PAs -‐increase in PAP -‐RV hypertrophy
The goal was to produce the desired effects above – and add the development of endothelial cell overgrowth to narrow the vascular lumen
-‐increased clinical relevance compared to either MCT / hypoxia alone -‐severe human PAH has luminal plexiform lesions that express angiogenesis factors
Hypoxia – Cofactor model
Hypoxia + Cofactor PAH Model* In human PAH, increased expression of VEGF receptor
-‐the lung endothelial cells expand in a monoclonal pagern -‐contain an inacCvaCng mutaCon of transforming growth factor receptor II
It was postulated that plexiform lesions arise from
-‐dysregulated angiogenesis common to neoplasCc processes Since VEGF is involved in maintenance, differenCaCon, funcCon of EC..
-‐hypothesis was to disrupt VEGF signaling – examine effects on EC VEGF antagonism + hypoxia results in endothelial cell death that selects for apoptosis resistant phenotype = overgrowth *TARASEVICIENE-‐STEWART et al, The FASEB Journal. 2001;15:427-‐438
Hypoxia – VEGF antagonist model
Hypoxia + VEGF Antagonist Model Worked with commercial athleCc training company to construct customized hypoxia chambers for animals at higher throughput Variable simulated alCtudes (sea level to 21,000 ^) IniCal experiments at 10% O2 proved fuCle in short term Final ‘alCtude’ of 12,500 ^ is used (13% O2) Senese et al, Journal of Pharmacological and Toxicological Methods, Volume 64, Issue 1, July–August 2011-‐
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(mm Hg)
Telemetered Hemodynamic Evaluation of Vehicle Administration in the Rat Pulmonary Arterial Hypertension Model Induced with Semaxanib (Day 1) and a Low
Oxygen Environment - Systolic Pulmonary Artery Pressure
Vehicle
Days of Hypoxia
Hypoxia – VEGF antagonist model
Hypoxia – VEGF antagonist model
Figure 3. Effect of semaxanib and a low oxygen environment on pulmonary arterial pressure in
rats. Data are presented as mean ± S.E.M. (n=10 per study, 3 studies).
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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
(mm
Hg)
Telemetered Hemodynamic Evaluation of Vehicle Administration in the RatPulmonary Arterial Hypertension Model Induced with Semaxanib (Day 1) and a Low Oxygen Environment - Systolic Pulmonary Artery Presssure
Vehicle Study 1 (n=10)
Vehicle Study 2 (n=10)
Vehicle Study 3 (n=10)
Days of Hypoxia
Hypoxia – VEGF antagonist model
0"
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20"
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60"
70"
SPAP
$($mm$Hg)$
Systolic$Pulmonary$Arterial$Pressure$(SPAP)$
G1"2"Days"(Normoxia)"
G2"4"Days"(Normoxia)"
G3"8"Days"(Normoxia)"
G4"15"Days"(Normoxia)"
G5"22"Days"(Normoxia)"
G6"29"Days"(Normoxia)"
G7"2"Days"(Hypoxia)"
G8"4"Days"(Hypoxia)"
G9"8"Days"(Hypoxia)"
G10"15"Days"(Hypoxia)"
G11"22"Days"(Hypoxia)"
G12"29"Days"(Hypoxia)"
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0.70#
0.80#
Fulton's)Index)(RV/LV+S))
G1#2#Days#(Normoxia)#
G2#4#Days#(Normoxia)#
G3#8#Days#(Normoxia)#
G4#15#Days#(Normoxia)#
G5#22#Days#(Normoxia)#
G6#29#Days#(Normoxia)#
G7#2#Days#(Hypoxia)#
G8#4#Days#(Hypoxia)#
G9#8#Days#(Hypoxia)#
G10#15#Days#(Hypoxia)#
G11#22#Days#(Hypoxia)#
G12#29#Days#(Hypoxia)#
Time Course: Pulmonary Artery Pressure Fulton’s Hypertrophy Index Days 2, 4, 8, 15, 22, 29
Hypoxia – VEGF antagonist model
IllustraCve image from rat αSMA/elasCn stain showing a completely muscularized arteriole (short arrow) and a parCally muscularized arteriole (long arrow). The long arrow points to the non-‐muscularized porCon of the parCally muscularized arteriole
Normal – 4 weeks
Hypoxia – VEGF antagonist model
IllustraCve image from rat αSMA/elasCn stain showing completely muscularized arterioles (arrows) and a parCally muscularized arteriole (P). Note the thick muscular walls relaCve to the size of the arteriolar lumens.
PAH – 4 weeks Untreated
Hypoxia – VEGF antagonist model
IllustraCve image from αSMA/elasCn stain showing parCally muscularized arterioles (arrows). The arrows point to the non-‐muscularized porCon of the parCally muscularized arterioles. The arteriolar walls are thinner than those of similarly sized arterioles in untreated
PAH – 4 weeks Treated
-‐ On Day 28, animals were instrumented for measurement of pulmonary arterial pressure and right ventricular hypertrophy -‐ Sildenafil rats were compared against normoxia controls and hypoxia/SU5416 vehicle treatments
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RV / (LV+
S) Vehicle normoxia (n=5)
Vehicle (n=10)
Sildenafil (n=10)
*
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Systolic Pulmon
ary Artery Pressure (m
m Hg)
Vehicle normoxia (n=5)
Vehicle (n=10)
Sildenafil (n=10)
*
-‐ Sildenafil significantly protects against development PAH in the 28 day hypoxia/SU5416 rat model. Bosentan has similar protecJve effects.
Hypoxia – VEGF antagonist model
-‐ On Day 28, established PAH animals were interrogated using an intervenJon treatment strategy. Treatment occurred from Day 28 to Day 56 -‐ Sildenafil and riociguat rats were compared against hypoxia/SU5416 vehicle treatments
-‐ Sildenafil and riociguat significantly reduce PAH in the 56 day hypoxia/SU5416 rat model using an intervenJonal treatment strategy.
Hypoxia – VEGF antagonist model
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Dose Group
SPAP
(mm Hg)
Systolic Pulmonary Arterial Pressure (SPAP)
Vehicle
Sildenafil 60 mg/kg/day
Riociguat 10 mg/kg/day
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Dose Group
SPAP
(mm Hg)
Systolic Pulmonary Arterial Pressure (SPAP)
Vehicle
Sildenafil 60 mg/kg/day
Riociguat 10 mg/kg/day
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120
Dose Group
SPAP
(mm Hg)
Systolic Pulmonary Arterial Pressure (SPAP)
Vehicle
Sildenafil 60 mg/kg/day
Riociguat 10 mg/kg/day
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"Dose"Group"
SPAP
$(mm$Hg)$
Systolic$Pulmonary$Arterial$Pressure$(SPAP)$
Vehicle"
Sildenafil"60"mg/kg/day"
Riociguat"10"mg/kg/day"
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#Dose#Group#
Fulton's)Index)(RV/LV+S))
Vehicle#
Sildenafil#60#mg/kg/day#
Riociguat#10#mg/kg/day#
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Dose Group
Fulton's Index (RV/LV+S)
Vehicle
Sildenafil 60 mg/kg/day
Riociguat 10 mg/kg/day
* *
*
Hypoxia – VEGF antagonist model
At Days 28 and 42, SPAP of MCT rat is strongly elevated from both normal and Day 21 rat. Sildenafil has strengthening protecJve effect at Days 28 and 42.
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Normal rat 21 days 28 days 42 days
SPAP
(mmHg
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The Effect of Sildenafil on SPAP caused by HxSu
Control
sildenafil
Hypoxia – VEGF antagonist model
AddiConal development of intravascular plexiform lesions – a hallmark of severe human PAH
PAH – 12 weeks (untreated)
Hypoxia – VEGF antagonist model
PAH – 12 weeks (sildenafil)
Hypoxia – VEGF antagonist model
PAH – 12 weeks (sildenafil)
Hypoxia – VEGF antagonist model
PAH – 12 weeks (sildenafil)
Early evidence suggests sildenafil confers a survival benefit in severe PAH in the absence of long term pathological protecQon – more work!
Immuno – VEGF antagonist model
T-‐cell deficient + VEGF receptor antagonist PAH Model* In certain human PAH cohorts, inflammaCon plays a major role in pathogenesis
-‐macrophages are prominent components of inflammatory infiltrates -‐produce leukotrienes, important mediators of inflammaCon
In athymic (T-‐cell deficient) rats, semaxanib induces strong PAH even in normoxia.
-‐in these rats (as in humans), macrophages accumulate around occluded pulmonary arterioles and release leukotriene B4 It was postulated that blocking LTB4 may miCgate development/progression of PAH
*TIAN et al, Science TranslaQonal Medicine. 2013;5(200):1-‐14
Immuno – VEGF antagonist model
-‐ Athymic nude rats were telemetrized with pulmonary artery pressure catheters
-‐ Following recovery, rats were dosed once with semaxanib and monitored for 42 days.
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Systolic)Pulmon
ary)Artery)Pressure)(m
m)Hg))
Days)
)Effect)of)Semaxanib)on)SPAP)in)the)Conscious,)Telemetrized)Athymic)
Nude)Rat)
Hypoxia – VEGF antagonist model
Figure 3. Effect of semaxanib and a low oxygen environment on pulmonary arterial pressure in
rats. Data are presented as mean ± S.E.M. (n=10 per study, 3 studies).
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160
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
(mm
Hg)
Telemetered Hemodynamic Evaluation of Vehicle Administration in the RatPulmonary Arterial Hypertension Model Induced with Semaxanib (Day 1) and a Low Oxygen Environment - Systolic Pulmonary Artery Presssure
Vehicle Study 1 (n=10)
Vehicle Study 2 (n=10)
Vehicle Study 3 (n=10)
Days of Hypoxia
Immuno – VEGF antagonist model
-‐ In a different study -‐ on Day 35, surviving animals were instrumented for measurement of pulmonary arterial pressure and right ventricular hypertrophy -‐ BestaJn (inhibits LTA4 hydrolase) and sildenafil rats were compared against vehicle treatments
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Systolic Pulmon
ary Artery Prssure (m
m Hg)
Vehicle n=4
Bestatin 1 mg/kg n=7
Sildenafil 60 mg/kg n=10
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RV / LV+S Vehicle n=5
Bestatin 1 mg/kg n=8
Sildenafil 60 mg/kg n=10
Immuno – VEGF antagonist model
-‐Both bestaJn and sildenafil conferred a protecJve effect on survival proporJons in this model -‐BestaJn observaJons are consistent with the literature, and the sildenafil data are the first reported with this model
Kiss et al; PLOS One, 8/18, 2014 – AnJ-‐inflamm effects of sildenafil in MCT PAH
Immuno – MCT model
-‐ Athymic nude rats dosed once with monocrotaline and monitored for 42 days.
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SPAP$(mmHg)$
Monocrotaline"(n=12)"
Vehicle"(n=9)"
0.0000#0.0500#0.1000#0.1500#0.2000#0.2500#0.3000#0.3500#0.4000#0.4500#
RV/LV+S'
Monocrotaline#(n=12)#
Vehicle#(n=9)#
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