Elsevier

Pregnancy Hypertension

Volume 22, October 2020, Pages 86-92
Pregnancy Hypertension

Esomeprazole and sulfasalazine in combination additively reduce sFlt-1 secretion and diminish endothelial dysfunction: potential for a combination treatment for preeclampsia

https://doi.org/10.1016/j.preghy.2020.07.013Get rights and content

Highlights

  • Combined esomeprazole and sulfasalazine additively decrease sFLT-1 secretion.

  • Combined esomeprazole and sulfasalazine additively reduce TNF-α induced VCAM and ET-1 mRNA expression.

  • Combined esomeprazole and sulfasalazine does not reduce sENG or ET-1 protein secretion.

Abstract

Development and repurposing of therapies that show promise in the prevention or treatment of preeclampsia would be a major advance for the obstetrics field. We recently identified esomeprazole and sulfasalazine as potential candidates for the treatment of preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and sENG and mitigate endothelial dysfunction in vitro.

Here we assessed whether esomeprazole and sulfasalazine in combination would additively attenuate the elevated release of anti-angiogenic factors and markers of endothelial dysfunction, key characteristics of preeclampsia. Primary placental tissue and cells, and primary endothelial cells were treated with esomeprazole and sulfasalazine alone and in combination. We assessed secretion of sFlt-1 and sENG and performed in vitro assays of endothelial dysfunction.

Combining esomeprazole and sulfasalazine in lower concentrations caused an additive reduction in sFlt-1 secretion in primary cytotrophoblasts, placental explants and endothelial cells. No additive reduction was observed in sENG secretion when esomeprazole and sulfasalazine were combined. Together, esomeprazole and sulfasalazine additively reduced TNF-α-induced VCAM and ET-1 mRNA expression, and monocyte adhesion to endothelial cells.

In conclusion, combining esomeprazole and sulfasalazine additively reduced secretion of sFlt-1 and markers of endothelial dysfunction. Combined administration of esomeprazole and sulfasalazine may provide a more effective treatment or prevention for preeclampsia compared to either as single agents.

Introduction

Preeclampsia is globally responsible for >70,000 maternal deaths and >500,000 neonatal deaths annually [1], [2]. There are no current efficacious medical therapies to halt disease progression and expectant management and delivery continues as the mainstay of treatment for this insidious syndrome [1], [3], [4], [5], [6].

Our understanding of the pathogenesis of preeclampsia is limited, however central to many of the leading theories is a defect in early trophoblast invasion of the maternal spiral arterioles perfusing the placenta, leading to inadequate blood flow and serious oxidative stress [7], [8]. Subsequently, excess production of the anti-angiogenic factors soluble fms-like tyrosine kinase 1 (sFlt-1) [9], [10], [11], [12] and soluble endoglin (sENG) [13] are released into the maternal circulation [8], [14], [15], [16]. The excess circulating levels of sFlt-1 sequester circulating pro-angiogenic factors such as placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), leading to impaired angiogenic signalling. Elevated levels of circulating sENG antagonizes transforming growth factor β (TGF-β). Together, high circulating levels of these anti-angiogenic factors result in widespread maternal endothelial dysfunction, leading to the multisystem organ injury that is observed clinically [3], [17], [18], [19]. The development of a medical treatment that is safe in pregnancy and able to reduce the excess release of sFlt-1 and sENG, preventing serious damage to the maternal endothelium, may provide a novel approach to treat or prevent preeclampsia.

We have published pre-clinical evidence demonstrating esomeprazole (as well as other proton pump inhibitors) and sulfasalazine have potential to treat, or prevent preeclampsia [20], [21]. Importantly, both appear to have a good safety profile in pregnancy [22], [23].

Esomeprazole is a proton pump inhibitor (PPI), a class of drug widely prescribed to treat serious gastric reflux. Large epidemiological studies have demonstrated that PPIs are safe in pregnancy, even when taken during the first trimester [22], [24]. PPIs potently decrease sFlt-1 and sENG secretion from primary placental cells and tissue as well as primary endothelial cells. Endothelial dysfunction, vasoconstriction and hypertension are hallmarks of preeclampsia. Our in vitro and ex vivo data demonstrate that PPIs rescue many aspects of endothelial dysfunction. Specifically, PPIs reduced expression of endothelial dysfunction markers; vascular cell adhesion molecule-1 (VCAM), and endothelin-1 (ET-1). Importantly, we also showed that PPIs are vasoactive in vivo, demonstrating their ability to prevent hypertension in a mouse model of preeclampsia [20]. Saleh et al, demonstrated that women taking a PPI during pregnancy had reduced circulating sFlt-1, sENG and ET-1 levels [25]. In a large epidemiological study, PPIs decreased preterm preeclampsia when administered in close proximity to disease onset, but not the overall incidence of preeclampsia [26]. We have tested the efficacy of esomeprazole to treat women diagnosed with early onset preeclampsia in a randomised control Phase II clinical trial and did not find a benefit as a lone agent [27], [28].

Sulfasalazine is an anti-inflammatory medication used to treat autoimmune diseases and is commonly continued in pregnancy with no adverse consequences for the fetus [23]. In vitro, sulfasalazine achieves similar results to those described above for esomeprazole. We have also demonstrated that sulfasalazine reduces expression of the epidermal growth factor receptor (EGFR), which is consistent with reduced sFlt-1 [29]. As such, our pre-clinical studies have demonstrated potential efficacy for sulfasalazine as a preventative or treatment for preeclampsia [21].

A strategy commonly used in drug discovery and clinical care is to combine therapeutic agents, to enhance clinical efficacy and/or to reduce the dose required in order to minimize possible side effects. We have recently reported on the advantage of combination esomeprazole and metformin for the treatment of preeclampsia [30]. Given esomeprazole and sulfasalazine successfully mitigate key pathogenic features central to preeclampsia [20], [21], we investigated whether combining lower concentrations of esomeprazole and sulfasalazine may be additive or synergistic (or neither) in reducing sFlt-1 and sENG secretion, and mitigating endothelial dysfunction, compared to either drug alone.

Section snippets

Isolation and treatment of primary human cytotrophoblasts

This study was approved by The Mercy Health Human Research Ethics Committee and all women gave informed written consent for sample collection (R11/34). All methods were performed in accordance with the University of Melbourne and Mercy Health guidelines and regulations. Human cytotrophoblasts were isolated from three normal term placentas (>37 weeks gestation), obtained at caesarean section as previously described [31]. Primary cytotrophoblasts were cultured in DMEM high Glutamax (Life

Combining esomeprazole and sulfasalazine additively reduced sFlt-1 secretion from placental and endothelial cells/tissues

We have previously shown both esomeprazole [20] and sulfasalazine [21] reduce sFlt-1 secretion. Here we test the two drugs in combination on primary cytotrophoblasts, placental explants and primary HUVECs, at low- to mid-range concentrations, that alone have previously shown no or little effect on sFlt-1 secretion.

In primary cytotrophoblasts, esomeprazole (50 μM) or sulfasalazine (50 μM) alone did not significantly alter sFlt-1 secretion (Fig. 1A). Combination esomeprazole and sulfasalazine

Discussion

We have previously demonstrated the potential for esomeprazole and sulfasalazine as novel therapeutic candidates for the treatment of preeclampsia [20], [21]. Both drugs are safe in pregnancy and have been shown to reduce the secretion of anti-angiogenic factors sFlt-1 and sENG and mitigate aspects of endothelial dysfunction.

The purpose of this study was to investigate whether combining these drugs may be additive or synergistic in reducing the secretion of sFlt-1 and sENG and rescuing

Conclusion

We have performed pre-clinical studies using primary human cells and tissues to show that combining esomeprazole and sulfasalazine is additive in reducing sFlt-1 secretion and endothelial dysfunction. These results demonstrate that there may indeed be merit in initiating animal studies and clinical trials to treat preeclampsia using combination therapy.

Sources of funding

This study was funded by the Norman Beischer Medical Research Foundation and a grant from the National Health and Medical Research Council of Australia (NHMRC #1101871). The NHMRC also provided salary support (TKL; #1159261 FB; #1142636 ST; #1136418 and NJH; #1146128). The funders had no role in study design, data collection, analysis or decision to publish.

Authors contributions

NJH and TKL designed the experiments. NB and NJH wrote the main manuscript. FB, TKL, NJH and ST provided intellectual input and assisted with the manuscript editing. FB, ST, TKL and NJH obtained funding. NB, SB, PC and TN were involved in data generation. All authors reviewed and edited the manuscript drafts.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We would like to thank the research midwives, Gabrielle Pell, Genevieve Christophers and Rachel Murdoch, Mercy Hospital for women Obstetrics and Midwifery staff and patients for participating in this research.

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