L‐arginine as an adjuvant drug in the treatment of sickle cell anaemia

Sickle cell disease (SCD) is an inherited disorder of haemoglobin synthesis that is determined by homozygosity of haemoglobin S (HbSS) and characterized by recurring episodes of vaso-occlusion, chronic inflammatory state, chronic haemolysis and progressive vasculopathy with resultant imbalance in the signalling mediated by nitric oxide (NO) (Sullivan et al, 2010). NO is a powerful vasodilator that acts by preventing the adhesion of leucocytes to the endothelium, inhibiting the expression of adhesion molecules and acting as an antiinflammatory substance (Kato et al 2007). In sickle cell anaemia (SCA), arginase – an enzyme present inside red blood cells, is released during haemolysis, catalysing the hydrolysis of L-arginine, the substrate for the production of NO in ornithine and urea, reducing the bioavailability of NO (Kato et al, 2007). The reduction of L-arginine also occurs following the increased consumption of NO as a result of the increase in reactive oxygen species (ROS), generated by the presence of free haemoglobin, ischaemic injury of recurrent reperfusion, pro-inflammatory state, and the high autoxidation of haemoglobin S (HbS) (Kato et al, 2007). Hydroxycarbamide (HC; also known as hydroxyurea) is a cytotoxic, mutagenic, recombinogenic and antineoplastic agent. It has been used to treat SCA by increasing the synthesis of HbF and total haemoglobin and reducing haemolysis (-Morris et al, 2008). HC also acts to reduce the expression of adhesion molecules, with anti-inflammatory and anti-aggregating properties, contributing to the decrease of vaso-occlusive episodes and reducing the need for blood transfusions, the frequency of hospitalizations, and mortality rate (-Morris et al, 2008). HC has also been attributed to affect NO metabolism, increasing production thereof via the cGMP cycle and consequently increasing HbF (Morris et al, 2003). HC therapy increases utilization of the arginine substrate, for the production of NO, by the activity of NOS (Nahavandi et al, 2000). L-arginine, a semi-essential amino acid, is a substrate for the endothelial nitric oxide synthase (eNOS) enzyme for the production of NO, and is reduced in SCA patients, thereby limiting the effectiveness of HC (Sullivan et al, 2010). The reduced overall bioavailability of arginine, measured by low plasma levels of L-arginine or L-ornithine, or L-ornithine and L-citrulline, is associated with increased mortality in SCA patients (Morris et al, 2005). The reduction of arginine levels in patients with SCA was demonstrated to be associated with endothelial damage, multiple organ injury, increased haemolysis and pulmonary hypertension, contributing to high mortality of these patients (Morris et al, 2008). The present study aimed to evaluate a therapeutic proposal for treating SCA, including supplementation with L-arginine as an adjuvant drug for treatment with HC. This was a randomized clinical trial that included 21 adult patients (9 men and 12 women, aged 20–40 years) with a clinical and laboratory diagnosis of SCD, confirmed by molecular biology, undergoing treatment with HC for more than one year at a referral university hospital in Fortaleza, Ceará, Brazil. After informed consent, the selected patients were randomly divided into Group I (Control group: HC only therapy, n = 09) and Group II (Study group; therapy with HC + L-arginine, n = 12), matched for age and sex. The patients selected for Group II were prescribed Reforgan (L-Arginine 250 mg), with a dose of one pill daily for 90 d, as a supplement to Hydréia (HC, dosage ranged from 500 to 1500 mg/day). Group II patients (HC + L-arginine) showed a significant increase in nitrite levels, HbF and reticulocytes, when compared to both baseline values and the Group I (HC only) at the various time points tested, demonstrating that supplementation with L-arginine increased the bioavailability of this substrate, leading to a better response to treatment with HC, as seen in Fig 1 (Table I). The increase in the levels of nitrite and HbF in patients after 12 weeks of use of HC + L-arginine, in relation to the baseline, demonstrates that such association induces a better therapeutic response to HC and confirms that the action of HC involves an NO-dependent pathway causing increased consumption of L-arginine, which is reduced in SCA patients and limits their response to HC. Our results support those of previous studies, which demonstrated that the in vitro induction of HF in progenitor cells occurs via NOSand soluble guanylate cyclase (Strouse et al, 2008), that there is chronic depletion of arginine levels in knockout sickle cell mice, and when treated with L-arginine, there is an increase in NO levels (Dasgupta et al, 2006). Patients using HC + L-arginine also showed an increase in reticulocyte counts at 12 weeks compared to the baseline, indicating that the arginine acts by stimulating erythropoiesis. Baliga et al (2010) evaluated the effect of the association of HC + L-arginine on the synthesis of fetal haemoglobin by erythroid colony-forming units, demonstrating excellent