Examining Drug Candidates for Pulmonary Arterial Hypertension: Ups and Downs of Multiple Models

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 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 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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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



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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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)



Days of Hypoxia


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SPAP

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G7"2"Days"(Hypoxia)"






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Fulton's)Index)(RV/LV+S))

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Time Course: Pulmonary Artery Pressure Fulton’s Hypertrophy Index Days 2, 4, 8, 15, 22, 29


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


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


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

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Sildenafil (n=10)

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-‐ Sildenafil significantly protects against development PAH in the 28 day hypoxia/SU5416 rat model. Bosentan has similar protecJve effects.


-‐ 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.


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Dose Group

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Systolic Pulmonary Arterial Pressure (SPAP)

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Sildenafil 60 mg/kg/day

Riociguat 10 mg/kg/day

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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

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The Effect of Sildenafil on SPAP caused by HxSu

Control

sildenafil


AddiConal development of intravascular plexiform lesions – a hallmark of severe human PAH

PAH – 12 weeks (untreated)


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


-‐  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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m)Hg))

Days)

)Effect)of)Semaxanib)on)SPAP)in)the)Conscious,)Telemetrized)Athymic)

Nude)Rat)


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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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




Days of Hypoxia


-‐ 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

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Vehicle n=4

Bestatin 1 mg/kg n=7

Sildenafil 60 mg/kg n=10

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Bestatin 1 mg/kg n=8

Sildenafil 60 mg/kg n=10


-‐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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Vehicle#(n=9)#

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Examining Drug Candidates for Pulmonary Arterial Hypertension: Ups and Downs of Multiple Models