Agents for inhibiting the adhesion of red blood cells to the endothelium in people with sickle cell disease

Abstract

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

To assess the effectiveness and safety of agents that inhibit the adhesion of red blood cells to the endothelium for managing acute painful vaso‐occlusive episodes in people with SCD.

Background

Please see the glossary for an explanation of terms used in this protocol (Appendix 1).

Description of the condition

Sickle cell disease (SCD) is one of the most common severe monogenic disorders inherited in an autosomal recessive fashion. It is estimated that approximately six million individuals who are affected reside in Africa, where more than 230,000 children are born with the disease each year, which constitutes about 80% of the global population with SCD (Modell 2008). The highest prevalence is in sub‐Saharan Africa with approximately 170,000 SCD births per year (Modell 2008; Rees 2010; Sheth 2013). By comparison, the yearly estimate of affected births in North America is 2600, in Europe 1300, and in Brazil 3500 (Cancado 2007; Modell 2008). People with SCD suffer from a multisystemic disease with recurring episodes of acute illness and progressive organ damage. It occurs due to a point mutation in the 17th nucleotide of the beta globin gene, with a substitution of thymine for adenine. Consequently, the sixth amino acid in the beta globin chain becomes valine instead of glutamic acid. This mutation results in the formation of haemoglobin S (HbS) that polymerises when deoxygenated, causing red blood cells to have a sickle shape. The rate and extent of HbS polymerisation is the main determinant of disease severity (Cancado 2007; Rees 2010).

Currently, several processes have been identified as having an important role in vaso‐occlusion, such as microvascular transit time (delay time), blood viscosity, dense irreversibly sickled cells, adhesion of sickle cells to endothelial cells and vascular matrix, vaso‐regulation, coagulation, inflammation and involvement of platelets and leukocytes (Chiang 2005; Frenette 2002; Kaul 2008; Steinberg 2014). Adhesion of red cells to the endothelium is an abnormal process and is followed by a propagation phase caused by the accumulation of poorly deformable red cells (Hebbel 2004). Many molecules including cytokines and chemokines have been implicated in the increased endothelial adherence (Kaul 2008; Steinberg 2014).

Clinical manifestations are driven by two major pathophysiological processes that overlap: haemolytic anaemia (destruction of red blood cells) and vaso‐occlusion. Haemolytic anaemia is associated with endothelial dysfunction and lower bioavailability of nitric oxide (Rees 2010). Vaso‐occlusion could result in acute pain from tissue ischaemia. This painful episode, known as a vaso‐occlusive crisis (VOC), is the most common complication and the leading cause of hospitalisation of people with SCD (Ballas 2010; Telen 2014). Diagnosis is based on the analysis of haemoglobin by electrophoresis and chromatography, with family studies and DNA tests as appropriate. Until now, hydroxyurea (HU) has been the only approved drug that could mitigate the clinical manifestations of SCD (Jones 2001). Although hydroxyurea is the standard treatment for the prevention and control of VOCs, not everyone responds to this treatment. Besides, many individuals express their concern about the toxic effects of HU, since it is a chemotherapeutic drug, and consequently, compliance to treatment is poor. Adherence is a major obstacle to the use of HU, with many families identifying forgetfulness and difficulty in obtaining refills as the key reasons for poor adherence (Estepp 2014).

In general, treatment of people with SCD has evolved in recent decades, and increased life expectancy has been observed. The main measures that are responsible for these improvements include newborn screening programmes, infection prophylaxis, prediction and prevention of stroke, improvement of the transfusion care, use of HU, and prevention and treatment of iron overload. Despite this progress, people with SCD still have recurring episodes of VOC, a poor quality of life and a lower life expectancy compared to the general population (Sheth 2013).

Description of the intervention

In the past, drugs to modulate foetal haemoglobin (i.e. hydroxyurea) have been the main focus of research, but increasing knowledge of the complex pathophysiology of SCD has led to the development of new drugs that target specific molecules involved in vascular injury (Vichinsky 2012). Vaso‐occlusion is associated with adhesion of cellular elements (red blood cells, white blood cells and platelets) to the endothelium with secondary inflammation. Interference with adherence of cellular elements to the endothelium, especially through key molecules, seems a strategic approach because it could mitigate vaso‐occlusion (Colin 2014) or prevent of VOCs (Kutlar 2014). Drugs which inhibit molecules involved in the complex mechanism of red blood cell adhesion to the endothelium include the following.

• Rivipansel (GMI‐1070): this drug is infused intravenously every 12 hours up to maximum of 15 doses. People aged 12 years or older and weighing over 40 kg receive an initial dose of 1680 mg followed by a dose of 840 mg every 12 hours. Those aged six to 11 years and weighing 40 kg or less, receive weight‐based dosing (mg/kg), initially of 40 mg/kg (maximum of 1680 mg) followed by a dose of 20 mg/kg (maximum of 840 mg) every 12 hours.

• SelG1: this drug is administered intravenously every month at a dose of 2.5 mg/kg or 5 mg/kg.

• Propranolol: it is administered orally at the standard dose of 40 mg every 12 hours.

• Poloxamer (MST‐188): this drug is administered intravenously as a continuous infusion 100 mg/kg for one hour followed by 30 mg/kg/hour for up to 48 hours.

• Pentosan polysulfate sodium (PPS): this drug is administered as a single daily oral dose of 300 mg.

Currently, 'anti‐adhesive therapy' is not a consensual class of drugs and there are other drugs which may inhibit the adhesion of red blood cells to the endothelium, but which have not yet been assessed in randomised controlled trials (RCTs), such as glycosylation inhibitors, anti‐p‐selectin aptamers, anionic polysaccharides, mitogen‐activated protein kinases (MEK) inhibitors (Ghoshal 2014; Gutsaeva 2011; Zennadi 2014). Since preclinical studies seem promising, the following drugs will also be evaluated:

• glycosylation inhibitors;

• anti‐p‐selectin aptamers;

• MEK inhibitors.

We suppose that several anti‐inflammatory agents may down‐regulate adhesion, but since they have multiple mechanisms of action, we believe their inclusion will introduce confounders that could interfere with data analysis. For example, HU the only drug approved for the treatment of SCD, also has several mechanisms of action (including anti‐adhesive properties) and has been evaluated alone in a Cochrane review (Jones 2001).

Since anticoagulants and antiplatelet drugs act in the haemostatic system, we believe that they should be assessed independently. Although they also interfere with cell adhesion, their inclusion will increase the data heterogeneity and, consequently, their interpretation. Furthermore, there is already a Cochrane review evaluating the use of low‐molecular‐weight heparins in SCD (van Zuuren 2013).

How the intervention might work

Abnormal adhesion is a complex mechanism that comprises a wide variety of adhesive interactions involving the red blood cells and the endothelium. Due to the complexity of VOC, a multi‐targeted approach will likely be required to achieve the best outcome (Hebbel 2008; Manwani 2013). Some of the main mechanisms implicated in SCD abnormal adhesion involve the following.

1. Selectins: the preliminary interactions between blood cells and vascular endothelium are mediated by selectins through rapidly reversible adhesive interactions, leading to rolling and tethering of cells on endothelial surfaces under conditions of shear stress. The blockade of pan‐selectin and p‐selectin appears beneficial (Kutlar 2012; Kutlar 2014; Okpala 2014; Telen 2014). This can be achieved using rivipansel (GMI‐1070) which is a small glycomimetic compound that was synthesized to block pan‐selectin, SelG1 which is a humanized monoclonal antibody directed against P‐selectin and pentosan polysulfate sodium (PPS) which is a semi‐synthetic hypersulfated polyxylan that effectively blocks cell adhesion to P‐selectin. Also, glycosylations inhibitors could inhibit p‐selectin expression on endothelial cells and anti‐p‐selectin aptamers could inhibit adhesion of red blood cells and leukocytes to endothelial cells (Ghoshal 2014; Gutsaeva 2011).

2. Beta adrenergic receptors: it is known that physiologic stress stimulates the release of epinephrine which in turn stimulates sickle red blood cell adhesion to endothelial cells (De Castro 2012; Steinberg 2014; Zennadi 2007); hence, beta blockers (e.g. propranolol) have a potential role as anti‐adhesive therapy by inhibiting cyclic adenosine monophosphate (c‐AMP)‐mediated sickle red blood cell adhesion.

3. Adhesive interactions (cell‐cell, cell‐protein, protein‐protein) (Orringer 2001): poloxamer (MST‐188) is a nonionic block co‐polymer surfactant that binds to hydrophobic surfaces of damaged cells to block cell‐cell adhesion through a broadly specific mechanism.

4. MEK inhibitors: They inhibit adhesion of sickle erythrocytes to endothelial cells acting through inactivation of extracellular signal regulated kinases (ERK) 1/2, which are abnormally activated in sickled red blood cells (Zennadi 2014).

Such adhesive events are thought to be critical to the vaso‐occlusive process, hence therapies that interfere in this mechanism are expected to reduce the main reason people with SCD require health care.

Why it is important to do this review

It is unclear whether therapies aimed at specific targets involved in the adhesion of red blood cells to the endothelium of people with SCD are effective and safe for managing vaso‐occlusion. This review aims to identify the best evidence of this new type of intervention for controlling VOCs.

Objectives

To assess the effectiveness and safety of agents that inhibit the adhesion of red blood cells to the endothelium for managing acute painful vaso‐occlusive episodes in people with SCD.

Methods

Criteria for considering studies for this review

Types of studies

All randomised controlled trials (RCTs) and quasi‐RCTs. We will only include quasi‐RCTs if there is sufficient evidence that the treatment and control groups were similar at baseline.

Types of participants

People of any age or gender with SCD (SS, SC, S‐beta⁰ thalassaemia and S‐beta⁺ thalassaemia) proven by electrophoresis, high performance liquid chromatography (HPLC), with family studies or DNA testing as appropriate. People with SCD on a chronic transfusion regimen will not be considered for analysis.

Types of interventions

We will include trials of people with SCD receiving any of the following interventions to inhibit the adhesion of red blood cells to the endothelium; we will compare each intervention with each other, standard treatment, placebo or no intervention.

• rivipansel (GMI 1070)

• SelG1

• propranolol

• poloxamer (MST‐188)

• pentosan polysulfate sodium (PPS)

• glycosylation inhibitors

• anti‐p‐selectin aptamers

• MEK inhibitors

Types of outcome measures

Primary outcomes

1. Rate of sickle cell‐related painful VOCs

2. Length of painful VOCs in hospitalised participants

3. Pain intensity assessed using validated scales (e.g. visual analog scale)

Secondary outcomes

1. Cumulative intravenous (IV) opioid consumption

2. Biomarkers of adhesion in plasma (measured by ELISA kits)

   1. soluble vascular cell adhesion molecule‐1 (sVCAM)

   2. intercellular adhesion molecule‐1 (sICAM)

   3. P‐selectin (sP‐selectin)

   4. E‐selectin (sE‐selectin)

3. Cutaneous microvascular blood flow and reactivity (evaluated by laser doppler velocimetry)

4. Adverse effects (for known adverse effects of the included drugs, please refer to the additional tables (Table 1))

   Table 1.

   Known side effects of included drugs

   	Rivipansel	SelG1	Propranolol	Poloxamer	PPS	Glycosylation inhibitors	Anti‐p‐selectin aptamers	MEK inhibitors
   Renal dysfunction	X	X	X	X	X	X	X	X
   Hepatic dysfunction	X	X	X	X	X	X	X	X
   Infections	X	X		X	X	X	X	X
   Bleeding	X	X		X	X	X	X	X
   Leukocytosis	X				X
   Allergic reactions	X	X
   Cardiovascular symptoms			X
   Bronchospasm			X
   Hypoglycemia						X

Search methods for identification of studies

Electronic searches

We will identify relevant studies from the Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register using the terms: (sickle cell OR (haemoglobinopathies AND general)) AND (Poloxamer OR Propanolol OR Pentosan polysulfate sodium OR Rivipansel OR SelG1 OR Glycosylation inhibitors Or Anti‐p‐selectin aptamers OR MEK inhibitors).

The Haemoglobinopathies Trials Register is compiled from electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL) (updated each new issue of The Cochrane Library) and weekly searches of MEDLINE. Unpublished work is identified by searching the abstract books of five major conferences: the European Haematology Association conference; the American Society of Hematology conference; the British Society for Haematology Annual Scientific Meeting; the Caribbean Health Research Council Meetings; and the National Sickle Cell Disease Program Annual Meeting. For full details of all searching activities for the register, please see the relevant section of the Cochrane Cystic Fibrosis and Genetic Disorders Group Module.

We will also search for relevant studies registered on the databases ClinicalTrials.gov (ClinicalTrials.gov) and WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp). The final search strategies will be published in the appendices.

We will not apply any language restrictions.

Searching other resources

We will check reference lists of articles, reviews and textbooks for possible relevant studies. Whenever necessary, we will also contact other researches or experts in the field for unpublished studies.

Data collection and analysis

Selection of studies

Two authors (AMFS and SAA) will independently assess and select potentially eligible studies for inclusion in the review, both for the initial screening and for the assessment of the full articles. We will resolve any disagreements by discussion and, whenever necessary, discussion with a third review author (MSF) (Higgins 2011b).

Data extraction and management

Two review authors (AMFS and EKMS) will use a piloted data extraction form to independently collect the following data:

1. source data;

2. demographic and participant data (age, sex, diagnostic criteria, type of SCD);

3. trial design (sequence generation, allocation sequence concealment, blinding);

4. intervention‐related data (type of intervention, route of delivery, dose, timing);

5. outcome‐related data (rate of sickle cell‐related painful VOCs, length of painful VOCs in hospitalised participants, pain intensity assessed using validated scales, cumulative IV opioid consumption, biomarkers of adhesion in plasma, cutaneous microvascular blood flow and reactivity, adverse effects);

6. results‐related data (number of participants allocated to each intervention group, sample size and missing participants).

We will resolve any disagreement by discussion, and if necessary arbitration by a third author (MSF). We will enter data into the Review Manager (RevMan) program (RevMan 2014). When necessary, we will send requests to trial authors for additional information or data (Higgins 2011b).

Assessment of risk of bias in included studies

Two review authors (AMFS and EKMS) will independently assess the risk of bias in the included trials. We will resolve any disagreements by discussion with a third review author (JAPB). According to the Cochrane Collaboration’s tool for assessing risk of bias, we will evaluate selection bias (random sequence generation and allocation concealment), performance bias (blinding of participants and personnel), detection bias (blinding of outcome assessment), attrition bias (incomplete outcome data), selective reporting bias and other sources of bias (including sponsorship bias). We will categorise each domain as having either a 'low', 'high' or 'unclear' risk of bias (Higgins 2011c).

We will consider other methods for assessment of reporting bias such as searching resources other than electronic databases for unpublished studies and requesting protocols from included studies to assess selective outcome reporting.

Measures of treatment effect

We will analyse continuous outcome data (duration of painful VOC in hospitalised participants, pain intensity (assessed using validated scales), cumulative IV opoid consumption, biomarkers of adhesion in plasma, cutaneous microvascular blood flow and reactivity) as a mean difference (MD) when two or more trials present data from the same instrument of evaluation. When trials express the same variables through different instruments and different units of measurement, we will use the standardized mean difference (SMD). We will consider 95% confidence intervals (CIs) for both cases. We will analyse dichotomous outcome data (adverse events) using risk ratios (RR) with 95% CIs. We will analyse count data (rate of sickle cell‐related painful VOCs) divided into counts of rare events and counts of common events; for counts of rare events we will focus on rates (i.e. rate of sickle cell‐related painful crisis) and for counts of common events we will treat these in the same way as continuous data (Deeks 2011).

Unit of analysis issues

We will consider the individual participant as the unit of allocation. However, since the primary outcome may occur in a participant more than once, we will perform this analysis using a rate ratio, comparing the rate of events in the two groups. For trials with a cross‐over design, we plan to use only first‐arm data (before participants have crossed over to the second treatment) due to the possibility of a carry‐over effect (Deeks 2011; Elbourne 2002; Higgins 2011d).

Dealing with missing data

Where possible, we will perform an intention‐to‐treat analysis including all participants randomised to any intervention. We will attempt to contact the original investigators of included trials if key data are missing, such as the number of events or participants, means or standard deviations (SDs). If necessary, we will impute any missing data with replacement values. For continuous outcomes, we will impute the mean observed value. We will perform a sensitivity analysis excluding the participants with missing data to assess the strength of the results. If it is not possible to impute missing data, we will consider the potential impacts on the findings of the review. We also will perform analyses to estimate the losses if they occurred at random or if they are related to treatment and outcomes. Then perform sensitivity analyses to assess how sensitive results are to reasonable changes in the assumptions that are made (Higgins 2011d).

Assessment of heterogeneity

We will identify heterogeneity by visual inspection of the forest plots and by using a standard Chi² test with a significant level of α = 0.1. We will quantify Inconsistency among the pooled estimates using the I² statistic ((Q ‐ df)/Q) x 100%, where Q is the Chi² statistic and df is its degrees of freedom. This clarifies the percentage of variability in effect estimates resulting from heterogeneity rather than sampling error. The thresholds for the interpretation of I² will be represented as follows (Deeks 2011; Higgins 2003):

• 0% to 40%: might not be important;

• 30% to 60%: may represent moderate heterogeneity;

• 50% to 90%: may represent substantial heterogeneity;

• 75% to 100%: considerable heterogeneity.

Assessment of reporting biases

If we include 10 or more trials in the review, we plan to assess reporting biases and small trial effects by drawing a funnel plot (trial effect versus trial size) (Sterne 2011).

Data synthesis

We will use the RevMan software to carry out quantitative analysis, based on the intention‐to‐treat principle. For dichotomous variables we will use the Mantel Haenszel method to perform the meta‐analysis. For continuous variables, we will use the inverse variance method. If the pooling of results is clinically appropriate and we do not find substantial statistical heterogeneity (I² under 50%), we will use the fixed‐effect model; however, if we identify substantial statistical heterogeneity (I² over 50%), we will use the random‐effects model (Deeks 2011). If heterogeneity is very high (I² over 80%), the appropriateness of performing the meta‐analysis will be discussed within the group.

Subgroup analysis and investigation of heterogeneity

Regardless of heterogeneity and the number of included trials, we plan to perform the following subgroup analyses:

• type of agent;

• dose.

Sensitivity analysis

We will assess the robustness of results performing sensitivity analysis using a fixed‐effect versus a random‐effects model, the inclusion or exclusion of trials with an overall high risk of bias or those trials that were not blinded. We will also perform a sensitivity analysis excluding the participants with missing data to assess the strength of the results (Deeks 2011).

Summary of findings table

We will present the main results of the review in 'Summary of findings' tables. These tables present key information concerning the quality of the evidence, the magnitude of the effects of the interventions examined, and the sum of the available data for the main outcomes (Schünemann 2011a). The 'Summary of findings' tables also include an overall grading of the evidence related to each of the main outcomes using the GRADE (Grades of Recommendation, Assessment, Development and Evaluation) approach. The GRADE approach defines the quality of a body of evidence as the extent to which one can be confident that an estimate of effect or association is close to the true quantity of specific interest. The quality of a body of evidence involves consideration of within‐trial risk of bias (methodological quality), directness of evidence, heterogeneity, precision of effect estimates and risk of publication bias (Schünemann 2011b). We plan to present the following outcomes in the 'Summary of findings' tables:

• rate of sickle cell related painful vaso‐occlusive crises;

• length of painful vaso‐occlusive crises in hospitalised participants;

• improvement in pain intensity assessed using validated scales (e.g. visual analog scale);

• adverse effects.

Appendices

Appendix 1. Glossary

Term	Explanation
adhesion	property of binding or remaining in proximity
amino acid	a biologically important organic compound composed of amine and carboxilic acid functional groups
aptamer	a single‐stranded oligonucleotide that binds molecular targets
autosomal recessive	one of several ways that a disease can be passed down through families; an autosomal recessive disorder means two copies of an abnormal gene must be present in order for the disease to manifest
beta‐adrenergic receptor	a class of receptors that are targets of the catecholamines, especially norepinephrine and epinephrine
chemokine	a family of small cytokines, or signalling proteins secreted by cells; the major role of chemokines is to act as a chemo‐attractant to guide the migration of cells
chemotherapeutic drug	a chemical substance used to treat disease, infections or other disorders, especially cancer
chromatography	the collective terms for a set of laboratory techniques for the separation of mixtures
cytokine	a cellular product that influences the function or activity of other cells
electrophoresis	the separation and identification of proteins and haemoglobin types based on their relative rates of migration through agarose or polyacrylamide gel in an applied electrical field
gene	a segment of a DNA molecule that contains all the information required for synthesis of a protein
globin	the protein constituent of haemoglobin
glycomimetic	refers to molecules that have structures similar to carbohydrates, but with some variation
glycosylation	the enzymatic process by which a sugar is covalently attached to a target protein
haemoglobin	the primary constituent of red blood cell cytoplasm; it transports molecular oxygen from the lungs to the tissues and returns carbon dioxide to the lungs
haemolytic anaemia	a form of anaemia due to haemolysis (the abnormal breakdown of red blood cells)
ischemia	a restriction of blood supply to tissues
monogenic disorder	a genetic disorder resulting from a single mutated gene
mutation	a random alteration in a gene or chromosome which results in a new trait or characteristic that can be inherited
nitric oxide	an important cellular signalling molecule involved in many physiological and pathological processes; it is a powerful blood vessel dilator
nucleotide	organic molecules that serve as the subunits of nucleic acids like DNA and RNA
polymerise	to add or condense smaller molecules to form a more complex one
selectin	any of a family of cell adhesion molecules
surfactant	a compound that lowers the surface tension between two liquids or between a liquid and a solid

Contributions of authors

All authors participated in the development of the protocol.

Roles and responsibilities
Task	Who will undertake the task?
Protocol stage: draft the protocol	AMFS, MSF, JAPB, EMKS, SAA
Review stage: select which trials to include (2 + 1 arbiter)	AMFS, SAA, MSF
Review stage: extract data from trials (2 people)	AMFS, EMKS
Review stage: enter data into RevMan	AMFS, EMKS
Review stage: carry out the analysis	AMFS, EMKS
Review stage: interpret the analysis	AMFS, MSF, JAPB, EMKS
Review stage: draft the final review	AMFS, MSF, JAPB, EMKS, SAA
Update stage: update the review	AMFS, MSF, JAPB, EMKS

Sources of support

Internal sources

• No sources of support supplied

External sources

• National Institute for Health Research, UK.

  This systematic review was supported by the National Institute for Health Research, via Cochrane Infrastructure funding to the Cochrane Cystic Fibrosis and Genetic Disorders Group.

Declarations of interest

AMFS, SAA and MSF are currently participating in the clinical trial involving anti‐p selectin monoclonal antibody (SelG1)in sickle cell disease financed by SELEXYS, whose grants will benefit the Anemia Research Lab from Universidade Federal de Sâo Paulo (UNIFESP).

EMKS and JAPB ‐ none known.

Notes

October 2016: This protocol has been withdrawn as it does not meet the current methodological standards of the Cochrane Collaboration.

Withdrawn from publication for reasons stated in the review