These slides were presented on 20 October, 2014 at the Safety Pharmacology Society meeting in Washington, D.C. Pulmonary hypertension continues to receive increasing interest as a therapeutic target. There are numerous animal models of this disease. Most are conducted in rodents, although a few large animal paradigms exist. As with any in in vivo model, there are pros and cons to each approach. We have been conducting research in this field for a number of years, performing thousands of PAH experiments. The data within were generated entirely at CorDynamics. Please contact us at [email protected]for further information, thank you for your interest.
Transcript
These slides were presented on 20 October, 2014 at the Safety Pharmacology Society meeting in Washington, D.C. Pulmonary hypertension continues to receive increasing interest as a therapeutic target. There are numerous animal models of this disease. Most are conducted in rodents, although a few large animal paradigms exist. As with any in in vivo model, there are pros and cons to each approach. We have been conducting research in this field for a number of years, performing thousands of PAH experiments. The data within were generated entirely at CorDynamics. Please contact us at [email protected] for further information, thank you for your interest.
Examining Drug Candidates for Pulmonary Arterial Hypertension: Ups and
Downs of Multiple Animal Models
Von Romberg (1891) – ‘pulmonary vascular stenosis’ increase in pulmonary blood pressure -pulmonary artery, pulmonary vein, or pulmonary capillaries -shortness of breath, dizziness, fainting -exacerbated by exertion -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 hospitalizations/yr, ~15,000 deaths/yr 1980s -untreated median survival of 2–3 years from time of diagnosis -cause of death -- cor pulmonale Recent -outcome study show 89% survival at 2 yrs with Rx -future expectation 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 injection into rats results in hepatic generation of toxic metabolite – MCT pyrrole Phase II metabolism of MCT is through glutathione conjugation Reactive metabolite is transported to lungs, injuring pulmonary vasculature
Monocrotaline Rat
Three weeks after MCT (60 mg/kg, SQ)
5HT2B antagonism in MCT rat
Platelets take up 5HT in blood, deliver to sites of microvascular injury and coagulation 5-HT is a mitogen for pulmonary endothelial cells, SMC and myofibroblasts Patients that ingested 5HT2B agonists develop PAH (fenfluramine) 5HT2B knockout mice resist development of hypoxic vasoconstriction C-122 is novel antagonist for 5HT2B receptors
5HT2B antagonism in MCT rat
Serotonin plays a role in proliferative and functional 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 condition -muscularization of PAs -increases PAP -RV hypertrophy -generally works across species -easy to interrogate prevention Cons of MCT model -dis-similar to human condition -lacks EC proliferation, lumen -additional organ toxicity -more of challenge to interrogate reversal
Hypoxia – Cofactor model
Hypoxia + Co-admin Induced PAH Model -exposure to hypoxia alone results in vasoconstriction of the pulmonary arterial tree in mammalian research species – (HPV)
Hypoxia alone
Hypoxia PAH Model -exposure to hypoxia alone results in vasoconstriction of the pulmonary arterial tree in mammalian research species – (HPV) -chronic hypoxia exposure results in moderate PAH
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Vehicle Normoxia 21 Days
Vehicle 10% Hypoxia 21 Days
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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 muscularization 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 pattern -contain an inactivating mutation of transforming growth factor receptor II It was postulated that plexiform lesions arise from -dysregulated angiogenesis common to neoplastic processes Since VEGF is involved in maintenance, differentiation, function 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 athletic training company to construct customized hypoxia chambers for animals at higher throughput Variable simulated altitudes (sea level to 21,000 ft) Initial experiments at 10% O2 proved futile in short term Final ‘altitude’ of 12,500 ft is used (13% O2) Senese et al, Journal of Pharmacological and Toxicological Methods, Volume 64, Issue 1, July–August 2011-
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Telemetered Hemodynamic Evaluation of Vehicle Administration in the Rat Pulmonary Arterial Hypertension Model Induced with Semaxanib (Day 1) and a Low
)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 Presssure
Vehicle Study 1 (n=10)
Vehicle Study 2 (n=10)
Vehicle Study 3 (n=10)
Days of Hypoxia
Hypoxia – VEGF antagonist model
Illustrative image from rat αSMA/elastin stain showing a completely muscularized arteriole (short arrow) and a partially muscularized arteriole (long arrow). The long arrow points to the non-muscularized portion of the partially muscularized arteriole
Normal – 4 weeks
Hypoxia – VEGF antagonist model
Illustrative image from rat αSMA/elastin stain showing completely muscularized arterioles (arrows) and a partially muscularized arteriole (P). Note the thick muscular walls relative to the size of the arteriolar lumens.
PAH – 4 weeks Untreated
Hypoxia – VEGF antagonist model
Illustrative image from αSMA/elastin stain showing partially muscularized arterioles (arrows). The arrows point to the non-muscularized portion of the partially 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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- Sildenafil significantly protects against development PAH in the 28 day hypoxia/SU5416 rat model. Bosentan has similar protective effects.
Hypoxia – VEGF antagonist model
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 protective effect at Days 28 and 42.
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The Effect of Sildenafil on SPAP caused by HxSu
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Hypoxia – VEGF antagonist model
Additional 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 protection – more work!
Immuno – VEGF antagonist model
T-cell deficient + VEGF receptor antagonist PAH Model* In certain human PAH cohorts, inflammation plays a major role in pathogenesis -macrophages are prominent components of inflammatory infiltrates -produce leukotrienes, important mediators of inflammation 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 mitigate development/progression of PAH *TIAN et al, Science Translational Medicine. 2013;5(200):1-14
Immuno – VEGF antagonist model
- On Day 35, surviving animals were instrumented for measurement of pulmonary arterial pressure and right ventricular hypertrophy - Bestatin (inhibits LTA4 hydrolase) and sildenafil rats were compared against vehicle treatments
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-possible survival bias in the vehicle group (literature suggests ~90-100 mm Hg)
Immuno – VEGF antagonist model
-Both bestatin and sildenafil conferred a protective effect on survival proportions in this model -Bestatin observations are consistent with the literature, and the sildenafil data are the first reported with this model
Kiss et al; PLOS One, 8/18, 2014 – Anti-inflamm effects of sildenafil in MCT PAH
Immuno – VEGF antagonist model
-Interestingly, the monocrotaline model also relies on an inflammatory component for its pathogenesis -As with the athymic rat/VEGF model, both bestatin and sildenafil conferred a protective effect on pulmonary arterial pressure
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Sildenafil 60 mg/kg/day
-The literature suggests that bestatin is ineffective in the hypoxia/VEGF model -We have not confirmed nor refuted that data to date
