The Role of Adenosine Signaling in Sickle Cell Therapeutics

Abstract

Recent data suggest a role for adenosine signaling in the pathogenesis of sickle cell disease (SCD). Signaling through the adenosine A_2A receptor (A_2AR) has demonstrated beneficial effects in SCD. Activation of A_2ARs decreases inflammation in mice and patients with SCD largely by blocking activation of invariant NKT cells. Decreased inflammation may reduce the severity of vaso-occlusive crises. In contrast, adenosine signaling through the A_2B receptor (A_2BR) may be detrimental for patients with SCD. Priapism and the formation of sickle erythrocytes may be a consequence of A_2BR activation on corpus cavernosal cells and erythrocytes, respectively. Whether adenosine signaling predominantly occurs through A_2ARs or A_2BRs may depend on differing levels of adenosine and disease state (steady state versus crisis). There may be opportunities to develop novel therapeutic approaches targeting A_2ARs and/or A_2BRs for patients with SCD.

Introduction

Sickle cell disease (SCD) is characterized by rigid, sickle-shaped erythrocytes, microvascular occlusion and tissue ischemia¹. Sickle erythrocytes initiate the development of vaso-occlusion that ultimately leads to tissue ischemia in a complex multi-cellular process. Tissue ischemia promotes an inflammatory response that is further amplified by ischemia re-perfusion injury (IRI) ². Thereafter, a vicious cycle of vaso-occlusion, tissue injury and inflammation is set into motion promoting further erythrocyte sickling^1,2. The consequences of vaso-occlusion are pain, end-organ damage and often premature death^3,4.

Therapies to prevent or treat sickle cell vaso-occlusion are limited. Broadly, treatments either prevent the formation of sickle erythrocytes (e.g., hydroxyurea) or interrupt the cellular interactions that follow red cell sickling and lead to vaso-occlusion⁵. Hydroxyurea is an anti-mitotic agent that disrupts the polymerization of sickle hemoglobin and, to date, is the only FDA-approved therapy for the prevention of painful vaso-occlusive crises (pVOC)⁶. Patient and provider barriers have limited the widespread use of hydroxyurea in patients with SCD, affecting the impact of the drug⁷. Hematopoietic stem cell transplantation is a potential cure; however, it is only an option for a few patients with SCD⁸.

In this review, we examine the role of adenosine signaling in SCD pathogenesis. There are opportunities to modulate adenosine pathways using therapies to prevent or treat SCD complications. As evidence has emerged about the importance of adenosine in SCD, separate lines of investigation have demonstrated protective and detrimental effects of adenosine in regards to disease severity. Recent data suggest that actions of adenosine mediated through the adenosine A_2A receptor (A_2AR) decrease inflammation, largely by selectively inhibiting the activation of a subset of lymphocytes called invariant NKT (iNKT) cells^9–11. In contrast, other studies have shown that adenosine signaling through the adenosine A_2B receptor (A_2BR) may contribute to the adverse processes of erythrocyte sickling¹² and priapism^13–15. Although much additional work is needed to fully elucidate the roles of adenosine signaling in SCD, targeting these pathways may produce novel therapeutic approaches.

Adenosine signaling pathway

Adenosine physiology

Adenosine signaling protects tissues by promoting vaso-dilation as well as decreasing heart rate and inflammation¹⁶. During periods of cellular hypoxia or stress, adenosine is released from cells along with the adenine nucleotides, ATP, ADP and AMP, which are converted to adenosine by ecto-nucleotidases. Binding of adenosine to four receptor subtypes, A₁, A_2A, A_2B, or A₃, elicits responses that are dependent upon the receptor subtypes found in various tissues (Table 1). Adenosine receptors are 7-transmembrane, G-coupled receptors that signal through adenylyl cyclase, affecting the production of cyclic AMP (cAMP), calcium, or the conductance of ion channels. A₁ and A₃ receptors couple to inhibitory G receptors (Gi) and decrease adenylyl cyclase activity, whereas A_2A and A_2B increase adenylyl cyclase activity by coupling to stimulatory G receptors (Gs or Go)¹⁶. The affinity for adenosine also differs among the receptor subtypes, affecting the concentration of adenosine necessary for activation. A₁ and A_2A are high affinity receptors, activating at lower concentrations of adenosine (~0.01 μM to 1 μM). A_2B is a low affinity receptor requiring 10 to 1000-fold higher levels of adenosine (~10 μM) for activation¹⁷. Downstream from adenylyl cyclase and cAMP, adenosine signaling modifies the activity of nuclear factor kappa-B (NF-κB), JAK-STAT and ERK pathways, regulating transcription and ultimately cellular functions¹⁸. Adenosine that accumulates during cellular stress is removed by uptake into cells and converted to AMP or inosine by adenosine kinase and adenosine deaminase (ADA), respectively. In patients with SCD, tissue injury may increase levels of plasma adenosine suggesting that adenosine pathways may influence SCD pathogenesis¹².

Table 1.

Adenosine receptor subtypes^17,32

Adenosine receptor subtypes
Characteristics	A1	A2A	A2B	A3
Predominant tissue/cell expression	Lung Heart Brain Leukocytes	Lung Brain Vasculature Leukocytes	Lung Colon Vasculature Leukocytes Erythrocytes Penis	Lung Liver Heart Brain Leukocytes
Actions	Negative chronotropic ↑inflammation	Vasodilation ↓inflammation	↑inflammation in lung and colon	↓inflammation
Affinity for adenosine	High	High	Low	High
Major disease associations	Sepsis	SCD Ischemia Arthritis Wound healing	SCD Asthma COPD IBD	Asthma Arthritis

Current therapeutic uses of adenosine and adenosine derivatives

Drugs that target adenosine receptors are part of current standard practice. Adenosine, dipyridamole and adenosine A_2AR agonists (e.g., regadenoson) are used clinically to induce cardiac hyperemia during myocardial stress testing via activation of coronary artery A_2ARs. Adenosine-mediated activation of A₁ receptors in the heart is a treatment for tachyarrhythmias. Theophylline, either alone or in complex with ethylenediame (aminophylline), non-selectively blocks A₁, A_2A and A_2B receptors and is used as a therapy for asthma¹⁶. A limitation of these therapies is lack of selectivity for the adenosine receptor subtypes, sometimes resulting in unwanted and potentially dangerous side effects. These include hypotension and tachycardia from A_2AR activation, bradycardia or heart-block from A₁ activation and bronchospasm from A_2BR activation in patients with asthma. Newer generation adenosine agonists and antagonists have greater receptor subtype selectivity thereby minimizing toxicities¹⁶. There is emerging cellular, animal and human data suggesting a role for the A_2A and A_2B receptors in the pathogenesis of SCD (Table 2). Adenosine-based therapies are currently being examined in patients with SCD (Clinicaltrials.gov #01788631).

Table 2.

Comparison of A_2AR and A_2BR data in sickle cell disease

Reference	Type of study	Transgenic SCD model	# SCD Patients	Agents evaluated	Key findings
A2AR
VOC
Wallace et al. Blood 201010	Investigation of transgenic SCD mice	NY1DD	-	A2AR agonist ATL146e	SCD causes induction of A2AR on iNKT cells A2AR agonist ATL146e reverses pulmonary dysfunction in SCD mice
Field et al. Blood 201311	Phase 1 clinical study	-	27	A2AR agonist regadenoson	A2AR agonist regadenoson decreases activation of iNKT cells in patients with SCD during VOC A2AR agonist regadenoson is safe in patients with SCD
Lin et al. PLosOne 201319	Investigation of transgenic SCD mice	NY1DD	8	-	SCD causes induction of A2AR on iNKT cells A2AR expression on iNKT cells is mediated through NF-κB
A2BR
Red blood cell sickling
Zhang et al. Nat Med 201112	Investigation of transgenic SCD mice, cultured human RBCs and SCD patient blood samples	Berkley	12	PEG-ADA, theophylline, A2BR antagonist MRS1754	Adenosine is elevated in the plasma of mice and patients with SCD A2BR on erythrocytes mediates sickling via decreasing hemoglobin oxygen binding affinity
Priapism
Mi et al. JCI 200815	Investigation of ADA deficient and transgenic SCD mice	Berkley	-	PEG-ADA, theophylline, A2BR antagonist MRS1706	Priapism is present in ADA−/− mice and corrected by PEG-ADA Interruption of A2BR activity on corpus cavernosal cells decreases priapism
Wen et al. J Sex Med 201014	Investigation of ADA deficient and transgenic SCD mice	Berkley	-	PEG-ADA	PEG-ADA therapy prevented episodes of priapism in SCD mice
Wen et al. FASEB 201013	Investigation of ADA deficient and transgenic SCD mice	Berkley	-	PEG-ADA, A2BR antagonist MRS1706	PEG-ADA therapy prevented penile fibrosis in SCD mice Interruption of A2BR in corpus cavernosal fibroblast cells prevents penile fibrosis

Role of A_2AR in sickle cell disease

A_2AR

A_2AR activation is well-known for producing vaso-dilation due to effects on vascular smooth muscle and some endothelial cells. In addition, A_2AR has a central role in the regulation of inflammation and immunity¹⁷. Ubiquitously expressed on neutrophils, monocytes, macrophages, T cells, NK cells and iNKT cells, adenosine signaling through the A_2AR has been shown to suppress key inflammatory and immune responses, including leukocyte activation, recruitment and cytokine production¹⁸. These immune suppressive effects of A_2AR activation are mediated by cAMP and protein kinase A (PKA)¹⁸. PKA signaling can inhibit other signaling pathways that activate inflammation mediated by NF-κB or the JAK-STAT pathway and serves to decrease transcription of key inflammatory genes¹⁹. The NF-κB pathway deserves special attention as it has been used as a marker of iNKT cell activity in clinical trials of the A_2AR agonist, regadenoson, in patients with SCD¹¹.

NF-κB is a critically important transcription factor that generally enhances inflammation²⁰. Comprised of a dimer of transcription factors from the RelA family of proteins (p50 or p52 and p65), NF-κB resides in the cytoplasm of cells bound to the inhibitory protein IκB. Upon activation of the NF-κB by numerous inflammatory mediators including tumor necrosis factor-α or interleukin-1, IκB is phosphorylated by IκB kinase, ubiquinated and degraded, thus liberating NF-κB to translocate into the nucleus and promote the transcription of pro-inflammatory genes. When NF-κB is released from IκB, the 65 kDa subunit (p65) can be phosphorylated on several sites including Ser536. The phosphorylation of p65 (phospho-p65) serves as a marker of NF-κB activity used in flow cytometry assays¹¹. In the case of A_2AR activation, in vitro data suggest that agonists of the A_2AR reduce IκB degradation, decreasing the ability of NF-κB to promote a pro-inflammatory cellular response²¹. NF-κB has also been shown to mediate the up-regulation of A_2AR in iNKT cells following activation¹⁹.

A_2AR agonist decreases inflammation following ischemia-reperfusion injury by interfering with iNKT cell activation

Murine models of liver and kidney transplant demonstrated that activation of A_2ARs by adenosine analogues administered during or after IRI markedly inhibit inflammation and secondary injury²². An investigation of the cell type primarily responsible for the protective effect of A_2AR activation implicated the iNKT cell as the primary target²². Although iNKT cells normally constitute < 1% of the lymphocyte population, iNKT cells can rapidly release large amounts of pro-inflammatory cytokines giving them a critical role in inflammation, despite representing a only small proportion of lymphocytes²³. Similar to B and T cells that produce adaptive immune responses, iNKT cell activation requires the engagement of an antigen presented on an antigen presenting cell²⁴. Unlike B and T cells, which undergo genetic recombination to generate diverse receptors that recognize various peptides, iNKT cells express a semi-invariant T cell receptor that non-specifically binds to lipid antigens presented on CD1d, a MHC class I-like molecule. Different lipids (glycolipids, phospholipids) have been shown to activate iNKT cells^24,25. The activation of iNKT cells is enhanced by cytokines produced by antigen presenting cells in response to Toll-like-receptor activation²⁶. Thus, the activation of iNKT cells is facilitated by innate immune responses stimulated by pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Upon CD1d-restricted activation, iNKT cells rapidly make mRNAs and release large quantities of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and interleukin-4 (IL-4)²⁷. IFN-γ stimulates the production in many cells of interferon-inducible CXCR3 chemokines, CXCL9, CXCL10 and CXCL11²⁸. IL-2 is known to induce CXCR3 receptors on lymphocytes²⁸. Thus, through rapid activation and generation of copious amounts of cytokines and chemokines, iNKT cells stimulate a pro-inflammatory cascade that may promote and sustain vaso-occlusion^9,10. Activation of A_2ARs, abundantly expressed on activated iNKT cells, reduces this inflammatory response and are critical to modulating the immune functions of iNKT cells^10,11.

A_2AR agonists decrease iNKT cell activation and reduce inflammation in SCD mice

In a series of experiments in an NY1DD mouse model of SCD, Linden and colleagues generated several lines of evidence implicating iNKT cells as critical to the process of sickle cell vaso-occlusion^9,10. Lung inflammation and injury were reduced when: 1) iNKT cells were antibody depleted or genetically knocked out, 2) activation or chemotaxis was inhibited and 3) upon administration of A_2AR agonists⁹. NY1DD mice treated with a continuous subcutaneous infusion of the A_2AR agonist ATL146e demonstrated a maximal improvement in lung function, histology and inflammatory cell infiltrate in 3 days at an infusion rate of 10 ng/kg/minute. The improvement was sustained up to the end of infusion at 7 days¹⁰. The infused dose of ATL146e only achieved plasma concentrations about 1 nM, and the mice did not demonstrate cardiovascular toxicities¹⁰. The absence of toxicity is in accord with prior studies demonstrating that the anti-inflammatory effects of A_2AR agonists occur at 10–100 fold lower concentrations compared to the cardiovascular effects²². Blockade or depletion of iNKT cells mitigated the beneficial effects of the A_2AR agonist, providing evidence that the anti-inflammatory actions of A_2AR activation are mediated largely through iNKT cells^9,10.

In the plasma of adult patients with SCD, circulating iNKT cells were also more likely to be activated and expanded compared to healthy controls⁹. There is selective expansion of iNKT cells among lymphocytes, from < 1% in control blood to an average of about 5% in the blood of SCD patients whose iNKT cells were also more like to express the activation markers CD69, intracellular IFN-γ, and CXCR3⁹.

Phase 1 study of the A_2AR agonist regadenoson in patients with SCD: study design and rationale

Based on the promising data from mice and patients with SCD suggesting that A_2AR agonists may interrupt activation of iNKT cells and potentially decrease sickle cell complications, a phase 1 trial was conducted of the A_2AR agonist regadenoson²⁹. FDA-approved for inducing cardiac hyperemia during myocardial imaging, regadenoson is a selective A_2AR agonist with 10-fold greater affinity for A_2A versus A₁ and few if any effects on A_2B or A₃ ^30,31. When used for myocardial imaging regadenoson is administered as a 400 μg bolus over 10 seconds³⁰. Bolus injection induces vaso-dilation and hyperemia in a time frame appropriate for capturing images before blood flow reverts to normal³¹. If the goal of administering regadenoson is to dampen the severity of a pVOC over several days, a continuous infusion would be necessary given its terminal half-life of 2 hours. When designing the study, three relatively low doses of regadenoson were selected based on data extrapolated from animal models²⁹. All of these doses produced the desired anti-inflammatory effects while avoiding cardiovascular toxicities²⁹. Using a traditional 3+3 study design, the dose levels were examined during a 12 hour infusion of regadenoson while patients with SCD were at steady state²⁹. Once the highest dose of infusional regadenoson (1.44 mcg/kg/hour) was found to be safe, SCD subjects were examined during a 24 or 48 hour infusion at steady state and then during a pVOC²⁹.

To evaluate the effects of regadenoson on iNKT cell activation, various activation markers were examined. Phosphorylation of the p65 subunit of NF-κB (phospho-p65 NF-κB) was identified as the most promising marker of iNKT cell activation because, as opposed to cell surface markers or cytokines, changes in phosphorylation are pre-transcriptional and thus occur quickly.

Phase 1 study of the A_2AR agonist regadenoson in patients with SCD: study results

Twenty-seven adult patients with SCD were administered regadenoson, 21 at steady state and 6 during pVOC¹¹. Circulating iNKT cells from adults with SCD during pVOC showed increased phospho-p65 NF-κB activation compared to steady state or healthy controls. When adults with SCD were administered a 24 hour infusion of the A_2AR agonist regadenoson during pVOC, the percentage of iNKT cells expressing increased phospho-p65 NF-κB decreased to levels similar to steady state patients and healthy controls. The effects of regadenoson were achieved at plasma concentrations that peaked at 2 ng/ml and were devoid of effects on heart rate or blood pressure. A large, randomized-controlled, phase 2 trial is in progress to evaluate the clinical efficacy of regadenoson during pVOC (Clinicaltrials.gov #01788631).

Role of A_2BR in sickle cell disease

A_2BR

The A_2BR is a lower affinity receptor that has well-described pro-inflammatory roles in the pathogenesis of asthma, chronic obstructive pulmonary disease and inflammatory bowel disease³². Higher levels of adenosine are necessary to activate the A_2BR (10–100 fold > A_2AR) and therefore signaling through A_2BR occurs selectively in stressed cells when adenosine is generated by ischemia, injury, or inflammation¹⁷. Although the distribution of the A_2BR is widespread on cells and tissues including smooth muscle cells, endothelial cells and macrophages, pathogenic inflammation is most notably promoted by the actions of the A_2BR on mast cells and intestinal epithelial cells³². Recent data suggest that activation of A_2BR on mast cells results in the production of cytokines with important roles in asthma pathogenesis, such as IL-4 and IL-13, and stimulates B cells to produce IgE³³. The role of A_2BR in asthma is also supported by data demonstrating increased inflammation in asthma patients after inhalation of adenosine^34,35. To this end, the non-selective adenosine receptor antagonist, theophylline, has a long-standing role in the management of asthma in part working through blockade of the A_2BR³⁶; although, its lack of selectivity is associated with side effects. A_2BR expression on intestinal epithelial cells is also important in disease pathogenesis promoting IL-6 production and resulting in intestinal inflammation, potentially contributing to the process of inflammatory bowel disease³⁷. More recently, A_2BR activation in the corpus cavernosum of the penis and erythrocytes has been shown to promote priapism^13–15 and red cell sickling¹², respectively, in a murine model of SCD.

Adenosine signaling through A_2BR is implicated in priapism and penile fibrosis

Studies in non-SCD mouse models demonstrating that intracavernosal injections of adenosine provoked priapism provided preliminary evidence for a role for adenosine signaling in the pathogenesis of priapism^38,39. More recent work has found that the actions of adenosine are mediated through A_2BR signaling on corpus cavernosum smooth muscle cells within the penis⁴⁰. In a series of experiments, investigators determined that ADA-deficient mice had higher intra-penile adenosine levels leading to cavernosal smooth muscle relaxation and priapism¹⁵. Administration of ADA or the A_2BR antagonists, theophylline or MRS1706, antagonized the effects of excess adenosine and reversed priapism^14,15. ADA^−/−/A_2BR^−/− mice were also protected from priapic episodes¹⁵. Many of the same findings were recapitulated in a transgenic SCD mouse model. SCD mice had higher plasma levels of adenosine than controls and similarly their episodes of priapism were counteracted by decreasing adenosine levels with ADA or administration of an A_2BR antagonist¹⁵. In SCD mice, there is also evidence that adenosine signaling through the A_2BR contributes to penile fibrosis, a consequence of priapism episodes that contributes to erectile dysfunction¹³. Taken together, these data suggest that interruption of adenosine signaling through A_2BR may have a role in the treatment of priapism and prevention of penile fibrosis in SCD.

Sickle erythrocyte formation promoted through A_2BR

Recently, another detrimental effect of A_2BR activation was described when Zhang and colleagues reported a novel mechanism of erythrocyte sickling in transgenic SCD mice caused by high plasma adenosine and activation of A_2BRs expressed on red cells¹². These investigators first discovered that in transgenic SCD mice a reduction in circulating adenosine levels following pegylated (PEG)-ADA administration was associated with a decreased number of sickle erythrocytes. To identify the adenosine receptor subtype responsible for promoting sickle cell formation, red cells from mice genetically deficient in one of the four receptor subtypes were activated with an adenosine analogue. The deleterious effects of adenosine were found to be mediated through the A_2BR due to an increase in intra-erythrocyte 2,3-DPG levels and a decreased hemoglobin-oxygen affinity. The resulting increased formation of deoxy-hemoglobin provides an explanation for the increased erythrocyte sickling. Translating these findings to humans, levels of plasma adenosine were found to be higher in patients with SCD versus healthy controls as were intra-erythrocyte 2,3-DPG levels (the latter more likely due to the effects of anemia). In vitro treatment of erythrocytes from patients with SCD with PEG-ADA or A_2BR antagonists reduced red cell sickling, consistent with the findings from the murine model.

Can adenosine have both protective and deleterious roles in SCD?

Recent data describing the role of adenosine signaling in SCD suggest discrepant effects on morbidity when the actions of adenosine are medicated through the A_2AR versus A_2BR⁴¹. On one hand, data show that activation of A_2AR decreases inflammation, largely through the inhibition of iNKT cell activation, potentially dampening the severity of pVOC^10,11. On the other hand, a separate line of research suggests that adenosine signaling through A_2BR promotes priapism^13–15 and erythrocyte sickling¹². Although further studies are needed to fully understand the seemingly conflicting roles of adenosine signaling in SCD, differences in adenosine receptor expression and affinity along with a better understanding of in vivo levels of adenosine in patients with SCD may help to reconcile the confusion.

Effects of adenosine levels and receptor density on A_2AR versus A_2BR signaling in SCD

Under steady-state conditions and potentially during pVOC, levels of adenosine may be sufficient to signal through the A_2AR, yet not high enough to trigger A_2BR signaling. In comparison to A_2BR, affinity of adenosine for A_2AR is 10 to 1000 times greater than A_2BR¹⁷. Moreover, the effects of adenosine are further potentiated in cells or tissue densely expressing adenosine receptors, as is the case for A_2AR expression on activated iNKT cells. Compared to CD4+ T cells, iNKT cells express 10 times more A_2AR and receptor expression increases further (100-fold) during pVOC^10,19. Thus, iNKT cells are exquisitely sensitive to inhibition through adenosine signaling due to dense expression of the high affinity receptor, A_2AR. Based on receptor affinity and density, a model for adenosine signaling emerges whereby differing levels of adenosine in a localized area may produce actions through A_2AR and/or A_2BR. Potentially, under conditions where levels of adenosine in SCD are not extremely high, the effects of adenosine are mediated through the A_2AR and only during the more extreme conditions of pVOC would the deleterious effects of the A_2BR activation be observed (Figure 1).

Figure 1. The proposed roles of the adenosine A_2A receptor (A_2AR) and the adenosine A_2B receptor (A_2BR) in the pathogenesis of sickle cell disease (SCD).

Sickle cell vaso-occlusion leads to tissue injury and liberation of adenosine. Left side of schema: expression of A_2AR are increased on iNKT cells and activation of A_2AR on iNKT cells leads to a reduction in pro-inflammatory mediators (IFN-γ, IL-4) and dampening in the severity of vaso-occlusion. A_2AR agonists (e.g., regadenoson) may promote the anti-inflammatory effects of the A_2AR. Right side of schema: Activation of A_2BR on erythrocytes results in increased levels of 2,3 DPG decreasing hemoglobin affinity for oxygen and contributing to red cell sickling. On corpus cavernosal cells of the penis, activation of A_2BR promotes priapism. Antagonists to A_2BR may decrease the formation of sickle erythrocytes and prevent priapic episodes. Potentially, dual therapy with A_2AR agonist/A_2BR antagonist would be an ideal for patients with SCD: anti-inflammatory, anti-sickling, anti-priapism.

Adenosine measurements have limitations

Levels of adenosine in patients with SCD have been shown to be higher than healthy controls and, likely, levels will rise further during pVOC¹². Adenosine measurements in the extracellular space reflect the sum of adenosine’s formation, transport and degradation⁴². During periods of microvascular occlusion and tissue ischemia, extracellular concentrations of adenosine may increase and activate adenosine signaling pathways to counteract further tissue damage by increasing blood flow and reducing heart rate and inflammation. Unfortunately, rapid cellular uptake and degradation cause adenosine to have a half life of approximately 5 seconds creating challenges to obtaining accurate measurements⁴². Further compounding errors in measurement is the compartmentalization of adenosine between the intravascular and interstitial spaces and the restricted, tissue-specific rise that may occur in adenosine levels following ischemia and injury⁴². Thus, measurement of adenosine levels in blood from the right arm may not accurately reflect the physiologic effects of adenosine in a patient who is experiencing ongoing vaso-occlusion in the vasculature of the left leg. A final consideration in the interpretation of adenosine levels in the preceding studies are the differences in adenosine biology between people and mice. Adenosine’s half-life is longer in mice than in people, and therefore blood levels are also higher in mice⁴¹. Under these conditions, adenosine signaling through A_2BR may be more pronounced in mouse models than in patients. A better understanding of adenosine levels in patients with SCD may clarify the role of A_2AR and A_2BR signaling under varied conditions and the contribution of this signaling to SCD morbidities.

Limitations of adenosine therapeutics in SCD

The main challenges for using adenosine analogues to treat and prevent vaso-occlusion are the need for a continuous intravenous infusion and unwanted side effects. The short half-life of regadenoson necessitates a continuous infusion, which requires stable intravenous access, and limits the role of regadenoson to the treatment of acute crises. Regadenoson has multi-phasic pharmacokinetics, but the terminal half-life is still only 2 hours³⁰. Current conceptual models of SCD describe ongoing vaso-occlusion with inflammation and end organ damage that is punctuated by severe pVOC that result in a hospitalization. Treatments aimed to shorten the duration of hospitalizations, such as regadenoson, have value as there is a higher risk of death during these acute events⁴, however, they will not affect the continuing daily damage that culminates in organ dysfunction. Preventing a major crisis is a better strategy to decease morbidity and mortality than treating one. To this end, a humanized monoclonal antibody that targets iNKT cells independently of the adenosine signaling pathways is currently under investigation in a phase 1 study (Clinicaltrials.gov #01783691). This investigational agent could potentially deplete iNKT cells on a longer-term basis than the effects of regadenoson and prevent crises. Another challenge to the use of adenosine signaling in the treatment of SCD is unwanted side effects due to a lack of adenosine receptor selectivity. Adenosine and dipyridamole cause activation of all adenosine receptor subtypes and are associated with severe side effects such as heart-block and hypotension. The A_2AR agonist regadenoson has been administered to patients with SCD without toxicity due to a high degree of selectivity for the A_2AR, along with the fact that lower concentrations of the drug are still able to achieve anti-inflammatory effects¹¹.

Future directions: combined A_2AR and A_2BR therapies for SCD?

Modulating the adenosine signaling pathway holds promise as a treatment for patients with SCD. Independent lines of investigation have provided evidence that the A_2AR and A_2BR are respectively protective and deleterious in the pathogenesis of vaso-occlusion. The combination of an A_2AR agonist with an A_2BR antagonist may be an ideal treatment in SCD. Thus far, the A_2AR agonist regadenoson is the only adenosine-based therapeutic that has been studied in patients with SCD¹¹. Another approach that has been suggested is PEG-ADA infusions to lower circulating adenosine levels and minimize signaling through the A_2BR. ADA is an FDA-approved therapy for patients with ADA deficiency⁴³. The possible shortcoming of this approach is that positive effects from adenosine signaling through the A_2AR might be negated with ADA therapy. A more novel and potentially effective approach to prevent and treat vaso-occlusion is dual therapy with A_2BR antagonists to prevent erythrocyte sickling and A_2AR agonists to decrease inflammation and dampen the severity of pVOC (Figure 1)⁴⁴. Highly selective A_2BR antagonists are currently in development for maintenance treatment of asthma^16,44. Clearly, rigorously-designed clinical trials demonstrating the clinical efficacy of A_2AR agonists and/or A_2BR antagonists need to be conducted prior to considering combination therapy, however, innovative approaches are needed in the treatment of SCD and multi-modal therapies may be necessary to demonstrate clinical benefit.

Key points.

• Activation of adenosine A_2A receptor (A_2AR) on invariant NKT cells decreases inflammation in a transgenic mouse model of sickle cell disease (SCD). The effects of regadenoson, an A_2AR agonist, are currently being examined in patients with SCD.

• The adenosine A_2B receptor (A_2BR) on red blood cells and corpus cavernosal cells of the penis has been implicated in the formation of sickle erythrocytes and priapism, respectively.

• These two independent lines of research examining the roles of A_2AR and A_2BR signaling in SCD may provide opportunities for new therapies.