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Biomarkers and recent advances in the management and therapy of sickle cell disease

[version 1; peer review: 2 approved]
PUBLISHED 12 Oct 2015
Author details Author details
OPEN PEER REVIEW
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Abstract

Although production of hemoglobin S, the genetic defect that causes sickle cell disease (SCD), directly affects only red blood cells, the manifestations of SCD are pervasive, and almost every cell type and organ system in the body can be involved. Today, the vast majority of patients with SCD who receive modern health care reach adulthood thanks to vaccine prophylaxis and improvements in supportive care, including transfusion. However, once patients reach adulthood, they commonly experience recurrent painful vaso-occlusive crises and frequently have widespread end-organ damage and severely shortened life expectancies. Over the last several decades, research has elucidated many of the mechanisms whereby abnormal red blood cells produce such ubiquitous organ damage. With these discoveries have come new ways to measure disease activity. In addition, new pharmaceutical interventions are now being developed to address what has been learned about disease mechanisms.

Keywords

sickle cell, anemia, biomarkers, red blood cells

Introduction and context

Although it has long been straightforward to define sickle cell disease (SCD) and its subtypes through biochemical and genetic analyses of hemoglobin and its encoding genes, understanding the pathophysiologic mechanisms leading to the disease’s protean manifestations has been more challenging. After decades of research, a great deal has been learned about the many pathways and processes affected downstream by the hemoglobin S (HbS) mutation, and, finally, new therapeutic approaches targeting these mechanisms are being developed. Nevertheless, a lack of even a basic ability to document and follow the processes leading to vaso-occlusive episodes persists. At this time, both the diagnosis of vaso-occlusion and the definition of its resolution rely exclusively on patient reports of pain.

The difficulty in obtaining objectively measurable biomarkers of disease activity and variability hampers many aspects of the management of SCD, including (1) diagnosis of acute vaso-occlusion, acute chest syndrome, and other sequelae of SCD; (2) understanding of the inciting events leading to vaso-occlusion and its complications in SCD; (3) personalized medicine through identification of particularly suitable or unsuitable candidates or events for targeted disease therapies; and (4) documentation of new drug effects and mechanisms of action.

Despite the fact that the abnormal hemoglobin that defines SCD is expressed only in erythrocytes, all blood cells as well as soluble blood elements and most other organ systems are affected by SCD. Patients with SCD have, at baseline, elevated leukocyte and platelet counts, abnormally increased leukocyte and platelet activation, abnormal activation of coagulation pathways, increased expression of multiple inflammatory markers, increased expression of soluble markers of endothelial activation and injury, and increased markers associated with a broad range of end-organ damage.

Many such “biomarkers” have been studied either in “steady-state” SCD or during acute vaso-occlusive episodes, and some have been shown to correlate with long-term survival. In addition, because vaso-occlusion may arise from a variety of inciting events (e.g., infection and physiologic stress), the mechanisms of vaso-occlusion may be variable among patients and vaso-occlusive events. Identification of biomarkers specific for processes contributing to vaso-occlusion could help determine which drugs might be most beneficial and might also elucidate the mechanism of action of new therapeutic agents. Biomarkers might also help determine when new therapeutic agents might be useful as prophylactic therapy to prevent progressive end-organ damage.

The past few decades, during which both mechanisms of vaso-occlusion and biomarkers have been identified, have also brought the dawn of targeted therapies for vaso-occlusion. Although none has yet to be proven useful or become US Food and Drug Administration (FDA)-approved since approval of hydroxyurea (HU) for the prevention of vaso-occlusion and acute chest syndrome, several promising targeted agents are in various stages of clinical trials. Many such studies are also examining biomarkers, as they are affected by both vaso-occlusion and the new potential therapeutic agents. Thus, it is reasonable to expect that progress in therapeutics and in the discovery of useful biomarkers will go hand-in-hand in the future, leading potentially to both targeted and personalized therapy for SCD.

Pathophysiology and biomarkers

Clinical measures and overall prognosis

Early studies of the natural history of SCD, largely in children and young adults, identified several markers of disease severity and poorer overall survival1,2. Frequency of vaso-occlusive episodes was a marker of poorer survival in patients with sickle cell anemia (homozygous for HbS) who were more than 20 years old2. High rates of vaso-occlusive episodes were also associated with higher hematocrit and lower fetal hemoglobin levels in that study. A second study of largely the same patient population further revealed that acute chest syndrome, renal failure, seizures, a high baseline white cell count (>15,000 cells/mm2), and low fetal hemoglobin were associated with an increased risk of early death; early mortality was again shown to be more frequent among the most symptomatic patients1. Finally, a more recent analysis of the cohort enrolled as newborns in the same study showed that more pronounced reticulocytosis increased the risk of stroke and mortality during childhood3.

More recent studies of factors associated with mortality in SCD have presumably reflected the era of HU therapy, and some have studied factors related to survival in different resource settings. Recent studies46 of adults have shown that more frequent episodes of vaso-occlusion remain a marker of increased mortality4,5, and sickle nephropathy is also a significant risk factor46. In addition, the presence of an elevated tricuspid regurgitant jet velocity (TRV) (≥2.5 m/sec), with or without catheterization-proven pulmonary arterial hypertension, is a very significant risk factor for accelerated mortality48. A history of cumulative end-organ damage and stroke was also associated with earlier mortality5.

Interest in pulmonary hypertension and SCD has also spurred investigation of markers thought to be related to endothelial damage and inflammation. Of these, vascular cell adhesion molecule 1 (VCAM-1) levels have been reproducibly associated with survival5,9.

Hemoglobin F (HbF) levels are often measured clinically, and several studies have supported the hypothesis that higher HbF levels lead to less severe SCD. HbF has higher oxygen affinity than HbA or HbS, and sickle red cells containing more HbF survive longer in the circulation10. Moreover, elevation of HbF is a key (though not the only) factor in the salutary effects of HU on SCD severity11.

Adhesion

Sickle red cells are abnormally adherent to many substrates, including endothelial cells12, leukocytes13, platelets14, and extracellular matrix molecules such as laminin13,15 and thrombospondin16,17. Hebbel and colleagues found evidence decades ago that patients with more adherent cells were more likely to suffer vaso-occlusive episodes18. However, while such adhesion almost certainly contributes to vaso-occlusive pathogenesis, measuring adhesion either ex vivo or in vivo remains difficult. Thus, assays of cell adhesion are often used for research purposes but have not been extensively explored as markers of disease.

Nevertheless, one measurable outcome of adhesion is the formation of circulating heterocellular aggregates that can be measured by flow cytometry, now a part of most clinical hospital laboratories. Sickle red cells, as well as leukocytes from patients with SCD, can be found in circulating aggregates involving both each other as well as platelets14,19. How such measures related to clinical status and outcomes in SCD remains to be better defined.

Inflammation

SCD is accompanied by a broad array of inflammatory processes. At steady state, in the absence of symptomatic vaso-occlusion, patients with SCD have increased numbers of activated leukocytes20, activated platelets, and formation of multicellular aggregates.

In addition, patients with SCD may have elevations of multiple inflammatory cytokines (Table 1), both in steady state as well as during vaso-occlusive events. Although not all studies demonstrate concordant findings, among the cytokines consistently found to be both elevated at steady state and then further elevated during vaso-occlusive events are interleukin-10 (IL-10), macrophage inflammatory protein 1α (MIP-1α), placenta growth factor (PlGF), prostaglandin E2 (PGE2), and soluble CD40 ligand (sCD40L). Current investigations are focusing on how these cytokines can contribute to the pathophysiology of vaso-occlusion.

Table 1. Expression of cytokines, acute-phase proteins, and inflammatory molecules in sickle cell disease.

Cytokine or moleculeExpression in steady-state
sickle cell disease
Expression during vaso-
occlusion
References
C-reactive proteinIncreasedCan be further increased102,103
Interferon-γNormal or increasedNot further increased compared
with steady state
104,105
Interleukin-1βNormal or increasedNormal or increased104107
Interleukin-2NormalNormal105
Interleukin-4IncreasedNot further increased compared
with steady state
105
Interleukin-6IncreasedFurther increased102,104,105,107
Interleukin-8IncreasedVariably further increased
compared with steady state
104,105,107109
Interleukin-10IncreasedFurther increased104,107
Monocyte chemoattractrant
protein-1 (MCP-1)
Variable102,104
Macrophage inflammatory
protein 1α (MIP-1α)
IncreasedFurther increased104
Neuropeptide substance P (SP)IncreasedFurther increased108
Pentraxin-3 (PTX3)NormalIncreased50
Placental growth factor (PlGF)IncreasedFurther increased110113
Prostaglandin E2 (PGE2)IncreasedFurther increased107,109,114
Soluble CD40LIncreasedFurther increased91
ThrombospondinIncreasedFurther increased111
Tumor necrosis factor-αNormal or increasedNormal or increased102,104107
TNFSF14 (LIGHT)Increased115

Coagulation

Coagulation pathways are broadly activated in patients with SCD21. Thus, SCD is considered a “hypercoagulable state”, and indeed there is a higher prevalence of pregnancy-related thrombosis and pulmonary emboli in patients with SCD than in age-matched African-American controls22,23. Clinically, levels of D-dimer are often chronically elevated and increase further during vaso-occlusive events24,25. Other biomarkers of activated coagulation, such as plasma levels of prothrombin fragment 1.2 (F1.2), thrombin-antithrombin (TAT) complexes, plasmin-antiplasmin complexes, and fibrinopeptide A, are also elevated in SCD. There is at least some evidence that the degree of elevation of D-dimer levels is predictive of the frequency of vaso-occlusive episodes26. Furthermore, the hypothesis that abnormal SCD red cells, and specifically those with increased phosphatidylserine (PS) exposure at their surfaces, are responsible for activation of coagulation is supported by the demonstration that the number of PS-positive sickle red cells is related to the degree of elevation of D-dimer, F1.2, and plasmin-antiplasmin complex levels27,28.

Investigators have also shown that there are elevated levels of tissue factor in the circulation in SCD2931 and that platelets are also activated in greater numbers32,33. Blood from patients with SCD also contains increased levels of microparticles derived from multiple cell types, including red cells, platelets, leukocytes, and endothelial cells. Tissue factor-expressing microparticles appear to be derived primarily from monocytes and endothelial cells34. Finally, recent evidence suggests that free plasma iron may also contribute to activated coagulation in SCD35.

Oxidant damage

Oxidant damage appears to occur at an accelerated rate in SCD, both within the red cell as well as in other tissues. Hemolysis results in the release of hemoglobin, which itself is a powerful oxidant. In addition, several investigators have reported reduced anti-oxidant compounds both within sickle red cells as well as in plasma. Plasma lipid peroxidation is higher in SCD patients than controls, and red cell content of glutathione reductase and superoxide dismutase is lower in sickle erythrocytes36. Depletion of glutathione has been associated with elevated TRV, itself a biomarker for pulmonary hypertension and early mortality37. Finally, some evidence suggests that reduction in expression of genes responsible for the synthesis of anti-oxidant compounds may also contribute to worsening anemia in SCD38. HU has been found to reduce markers of oxidative stress in SCD39,40.

Biomarkers and stroke

Stroke is a common and potentially devastating problem in SCD, and strokes start to occur in very young children41. Once a stroke occurs, patients are at great risk for recurrent strokes, which can be largely prevented by chronic transfusion but have a high frequency of recurrence without continuing transfusion42. Transcranial Doppler (TCD) measurements have been known to provide a good measurement of the risk of stroke in children with SCD since 1992, when children with abnormal TCDs were shown to be at 44 times the risk for stroke than children with normal TCDs43. The Stroke Prevention Trial in Sickle Cell Anemia (STOP) showed that regular transfusion could reduce the risk of a first stroke by 92%, compared with non-transfused children with abnormal TCDs, who had about a 10% incidence of stroke annually44. However, a subsequent study of children who had already undergone at least 30 months of transfusion for abnormal TCDs failed to show that such transfusion could be safely stopped45. Thus, while TCDs are now standard of practice in pediatric care of SCD, in order to identify children at risk for primary strokes, they do not allow avoidance of transfusion for the relatively large number of children who would not ultimately have a stroke, despite their abnormal TCDs. Efforts to better define the at-risk population, such as through identification of genetic risk factors or other biomarkers, have not yet defined a solution to this problem.

Biomarkers of acute chest syndrome

Acute chest syndrome (ACS) is one of the most feared complications of vaso-occlusive episodes and is highly associated with mortality. Secretory phospholipase A(2) (sPLA(2)) levels become quite elevated in about 80% of patients with ACS46,47 and often become quite elevated shortly before patients become symptomatic47,48. Thrombospondin-1 levels have also been reported to become markedly increased in ACS49, as have levels of pentraxin-350. C-reactive protein has been reported to parallel sPLA(2) in the context of vaso-occlusion and ACS51. At least one study has attempted to prevent ACS with transfusion in patients with high sPLA(2) levels52, but a definitive study has not been conducted.

Biomarkers and pulmonary hypertension

Pulmonary hypertension has become recognized as a major risk factor for death in adults with HbSS and HbSβ0 thalassemia8. However, the most widely available screening test for pulmonary hypertension—echocardiography—does not reliably reflect pulmonary arterial pressures, as measured by right heart catheterization. In fact, right heart catheterization may confirm pulmonary hypertension in only 10% to 25% of patients with elevated TRV53,54, giving echocardiography a positive predictive value of only 25% to 32%55. Nevertheless, a TRV of over 2.5 to 3 m/sec has been an indicator of risk for mortality in several studies6,7,56 despite its lack of reliability as an indicator of catheterization-measurable pulmonary arterial hypertension6. Thus, the recent National Institutes of Health guidelines for sickle cell anemia management did not recommend for or against routine echocardiographic screening for pulmonary hypertension in asymptomatic adults with SCD57. In addition, several studies have shown that elevated pro-brain natriuretic peptide (pro-BNP) is associated with a high risk of mortality, especially in combination with a TRV of at least 3 m/sec6. VCAM-1, a marker of endothelial activation and damage, is also present at higher levels in SCD patients with pulmonary hypertension58. VCAM-1 is also reproducibly associated with poorer survival5,9.

Biomarkers and therapeutic approaches

Although the biomarkers discussed above do not represent the totality of biomarkers explored and found to be possibly informative in the context of SCD, they do point to the panoply of pathophysiologic mechanisms now appreciated as active in SCD. Adhesion, inflammation, coagulation, and oxidative damage are likely the most important, though not the only, contributors to the development of vaso-occlusion and organ damage in this disease. Moreover, we do not completely understand how the processes implicated by the evidence of biomarkers produce the varied physiologic events we observe. For example, SCD involves both large-vessel events (e.g., strokes) as well as occlusion of microvascular structures, which is believed to be involved in typical painful vaso-occlusive events. Nevertheless, current efforts to develop new therapies for SCD are concentrating on the processes reflected by the biomarkers discussed above. In the future, biomarkers may assist us in personalizing treatment according to the predominant mechanisms involved in a particular disease sequela or event.

New and targeted drugs in development

Since SCD was recognized and defined in the early 20th century, survival and quality of life have until recently improved primarily because of advances in supportive care, including penicillin prophylaxis, routine immunizations, transfusion for stroke and acute chest syndrome, and hydration and narcotic therapy for vaso-occlusive episodes. This changed after the Multicenter Study of Hydroxyurea59, which showed that HU reduced the frequency of both vaso-occlusive episodes and acute chest syndrome while increasing both total hemoglobin levels and hemoglobin F percentages and decreasing neutrophil counts. However, the study was not designed to, and did not, determine the mechanism whereby HU reduced vaso-occlusive and acute chest syndrome events. In this setting, the FDA approved the drug for use in adults with SCD in 1998. Subsequent studies have confirmed that the drug is cost-effective60 and improves long-term survival6163. The drug has also proven to be safe and effective in children64,65. Nevertheless, the consensus remains that the drug is underutilized in both children and adults (http://consensus.nih.gov/2008/sicklecellstatement.htm). One recent study suggested that, outside a center dedicated to treating SCD, only about 25% of patients meeting the original study criteria actually receive HU66. Moreover, the mechanisms whereby HU has its beneficial effects remain only partly understood.

Not only have new therapies for SCD not arrived since HU gained FDA approval in 1998, but truly curative therapies have been difficult to achieve (Table 2). Hematopoietic stem cell transplantation and gene therapy offer the best chances for cure but each presents numerous challenges that have been difficult to overcome. Although there were initial successes in young pediatric patients, successful hematopoietic stem cell transplantation with good survival and tolerable graft-versus-host disease in adults proved much harder to achieve. Nevertheless, such transplants can now be confidently undertaken6769, and many ongoing clinical trials are looking at ways to improve both engraftment and the availability of donors.

Table 2. New therapeutic approaches under investigation or recently investigated for sickle cell disease.

Classification and agentRationale/GoalClinicalTrials.gov registration
Hematopoietic stem cell
transplantation
Replace stem cells leading to production of HbS with cells
that produce either normal or anti-sickling Hb (such as HbF)
00408447, 01461837, 00977691,
01877837, 02065596, and 00152113
Gene transfer or correctionReplace or correct the HbS gene with a gene encoding either
normal or anti-sickling Hb (such as HbF)
02186418, 02140554, and 02151526
Nutritional supplements
     •   L-glutamineLower energy requirements to improve growth and strength01179217, 00131508, 00586209,
and 00125788
     •   NiacinImprove blood flow00508989
     •   Vitamin DCorrect chronic vitamin D deficiency common in children with
SCD
01276587
     •   Alpha-lipoic acid and
acetyl-L-carnitine
Anti-oxidant01054768
     •   ArginineImprove availability of NO00513617
Anti-inflammatory
     •   RegadenosonDownregulate the activity of iNKT cells01566890 and 01085201
     •   NKTT120Deplete iNKT cells01783691
     •   ZileutonReduce inflammation01136941
     •   MontelukastReduce soluble vascular cell adhesion molecule-1 (sVCAM-1)01960413
     •   IVIgShorten VOC01757418
Anti-adhesive
     •   PropranololReduce activation of red cell adhesion receptors activated via
beta adrenergic receptor signaling pathways
01077921 and 02012777
     •   SelG1Block adhesion via P-selectin to prevent VOC episodes01895361
     •   PF-04447943Phosphodiesterase-9 inhibitor02114203
     •   RivipanselReduce adhesion and inflammation dependence on E-selectin01119833 and 02187003
Anti-sickling
     •   MP4COReduce sickling by delivering CO01356485
     •   SCD-101Reduce sickling02380079
     •   Sanguinate (PEG-bHb-CO)Reduce sickling by delivering CO01848925
     •   AES-103Reduce sickling01987908
Alter hemoglobin expression
     •   VorinostatIncrease HbF01000155
     •   HU with Mg PidolateIncrease both HbF and cell hydration00143572
     •   HQK-1001Increase HbF01601340
     •   PanibostatIncrease HbF01245179
     •   DecitabineIncrease HbF01375608 and 01685515
     •   PomalidomideIncrease HbF0522547
Anti-coagulant and anti-platelet
     •   TicagrelorDecrease vaso-occlusive episodes and pain02214121
     •   PrasugrelDecrease vaso-occlusive episodes and pain01167023, 01794000, and 01476696
     •   N-acetyl cysteineDecrease VWF levels, VWF total activity, ULVWF multimers,
and VWF functions
01800526
     •   AspirinDiminish the incidence and progression of cognitive deficits
as well as occurrence of overt and silent stroke
00178464
     •   EptifibatideReduce platelet activation and release of pro-inflammatory
cytokines
00834899
Other
     •   Inhaled NOImprove pulmonary hypertension00023296
     •   ICA-17043Reduce hemolysis and VOC by increased cell hydration00040677
     •   Intravenous NOVaso-dilation00095472
     •   LosartanImprove SCD nephropathy02373241
     •   6R-BH4 (sapropterin
dihydrochloride)
Improve endothelial dysfunction00445978, 00403494, and 00532844
     •   Magnesium sulfateReduce length of stay and pain in children with acute VOC00313963
     •   Varespladib (A-001)Decrease acute chest syndrome01522196 and 00434473
     •   AtorvastatinImprove endothelial dysfunction00072826
     •   SimvastatinImprove endothelial dysfunction00508027
     •   BosentanImprove pulmonary hypertension00313196, 00360087, and 00310830
     •   ClotrimazoleReduce hemolysis and anemia by blocking Gardos channel00004404 and 00004492

CO, carbon monoxide; Hb, hemoglobin; HbF, hemoglobin F; HbS, hemoglobin S; HU, hydroxyurea; iNKT, invariant natural killer T; NO, nitric oxide; SCD, sickle cell disease; ULVWF, ultra-large von Willebrand factor; VOC, vaso-occlusive crisis; VWF, von Willebrand factor.

Gene therapy is an even more challenging but ultimately less toxic approach to achieving curative therapy for SCD. The general approach has been to develop methods for inserting into autologous hematopoietic stem cells either a gene encoding normal β-globin or a globin chain with anti-sickling properties, such as γ-globin (that leads to production of HbF). Another approach that has been explored involves methodologies for “correcting” the faulty β-globin gene. These approaches, however, either are still in development or are in very early clinical trials.

Inhibitors of cell activation and adhesion

One of the most attractive therapeutic targets in SCD is cell adhesion. Although SCD severity was first linked to the degree of red cell adhesion exhibited by patients’ red cells, it is now apparent that leukocyte and platelet activation and adhesion also contribute to pathophysiology. Therefore, cell adhesion has become a primary target for the development of new therapeutic approaches. In general, such approaches may involve inhibition of cell-cell interactions generally, specific inhibition of adhesion receptors, or interference with the signaling mechanisms that lead to activation of adhesion receptors.

Non-specific inhibition of cell adhesion by poloxamer-188 was studied in a multicenter randomized phase III trial involving both children and adults to determine its effect on duration of painful vaso-occlusive episodes. Although the results were statistically significant, differences in time to resolution of painful episodes were small but were slightly greater in children70. Another phase III study of this drug, using a somewhat different study design, is now under way (NCT01737814, ClinicalTrials.gov).

Another anti-adhesion therapeutic has been developed to address adhesive interactions involving primarily E-selectin. This drug (GMI-1070, now known as rivipansel) was quite successful in abrogating vaso-occlusion in sickle mice71. In a subsequent phase I study of SCD patients in steady state, rivipansel was well tolerated and appeared to improve blood flow in a subset of patients. Perhaps most interesting, however, was the drug’s effect on biomarkers: the drug was associated with significant decreases in biomarkers of endothelial activation, including sE-selectin, sP-selectin, and soluble intercellular adhesion molecule-1 (sICAM). Markers of leukocyte activation and coagulation were also decreased72. A phase II study of the drug in patients experiencing painful vaso-occlusive episodes showed large and consistent decreases in all measures of time to crisis resolution, although these were not statistically significant. Moreover, opiate usage was markedly and statistically significantly decreased with drug versus placebo73. A phase III study is expected to be under way shortly (NCT02187003, ClinicalTrials.gov).

Other drugs, both new and old, also have potential anti-adhesive effects that could be useful in SCD. P-selectin is known to contribute to adhesion of both sickle red cells and leukocytes to endothelial cells7476, and heparins have both anti-P-selectin as well as anticoagulation effects75. In fact, heparin blocks P-selectin-mediated adhesion at levels considerably lower than those needed for anticoagulation77. A small phase 2 study of pentosan polysulfate sodium has shown promising results, in that a single oral dose improved microvascular blood flow, and repeated daily doses were associated with decreased plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1)77. Another compound chemically related to low-molecular-weight heparin (LMWH), sevuparin (Dilaforette), has shown promising results in vitro and in vivo in a mouse model of vaso-occlusion78, and plans are under way to study the drug in SCD. Another P-selectin-targeted drug, SelG1 (Selexys Pharmaceuticals), is currently in clinical trial for use as a prophylactic agent to prevent vaso-occlusive crises (NCT01895361, ClinicalTrials.gov).

Finally, several studies in animals and patients have addressed the possibility that downregulation of signaling pathways may decrease cell adhesion. Several red cell adhesion receptors, including the BCAM/Lu receptor for laminin79 and the ICAM-4 receptor for integrins80,81, are activated downstream of β-adrenergic receptor signaling pathways. Animal studies and a phase 1 trial of propranolol showed that propranolol decreased sickle red cell adhesion measured in vitro and decreased vaso-occlusion in mice in vivo81,82. In addition, the ERK signaling pathway appears to be involved in sickle red cell adhesion83,84, and the ability to affect this pathway via MEK inhibition is now being explored85.

Anticoagulants

Given the abundant data that coagulation pathways are abnormally activated in SCD, early studies explored the possibility that anticoagulation might have a beneficial effect in SCD, but most of those studies were too small or time-limited to be definitive. Using acenocoumarol, one study showed that achieving an international normalized ratio (INR) of 1.64 (range of 1.18–2.2) was associated with normalization of the F1 + 2 level and therefore concluded that low-intensity oral anticoagulation could normalize the hypercoagulability in SCD86. Newer studies have again approached the potential usefulness of anticoagulation in SCD. A randomized double-blind clinical trial of an LMWH, tinzaparin, versus placebo was conducted during the management of acute painful vaso-occlusive episodes. This 253-patient study administered tinzaparin subcutaneously at 175 IU/kg once daily, along with usual supportive care and analgesia. Although the endpoints and criteria for discharge were different from those usually used in the United States and other Western countries, analysis demonstrated a statistically significant reduction in several measures of time to resolution87. Another double-blind prospective study randomized SCD patients hospitalized for pain episodes to receive prophylactic LMWH (dalteparin 5,000 IU subcutaneously daily) or placebo. Although this study did not meet its target enrollment, the group receiving dalteparin had a greater decrease in pain scores at day 3 than did the placebo group (NCT01419977, ClinicalTrials.gov), although these results are unpublished to date. Another study used low-dose warfarin during vaso-occlusive crisis and studied D-dimer levels as their primary endpoint. They found that patients on warfarin had significantly lower D-dimer levels than patients not receiving the drug88; however, effects on clinical endpoints, such as time to resolution of painful episode, were not reported. Finally, another study of acenocoumarol showed that treatment to INR values of 1.6 to 2 failed to lower the plasma levels of endothelial activation markers in SCD89, raising questions about the likely clinical utility of anticoagulation to prevent SCD-related vascular events. Nevertheless, studies of the newer direct factor X inhibitors (apixaban and rivaroxaban) are currently planned or ongoing (NCT02179177 and NCT02072668, respectively, ClinicalTrials.gov). In addition, a study is under way to determine the feasibility of performing a larger multicenter phase III trial to assess the effects of unfractionated heparin in acute chest syndrome (NCT02098993, ClinicalTrials.gov).

Anti-platelet agents have also received attention and continue to be studied in the context of SCD. Eptifibatide is an anti-platelet agent that binds to the αIIbβIII integrin on platelets and decreased in platelet aggregation and sCD40L levels in patients with SCD in a phase I study90. sCD40L is a pro-inflammatory cytokine released by platelets and chronically elevated in SCD plasma91. However, use of eptifibatide in a small pilot study showed that eptifibatide did not improve the times to crisis resolution or hospital discharge92. Another anti-platelet agent being studied in SCD is prasugrel. Recently, a multicenter phase 2 study of prasugrel versus placebo in adult patients with SCD showed that the drug could be safely used, and although it did not achieve statistically significant reductions in pain scores, it did reduce both platelet surface P-selectin and plasma soluble P-selectin levels, compared with placebo93.

Anti-inflammatory agents

Inflammatory pathways in SCD are both the result of red and white cell adhesion to endothelial cells and to each other as well as promoters of such adhesion. In addition, transient vaso-occlusion leads to hypoxia/reperfusion injury with a robust inflammatory component. Therefore, targeting inflammatory pathways is a rational approach to treating or trying to prevent vaso-occlusion in SCD. Corticosteroids have been investigated but with mixed results. Newer therapies are now addressing the role of invariant natural killer T (iNKT) cells, which are known to play an important role in ischemia/reperfusion injury and which are increased in both number and activity in SCD94. In sickle mice, inhibition of iNKT cell activity with regadenoson, an adenosine A2A receptor agonist, led to a reduction in pulmonary inflammation and injury94. A phase 1 study in patients also showed promising results, including reduction in phospho-NF-kappa-B p65 activation in iNKT cells, compared with pretreatment baseline during vaso-occlusion95. The phase 2 study of regadenoson for treatment of vaso-occlusion is ongoing (NCT01788631, ClinicalTrials.gov). Another drug targeting iNKT cells is NKTT 120, a humanized monoclonal antibody against iNKT cells. After an initial study indicating safety, further ascending-dose phase 1 studies are being conducted to evaluate the safety, pharmacokinetics, pharmacodynamics, and biologic activity of the drug (NCT01783691, ClinicalTrials.gov).

Other therapeutic approaches

Although the salutary effect of HU on SCD may be due to a more complex mechanism than its ability to raise HbF levels in about 50% of patients, it is also clear that not everyone responds to HU either clinically or with appreciably higher HbF levels. Therefore, other ways to increase HbF continue to be explored. Early studies showed that decitabine could substantially increase HbF levels in HU-non-responders96 and that such increased HbF levels were sustainable with repeated intermittent treatment97. Decitabine continues to be studied, both alone (NCT01375608, ClinicalTrials.gov) and in combination with tetrahydrouridine, a competitive inhibitor of cytidine deaminase that is being studied in an effort to improve oral bioavailability of decitabine98 (NCT01685515, ClinicalTrials.gov). Other agents that are being studied for their effects on HbF levels include panobinostat, vorinostat, pomalidomide, arginine butyrate, and HQK-1001 (2,2-dimethylbutyrate)99101. However, in a phase 2 study, HQK-1001 was associated with only a modest HbF response and a paradoxical increase in vaso-occlusive episodes101.

Implications for the future

The complexity of the pathogenesis of SCD has resulted in a plethora of potential druggable targets in our effort to ameliorate the disease’s sequelae. However, this same complexity has also prevented us from knowing which targets are optimal ones. Biomarkers may be helpful in patient selection for both research studies or therapy, but proof that changes in biomarkers are associated with clinical improvement remains elusive in most instances. Therefore, until we achieve wide availability of curative therapies through either hematopoietic stem cell transplantation or gene therapy, we need to continue our search for therapies that can be provided either to prevent or treat acute disease manifestations, such as vaso-occlusive pain, and chronic organ damage, such as sickle cell nephropathy and pulmonary hypertension. These goals, however, are still not quite within reach.

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Telen MJ. Biomarkers and recent advances in the management and therapy of sickle cell disease [version 1; peer review: 2 approved]. F1000Research 2015, 4(F1000 Faculty Rev):1050 (https://doi.org/10.12688/f1000research.6615.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 12 Oct 2015
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22
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Reviewer Report 12 Oct 2015
Emily Meier, Children's National Medical Center, District of Columbia, Washington, 20010, USA 
Approved
VIEWS 22
I confirm that I have read this submission and believe that I have an ... Continue reading
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HOW TO CITE THIS REPORT
Meier E. Reviewer Report For: Biomarkers and recent advances in the management and therapy of sickle cell disease [version 1; peer review: 2 approved]. F1000Research 2015, 4(F1000 Faculty Rev):1050 (https://doi.org/10.5256/f1000research.7103.r10775)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Views
24
Cite
Reviewer Report 12 Oct 2015
James G. Taylor VI, Genomic Medicine Section, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, 20892-1476, USA 
Approved
VIEWS 24
I confirm that I have read this submission and believe that I have an ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Taylor VI JG. Reviewer Report For: Biomarkers and recent advances in the management and therapy of sickle cell disease [version 1; peer review: 2 approved]. F1000Research 2015, 4(F1000 Faculty Rev):1050 (https://doi.org/10.5256/f1000research.7103.r10774)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Comments on this article Comments (0)

Version 1
VERSION 1 PUBLISHED 12 Oct 2015
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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