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