RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats

Laxminarayan Bhata*, Jon Hawkinsonb, Marc Cantillona, Dasharatha G Reddya, Seema R Bhata, Charles E. Laurentc, Annie Bouchardc, Marzena Biernatc, Dany Salvailc

a Reviva Pharmaceuticals, Inc., Santa Clara, CA, USA

b Institute for Therapeutics Discovery & Development and Department of Medicinal Chemistry, University of Minnesota

c IPS Therapeutique Inc., Sherbrooke, Quebec, Canada

* Corresponding author at: 1250 Oakmead Parkway, Suite 210, Sunnyvale, CA 94085, USA.

Tel.: +1 (408) 816.1470 Fax:  +1 (408) 904 6270.

Email address: lbhat@revivapharma.com (L. Bhat).

 

Eur J Pharmacol. 2017 Sep 5; 810:83-91. doi: 10.1016/j.ejphar.2017.05.052. Epub 2017

May 31. PubMed PMID: 28576407.

 

ABSTRACT

RP5063, a multimodal dopamine (DA) and serotonin (5-HT) modulator with high affinity for DA2/3/4 and 5-HT2A/2B/7 receptors and moderate affinity for SERT, is a novel therapeutic of special interest in the treatment of pulmonary arterial hypertension (PAH). Evidence indicates that therapeutics targeting the 5-HT2A/2B receptors can influence the pathogenesis of PAH. However, the therapeutic effect of RP5063 in patients with PAH has yet to be investigated. A Sugen 5416-hypoxia (SuHx)–induced PAH model was used to evaluate twice-daily (b.i.d.) RP5063 at 10 mg/kg (RP-10) and 20 mg/kg (RP-20), as compared with positive (sildenafil 50 mg/kg b.i.d.; Sil-50) and negative controls (SuHx+vehicle; SuHx+veh), in 24 adult male Wistar-Kyoto rats. RP5063 showed significantly lower systolic pulmonary arterial (both doses) and systolic right ventricular (RP-10) pressures, and improvement in oxygen saturation (RP-20). It significantly reduced small-vessel wall thickness (RP-20), lowered the percentage of muscular vessels (both doses). Both doses limited arterial obliteration due to endothelial cell proliferation, prevented plexiform lesion formation, and stemmed the release of leukotriene B4. Sildenafil, an active comparator, dosed at 50 mg/kg b.i.d., also showed significant effects on vessel structure and improved oxygen saturation.  However, Sildenafil did not demonstrate any significant effect on arterial obliteration, plexiform lesion development, or pulmonary arterial or right ventricular pressure. As PAH gains in severity, the impact of RP5063 inhibition of 5HT2B increases, preventing arterial constriction and improving pulmonary hemodynamics. Due to its functional, structural, and chemokine effects, RP5063 represents a promising candidate for investigation in late-phase PAH.

KEYWORDS: 5-hydroxytryptamine 2B receptor; Anti-proliferative agent; Pulmonary arterial hypertension; RP5063; Rat Sugen 5416-hypoxia model; Vascular remodeling

PMID: 28576407 DOI: 10.1016/j.ejphar.2017.05.052

Reprinted from the European Journal of Pharmacology, 810, Bhat L, Hawkinson J, Cantillon M, et al. RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats, Pages No. 83-91, Copyright (2017), with permission from Elsevier.


 

SUPPLEMENT

Pulmonary arterial hypertension (PAH), a progressive condition that affects 15 to 50 individuals per 1,000,000, has survival rate at 5 and 7-year post diagnosis of approximately 50% [1,2]. The condition is defined by increased pulmonary artery pressure (PAP) due to pulmonary arterial constriction/remodeling that leads to right ventricle (RV) pressure overload, hypertrophy, and failure, which results in death [3-6]. Present treatment fails to ameliorate the underlying cytoproliferation that influences the pulmonary vascular structure [7].

RP5063 is a multimodal dopamine (DA) / serotonin (5-HT) modulator with high affinity for DA2/3/4 and 5-HT2A/2B/7 receptors and moderate affinity for SERT. Evidence indicates that targeting the 5-HT2A/2B receptors can influence PAH disease pathogenesis [8-11]. In a study of PAH induced by Sugen 5416 and hypoxia (SuHx) in adult male Wistar-Kyoto rats, RP5063 mitigated functional symptoms, attenuated underlying histologic pathology, and prevented the release of the chemokine, Leukotriene B4 (LTB4).

UNDERSTANDING THE SUGEN-HYPOXIA (SU-HX) MODEL

Sugen-Hypoxia is a widely used model to characterize PAH.  SU5416, a vascular endothelial growth factor (VEGF)-receptor antagonist, known to cause pulmonary endothelial lesions in the pulmonary vasculature, facilitates pulmonary artery vasoconstriction resulting from both the hypoxic conditions and the endothelial damage that decreases nitric oxide and endothelium-derived relaxing factor levels [12-13]. By obstructing the pulmonary arterioles and limit pulmonary blood flow, these changes increase PAP, along with RV afterload and hypertrophy.

This model highlights the etiologic mechanisms involved in the endothelial cell hyperproliferation that characterizes the plexogenic arteriopathy in the peripheral pulmonary arteries in human PAH [14]. It emanates from the concept that vascular endothelial growth factor (VEGF) is an important maintenance and differentiating factor for vascular endothelial cells. In chronically hypoxic rats, Su5416, by antagonizing the VEGF-receptor, causes severe, irreversible PAH with intimal lesions, characterized as precapillary, small-vessel arterial endothelial proliferations [14]. VEGF receptor blockade and chronic hypoxia results in these lesions and a persistent and progressive PAH, which lead to RV remodeling and failure, even after removal of the hypoxic stimulus.

RELEVANT METHODOLOGY

In this parallel design study, Sugen solution (SU 5416, 10 mg/mL diluted in DMSO) dosed at 20 mg/kg was administered on Day 0 subcutaneously to the animals (Groups 2–5). Animals in these groups were kept under hypoxic conditions (FiO2 of 10%) for 3 weeks, then in ambient oxygen levels (FiO2 of 21%) for 2 weeks following. From the end of Week 2 (Day 14) to the end of Week 5 (Day 35), animals were gavaged twice daily: 1) Group 2 (vehicle, SuHx+veh; n=8); 2) Group 3 (RP5063 10 mg/kg (RP-10); n=10); 3) Group 4 (RP5063 20 mg/kg (RP-20); n=10); and 4) Group 5 (sildenafil 50 mg/kg (Sil-50); n=5).

On the day of terminal surgery (Day 35), hemodynamic parameters were recorded, a blood sample was collected, and tissues (traches, lungs, heart, and liver) were harvested. Internal arterial pressures were obtained using a flexible fluid filed catheter attached to a pressure transducer.

Hemodynamic parameters measured including electrocardiography, heart rate, and blood oxygen saturation (SO2), systemic arterial blood pressure (SAP), RV systemic pressure (RVSP), and PAP. Mean SAP (mSAP) and mean PAP (mPAP) were calculated from systolic and diastolic pressure recordings. To assess histology, the middle left lung lobe was prepared for analysis of blood vessel size and composition. Microphotographs were examined for plexiform-like lesions and luminal obliterations.  Blood samples were obtained on Days 0, 21, 28, and 35. One-half of each sample was saved for analysis of LTB4, quantified for the no-SuHx, SuHx+veh, and RP5063 10- and 20-mg groups at all time points.

 

 

Fig. 1. Effect of RP5063 on pulmonary hemodynamics in SuHx-PAH rats at time of surgery (Day 35). Panels the meanPAP (A, left) and the right ventricular systolic pressure (RVSP; B, right).

* p<0.05; as compared with No-SuHx.

# p<0.05; as compared with SuHx+vehicle.

No-SuHx — Normoxic control, SuHx+veh —SuHx + vehicle, RP-10 — RP5063 10 mg/kg b.i.d.,

RP-20 — RP5063 20 mg/kg b.i.d., Sil-50 — sildenafil 50 mg/kg b.i.d.

Reprinted with modifications from the European Journal of Pharmacology, 810, Bhat L, Hawkinson J, Cantillon M, et al. RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats, Pages No. 83-91, Copyright (2017), with permission from Elsevier.

 

RP5063 TREATMENT MITIGATES FUNCTIONAL EFFECTS

Specific to pulmonary hemodynamics (Fig. 1A–B), both RP5063 groups showed improvement in PAPSyst and the 10-mg enhanced RVSP over the SuHx+veh control, though the mPAP did not reach significance. They displayed significantly lower PAPSyst at 32.2 mm Hg (Δ –25.1%) and 32 mm Hg (Δ –25.5%), respectively (p<0.05). In addition, the 10-mg group reflected a significant reduction in RVSP as compared with the SuHx+veh group (32.8 versus 42.8 mm Hg; Δ –23.3%; p=0.011).

Both the 20-mg group and sildenafil group showed SO2 improvements over the SuHx+veh group (Fig. 2A). SO2 in 20 mg group was 3% higher at 96.9% and the sildenafil group was 3.1% higher at 97% (p<0.05).

Treatment resulted in higher weight gain, as compared with SuHx+veh control; the RP5063 10 and 20 mg groups, and sildenafil groups reflected 66.7 g, 50.8 g, and 64.0 g increases in weight, respectively. RP5063 treatment resulted in lower lung weight, with the relative lung weights for sildenafil, and RP5063 10 and 20 mg groups were 0.71%, 0.65%, and 0.62%, respectively.

Specific to Fulton’s index (Fig. 2B), an indicator of right-ventricular hypertrophy, both RP5063 and Sildenafil groups displayed decreases, as compared to SuHx+veh, though not significant.

Some of the functional improvements, such as pulmonary hemodynamic and Fulton’s index, as compared with control could have been more dramatic, as in contrast to the effects seen in a companion study utilizing monocrotaline [15]. These effects might have been limited by the study design, in addition to the pathologic process initiated by SuHx. In this study, treatment in the SuHx was delayed for two weeks following Sugen induction and given for only three weeks.  This delay in treatment initiation could be significant, as some of the underlying pathology might have been well established. For example, endothelial dysfunction and vascular smooth muscle (VSM) remodeling processes should be quite evident by this time. Hence, these two factors, delay and shorter duration of treatment, could have limited these hemodynamic effects. Thus, treatment acted more as a remedy to the established structural and functional changes that had been stimulated, rather than as a preventive intervention by being administered starting at Day 0.

 

 

 

Fig. 2. Effect of RP5063 in SuHx-PAH rats on (A) blood oxygen saturation levels and (B) RV hypertrophy as measured by the Fulton’s index in SuHx-PAH rats at the time of surgery (Day 35).

# p<0.05; as compared to SuHx+veh.

No-SuHx — Normoxic control, SuHx+veh —SuHx + vehicle, RP-10 — RP5063 10 mg/kg b.i.d.,

RP-20 — RP5063 20 mg/kg b.i.d., Sil-50 — sildenafil 50 mg/kg b.i.d.

Reprinted with modifications from the European Journal of Pharmacology, 810, Bhat L, Hawkinson J, Cantillon M, et al. RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats, Pages No. 83-91, Copyright (2017), with permission from Elsevier.

 

RP5063 ATTENUATES PULMONARY VESSEL HISTOLOGY

Treatment with RP5063 was able to affect the blood vessel composition (Fig. 3A). RP5063-treated animals showed a lower percentage of muscular tissue than SuHx+veh by 25.8%, and 48.4% in the 10 and 20 mg/kg b.i.d. groups, respectively (p<0.001). Correspondingly, the RP5063 groups displayed a higher percentage non-muscular tissue than the SuHx+veh group by 20.14% and 43.17% in the 10 and 20 mg/kg b.i.d. groups, respectively (p<0.001).

RP5063 also impacted vessel wall thickness (Fig. 3B). The 20-mg group decreased wall thickness by 18.96% (p˂0.01), while sildenafil group did the same by 25.48% (p<0.01), compared with the SuHx+veh group. The wall thickness for small vessels was less in the 20-mg group by 18.96% (p˂0.01) and for the sildenafil 50 mg group by 25.48% (p<0.01). These effects produced by RP5063 were at doses 2.5 to 5-fold lower in strength than that used with sildenafil.

 

 

 

Fig. 3. Effect of RP5063 and sildenafil on pulmonary artery blood vessel composition (A) and wall thickness (B).

* p˂0.05, ** p˂0.01, *** p˂0.001; as compared with No-SuHx

# p˂0.05, ## p˂0.01, ### p˂0.001; as compared with SuHx+veh

No-SuHx — Normoxic control, SuHx+veh —SuHx + vehicle, RP-10 — RP5063 10 mg/kg b.i.d.,

RP-20 — RP5063 20 mg/kg b.i.d., Sil-50 — sildenafil 50 mg/kg b.i.d.

Reprinted from the European Journal of Pharmacology, 810, Bhat L, Hawkinson J, Cantillon M, et al. RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats, Pages No. 83-91, Copyright (2017), with permission from Elsevier.

 

RP5063 EFFECTS ON PLEXIFORM LESIONS

In this model, SU 5416 (Fig. 4 A-E) induced combinations of small pulmonary medial and adventitial thickening and some arteriopathy, including concentric neointimal and complex plexiform-like lesions. The lungs of SU 5416-treated rats showed a range of luminal obliterations, from light to severe, in medium-sized and small pulmonary arteries, all caused by endothelial cell proliferation. Complex lesion formation was observed both within the vessel lumen (essentially obliterating the luminal space), and outside the vessel (aneurysm-like). Animals in the SuHx+veh group presented with significant vascular walls, characterized by vascular smooth muscle (VSM) cell hypertrophy and hyperplasia (Fig. 4B), as compared with the non-induced control (Fig 4A). Furthermore, a number of plexiform lesions, a hallmark characteristic of clinical PAH, were observed.

Comparatively, the remodeling of the pulmonary vasculature in the RP5063-treated animals was less severe; VSM cell hyperplasia was essentially nonexistent, with cell hypertrophy accounting for most of the intimal thickening in treated animals. No plexiform lesion could be observed in any of the treated animals. As a result of the diminished endothelial hyperplasia, and VSM cell hypertrophy and hyperplasia, vascular luminal diameters remained greater, limiting the obliteration of the blood vessels by plexiform lesions and vascular remodeling.

Endothelial cell obliteration of small and medium pulmonary vessels occurred after the animals were returned to normoxic conditions. The animals in the SuHx+veh group exhibited between 10% and 15% arterial obliteration due to endothelial cell proliferation. Arterial obliteration was observed in 8% of the vessels with both doses of RP5063, while 13% of the vessels were affected in sildenafil group.

 

 

Fig. 4. Representative photomicrographs of RP5063 effects in SuHx-PAH rats. Panels A-E show a cross section of the left lung stained with H&E in control No-SuHx animals (A), in SuHx+veh (B), in treated animals with RP-10 (C) and RP-20 (D), as well as animals treated with Sil-50 (E). The arrow in panel B points to a plexiform-like lesion, a hallmark characteristic of severe PAH.

Reprinted from the European Journal of Pharmacology, 810, Bhat L, Hawkinson J, Cantillon M, et al. RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats, Pages No. 83-91, Copyright (2017), with permission from Elsevier.

 

RP5063 PREVENTS CHEMOKINE AND CYTOKINE RELEASE

Exploratory evidence suggests from this study and that involving the monocrotaline model may facilitate a marked downregulation of chemokines and inflammatory cytokines [15].

It is important to recognize that inflammation is an important characteristic of PAH, and elevated plasma levels of cytokines/chemokines are reported in patients with idiopathic PAH [16].

Because the SuHx model did not exhibit a significant inflammatory component that would involve proinflammatory cytokines, RP5063 only demonstrated notable changes in levels of LTB4. This chemokine causes pulmonary vascular remodeling by directly injuring luminal endothelial cells, promoting the growth of the smooth muscle cell layer of pulmonary arterioles, and enhancing human pulmonary artery adventitial fibroblast proliferation, migration, and differentiation. [17, 18].

The SuHx+veh group showed higher levels of LTB4 than the no-SuHx group at induction (Fig. 5), peaking on Day 21, and remaining high on Days 28 and 35. Both RP5063 doses prevented its release to more than one-half the optical density level seen on Day 21 within the SuHx group. This effect persisted to Day 28 with both doses and to Day 35 with 20 mg group. The effect of RP5063 on LTB4 suggests that an additional mechanism might be involved with the amelioration of the structural and functional changes related to PAH.  In comparison with the levels with RP5063, those in the sildenafil group were 1.5- and 2.5-fold higher on Days 21 and 28. The impact of RP5063 may be significant to the disease process.

Interestingly, in the monocrotaline model, one that is representative of idiopathic PAH and involves an early inflammatory phase, all doses of RP5063 (as compared with the induced group and vehicle) reflected lower levels of the proinflammatory cytokines, TNF-a, IL-b, and IL-6, over the final 2 weeks of treatment [15]. The 10-mg dose also produced a significant effect on IL-6 levels (p<0.05). Inflammatory cytokines (e.g., monocyte chemoattractant protein 1, TNF-a, IL-b, IL-6) play a role in idiopathic PAH development, especially related to the vascular cell proliferation and remodeling of the pulmonary arteries, and some can predict survival (e.g., IL-6, IL-8, IL-10, and IL-12p70) [16, 19, 20]. Pathologic specimens in PAH model show that perivascular inflammatory cells (e.g., macrophages, dendritic cells, T and B lymphocytes, mast cells) accumulate due to the increased levels of cytokines and chemokines [21].

 

 

 

Fig. 5. Changes in LTB4 levels in SuHx-PAH rats.

b.i.d. — twice daily, IL — interleukin, LT — leukotriene, OD — optical density value,

No-SuHx — Normoxic control, SuHx+veh —SuHx + vehicle, RP-10 — RP5063 10 mg/kg b.i.d.,

RP-20 — RP5063 20 mg/kg b.i.d., Sil-50 — sildenafil 50 mg/kg b.i.d.

Reprinted from the European Journal of Pharmacology, 810, Bhat L, Hawkinson J, Cantillon M, et al. RP5063 is effective in Sugen-hypoxia–induced pulmonary arterial hypertension in rats, Pages No. 83-91, Copyright (2017), with permission from Elsevier.

 

 

PHARMACOLOGIC RATIONALE

Dysfunctional 5-HT and/or endothelin signaling in PAH can lead to vascular fibrosis, vasoconstriction, and proinflammatory cytokine/chemokine increase (e.g., TNF-α, IL-6, IL-8, LTB4, etc.). Clinical studies have reported that in patients a 10- to 13-fold increased level of 5-HT in PAH patients [22] and a higher expression of 5-HT1B/2A/2B/7 in the pulmonary arterial/vascular system [23]. The 5-HT1B/2A receptors have been reported to play major roles in the regulation of pulmonary vasoconstriction and vasodilation. By binding to the 5-HT2B receptor, RP5063 produced limited pulmonary smooth muscle hyperplasia in a dose-related manner, which complemented its dilatory effects.

RP5063 mitigates the effects of 5-HT in the pulmonary vasculature due to its high affinity for 5-HT2A/2B/7 receptors [24]. RP5063 can prevent the vasoconstriction via 5-HT2A signaling and mitigate cell proliferation and vascular remodeling facilitated by 5-HT2B signaling [8, 25]. It appears that through the modulation of these 5-HT2 receptors that RP5063 might be offer the potential to limit fibrosis and restore tissue regeneration [26].  In addition, modulatory of effects on 5-HT7  receptor appears to play a role in immune and inflammatory responses including the release of proinflammatory cytokines and the maturation migratory properties of  dendritic cells [27, 28].

TRANSLATIONAL ASPECTS

A final consideration around this preclinical research relates to the potential translation benefits in the clinic. Most notable are the need for treatments that can be co-administered with present treatments for PAH and the ability to address associated psychological co-morbidities associated with this condition.

Due to the progressive nature of PAH, multiple combinations have been used. In one analysis, approximately 56% of patients were taking additional therapy within 2 years of initial diagnosis [29]. Since currently targeted pathways overlap, present combination strategies have yet to yield consistent clinical results [30,31]. Most of the present treatment fail to address the ongoing cytoproliferative processes that modify the pulmonary vascular structure.

To this need, RP5063 could provide significant benefit by suppressing the underlying structural processes associated with idiopathic PAH by mitigating the influence of 5-HT on 5-HT2A/2B/7 receptors that impact underlying structural processes including its effects on various proinflammatory cytokines and chemokines.  Thus, future investigations should evaluate the contribution of RP5063 in combination with present treatments including as either a two-drug or three-drug combination: 1) endothelin receptor antagonists; 2) PDE-5 inhibitors; and 3) exogenous prostacyclin and analogs.  Preliminary preclinical evidence points to this potential with RP5063 [32].

A second need relates to the ability to manage associated co-morbidities.  Approximately 35% of patients with pulmonary hypertension exhibit some form of mental disorder [33, 34]. Studies have reported acceptance of risk of death, anxiety, cognitive sequela, depression, panic disorder, memory deficits, work disability, and concurrent use of antidepressant (25%) and antipsychotic (24.1%) agents in these patients [33, 35, 36]. As brain serotonergic pathway dysregulation plays a significant role in mood and psychotic disorders, the possibility exists that underlying imbalance in serotonergic function may linking mental disorders with PAH [37]. Interestingly, RP5063 has been demonstrated effectiveness in managing patients with acute schizophrenia and schizoaffective disorder in phase 2 clinical evaluation [24].  Coupled with its preclinical activity in PAH, these observations in patients offers the clinician the potential of a treatment that might offer the potential to address not only the primary condition of PAH, but also associated mood and behavioral co-morbidities.

 

CONCLUSIONS

The Su-Hx is a common model to evaluate PAH and reflects this condition in patients [13,14]. In this model, RP5063 mitigated relevant functional, structural, and chemokine changes. These data are supported by corroborating studies evaluating RP5063 using the monocrotaline-induced model of PAH [15, 32]. These effects appear to be mediated particularly via modulation of the 5-HT2A/2B/7 receptors in particular. These observations support the potential of RP5063 as a promising therapeutic option with the potential of a disease-modifying treatment for PAH that offers the potential to play a role as co-administered treatment to standard PAH therapies and to address associated mood and behavior co-morbidities. RP5063 has received a FDA Orphan Designation for further exploration in a phase 2 study for the management of PAH.

 

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