Endothelin receptor antagonists for persistent pulmonary hypertension in term and late preterm infants

  • Protocol
  • Intervention

Authors

  • Kiran More,

    Corresponding author
    1. Princess Margaret Hospital and King Edward Hospital, Department of Neonatology, Subiaco, Western Australia, Australia
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  • Gayatri Athalye-Jape,

    1. Princess Margaret Hospital and King Edward Hospital, Department of Neonatology, Subiaco, Western Australia, Australia
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  • Shripada C Rao,

    1. King Edward Memorial Hospital for Women and Princess Margaret Hospital for Children, Centre for Neonatal Research and Education, Perth, Western Australia, Australia
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  • Sanjay K Patole

    1. King Edward Memorial Hospital, School of Paediatrics and Child Health, School of Women's and Infant's Health, University of Western Australia, Perth, Western Australia, Australia
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Abstract

This is the protocol for a review and there is no abstract. The objectives are as follows:

  1. To assess the efficacy and safety of ETRA in the treatment of PPHN in full-term and late preterm infants.

  2. To assess the efficacy and safety of selective (which block only the ETA receptors) and non-selective (which block both ETA and ETB receptors) ETRA separately.

Background

Description of the condition

Persistent pulmonary hypertension of the newborn (PPHN)

Persistent pulmonary hypertension of the newborn (PPHN) or persistent fetal circulation (PFC) is a clinical syndrome characterised by failure of the lung circulation to achieve or sustain the normal drop in pulmonary vascular resistance (PVR) at birth (Gersony 1984). Incidence of PPHN is estimated at two per 1000 live births and is associated with substantial morbidity and mortality (Steinhorn 2010). PPHN represents a failure of the unique adaptations that occur in the pulmonary circulation at birth (Konduri 2009). The fetal lung is a fluid filled organ that does not participate in gas exchange and offers high resistance to blood flow. Low oxygen tension present during fetal life and high levels of endogenous vasoconstrictors like endothelin-1 and thromboxane facilitate the maintenance of high PVR (Lakshminrusimha 1999). A dramatic decrease in PVR occurs immediately after birth as the lungs take over the gas exchange function. Several mechanisms contribute to the normal fall in PVR at birth, including increased oxygen tension, ventilation and shear stress. These physiological stimuli lower the PVR through increased release of potent endogenous vasodilators, such as nitric oxide (NO) and prostacyclin (PGI2), and decreased activity of vasoconstrictors such as endothelin (ET)-1 (Levy 2005; Levy 2005a). Some neonates fail to achieve the normal decrease in PVR at birth, resulting in PPHN (Rao 2010). Affected neonates are hypoxaemic because of right-to-left shunt through the ductus arteriosus and foramen ovale leading to a vicious cycle of hypoxaemia, acidosis and further pulmonary vasoconstriction (Greenough 2005). In advanced stages of PPHN, progressive pulmonary vascular functional and structural changes ultimately cause increased pulmonary vascular impedance, right-ventricular failure and death.

Echocardiography is an important tool in the diagnosis of PPHN. The main findings are presence of right to left shunting at the atrial level or via the patent ductus arteriosus, near or suprasystemic pulmonary pressures and the presence of tricuspid regurgitation (Rao 2010; Stayer 2010; Dhillon 2012).

Management of PPHN includes prevention and/or treatment of active pulmonary vasoconstriction, support of right-ventricle function, treatment of the underlying disease and promotion of regressive remodelling of structural pulmonary vascular changes (Oishi 2011). The vital role of nitric oxide (NO)-cGMP signalling in the regulation of the perinatal lung circulation, leading to the development and successful application of inhaled nitric oxide (iNO) therapy for PPHN is now well established (Abman 2007). Approximately 40% of patients will not respond or sustain a response to iNO (Steinhorn 2010) and hence newer alternatives or adjuncts to iNO are being explored.

Description of the intervention

Endothelin receptor antagonists (ETRAs)

Endothelin-1 was discovered by Japanese scientists, Yanagisawa and co-workers, in 1988 (Yanagisawa 1988); they described it as one of the most potent vasoconstrictors. It was subsequently characterised as the most potent vasoconstrictor ever identified, being 100 times more potent than noradrenaline (Barton 2008). Endothelins are a family of 21 aminoacid peptides that are secreted mainly by the systemic and pulmonary vascular endothelial cells. To a lesser degree, they are also secreted by other cell types including smooth muscle cells of the pulmonary artery and fibroblasts. There are three isoforms, ET-1, ET-2 and ET-3, of which ET-1 is biologically the most active. ET-1 is produced in response to various stimuli such as hypoxia, hyperoxia, reactive oxygen species, cytokines, catecholamine stress etc. (Abman 2009). It is synthesised from its biologically inactive precursor by the endothelin-converting enzyme (Davenport 2006). ET-1 is also a smooth muscle mutagen (Abman 2009; Galie 2004).

ET-1 acts via two G protein coupled receptors: ETA receptors which are located primarily on the vascular smooth muscle cells and ETB receptors which are expressed both on vascular endothelium and vascular smooth muscle cells. They are present in both pulmonary as well as systemic vasculature. The activation of ETA receptors mediates vasoconstriction, smooth muscle proliferation, hypertrophy, cell migration and fibrosis, whereas the activation of ETB receptors stimulates the release of potent vasodilators (NO and PGI2), which also exhibit anti-proliferative properties and prevent apoptosis (La 1995; Dakshinamurti 2005; Abman 2009; Rao 2010).

ET-1 plays an important role in the pathogenesis of pulmonary arterial hypertension in adult and paediatric populations. In a very important study in this field, Giaid and co-workers (Giaid 1993) studied the distribution of endothelin-1-like immunoreactivity (by immunocytochemical analysis) and endothelin-1 messenger RNA (by in situ hybridisation) in the lung specimens of 15 control subjects, 11 patients with plexogenic pulmonary arteriopathy (out of which seven had primary pulmonary hypertension) and 17 patients with secondary pulmonary hypertension. In the controls, endothelin-1-like immunoreactivity was rarely seen in the pulmonary vascular endothelial cells. In patients with pulmonary hypertension, endothelin-1-like immunoreactivity was abundant, predominantly in endothelial cells of pulmonary arteries with medial thickening and intimal fibrosis. Likewise endothelin-1 messenger RNA was increased in patients with pulmonary hypertension and was expressed primarily at sites of endothelin-1-like immunoreactivity. They concluded that pulmonary hypertension is associated with increased expression of endothelin-1 in vascular endothelial cells, suggesting that the local production of endothelin-1 may contribute to the vascular abnormalities associated with this disorder (Giaid 1993). Plasma ET-1 are known to be elevated in adults and children with pulmonary hypertension and correlate with increased morbidity and mortality (Allen 1993; Shao 2011).

ET-1 is also known to play an important role in the pathogenesis of PPHN in newborn infants. During fetal life, ET-1 and its receptors are strongly expressed in the pulmonary circulation early during lung development. Basal ET-1 activity contributes to the high pulmonary vascular resistance in utero (Abman 2009) via predominant action on the ETA receptors. Activation of the ETB receptor causes vasodilatation by release of nitric oxide and partly contributes to pulmonary vasodilatation that occurs at birth. In PPHN, the balance is in favour of ETA receptor activation leading to increased pulmonary vascular resistance. In a case control study, Rosenberg and co-workers (Rosenberg 1993) found that plasma ET-1 concentrations were significantly higher in neonates with PPHN compared to those with hyaline membrane disease or cord blood of healthy newborn infants. They also reported that plasma ET-1 levels correlated with disease severity in PPHN. In another case control study Endo and co-workers also found that plasma ET-1 concentrations were significantly higher in the PPHN group than in the control group at < 12 hours and 24 hours of age (Endo 2001). de Lagausie and co-workers (de Lagausie 2005) studied the ETA and ETB receptor protein expression using immunohistochemistry in 10 lung specimens obtained from newborns with congenital diaphragmatic hernia (CDH) and four normal lung specimens. ETA and ETB mRNAs were quantified using real-time RT-PCR in laser-micro-dissected pulmonary resistive arteries. In the lungs of newborns with CDH, immunohistochemistry of both ETA and ETB receptors demonstrated over-expression in the thickened media of pulmonary arteries. Using laser micro-dissection and real-time RT-PCR, higher levels of ETA and ETB mRNA were found in CDH pulmonary arteries than in controls: this increase was more pronounced for ETA mRNA.They concluded that dysregulation of ET-1 receptors may contribute to PPHN associated with CDH (de Lagausie 2005).

Hence blocking the ET receptors using endothelin receptor antagonists (ETRA) may be of benefit in the treatment of PPHN. Bosentan and tezosentan are non-selective ETRAs because they block both the ETA and ETB receptors, whereas sitaxentan and ambisentran are selective ETA receptor inhibitors (Luscher 2000; Geiger 2006; O'Callaghan 2011). The ETRAs are administered either orally or intravenously (Torre Amione 2003). The inhalation route has also been tried in animal models of pulmonary hypertension (Persson 2009).

How the intervention might work

The biology of ET receptors is complex and incompletely understood (Abman 2009). ETRAs appear to work by blocking the endothelin receptors thereby leading to decreased pulmonary vascular resistance and decreased medial hypertrophy of pulmonary vasculature. Newborn animal studies have shown that ETRAs reduce pulmonary hypertension secondary to sepsis (Peng 2003) and meconium aspiration syndrome (Kuo 2001). Ambalavanan and co-workers studied hypoxia-induced pulmonary vascular remodeling (HPVR) leading to PPHN or cor pulmonale in a newborn mouse model. They demonstrated that HPVR secondary to chronic hypoxia can be completely prevented and partially reversed by ET(A) blockade (Ambalavanan 2005).

The most important potential adverse effect of ETRA is hypotension because ETRAs block the endothelin receptors not only in the pulmonary circulation, but also in the systemic circulation. Other potential adverse effects of ETRA are liver dysfunction, cardiac arrhythmias and hypoxaemia. However, a systematic review of observational studies of bosentan in a paediatric population concluded that it is a well tolerated and effective therapy for paediatric pulmonary hypertension (Beghetti 2009).  

Why it is important to do this review

The current treatment for PPHN involves conventional/high-frequency ventilation, administration of oxygen, sedation, iNO and the phosphodiesterase 5 (PDE) inhibitor, sildenafil (Rao 2010; Shah 2011). Approximately 40% of patients do not respond or will not sustain a response to iNO (Steinhorn 2010); iNO is also very expensive. Hence, alternatives to iNO in the management of PPHN are being explored.

Experience with the use of ETRA in adult patients with pulmonary arterial hypertension

A recently published Cochrane review in adult patients with pulmonary hypertension included 12 randomised controlled trials involving 1471 patients (Liu 2013). They concluded that ETRAs can increase exercise capacity, improve World Health Organization (WHO)/New York Heart Association (NYHA) functional class, prevent WHO/NYHA functional class deterioration, reduce dyspnoea and improve cardiopulmonary haemodynamic variables in patients with pulmonary arterial hypertension with WHO/NYHA functional class II and III (Liu 2013).

Experience with the use of ETRA in paediatric patients with pulmonary arterial hypertension

Observational studies have shown that ETRAs may improve the outcomes of children with pulmonary hypertension associated with congenital heart disease (Nemoto 2007; Goissen 2008; Hirono 2010 ), congenital diaphragmatic hernia (Stathopoulos 2011) and bronchopulmonary dysplasia (Krishnan 2008). A systematic review of observational studies also suggested a beneficial effect of ETRA in paediatric pulmonary hypertension (Beghetti 2009). The details of these studies are given below.

Goissen and co-workers (Goissen 2008) reported two cases of PPHN complicating transposition of great arteries with intact ventricular septum that were refractory to multiple therapies and resolved 48 hours after initiation of bosentan therapy.

Nemoto and co-workers (Nemoto 2007) reported on successful application of bosentan in the treatment of pulmonary hypertension early after surgical correction of congenital heart disease. Two infants (five and nine-month old girls who underwent repair of complete atrioventricular septal defect and closure of ventricular septal defect, respectively) suffered from pulmonary hypertension-related symptoms after extubation despite oral sildenafil treatment. After administration of bosentan, the symptoms were dramatically relieved. Bosentan caused a transient increment of liver transaminase in the five-month old girl.

Hirono and co-workers (Hirono 2010) investigated the efficacy of bosentan in patients with single-ventricle physiology who were unable to undergo right-sided heart bypass surgery because of high pulmonary vascular resistance and pulmonary artery pressure. Eight patients with single-ventricle physiology (two male and six female; aged seven months to five years, median one year) were enrolled. Right-sided heart bypass surgery was contraindicated for all patients because of high pulmonary vascular resistance and pulmonary artery pressure. Bosentan therapy successfully reduced pulmonary artery pressure and pulmonary vascular resistance in all patients. All patients had improved clinical symptoms and underwent successful Fontan operations.

Stathopoulos and co-workers (Stathopoulos 2011) studied the effects of tezosentan, a non-selective ETA and ETB receptor antagonist on the cardiopulmonary profile in a fetal lamb model of CDH in utero. A diaphragmatic hernia was surgically created at day 75 of gestation. During 45 minutes of tezosentan perfusion at 135 days of gestation, cardiopulmonary haemodynamic parameters were measured. Age-matched fetal lambs served as controls. In the CDH group, pulmonary artery pressures decreased in the tezosentan group but not in controls.

Krishnan and co-workers (Krishnan 2008) reported a series of six children with chronic lung disease (CLD) and severe pulmonary hypertension treated with bosentan (six of six) and sildenafil (four of six). Vascular reactivity was assessed by cardiac catheterisation prior to and after six months of therapy. Serial echocardiography was also used to assess response. Patients were treated for 2.1 to 2.9 years. They found improvement in oxygenation, symptoms, echocardiographic parameters and haemodynamics by cardiac catheterisation. Transiently elevated liver enzymes were noted, associated with viral respiratory infections in two subjects; no other adverse effects were noted.

Beghetti and coworkers (Beghetti 2009) conducted a systematic review to evaluate the safety and effectiveness of bosentan in paediatric pulmonary arterial hypertension. Twenty-one clinical studies (2000 to 2007) were selected: one interventional prospective, six prospective observational, five retrospective and nine case reports/case series. They concluded that experience from observational studies suggests that bosentan is a well-tolerated and effective therapy for paediatric pulmonary hypertension. Bosentan appeared to improve long-term functional status and haemodynamics in children with pulmonary arterial hypertension. Adverse events, including liver enzyme elevations, also appeared to be less frequent than reported in the adult clinical trials.

Experience with the use of ETRA in animal models of PPHN

Tezosentan has been shown improve survival and decrease pulmonary artery pressure in a porcine model of acute pulmonary arterial hypertension after meconium aspiration (Geiger 2006; Geiger 2008).

Pearl and co-workers showed that bosentan prevents hypoxia-reoxygenation-induced pulmonary hypertension and improves pulmonary function in neonatal piglets (Pearl 1999).

Clinical experience with the use of ETRA in neonatal population

Clinical experience with ET-1 receptor antagonists in the neonatal population is limited. Nakwan et al have reported the benefits of bosentan in a neonate with PPHN (Nakwan 2009). Radicioni and co-workers reported the use of oral bosentan as adjunct therapy to iNO and oral sildenafil in a preterm infant with PPHN after preterm rupture of membranes (PPROM) (Radicioni 2011). Goissen and co-workers have also reported the benefits of bosentan in PPHN associated with congenital heart disease (Goissen 2008).

Considering that ETRAs have shown to be of benefit in adult and paediatric patients with pulmonary arterial hypertension and animal models of PPHN, there may be a role for them in the management of PPHN. Hence, we will conduct this systematic review.

Objectives

  1. To assess the efficacy and safety of ETRA in the treatment of PPHN in full-term and late preterm infants.

  2. To assess the efficacy and safety of selective (which block only the ETA receptors) and non-selective (which block both ETA and ETB receptors) ETRA separately.

Methods

Criteria for considering studies for this review

Types of studies

Randomised, cluster-randomised or quasi-randomised controlled trials will be eligible for inclusion.

Types of participants

Late preterm (born at 34+0 to 36+6weeks) and term infants (born at 37+0 to 41+6 weeks) until postmenstrual age (PMA) up to 44 weeks with PPHN will be eligible for inclusion.

The diagnosis of PPHN could be either clinical or based on echocardiography. Clinical diagnosis of PPHN is considered when there is hypoxaemia refractory to oxygen therapy and mechanical ventilation (Roberts 1997). The echocardiographic diagnosis of PPHN is made by demonstrating the presence of extrapulmonary right to left shunting at the ductal or atrial level, near or suprasystemic pulmonary arterial pressures and Doppler evidence of tricuspid regurgitation (Stayer 2010; Dhillon 2012).

Types of interventions

  1. ETRAs - any dose, frequency, duration, timing, mode of delivery.

  2. ETRAs used alone or combined with other pulmonary vasodilator medications. The other pulmonary vasodilator medications are inhaled nitric oxide, inhaled or systemic prostaglandins, milrinone or sildenafil.

Comparisons
  1. 'ETRA' versus 'placebo'.

  2. 'ETRA' versus 'no pharmacological pulmonary vasodilators'.

  3. 'ETRA' versus 'other pulmonary vasodilator medications'. The other pulmonary vasodilator medications are inhaled nitric oxide, inhaled or systemic prostaglandins, milrinone or sildenafil.

  4. 'ETRA as an adjunct to iNO' versus 'iNO alone'.

  5. 'ETRA as an adjunct to iNO' versus 'placebo and iNO'.

  6. 'ETRA as an adjunct to other pulmonary vasodilators' versus 'other pulmonary vasodilators and placebo'.

  7. 'ETRA as an adjunct to other pulmonary vasodilators' versus 'other pulmonary vasodilators without the use of placebo'.

The dose, route and frequency of other pulmonary vasodilators will be as per published standard neonatal pharmacopeia guidelines.

Types of outcome measures

Primary outcomes
  1. Death from any cause prior to hospital discharge.

  2. Death from any cause prior to 28 days of life (neonatal period).

  3. Death from any cause prior to hospital discharge or need for extracorporeal membrane oxygenation (ECMO).

  4. Requirement for ECMO prior to hospital discharge (criteria for ECMO as per specifications of the included trials).

  5. Improvement in oxygenation index (OI = FiO2 x MAP x 100 / PaO2 ) by a minimum of 20% (as a dichotomous variable) within 24 hours.

  6. Effects on oxygenation index after 30 to 60 minutes of therapy (both absolute values and change from baseline).

  7. Length of hospitalisation (days).

Secondary outcomes
  1. Cerebral palsy: defined as non-progressive motor impairment characterised by abnormal muscle tone and decreased range or control of movements (diagnosed on or before 24 months of age).

  2. Deafness: defined as bilateral sensorineural hearing loss requiring hearing aids (diagnosed on or before 24 months of age).

  3. Blindness: defined as a corrected visual acuity less than 20/200 (diagnosed on or before 24 months of age).

  4. Mild cognitive impairment at 18 to 24 months of age: defined as a Mental Development Index score of greater than 1 standard deviation (SD) below the mean on the Bayley Scales of Infant Development II (BSID II) or a cognitive score greater than 1 SD below the mean on BSID III.

  5. Significant cognitive impairment at 18 to 24 months of age: defined as a Mental Development Index (MDI) score of greater than 2 SD below the mean on BSID II or a cognitive score greater than 1 SD below the mean on BSID III.

  6. Major neurodevelopmental disability at 18 to 24 months of age: defined as one or more of the following: a) BSID III cognitive score and/or language score greater than 2 SD below the normative mean at 18 to 24 months of age; b) BSID II MDI and/or Psychomotor Developmental Index (PDI) scores greater than 2 SD below the normative mean; Griffiths GQ scores greater than 2 SD below the mean at 18 to 24 months of age; non-ambulant cerebral palsy (CP); blindness or sensorineural deafness.  Any other clinically important outcome reported by authors (not pre-specified).

  7. Systemic hypotension requiring the administration of inotropes (after introduction of the trial medication).

  8. Hepatotoxicity (elevated liver enzymes and or conjugated hyperbilirubinaemia).

  9. Cardiac arrhythmias.

Safety

Potential adverse effects of ETRA (e.g. systemic hypotension, elevated serum bilirubin, abnormal liver enzymes and bleeding tendency) during initial hospitalisation will be reviewed.

Search methods for identification of studies

We will search MEDLINE (1966 to 2013) for Clinical Trials (MeSH) OR Controlled Clinical Trials (MeSH) OR Randomised Controlled Trials (MeSH) using following terms:

Population: Infant-Newborn (MeSH) OR Infant, Newborn, Diseases (MeSH) OR newborn (text word) OR infant (text word) OR neonate (text word) AND Hypertension, pulmonary (MeSH) OR persistent fetal circulation syndrome (MeSH) OR Respiratory failure OR Hypoxemia

Intervention: (("Receptors, Endothelin/antagonists and inhibitors"[MeSH]) OR "bosentan" [Supplementary Concept]) OR "sitaxsentan" [Supplementary Concept]. We will also search using the following text words: Selective Endothelin A(ETA) receptor antagonists (Sitaxentan, ambrisentan, atrasentan, BQ-123, Zitobentan), Dual endothelin receptor antagonists (Bosentan, tezosentan), and  Selective ETB receptor antagonists (BQ-788 and A192621).

Comparison: Placebo or no pharmacological pulmonary vasodilators.

Outcome: Death, ECMO, cognitive impairment, cerebral palsy, deafness or hearing impairment, blindness or visual impairment, developmental delay, adverse effects.

Electronic searches

Other databases that we will search include: the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, current issue); EMBASE (1980 to present) and CINAHL (1982 to present). We will search the reference lists of identified trials and electronically published abstracts from the annual meetings of the American Society for Pediatric Research. We will also search Clinical trials.gov and the WHO International Clinical Trials Registry Platform (http://www.who.int/ictrp/en/) websites to identify ongoing studies. We will not apply any language restrictions.

Searching other resources

We will search reference lists of the identified articles. We will search the Science Citation Index (Web of Science) for quotations of any identified and included trial.

Data collection and analysis

We will use the standard method of conducting a systematic review, as described in the Cochrane Handbook for Systematic Reviews of Interventions, version 5.1.0 (Higgins 2011). 

Selection of studies

The study selection will be done independently by authors K. More and G. Jape. We will exclude studies that are not randomised/quasi-randomised. Authors K. More and G. Jape will independently assess each study to determine whether it meets the pre-defined selection criteria, and resolve any differences by discussion with all members of the review team.

Data extraction and management

Two review authors (K. More, G. Jape) will independently extract the data and enter it into a piloted data extraction form. Co-authors (S. Patole, S. Rao) will resolve any differences by group discussion.

Assessment of risk of bias in included studies

The following headings and associated questions (based on the questions in the 'Risk of bias' table) will be evaluated by review authors K. More and G. Jape and entered into the 'Risk of bias' table. We will resolve differences of opinion by discussion with S. Patole and S. Rao.

Selection bias (random sequence generation and allocation concealment)
Adequate sequence generation?

For each included study, we will categorise the risk of bias regarding sequence generation as:

  • low risk - adequate (any truly random process e.g. random number table; computer random number generator);

  • high risk - inadequate (any non-random process e.g. odd or even date of birth; hospital or clinic record number);

  • unclear risk - no or unclear information provided.

Allocation concealment?

For each included study, we will categorise the risk of bias regarding allocation concealment as:

  • low risk - adequate (e.g. telephone or central randomisation; consecutively numbered sealed opaque envelopes);

  • high risk - inadequate (open random allocation; unsealed or non-opaque envelopes; alternation; date of birth);

  • unclear risk - no or unclear information provided.

Blinding?
Performance bias

For each included study, we will categorise the methods used to blind study personnel from knowledge of which intervention a participant received. (As our study population will consist of neonates they would all be blinded to the study intervention):

  • low risk - adequate for personnel (a placebo that could not be distinguished from the active drug was used in the control group);

  • high risk - inadequate - personnel aware of group assignment;

  • unclear risk - no or unclear information provided.

Detection bias

For each included study, we will categorise the methods used to blind outcome assessors from knowledge of which intervention a participant received. (As our study population will consist of neonates they would all be blinded to the study intervention). We will assess blinding separately for different outcomes or classes of outcomes. We will categorise the methods used with regards to detection bias as:

  • low risk - adequate; follow-up was performed with assessors blinded to group;

  • high risk - inadequate; assessors at follow-up were aware of group assignment;

  • unclear risk - no or unclear information provided.

Incomplete data addressed?
Attrition bias

For each included study and for each outcome, we will describe the completeness of data including attrition and exclusions from the analysis. We will note whether attrition and exclusions were reported, the numbers included in the analysis at each stage (compared with the total randomised participants), reasons for attrition or exclusion where reported, and whether missing data were balanced across groups or were related to outcomes. Where sufficient information is reported or supplied by the trial authors, we will re-include missing data in the analyses. We will categorise the methods with respect to the risk attrition bias as:

  • low risk - adequate (< 10% missing data);

  • high risk - inadequate (> 10% missing data);

  • unclear risk - no or unclear information provided.

Free of selective reporting?
Reporting bias

For each included study, we will describe how we investigated the risk of selective outcome reporting bias and what we found. We will assess the methods as:

  • low risk - adequate (where it is clear that all of the study's pre-specified outcomes and all expected outcomes of interest to the review have been reported);

  • high risk - inadequate (where not all the study's pre-specified outcomes have been reported; one or more reported primary outcomes were not pre-specified; outcomes of interest are reported incompletely and so cannot be used; study fails to include results of a key outcome that would have been expected to have been reported);

  • unclear risk - no or unclear information provided (the study protocol was not available).

The most ideal way to assess reporting bias is to obtain the full trial protocol. However, on a practical level, it is may not be feasible to obtain study protocols. If the trial protocol is published online, we will use it. We will also look at the clinical trial registries to find relevant information regarding pre-specified outcomes.

If such information is not available, we will assess the reporting bias by carefully reading the manuscript to see if all the pre-specified outcomes mentioned in the aims and objectives section are described in the results section.

Free of other bias?
Other bias

For each included study, we will describe any important concerns we have about other possible sources of bias (for example, whether there was a potential source of bias related to the specific study design or whether the trial was stopped early due to some data-dependent process). We will assess whether each study was free of other problems that could put it at risk of bias as:

  • low risk - no concerns of other bias raised;

  • high risk - concerns raised about multiple looks at the data with the results made known to the investigators, difference in number of patients enrolled in abstract and final publications of the paper;

  • unclear - concerns raised about potential sources of bias that could not be verified by contacting the authors.

Overall risk of bias

We will make explicit judgements about whether studies are at high risk of bias, according to the criteria given in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). We will assess the likely magnitude and direction of the bias and whether we consider it is likely to impact on the findings. We will explore the impact of the level of bias through undertaking sensitivity analyses - see 'Sensitivity analysis'.

Measures of treatment effect

We will express treatment effect for dichotomous outcomes as risk ratio, risk difference and number needed to treat to benefit (NNTB), with 95% confidence intervals. We will express treatment effect for continuous outcomes as mean difference, with 95% confidence intervals. For harmful effects, we will use risk ratio, risk difference and number needed to treat to harm (NNTH), with 95% confidence intervals.

Unit of analysis issues

If available, we will combine results from cluster trials with other trials using generic inverse variance methods.

Dealing with missing data

If participant dropout leads to missing data then we will attempt to conduct an intention-to-treat analyses. We will also endeavour to obtain missing data from the trial authors.

Assessment of heterogeneity

We will assess statistical heterogeneity using the I2statistic. We will grade the degree of heterogeneity as follows: < 25% no heterogeneity, 25% to 49% low heterogeneity; 50% to 74% moderate heterogeneity, > 75% high heterogeneity. If substantial heterogeneity exists (I2 > 50%) between studies for the primary outcomes, we will explore reasons for heterogeneity.

Assessment of reporting biases

We will assess publication bias using a funnel plot (Egger 1997) if at least 10 studies are included in the meta-analysis.

Data synthesis

For studies with similar type of intervention, we will perform a meta-analysis to calculate a weighted treatment effect across trials using fixed-effect model. We will express the results as typical risk ratios (RR) with 95% confidence interval (CI) for dichotomous outcomes, numbers needed to treat to benefit (NNTB) and numbers needed to treat to harm (NNTH).

We will use mean differences (MD) with 95% confidence interval (CI) to express results for continuous outcomes.

Subgroup analysis and investigation of heterogeneity

We will perform subgroup analyses for:

  • selective and non -selective ETRA. Theoretically, selective blocking of ETA may be preferable because vasoconstriction is mediated via ETA receptors.

Sensitivity analysis

We will perform sensitivity analyses to examine the effects of excluding studies with high risk of selection bias (sequence generation and allocation concealment).

Acknowledgements

None noted.

Contributions of authors

Authors Kiran More and Gayatri Athalye-Jape wrote the first draft of the protocol and also decided literature search strategies and other methodology.

Author Shripada Rao reviewed the search terminology and other methodology and supervised the first draft and final draft.

Author Sanjay Patole was responsible for the concept, design and edited the first and the final draft of protocol.

Declarations of interest

Kiran More: none known

Gayatri Athalye-Jape: none known

Shripada Rao: none known

Sanjay Patole: none known

Sources of support

Internal sources

  • No sources of support supplied

External sources

  • Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health, Department of Health and Human Services, USA.

    Editorial support of the Cochrane Neonatal Review Group has been funded with Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health, Department of Health and Human Services, USA, under Contract No. HHSN275201100016C.

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