PHARMACOTHERAPY IN SICKLE CELL DISEASE – STATE OF THE ART AND FUTURE PROSPECTS

Abstract

In the last decade, the care of patients with sickle cell disease (SCD) has undergone important advances with better understanding of disease pathophysiology and improvement in standards of care, especially among paediatric patients. Although many new drugs are currently being investigated and are at different stages of development, the pace of drug discovery and utilization has been slow and suboptimal. Hydroxycarbamide (hydroxyurea) has been investigated and utilized for at least two decades. Hydroxycarbamide’s efficacy has been demonstrated, albeit with different levels of evidence, in paediatric and adult populations, and yet clinician and patient acceptance and use have been far from ideal. In this review we discuss the current usage of hydroxycarbamide and its possible future indications in SCD, as well as the use of new compounds that have very different mechanisms of action, which may prove safe and efficacious when used alone or in combination in patients with SCD.

INTRODUCTION

Sickle Cell Disease (SCD) is a genetic condition that is the result of a single point mutation in the β-globin gene (HBB, β^s), producing the abnormal Haemoglobin (Hb) S. Although Hb S sickling and vaso-occlusion are involved in the pathophysiology of the disease, a much more complex array of events has been implicated. Ongoing haemolysis, nitric oxide (NO) deficiency with consequent vascular instability, (Pawloski et al, 2005) red blood cell (RBC) dehydration, (Hofrichter et al, 1974) RBC adherence to the endothelium, pro-inflammatory events, and activation of the coagulation system,(Hebbel et al, 2004) all have roles in the pathogenesis of this disease. This multifactorial aspect of SCD opens great avenues for the investigation of drugs with different physiological targets.

The care of patients with SCD has undergone important advances in recent years, with institution of newborn screening programs in many countries, provision of pneumococcal vaccination and prophylaxis with penicillin in childhood, and better understanding of the effects of efficacious drugs, such as hydroxycarbamide (also known as hydroxyurea). The development of effective drugs to treat sickle cell patients has not kept pace with discoveries and advances in the field, however. New drugs are currently being examined as possible alternatives for pharmacotherapy, and are at different stages of basic and clinical investigation. The concepts of individualized therapy and combination therapy have been further explored in more recent years, also offering promise to improve the care of patients with SCD. This review discusses the current use of established therapies, along with a “look ahead” to prospective drugs for the treatment of SCD.

INDUCERS OF HAEMOGLOBIN F

Elevated Hb F levels in patients with SCD have long been recognized as a prognostic factor of clinical severity. Sustained Hb F levels ≥20% are associated with reduced clinical events. (Powars et al, 1984) In addition, low Hb F concentration is recognized as a predictor of early mortality among patients with SCD. (Platt et al, 1994) Hb F α₂/γ₂ homotetramers and α₂/γ/β^s heterotetramers do not participate in the polymerization process, therefore an increase in the intracellular concentration of Hb F offers a molecular advantage for the prevention of sickling. Different drugs have the ability to increase Hb F production, using different mechanisms of action. Any drug that substantially increases the HbF production and promotes high and homogenous distribution of HbF among cells (pancellular Hb F distribution) has the potential to improve clinical outcomes in patients with SCD.

Hydroxycarbamide

The only Federal Drug Administration (FDA)-approved drug for use in patients with SCD, hydroxycarbamide, is an antimetabolite chemotherapeutic agent known to stimulate fetal haemoglobin production, therefore offering therapeutic benefit in this disease. Hydroxycarbamide has the ability to raise Hb concentration and Hb F values, as well as promote a parallel increase in red cell mean corpuscular volume (MCV). (Ferster et al, 2001;Kinney et al, 1999) The myelosuppressive and cytotoxic effects of hydroxycarbamide induce erythroid regeneration and the recruitment of earlier progenitors programmed to produce higher levels of Hb F. (Dover et al, 1986) The exact mechanism by which hydroxycarbamide increases Hb F levels seems to be mediated through a NO-dependent activation of soluble guanylyl cyclase in erythroid cells. (Cokic et al, 2008;Cokic et al, 2003) Hydroxycarbamide has additional effects, including lowering white blood cell count (WBC), reticulocytes, and platelets, increasing NO production, (Nahavandi et al, 2002) improving RBC hydration, (Orringer et al, 1991) and decreasing RBC adhesiveness to endothelium. (Hillery et al, 2000)

Hydroxycarbamide can decrease the rate of pain and acute chest syndrome (ACS) events in severely affected adults and children. (Charache et al, 1995;Ferster et al, 2001;Jayabose et al, 1996) The usual starting dose is 15 to 20 mg/kg/day given once daily orally. Hydroxycarbamide is also utilized in the pre-conditioning regimen of bone marrow transplantation of patients with SCD and its use seems to be associated with a lower incidence of rejection and engraftment failure. (Brachet et al, 2004) In addition, there is a growing body of evidence pointing toward end organ protection with early hydroxycarbamide therapy initiation. In young children with sickle cell anaemia, splenic function was preserved and in some cases regained when hydroxycarbamide was escalated to the maximum tolerated dose (approximately 30 mg/kg/day). (Hankins et al, 2008a;Hankins et al, 2005) Similar beneficial findings in other organs have also been reported, such as improvement of proteinuria, resolution of hypoxaemia, and protection against recurrent stroke. (Fitzhugh et al, 2005;Singh et al, 2008;Ware et al, 2004) Two ongoing randomized multicenter trials are investigating the role of hydroxycarbamide in organ preservation in paediatric patients: the first one, the BABY HUG study (www.clinicaltrials.gov # NCT00006400), is investigating the potential effects of hydroxycarbamide in splenic and renal function preservation in very young children, and the second one, the SWiTCH (Stroke With Transfusions Changing to Hydroxyurea) study (www.clincaltrials.gov # NCT00122980), is examining hydroxycarbamide for secondary stroke prevention. These two important trials may expand the role of hydroxycarbamide therapy beyond reducing the frequency of vaso-occlusive events, to include the protection of organs from the progressive and inevitable damage caused by SCD.

Limited information about the pharmacokinetics of hydroxycarbamide is available, however, in a small cohort of SCD patients there were no significant differences in pharmacokinetic parameters among adult and paediatric individuals. (De Montalembert et al, 2006) Because hydroxycarbamide undergoes renal clearance, its dose must be adjusted in individuals with renal impairment. A reduced initial dose of hydroxycarbamide (7.5 mg/kg/day) is recommended to individuals with a creatinine clearance <60mL/min. (Yan et al, 2005) Close monitoring of myelotoxicity is essential in patients with renal impairment who are treated with hydroxycarbamide.

Long-term safety of hydroxycarbamide continues to be scrutinized, as concerns with genotoxicity and carcinogenesis continue to be a topic of discussion among clinicians and patients. Fears about cancer development have not been substantiated, as no evidence to date points toward an increased rate of malignancy associated with hydroxycarbamide therapy. (Brawley et al, 2008) However, continued investigation in this area is important. Results of an ongoing study examining the long-term effects of hydroxycarbamide in children with SCD should provide important data regarding long-term risks and benefits of hydroxycarbamide in the paediatric population (www.clinicaltrials.gov # NCT00305175).

Despite years of research and extensive evidence of its efficacy, hydroxycarbamide remains a vastly underutilized drug. To address this underutilization, a number of hurdles must be overcome, including concerns of patients, families, and providers about cancer, birth defects, infertility, and questions regarding the drug’s long-term benefits and toxicities. (Zumberg et al, 2005;Lanzkron et al, 2008) Perhaps more importantly, lack of awareness of this therapeutic option, and communication gaps between clinicians and patients constitute barriers to appropriate hydroxycarbamide utilization. A recent consensus conference at the National Institutes of Health (NIH), which brought together experts in the field to discuss hydroxycarbamide therapy for patients with SCD, affirmed its underutilization, despite sufficient evidence of its efficacy. (Brawley et al, 2008) During this meeting, barriers were identified and specific recommendations were provided.

Decitabine

Decitabine and its analogue, 5-azacytidine, are cytidine surrogates. Once incorporated into the DNA, they form covalent bonds with DNA methyltrasferase (DNMT), leading to depletion of this enzyme, and resulting in DNA hypomethylation. (Creusot et al, 1982) Hypomethylation of the HBG (γ-globin gene) promoter triggers its expression and induces γ-globin synthesis, resulting in the so called “γ-globin reverse switch”, the postulated mechanism of action for increased Hb F production. (DeSimone et al, 1983;Charache et al, 1983) In addition, these compounds induce selective degradation of DNMT1, also resulting in re-expression of γ-globin genes. (Ghoshal et al, 2005)

Following early reports of success in baboons, a remarkable increase in F-cell and Hb F production was observed in patients with SCD and thalassaemia with 5-azacytidine use, in addition to a reduction in the proportion of dense RBCs in patients with SCD. (DeSimone et al, 1982; Ley et al, 1983a, b;Dover et al, 1985) These initial reports were very encouraging, however concerns related to a presumed 5-azacytidine proclivity to causing malignant transformation in rats, halted future investigation. (Carr et al, 1984) The 5-azacytidine analogue, decitabine (2-deoxy 5-azacytidine), was shown to promote similar molecular and cellular effects to its counterpart, with no apparent tumorigenic risks. (Koshy et al, 2000;DeSimone et al, 2002) Prompted by the fact that not all hydroxycarbamide-treated patients will attain optimal response (Hb F ≥20%), even in the setting of good adherence (i.e.: hydroxycarbamide low responders), more effort recently has been placed into the development of decitabine as an alternative or adjunct therapy in SCD. Low dose subcutaneous use of decitabine (0.2 mg/kg 1–3 times/week) was tested in small group of adult patients who responded poorly to hydroxycarbamide and promoted a marked increase in Hb F, F cell proportion, and Hb concentration, and decreased reticulocyte and absolute neutrophil counts. (Saunthararajah et al, 2003) In this study, however, the platelet count increased with decitabine therapy, an incompletely understood effect that may trigger undesirable clotting activation and will require close monitoring in subsequent studies. A report of 4 adult patients with multiple complications of SCD described clinical benefit of decitabine in reducing vaso-occlusive events and improved symptoms of heart dysfunction. (Saunthararajah et al, 2008)

Decitabine has only been tested parenterally (IV or SQ) in human subjects. An oral form of decitabine was tested in baboons and seemed to offer the same benefits as the parenteral formulations, (Lavelle et al, 2007) and would improve the likelihood of this drug becoming an acceptable therapy for SCD. The long-term clinical effects (e.g., reduction in vaso-occlusive events and protection against end organ damage) and long-term side effects of decitabine (e.g., malignancy and male infertility) have not yet been investigated. There have been numerous obstacles to the development of decitabine, but the drug may provide significant benefit to patients with SCD, especially among those with biological impairment in Hb F production during hydroxycarbamide therapy. Clinical investigation of decitabine in larger groups and for longer periods is, therefore, warranted.

Butyrate

Short chain fatty acid butyrates inhibit histone deacetylase and promote elevated levels of core histone acetylation, affecting chromatin structure and transcription rates of γ-globin genes. After de-repressing γ-globin genes, increased gene transcription and γ-globin synthesis will follow, thereby increasing Hb F levels. (McCaffrey et al, 1997) Initial observational studies in babies of women with diabetes found an association between high plasma levels of amino-butyric acid and a delayed switch from γ-globin to β-globin production. (Perrine et al, 1985) Several experiments in animal models and erythroid cultured cells confirmed the ability of butyrate or butyrate analogues to induce γ-globin production. (Constantoulakis et al, 1989;Perrine et al, 1988) Studies in patients with SCD and β-thalassaemia using continuous IV infusion of butyrate showed increased Hb F production with subsequent decline and no substantial increase in Hb concentration, however. (Collins et al, 1995) This disappointing finding was probably due to the cumulative erythroid antiproliferative effects of butyrate. More encouraging results were obtained with “pulse” butyrate therapy (250 – 500 mg/kg/day IV infused over 6 to 12 hours at a maximum of 6 days per month) with sustained Hb F production and higher Hb concentration. (Atweh et al, 1999;Hines et al, 2008;Ikuta et al, 1998)

The magnitude of Hb F production with butyrate use seems dependent on elevated levels of Hb F pre-therapy (Atweh et al, 1999) that reflect increased baseline activity of fetal globin genes. Thus, young children would be good candidates for butyrate therapy due to their elevated Hb F levels. In fact, this may be the case, as children with Hb SS had significant increases of F-reticulocytes and Hb F levels when given relatively low doses of oral sodium phenylbutyrate. (Resar et al, 2002)

The disadvantages of butyrate centre on its short half-life, requiring continuous IV dosages through a central line. Oral butyrate analogues would overcome this problem, but they require very high doses (as many as 40 tablets per day, in addition to having an unpleasant taste). (Collins et al, 1995) Taken together, the route and inconvenience of oral use will limit patient acceptance, and low adherence rates can be anticipated. But, if used for a definite period of time (from a few weeks to a few months), butyrate may be better accepted by patients. One possible scenario would be in the care of indolent leg ulcers, since butyrate was shown to heal intractable cases in patients with SCD. (Sher & Olivieri, 1994) In fact, a current phase II clinical trial is evaluating the role of arginine butyrate in patients with SCD and refractory leg ulcers (www.clinicaltrials.gov # NCT00004412). New short chain fatty acid derivatives with greater bioavailability and longer half-lives are currently under investigation in animal models. If their development is successful, these improvements may facilitate patient compliance. (Pace et al, 2002)

Erythropoietin

Recombinant human erythropoietin is another compound with Hb F augmenting properties. In vitro and in vivo experiments have demonstrated erythropoietin’s ability to raise the percentage of F cells and Hb F concentration when used alone. (Al-Khatti et al, 1987;Stamatoyannopoulos et al, 1989) Subsequently, studies combining hydroxycarbamide and erythropoietin in adults with SCD showed mixed results in promoting a significant increase in F cells. (el-Hazmi et al, 1995a;Goldberg et al, 1990) However, insufficient doses of erythropoietin, the schedules used for drug combination, and the presence of iron deficiency may have obscured erythropoietin’s effect. Studies using higher doses of erythropoietin, sequential use of drugs (4 days of hydroxycarbamide followed by 3 days of erythropoietin) and iron supplementation demonstrated an increase in F cells and Hb F production. (Nagel et al, 1993;Rodgers et al, 1993;McDonagh et al, 1992) In patients receiving erythropoietin in combination with hydroxycarbamide, high levels of F-cells and Hb F were achieved very rapidly (within 4 weeks). When erythropoietin was discontinued and hydroxycarbamide alone was continued, the effect was sustained. (el-Hazmi et al, 1995b) It is conceivable that the benefit of recombinant erythropoietin lies in accelerating the response to hydroxycarbamide, however, given the body of information gained over years with hydroxycarbamide use, the end result of sustained Hb F production seems to be similarly achieved even without upfront erythropoietin use. (Zimmerman et al, 2004) The use of erythropoietin in combination with hydroxycarbamide may still find its place among patients with poor Hb F response due to decreased endogenous erythropoietin production (e.g., renal insufficiency). In a recent report of the NIH experience, 13 adult patients, either hydroxycarbamide-intolerant (due to reticulocytopenia) or with renal insufficiency, benefited from the combination of erythropoietin and higher hydroxycarbamide doses. This resulted in increased Hb F production and few side effects. (Little et al, 2006) This report illustrates the important role of erythropoietin in allowing patients to tolerate hydroxycarbamide dose escalation, while preventing erythroid toxicity from hydroxycarbamide in the setting of endogenous erythropoietin deficiency. At present, an NIH Phase I clinical trial is investigating the role of erythropoietin in combination with hydroxycarbamide in adult patients with SCD and renal dysfunction and/or evidence of pulmonary hypertension (www.clinicaltrials.gov # NCT00270478). This study will be important to help define the optimal role of erythropoietin in the management of patients with SCD. In addition, this study may offer some information regarding the possible increased risk of thrombosis with erythropoietin use, a finding recently observed in patients with cancer. (Bennett et al, 2008)

AGENTS THAT PREVENT RED BLOOD CELL DEHYDRATION

The propensity of the RBC to undergo sickling is uniquely dependent on the intracellular concentration of deoxy Hb S; a higher Hb S concentration markedly reduces the lag time to polymer formation, thereby accelerating sickling. (Eaton & Hofrichter, 1987;Ferrone et al, 1985) Hb S concentration is directly dependent upon cellular hydration status, therefore dehydrated RBCs have higher intracellular Hb S concentrations. Three main pathways are involved in red cell dehydration: the calcium-activated potassium efflux channel (Gardos channel), (Vandorpe et al, 1998) the KCl co-transporter, (Brugnara et al, 1986) and the Na⁺ pump. (Joiner et al, 1986) Inhibition of any of these pathways can potentially prevent RBC dehydration and provide clinical benefit.

ICA- 17043 (Senicapoc)

In vivo and in vitro experiments using the imidazole antimycotic drug clotrimazole showed blockade of Gardos channel, increased K⁺ content, and reduced mean corpuscular haemoglobin concentration (MCHC) and erythrocyte density. (De Franceschi L. et al, 1994;Brugnara et al, 1996) Side effects of clotrimazole including nausea, vomiting, and transaminitis, however, precluded its long-term clinical use. The encouraging results of clotrimazole in the prevention of RBC dehydration triggered an active search for a compound with similar membrane effects as clotrimazole, but without its undesirable side effects.

ICA-17043 was first shown to modulate RBC membrane cation permeability by decreasing the Gardos channel activity and calcium-induced K⁺ efflux. (Stocker et al, 2003) In this study, the membrane effect was demonstrated in a dose-dependent manner in both mice and humans. ICA-17043 was also shown to reduce RBC density in mice in comparison with untreated controls, even in the setting of hypoxia-induced RBC dehydration. (Stocker et al, 2003) These preliminary results, coupled with the long half-life of ICA-17043 (allowing once-daily dosing) and minimal side effects, (Ataga et al, 2006) fostered further investigation in a clinical trial of adults with SCD. A phase II clinical trial randomized 90 adult patients with SCD to receive a loading dose of ICA-17043 of 150 mg followed by 10 mg daily, a lower loading dose of 100 mg followed by 6 mg daily, or placebo. (Ataga et al, 2008) The duration of therapy in this phase II clinical trial was 12 weeks, and hematological efficacy was demonstrated in the higher dose treatment arm by a significant increase in Hb concentration, and a decrease in lactate dehydrogenase (LDH), bilirubin, reticulocyte count, and dense RBCs. In addition, the activity of the Gardos channel decreased. Although it reduced haemolysis, ICA-17043 failed to reduce the frequency of vaso-occlusive painful events. This finding led to an early closure of a phase III clinical trial of ICA-17043, in which vaso-occlusive events were the primary outcome measure. The clinical benefit of ICA-17043 remains unanswered at this point. Clinical efficacy of ICA-17043 could still be shown in clinical trials evaluating haemolysis-associated vasculopathy such as pulmonary hypertension, stroke, and priapism. The challenges involved in designing a trial with these endpoints are numerous, but such trials are extremely important to determine if the laboratory efficacy of ICA-17043 will indeed translate into clinical benefit.

Magnesium Pidolate

Magnesium (Mg⁺⁺) is an important regulator of cellular cation transporters, such as the KCl co-transporter. (De Franceschi et al, 1996) Increased intracellular Mg⁺⁺ inhibits K⁺ efflux from the sickle erythrocyte and consequently prevents RBC dehydration. (Brugnara & Tosteson, 1987) Patients with SCD may have decreased Mg⁺⁺ levels in plasma and erythrocytes. (Olukoga et al, 1990) Their urinary excretion of Mg⁺⁺ is elevated, suggesting a high rate of clearance and net movement of Mg⁺⁺ from the erythrocyte to the plasma. (Olukoga et al, 1993)

Preliminary studies in transgenic sickle mice have shown that magnesium supplementation can significantly reduce KCl co-transport activity (measured by cell K⁺ efflux), and thereby decrease MCHC, red cell density, and reticulocyte count, when compared to mice receiving a low magnesium diet. (De Franceschi et al, 1996) Administration of magnesium supplementation to adult patients with SCD has been associated with minimal toxicity (mostly diarrhoea) and possibly a reduced frequency of vaso-occlusion. (De Franceschi et al, 1997;De Franceschi et al, 2000)

The combination of magnesium pidolate and hydroxycarbamide was investigated in a phase I clinical trial in children with Hb SS who had been on a stable dose of hydroxycarbamide for at least 6 months. (Hankins et al, 2008b) In this study, the maximum tolerated dose (MTD) of magnesium pidolate was determined to be 125 mg/kg/day (0.9 mEq/kg/day) divided into two daily doses when used in combination with hydroxycarbamide therapy. Toxicities from magnesium pidolate were mostly gastrointestinal (diarrhoea and abdominal pain), and most importantly, there was no enhancement of hydroxycarbamide toxicity with the addition of magnesium pidolate over a 24-week treatment period. A significant reduction of KCl co-transport activity occurred after introduction of oral magnesium pidolate, supporting previous reports of its membrane effects in adults with SCD. A phase II double-blinded, placebo controlled clinical trial is investigating the effectiveness of hydroxycarbamide and magnesium pidolate alone and in combination in reducing RBC density among patients with Hb SC disease (www.clinicaltrials.gov # NCT00532883). Another phase II clinical trial is underway to investigate the role of magnesium pidolate in preventing RBC dehydration and reducing the frequency of painful events in adults and children with Hb SC (www.clinicaltrials.gov # NCT00040456). These two trials should provide preliminary information on the effects of magnesium pidolate, used alone or in combination with hydroxycarbamide, on laboratory measures of red cell dehydration, setting the stage for phase III trials investigating clinical benefits.

VASODILATORS

Vaso-occlusion is the hallmark of SCD pathophysiology, making the promotion of vaso-dilation a desirable effect in the treatment of this disease. NO is one of the most potent naturally occurring vasodilator agents. Endogenous NO is formed from L- arginine by NO synthase. NO mediates vaso-relaxation via formation of cyclic guanosine monophosphate (cGMP), which signals intracellular Ca⁺⁺ sequestration and vasodilation. The ongoing intravascular haemolysis that occurs in SCD rapidly inactivates NO via destruction by cell-free haemoglobin and also by a reaction between ferrous iron and ferric iron, which forms nitrosylhaemoglobin that consumes NO. (Reiter et al, 2002) Haemolysis also releases arginase, an enzyme that leads to depletion of arginine, the substrate for NO production. Pulmonary hypertension, stroke, priapism and leg ulcers have all been linked to haemolysis-induced vasculopathy in SCD, and haemolysis with NO depletion seems to play a central role in their development. (Kato et al, 2006)

Inhaled Nitric Oxide

The first clinical use of NO in SCD was reported in 1997 in two children who developed ACS with respiratory failure and pulmonary hypertension despite aggressive medical therapy, including positive pressure ventilation and exchange transfusion. (Atz & Wessel, 1997) These children were treated with inhaled NO and had rapid and significant pulmonary vasodilation and improved oxygenation. In another report, a teenager with SCD and a severe episode of ACS showed a remarkable improvement of the alveolar-arterial oxygen gradient and marked reduction in the pulmonary arterial pressure and pulmonary vascular resistance with inhaled NO. (Sullivan et al, 1999) In a prospective, double-blinded, placebo-controlled randomized clinical trial, 20 paediatric patients aged 10–21 years were treated with inhaled NO (80 ppm with 21% inspired oxygen) vs. placebo (21% inspired oxygen) for treatment of vaso-occlusive crisis. (Weiner et al, 2003) In this study, the decrease in pain measured by visual analog scale was not significant, however there was a significant reduction in morphine use after 6 h (mean cumulative dose 0.29 vs. 0.44 mg/kg, p = 0.03). Two phase II, double-blinded, randomized, placebo-controlled studies of inhaled NO for acute treatment of pain crisis (www.clinicaltrials.gov #NCT00094887 and NCT00142051) are currently in progress and if successful, may offer an additional option for the acute management of pain in SCD.

Arginine

L-arginine is deficient in adults with steady state Hb SS disease, despite adequate dietary intake, and decreases to even lower levels during a vaso-occlusive event. (Enwonwu et al, 1990;Lopez et al, 2003) The significant correlation between lower arginine/ornithine ratio and marked anaemia (due to haemolysis) suggests that more arginine is metabolized via the arginase pathway in patients with a higher hemolytic rate. (Schnog et al, 2004) Furthermore, plasma arginase levels are significantly increased in patients with SCD, with the highest activity found in patients with pulmonary hypertension. (Morris et al, 2005)

In children with Hb SS disease, steady state arginine and NO levels are normal at baseline, however, similar to adults, decrease during vaso-occlusive events. (Morris et al, 2000a) Low arginine levels during vaso-occlusive events are thought to reflect acute substrate depletion resulting in decreased NO production. To test this hypothesis, oral arginine was given to SCD patients both at steady state and during vaso-occlusive events, and to normal controls. Patients with vaso-occlusive events had a significant increase in NO levels after L-arginine supplementation in comparison with the other two groups. (Morris et al, 2000b) In another study using oral arginine supplementation in SCD patients, a 15% reduction in mean estimated pulmonary artery pressure was observed in 10 patients with pulmonary hypertension (p = .002). (Morris et al, 2003a) The combination of hydroxycarbamide and arginine was investigated in five patients with SCD, who received both drugs, and showed an increase in NO levels. (Morris et al, 2003b) These preliminary encouraging results of oral arginine supplementation led to a multicenter, double blinded, placebo-controlled, phase II study of children with SCD (www.clinicaltrials.gov # NCT00513617). In this study, subjects received oral arginine for 3 months (0.05 g/kg/day or 0.1 g/kg/day), however no significant increase in arginine level or change in any of the other laboratory outcomes (NO level, Gardos channel activity, and RBC density) was observed in a preliminary report of this study. (Styles et al, 2007a) These results have dampened enthusiasm for this agent, but a phase II trial to determine if oral arginine will increase NO in SCD patients during ACS events is currently underway (www.clinicaltrials.gov #NCT00029731).

Sildenafil

Sildenafil is an oral phophodiesterase-5 inhibitor. In tissues that express phosphodiesterase-5, such as the lungs and corpora cavernosa, sildenafil amplifies NO signaling by inhibiting hydrolysis of cGMP, the mediator of NO signal transduction. (Atz & Wessel, 1999) In addition, sildenafil decreases glycoprotein IIb/IIIa-dependent platelet activation in patients with SCD and pulmonary hypertension. (Villagra et al, 2007) The clinical efficacy of sildenafil includes a report of reduction of tricuspid velocity gradient and increased 6-min walk test in a 38-year-old man with HbSβ⁰ thalassaemia, (Derchi et al, 2005) and in a series of 12 patients with SCD and pulmonary hypertension treated for 6 months. (Machado et al, 2005) Furthermore, sildenafil has also been used to treat and prevent priapism in SCD. Three patients, including one with refractory priapism, had either substantial (one patient) or complete resolution (two patients) of symptoms within 90 min of sildenafil administration. (Bialecki & Bridges, 2002) In two other reports, sildenafil was used in 4 patients with recurrent priapism and prevented recurrences in 3 of them. (Bialecki & Bridges, 2002;Burnett et al, 2006) Currently, two trials are assessing the role of sildenafil in the cardiopulmonary function and exercise endurance of patients with SCD with or without pulmonary hypertension (www.clinicaltrials.gov #NCT00352430 and NCT00492531).

Statins

Independent of their cholesterol-lowering effects, statins have been shown to prevent damage to blood vessels by several mechanisms, including up-regulation of endothelial NO. Given that NO metabolism is altered in SCD, statins may have a beneficial role in this disease. In one study of transgenic sickle mice, expression of tissue factor in endothelial cells was inhibited by lovastatin. (Solovey et al, 2004) Currently, there is a phase I/II study investigating the role of simvastatin in vasoreactivity, endothelial adhesion, and inflammation in patients with SCD (www.clinicaltrials.gov #NCT00508027).

OTHER AGENTS

Anti-adhesive Agents

SCD is a chronic inflammatory state in which leucocyte-endothelium interactions play a role in vaso-occlusion. Nuclear factor-kappa B (NFKB) is a transcription factor that activates expression of endothelial adhesion molecules in transgenic sickle mice. In sickle mice, NFKB inhibition by sulfasalazine led to decreased leucocyte adhesion and improved microvascular blood flow. (Kaul et al, 2004) In humans, sulfasalazine (1 g orally every 8 hours) significantly reduced expression of vascular endothelial adhesion molecule, intracellular adhesion molecule and E-selectin on circulating endothelial cells. (Solovey et al, 2001) There are no clinical trials currently reported using sulfasalazine or any other NFKB inhibitor in patients with SCD.

Purified poloxamer 188 is a nonionic block copolymer surfactant that improves microvascular flow by lowering viscosity, decreasing RBC aggregation, and decreasing friction between erythrocytes and the vessel wall. In a phase II trial, 50 patients with SCD and vaso-occlusive crisis were treated with 48-h infusions of poloxamer (60-min loading dose of 300 mg/kg followed by a 47-h maintenance infusion of 30 mg/kg/h) or placebo. (Adams-Graves et al, 1997) In this study, the median durations of painful episodes and analgesic use were significantly reduced among patients who received poloxamer. In another multicenter, double-blinded, placebo controlled trial, poloxamer 188 was given as a 48-h infusion during vaso-occlusive events in hospitalized patients. (Orringer et al, 2001) A 9-h mean reduction in the duration of pain and a greater proportion of patients achieving pain resolution was observed in the poloxamer group. An ongoing phase III randomized trial is assessing the efficacy of poloxamer 188 in reducing the duration of painful vaso-occlusive crisis in patients with SCD (www.clinicaltrials.gov # NCT00004408).

Nix-0699 (Nicosan®, Hemoxin®)

Nix-0999 is an herbal preparation comprised of extracts from 4 different kinds of plants: Piper guineense, Pterocarpus osun, Eugenia caryophyllum, and Sorghum bicolor. This drug was tested in adult and paediatric patients with Hb SS at the dose of 12 mg/kg/day, in a randomized, placebo-controlled, cross-over design trial, and showed a decreased incidence of severe painful events in the treatment group. (Wambebe et al, 2001) Although there was a significant decrease in events reported by pain diaries, this difference only occurred after the crossover point. (Cordeiro & Oniyangi, 2004) Following this clinical trial, in vitro studies using blood samples from SCD patients and in vivo experiments using transgenic mice showed a reduction in the percentage of sickle cells, increase in the delay time to polymerization, and increased solubility of deoxy-Hb S. (Iyamu et al, 2002;Iyamu et al, 2003) Furthermore, this drug was found to promote an accentuated left shift of the dissociation curve of deoxy Hb S. (Iyamu et al, 2002) However, no studies to date have showed significant changes in haematological parameters secondary to Nix-0699 use, such as Hb concentration, MCV, MCHC, reticulocytes, or white blood cell count, indicating a mechanism of action distinct from drugs stimulating Hb F production or preventing RBC dehydration. Increased Hb S oxygen affinity is the presumed mechanism of action of this drug, however in-vivo studies failed to reproduce the effect seen in in-vitro experiments. (Iyamu et al, 2003) Currently, the exact mechanism of action of this drug remains unclear, and the postulated direct interaction process by which Nix-0699 decreases Hb S polymerization and sickling has not yet been elucidated. Furthermore, the precise active ingredient(s) of Nix-0699 have not been determined.

Nix-0699 was designated as an orphan drug by the European Medicine Evaluation Agency in 2005 and the US FDA in 2006. Even though its mechanism of action and toxicity profile has not yet been clarified, Nix-0699 was approved in Nigeria in 2006, and is commercially available in this country. The lack of understanding of Nix-0699’s mechanism of action seems problematic and its effects on other tissues and its long-term safety profile are unknown. Although a promising drug, further prospective controlled studies evaluating NIX-0699’s efficacy and toxicity, and identification of the active compound, are necessary to establish its clinical benefit.

Secretory Phospholiapse A₂ Inhibitors

Elevation in the inflammatory mediator secretory phospholiapse A₂ (sPLA₂) often precedes the development of ACS in SCD patients who are admitted for vaso-occlusive events. (Styles et al, 2007b) An ongoing phase II double-blinded, randomized, parallel group, placebo-controlled, dose escalation study is investigating the role of Varespladib in patients with elevated sPLA₂ and SCD who are at risk for developing ACS (www.clinicaltrials.gov # NCT00434473).

Anti-thrombotic Agents

Unfractionated heparin has been shown to inhibit P-selectin-mediated adherence of sickle cells to thrombin-treated human vascular endothelial cells, as well as thrombospondin-mediated adhesion of sickle cells to human umbilical vein endothelial cells. (Gupta et al, 1999;Matsui et al, 2002) A randomized double-blind trial of tinzaparine (a low-molecular-weight heparin) investigated 263 patients with SCD and vaso-occlusive events and showed significant reduction in number of days with severe pain scores, overall duration of painful crisis, and duration of hospitalization. (Qari et al, 2007) Currently there is a Phase I/II trial of aspirin in children with SCD with the aim of evaluating the safety and tolerability of daily low-dose aspirin in children with HbSS and HbSβ°thalassemia (www.clinicaltrials.gov # NCT00178464).

Immunomodulatory Drugs

In an in vitro study, pomalidomide and lenalidomide induced Hb F expression in healthy and SCD CD34+-derived erythrocytes, and when used in combination with hydroxycarbamide, showed synergistic effect in the production of Hb F. (Moutouh-de Parseval et al, 2008)

Corticosteroids

Steroids have been used in the acute treatment of ACS, however, there is still controversy regarding their use in SCD. Steroids have been shown to have short-term beneficial effects in patients with painful crises and ACS (reduction of duration of hospitalization and severity of the ACS events), however an increased risk of rebound painful events has been reported. (Bernini et al, 1998;Darbari et al, 2008;Griffin et al, 1994;Strouse et al, 2008) Currently there is an ongoing phase III randomized trial of high dose methylprednisolone followed by prednisone taper for treatment of vaso-occlusive crisis in SCD (www.clinicaltrials.gov, NCT00263562).

FUTURE DIRECTIONS

Because several different mechanisms are involved in the pathophysiology of SCD, combination drug therapy with drugs aimed at different targets becomes enticing. Magnesium pidolate and hydroxycarbamide, for instance, work through different mechanisms and have non-overlapping toxicities, and both treatments have the potential to prevent or improve vaso-occlusion and haemolysis. The paradigm of drug combination is not new and has successfully been employed in Infectious Diseases and Oncology for many years. The option of combining Hb F inducers with other agents, such as vasodilators and compounds that prevent endothelium adhesion or improve RBC dehydration, seems logical, promising, and deserving of further investigation. In addition, combining drugs that have different mechanisms of action but result in enhanced Hb F production may potentiate the final Hb F concentration and improve the Hb F distribution among sickle erythrocytes (i.e., pancellular Hb F distribution). Since hydroxycarbamide is the most investigated drug among those described in this review, it is likely to be utilized in future trials of drug combinations. A summary of drug dosage utilized in prior clinical trials, their clinical efficacy, and toxicities is presented in Table I. In addition, examples of possible drug combinations to be investigated for the treatment of SCD are shown in Table II.

Table I.

Summary of Dosage, Efficacy, and Toxicity of Drugs Investigated in Sickle Cell Disease

Drug	Initial Dose	Side Effects	Clinical Efficacy	Ongoing Clinical Trial (s) (www.clinicaltrials.gov # as of January 2009)
Hydroxycarbamide	20 mg/kg once daily PO, escalated to MTD* (30 mg/kg/day) by 5 mg/kg every 8 weeks (Zimmerman et al, 2004) Renal impairment (creatinine clearance < 60mL/min) 7.5 mg/kg/day orally (Yan et al, 2005)	Reversible and dose- dependent myelosuppression. Nail bed and mucosal hyperpigmentation Uncertain risk of male infertility Uncertain risk of cancer (likely improbable)	Reduction of painful and ACS events, and need for blood transfusion Reduction of mortality Possible end-organ damage protection (brain, spleen, lungs)	NCT00480974 NCT00270478 NCT00305175 NCT00784082 NCT00006400 NCT00001197 NCT00350844 NCT00350844 NCT00004404 NCT00004492 NCT00122980
Decitabine	0.2 mg/kg SQ 1–3 times/week (Saunthararajah et al, 2003;Saunthararajah et al, 2008)	Myelosuppression Uncertain risk of cancer	Possible reduction of pain and symptoms of anemia (Saunthararajah et al, 2008)	None
Butyrate	Pulse therapy: 250 – 500 mg/kg/day IV over 6 to 12 hours, max 6 days/month (Atweh et al, 1999;Hines et al, 2008;Ikuta et al, 1998)	Gastric discomfort Body odor Uncertain risk of cancer	Possible improvement of leg ulcers	NCT00004412
Erythropoietin	100 U/kg SQ twice a week Increase by 100 U/kg/dose every 4–6 weeks (Little et al, 2006) Consider adding iron Supplementation	Bone pain, increased blood viscosity causing painful events (Goldberg et al, 1990) Uncertain risk of thrombosis (Bennett et al, 2008)	Reduction of erythroid suppression in patients treated with hydroxycarbamide	NCT00270478
ICA- 17043	150 mg PO loading dose, followed by 10 mg PO once daily (Ataga et al, 2008)	Diarrhoea and nausea	Unknown	None
Magnesium Pidolate	125 mg/kg/day (Hankins et al, 2008b)	Diarrhoea and abdominal pain	Possible reduction of vaso-occlusive events	NCT00040456 NCT00532883
Nitric Oxide	80 ppm inhaled with 21% inspired oxygen (Weiner et al, 2003)	None reported	Possible reduction of vaso-occlusive events	NCT00094887 NCT00142051
Arginine	0.05 – 0.1 g/kg PO once daily (Styles et al, 2007a)	None reported	Unknown	NCT00029731
Sildenafil	25 mg PO three times daily, escalated to a max of 100 mg PO three times daily (Machado et al, 2005)	Headaches Eyelid oedema	Possible improvement of pulmonary hypertension and priapism	NCT00352430 NCT00492531

Notes: MTD = maximum tolerated dose, ACS = acute chest syndrome, ANC= absolute neutrophil count, ARC = absolute reticulocyte count

Table II.

Possibilities for Drug Combination in Sickle Cell Disease

Mechanism of action	Drug combination
Induction of Hb F primarily	Hydroxycarbamide + Short Chain Fatty Acids
	Hydroxycarbamide + Decitabine*
	Short Chain Fatty Acids + Decitabine
	Hydroxycarbamide + Erythropoietin
Induction of Hb F and reduction of RBC dehydration	Hydroxycarbamide + Mg Pidolate
	Hydroxycarbamide + ICA-17043
Induction of Hb F and vasodilation	Hydroxycarbamide + Arginine
	Hydroxycarbamide + NO
	Hydroxycarbamide + Sildenafil

Notes:

^*Hydroxycarbamide and Decitabine should probably be used sequentially rather than concomitantly, given reports that hydroxycarbamide may possibly impair the effect of decitabine when both drugs are given simultaneously. (Choi et al, 2007)

A concept that deserves further investigation in SCD is individualized therapy. Hydroxycarbamide, for instance, has a great heterogeneity in its response and not all individuals respond with the same degree of haematological effects (e.g., different degrees of Hb F production, MCV increase, etc.). Contrasting with studies in adults where some patients were unable to sustain a hydroxycarbamide response, (Steinberg et al, 1997) children seem to attain higher Hb F values. (Zimmerman et al, 2004) Better compliance, higher dosing, higher starting Hb F values, less gene silencing, and/or increased marrow reserve may be related to the superior results, however further investigation is needed. Different pharmacokinetic and pharmacogenomic profiles may also account for this heterogeneity in hydroxycarbamide response, suggesting new and exciting avenues for further investigation. Several single nucleotide polymorphisms (SNPs) have been found in association with the degree of Hb F production after 2 years of hydroxycarbamide therapy in adults with SCD, suggesting a genetic modulation of this drug’s response. (Ma et al, 2007) The heterogeneity of drug response in SCD offers opportunities for investigating the best drug or drug combination for a particular disease complication. For instance, a drug or drug combination for stroke prevention may not be effective in management of pulmonary hypertension. Furthermore, age may be another factor to be considered when individualizing therapy in SCD. A myriad of possible scenarios can be explored in which the best drug or drug combination could be matched to an individual’s phenotype, age, and gene expression profile.

An ideal drug for SCD should be effective, non-toxic, and easy to use (oral route preferentially with few daily doses and a small number of capsules/tablets per day). Hydroxycarbamide seems to fit all these conditions, and yet, it is underutilized by many children and most adults with SCD. Barriers need to be overcome to allow this drug to journey from efficacy to effectiveness. Furthermore, clinical trials investigating new drugs in SCD must include paediatric patients, so the needs of these vulnerable individuals are not ignored. A great need to develop suitable, safe, and effective drugs for children exists, which could lead to more appropriate paediatric formulations (adequate volume of suspensions, powder preparation, palatable drugs, etc.), and lesser utilization of off-label drugs. (Zucker & Rago, 2007) Meanwhile, investigators need to press on in their quest for new drugs and drug combinations, so the approaching 100^th year of this disease’s discovery can mean a new and hopeful era for people living with SCD.