Improvements in haemolysis and indicators of erythrocyte survival do not correlate with acute vaso-occlusive crises in patients with sickle cell disease: a phase III randomized, placebo-controlled, double-blind study of the gardos channel blocker senicapoc (ICA-17043)


Jonathan Stocker, PhD, ClinPharm Consulting, LLC, 2525 Meridian Parkway, Suite 280, Research Triangle Park, NC 27713, USA.


Red blood cell (RBC) hydration is regulated in part by the Ca2+-activated K+ efflux (Gardos) channel. Senicapoc selectively blocks potassium efflux through the Gardos channel, reducing RBC dehydration and haemolysis, and increasing haemoglobin levels in sickle cell disease (SCD). This randomized, placebo-controlled trial was designed to determine the safety and clinical efficacy of senicapoc in SCD patients. One hundred and forty-five patients were randomized to receive senicapoc and 144 patients to receive placebo for 52 weeks. Consistent with a previous study, patients in the senicapoc group had significantly increased haematocrit, haemoglobin, and decreased numbers of both dense erythrocytes and reticulocytes when compared to the placebo group. The unblinded Data Monitoring Committee terminated this study early due to a lack of efficacy when it determined that, despite improvements in anaemia and haemolysis, no significant improvement in the rate of sickle cell painful crises was observed in patients treated with senicapoc compared to those on placebo (0·38 vs. 0·31, respectively). Comparisons of the times to first, second and third crises between the senicapoc and placebo groups were not statistically significant. Nausea and urinary tract infections occurred more frequently in the senicapoc group than placebo. Serious adverse events were similar in the two groups.

Polymerization of sickle haemoglobin (HbS) in sickle cell disease (SCD) produces abnormal erythrocytes that impair circulation and cause pain, tissue and organ damage and hypoxia. The extent of polymerization depends on intracellular concentrations of HbS. Small increases in intracellular HbS concentrations, such as those that occur upon pathological sickle cell dehydration, greatly increase the likelihood and speed at which haemoglobin polymers form in circulation (Eaton et al, 1976). Inhibition of sickle cell dehydration has been demonstrated to reduce dense cell formation and improve anaemia in patients (Ataga et al, 2008). Our proposed hypothesis was that these effects of Gardos channel block would result in an improvement in the clinical manifestations of SCD, including vaso-occlusive crisis (Stuart & Nagel, 2004).

Sickle cell dehydration is regulated in part by the Ca2+-activated K+ efflux (Gardos) channel that is identical to the human intermediate-conductance potassium channel (KCa3.1) (Ishii et al, 1997). The Gardos channel plays a role in ion homeostasis and volume regulation of normal erythrocytes and contributes to cell dehydration following its activation (Cala, 1983; Canessa, 1991). Activation of the Gardos channel, which follows increased levels of cytosolic calcium, causes K+ and water efflux leading to dehydration and increased cellular HbS concentration. In both animal models of SCD and patients with SCD, blocking of the Gardos channel has been shown to inhibit K+ and water loss and to prevent polymerization of HbS (Brugnara et al,1993; De Franceschi et al, 1994).

Senicapoc (ICA-17043) is an ion-channel blocker that selectively blocks potassium efflux through the Gardos channel in human RBCs (McNaughton-Smith et al, 2008). Senicapoc prevents erythrocyte dehydration in vitro and reduces erythrocyte dehydration in vivo as indicated by significant increases in intracellular K+ and haematocrit and decreases in erythrocyte density in a mouse SCD model (Stocker et al, 2003). A phase I dose-escalation and pharmacokinetic study in patients with SCD showed that once-daily oral dosing with senicapoc was well tolerated and adequate to maintain steady-state plasma concentrations (Ataga et al, 2006). A phase II study in patients with SCD showed that oral administration of senicapoc for 12 weeks reduced haemolysis, as indicated by increased haemoglobin levels and concomitant decreases in lactate dehydrogenase (LDH), indirect bilirubin and numbers and percentages of reticulocytes (Ataga et al, 2008). A decrease in the number of circulating dense erythrocytes was also noted (Ataga et al, 2008). Senicapoc was well tolerated in patients with SCD during the 12 weeks of dosing.

The objectives of this phase III randomized, placebo-controlled trial were to determine whether senicapoc administration was associated with an improvement in the rate of painful crises in patients with SCD and to define the safety and tolerability of chronic (52 weeks) senicapoc use.


A randomized, double-blind, placebo-controlled, parallel-group study was conducted at 75 study centres in the United States, Jamaica, Brazil, France, Trinidad and the United Kingdom. The protocol (ICA-17043-10; CTR# NCT00102791) was approved by study centre review boards or ethics committees and all participants gave written informed consent.


Men and women, 16–65 years of age, with a confirmed diagnosis of SCD (including HbSS, HbSC, HbSβ0-thalassaemia and HbSβ+-thalassaemia) were eligible for the study if they had suffered at least two acute sickle-related painful crises in the previous 12 months that required a visit to a medical facility. Normal physical examinations, chest X-ray, 12-lead electrocardiogram (ECG) and acceptable clinical laboratory evaluations (including serum chemistries, haematology and urinalysis) were required before assigning patients to treatment groups. Women were required to use approved methods of birth control if capable of becoming pregnant. Patients were permitted to receive concomitant therapy with hydroxycarbamide (HC) if they had received HC for the preceding 12 months and their dose was stabilized for at least 3 months prior to the study.

Exclusion criteria included any unstable cardiovascular, neurological, endocrine, hepatic, or renal disorders unrelated to SCD. Patients were not enrolled if they had received previous treatment with senicapoc, had an allergic reaction that could indicate hypersensitivity to senicapoc (e.g., to clotrimazole), had haemoglobin levels <40 or >110 g/l, participated in a chronic transfusion programme, had a cancer diagnosis within the last 5 years, or had a history of hepatitis B or C or human immunodeficiency virus infection.

Study design and treatment schedule

The study consisted of a screening phase, 52-week treatment phase, and follow-up evaluation 8 weeks after the end of the study for a safety assessment. Patients who met enrollment criteria during screening were stratified based on HC use and the number (2–4 or ≥5) of acute sickle cell-related painful crises in the past 12 months. Patients were randomized within 30 d of screening to the placebo or senicapoc treatment groups (1:1). Patients received a loading dose of senicapoc 20 mg (or matching placebo) twice daily for 4 d followed by a maintenance dose of senicapoc 10 mg (or placebo) once daily for the remainder of the treatment phase. Patients returned to the clinic on day 8 and week 4, and then every 4 weeks until end of study for efficacy and safety evaluations.

Treatment assignment was determined using a central automated randomization protocol and an integrated voice response system. Senicapoc and placebo were identical in appearance and packaged in identical blister cards to maintain blinding. All site and study personnel were blinded to treatment assignment. Patients were instructed to take the loading dose of study medication (two tablets in the morning and two tablets in the evening) and the maintenance dose (one tablet) at approximately the same time each day. The senicapoc doses chosen for the study were based on previous studies in patients with SCD (Ataga et al, 2006, 2008).


The primary efficacy endpoint was the comparison across treatment groups of the frequency of acute sickle cell-related painful crises (SCPC) that occurred during the treatment phase. A painful crisis was defined as an episode of acute pain with no cause other than a vaso-occlusive event that required a medical facility visit and treatment with oral or parenteral narcotics, or parenteral non-steroidal anti-inflammatory drugs (NSAIDs). Included in the definition of painful crisis were acute chest syndrome, hepatic sequestration, splenic sequestration, priapism, stroke and death (with the exception of homicide, suicide, or accidental death). To ensure consistency across sites, all protocol-defined sickle-related painful crises identified by the Investigators that resulted in a visit to a medical facility were adjudicated by an independent, blinded, Crisis Review Committee (CRC). The CRC was comprised of five physicians with expertise in SCD. CRC members also reviewed adverse event listings to identify potentially unreported crisis events. Efficacy analysis was performed on the adjudicated data. The SCPC rate was defined as the total number of adjudicated painful crises divided by the number of months in which the patient received treatment.

Secondary endpoints included the time (in months) to first, second and third painful crises. The effect of senicapoc on haematological parameters was assessed by measuring the laboratory markers of haemolysis [indirect bilirubin and lactate dehydrogenase (LDH)], the number of reticulocytes and dense erythrocytes (defined as the % erythrocytes with haemoglobin concentrations >410 g/l), haemoglobin, haematocrit and erythrocyte count.

Quality of life assessments were made using the Functional Assessment of Chronic Illness Therapy-Fatigue Scale (FACIT-Fatigue).The FACIT-Fatigue was collected at Screening, Baseline (Day 1), and every 4 weeks thereafter until Week 52, or at the time of discontinuation from the study. Health economic endpoints included determination of the average number of days in the hospital per subject, number of patients who received a blood transfusion, average number of transfusions per subject, and average number of units per transfusion (for those subjects who received at least one transfusion).

A Maximum Crisis Morbidity Rank (MCMR) for each subject’s SCPCs was assessed using the (Sickle Cell) Crisis Morbidity Index (CMI), a six-point scale with a ranking from 0 to 5 based on the following: 0 – no painful crises during the study; 1 – mild to severe painful crisis requiring a clinic or emergency room (ER) visit; 2 – painful crisis requiring a hospitalization; 3 – transfusion or priapism requiring clinic or ER visit; 4 – Acute chest syndrome, hepatic sequestration, splenic sequestration, myocardial infarction, or stroke; and 5 – Death (excluding homicide, suicide, or accidental death). The MCMR for a subject was defined as the maximum value for the Crisis Morbidity Index that a subject had for all of their adjudicated SCPCs. The MCMR was developed by Icagen, Inc. (Research Triangle Park, NC, USA) in consultation with several sickle cell disease experts.

Mean senicapoc plasma concentrations were determined from blood samples collected at selected sites over the length of the 52-week Treatment Phase. Sporadic collection of plasma samples was planned to provide data for a population pharmacokinetic analysis. Senicapoc levels were measured using a high performance liquid chromatography/mass spectrometric assay developed and validated by Pharmaceutical Product Development, Inc (PPD) (Middleton, WI, USA).

Safety and tolerability

Safety assessments were performed throughout the study and included: physical examinations, vital signs (blood pressure, pulse rate, oral body temperature), clinical laboratory tests (haematology, serum chemistry, urinalysis), 12-lead ECGs and reported or observed adverse events.

Statistical analyses

A sample size of 133 patients per group was selected in order to have 90% power to detect at least a 33% difference (considered to be clinically significant) in the painful crisis rates between the active treatment and placebo groups. A target of enrolling 150 subjects per group was set to compensate for potential early patient dropout.

For the primary endpoint, differences between the two randomized treatment groups were assessed using a Mantel-Haenszel Mean-Score Chi-Square test. Differences between the randomized treatment groups with respect to the time in months to the first SCPC were analysed with Kaplan–Meier survival distributions and stratified log-rank tests. The correlation between the change from Baseline to Endpoint in each of the haematological parameters and rates of SCPC were assessed within each randomized treatment group using Spearman correlations. For quality-of-life assessments, differences in response between the two treatment groups were tested at each post-Baseline visit and Endpoint using the Mantel-Haenszel Chi-Square test. Health economic information was summarized using descriptive statistics. Statistical comparisons were made using a stratified Wilcoxon test. Differences in the MCMR between the study groups were tested using the Mantel-Haenszel Mean Score Chi-Square test. Mean plasma concentrations were summarized using descriptive statistics. A P value of <0·05 was considered significant for the primary endpoint, whilst statistical significance was set at <0·01 for secondary endpoints.

Interim analyses/study termination

A data monitoring committee (DMC) met and reviewed one interim analysis for efficacy on July 31, 2006 and recommended that patients not on concurrent HC be terminated from the study and all future patients enrolled should be on concurrent HC therapy. At a subsequent meeting on April 2, 2007, the DMC recommended termination of the trial due to the unlikelihood of achieving the primary efficacy endpoint.


Patient characteristics

Two hundred and ninety-seven patients with SCD were randomized in the study between February 2005 and April 2007. While the majority of patients resided in the United States, patients were enrolled from a total of six countries: USA, 240; Jamaica, 19; Brazil, 18; France, 10; Trinidad, seven; and the UK, three. Ninety-seven percent of patients (289/297) received at least one dose of study medication and were included in the modified intent-to-treat (ITT) population for efficacy. One patient randomized to placebo actually received senicapoc in error and this is accounted for in analysis of the safety population.

Figure 1 describes the distribution and progression of patients in the study. Fifty-six percent (163/289) of patients were in the HC cohort and 44% (126/289) were in the non-HC cohort. The difference in proportion of patients in the two groups was due to enrollment of only patients on concurrent HC following the first interim analysis by the DMC as described in the Methods section. Similar percentages of patients in the two cohorts were randomized to the placebo and senicapoc groups: 79 (48%) and 84 (51%) patients respectively, were randomized to the placebo and senicapoc groups in the HC cohort, and 65 (52%) and 61 (48%) patients respectively were randomized to the placebo and senicapoc groups in the non-HC cohort. The number of patients discontinued from the study reflects the discontinuation of the non-HC cohort following the interim analysis and subsequent early study termination.

Figure 1.

 Patient progression in the study.

The mean duration of study drug exposure was similar between the senicapoc and placebo treatment groups (placebo: 218 d; senicapoc: 227 d). More than 50% of patients overall were exposed to study medication for at least 7 months (placebo: 54%; senicapoc: 61%). More than 25% of patients completed the study and were exposed to study medication for 1 year.

Baseline characteristics of study patients are shown in Table I. Demographics and baseline characteristics for patients in the placebo and senicapoc groups were similar, as were the characteristics within the non-HC and HC cohorts (not shown) with the exception that 90% of HC cohort patients were of the HbSS genotype, while 76% of patients in the non-HC cohort were HbSS. Average patient age was 29 years old, 46% of patients were male, and 98% were black with a mean body mass index of 23·4 kg/m2. Sixty-two percent of patients (179/289) had a history of 2–4 crises and 38% of patients (110/289) had ≥5 crises that required a visit to a medical facility in the 12 months preceding the study. The distribution of these patients in the placebo and senicapoc groups is shown in Table I. Mean baseline haematological parameters were also found to be similar across the placebo and senicapoc groups.

Table I.   Patient demographics and baseline characteristics, intent-to-treat population.
ParameterSenicapoc = 145Placebo N = 144
  1. *61% of HbSS occurred in the HC cohort vs. 39% in the non-HC cohort.

  2. SCPC, sickle cell-related painful crises; LDH, lactate dehydrogenase.

Age, mean years ± SD28·5 ± 9·929·5 ± 10·5
Sex, n (%)
 Women82 (57)78 (54)
 Men63 (43)66 (46)
Race, n (%)
 Black134 (92)141 (98)
 Multiracial6 (4)3 (2)
 Caucasian3 (2)0
 Other2 (1)0
Hydroxycarbamide therapy, n (%)84 (58)79 (55)
SCPC history in past 12 months, n (%)
 2–486 (59)93 (65)
 >559 (41)51 (35)
Sickle cell disease genotype, n (%)
 HbSS*122 (84)123 (85)
 HbSC7 (5)9 (6)
 HbSβ0-thalassaemia13 (9)8 (6)
 HbSβ-thalassaemia2 (1)2 (1)
 Other1 (<1)2 (1)
Haematological parameters, mean ± SE
 Haemoglobin (g/l)90·6 ± 0·989·6 ± 1·0
 Haematocrit (%)26·95 ± 0·2826·72 ± 0·27
 Erythrocyte count (1012/l)2·90 ± 0·052·84 ± 0·05
 Reticulocyte count (%)11·07 ± 0·4110·92 ± 0·36
 Haemoglobin F (%)10·74 ± 1·269·73 ± 1·04
 Dense erythrocytes (%)7·05 ± 0·377·15 ± 0·42
 LDH (u/l)407·59 ± 13·99408·03 ± 14·34
 Indirect bilirubin (μmol/l)44·6 ± 3·448·1 ± 3·2


Frequency of sickle cell-related painful crises.  Seven hundred SCPCs (adjudicated) were identified during the conduct of the study. The CRC classified 84% of investigator-identified events as SCPCs. The majority of the SCPC in both the placebo and the senicapoc groups were categorized as pain-only crises (626, 89·4%) with much smaller numbers of acute chest syndrome (67, 9·6%), hepatic sequestration (1, 0·1%), priapism (4, 0·6%) and stroke (2, 0·3%). There were no episodes of splenic sequestration. A summary of the frequency of SCPCs is provided in Table II.

Table II.   Incidence of sickle cell-related painful crises (SCPCs) for groups combined and stratified by hydroxycarbamide (HC) therapy, intent-to-treat population.
 All patientsHC cohortNon-HC cohort
Senicapoc N = 145Placebo N = 144Senicapoc N = 84Placebo N = 79Senicapoc N = 61Placebo N = 65
N (%)N (%)N (%)
  1. All events included in this table were adjudicated by an independent, blinded, Crisis Review Committee (CRC) as described in the Methods section.

All SCPCs106 (73)89 (62)65 (77)53 (67)41 (67)36 (55)
Pain only98 (68)80 (56)60 (71)49 (62)38 (62)31 (48)
Acute chest syndrome27 (19)24 (17)19 (23)16 (20)8 (13)8 (12)
Hepatic sequestration1 (<1)0001 (2)0
Priapism2 (1)1 (<1)1 (1)01 (2)1 (2)
Stroke1 (<1)1 (<1)1 (1)001 (2)

Crises rates for the senicapoc and placebo groups within the ITT population, within the HC and non-HC cohorts, and within the stratum of baseline crisis rates are summarized in Table III. The difference in crisis rates between the senicapoc and placebo groups for the ITT population was not statistically significant (0·38 vs. 0·31, respectively, P = 0·054). Rates between treatment groups were also not statistically different within the HC cohort, but were significantly different in the non-HC cohort, where the crisis rate was higher in the senicapoc group compared to placebo (0·37 vs. 0·29, P = 0·037). Within the non-HC cohort, the crisis rate was significantly higher in the senicapoc group compared to placebo in patients with a crisis history of 2–4 (0·31 vs. 0·23, P = 0·042). For subjects with a history of five or more crises there was no difference in crises rates between the two treatment groups (P = 0·405) regardless of stratification for HC (P = 0·599) or non-HC use (P = 0·435).

Table III.   Rates of sickle cell painful crises (SCPCs) for groups combined and stratified by hydroxycarbamide (HC) therapy, intent-to-treat population.
 All PatientsHC cohortNon-HC cohort
All patients14514484796165
 Mean SCPC rate (SE)0·38 (0·03)0·31 (0·04)0·39 (0·04)0·33 (0·05)0·37 (0·05)0·29 (0·08)
Crisis history strata
 2–4 crises Hx869348503843
  Mean SCPC rate (SE)0·27 (0·04)0·21 (0·05)0·23 (0·04)0·19 (0·04)0·31 (0·07)0·23 (0·10)
 ≥5 crises Hx595136292322
  Mean SCPC rate (SE)0·56 (0·06)0·50 (0·07)0·61 (0·08)0·56 (0·09)0·47 (0·09)0·41 (0·11)

When the analysis of crises rates was limited to patients with sickle cell anaemia (genotype HbSS, n = 245), the crisis rate among patients randomized to senicapoc was significantly higher than that among patients randomized to placebo (0·41 vs. 0·30, P = 0·004) overall and for patients in the non-HC cohort (0·40 vs. 0·31, P = 0·02). There was no statistically significant difference when patients in the HC cohort were compared (0·41 vs. 0·29, P = 0·083).

Haematological endpoints. Table IV summarizes the changes from baseline at the end of the study for markers of haemolysis and erythrocyte parameters in the senicapoc and placebo groups. Statistically significant increases from baseline occurred in the senicapoc group compared to placebo for haemoglobin, haematocrit and erythrocyte counts. There were statistically significant decreases from baseline in LDH and indirect bilirubin in the senicapoc group, suggesting decreased haemolysis following treatment with senicapoc. The number and percentage of reticulocytes in the senicapoc group also decreased from baseline following treatment with senicapoc. There were significantly decreased numbers of dense erythrocytes in the senicapoc group compared to placebo. Most of the changes from baseline for the haematological parameters were statistically significant by the Day 8 visit, were maintained throughout the treatment period, and had returned to baseline levels by 8 weeks after termination of study medication. Similar differences in haematological parameters between placebo and senicapoc groups were maintained within both the HC and non-HC strata with the exception of the percentage of dense erythrocytes.

Table IV.   End of study changes from baseline in indicators of erythrocyte pathophysiology, intent-to-treat population.
 Mean change from baseline (SE)p-value
Senicapoc N = 145Placebo N = 144
Haemoglobin (g/l)5·9 (1·0)−1·0 (1·0)<0·001
Haematocrit (%)1·35 (0·28)−0·55 (0·22)<0·001
Erythrocyte count (1012/l)0·23 (0·04)−0·02 (0·04)<0·001
Reticulocyte count (%)−2·46 (0·42)0·79 (0·30)<0·001
Haemoglobin F (%)0·06 (0·74)0·82 (0·95)0·320
Dense erythrocytes (%)−0·09 (0·47)1·65 (0·40)0·003
LDH (u/l)−58·7 (10·6)13·2 (12·4)<0·001
Indirect bilirubin (μmol/l)−16·6 (2·1)−0·3 (1·7)<0·001

Other endpoints.  There were no statistically significant difference between the senicapoc and placebo treatment groups for the mean MCMR score (1·8 vs. 1·5 respectively), regardless of concurrent HC treatment. Kaplan–Meier survival distributions and stratified log-rank tests for time to first, second, or third crisis were not significantly different between groups.

While FACIT-Fatigue scores generally increased from Baseline, no statistically significant differences were observed in changes from Baseline to End of Study assessments for the senicapoc versus placebo treatment groups, either overall (1·3 vs. 2·0 respectively) or when analysed by HC or non-HC strata.

A statistically significant increase in the number of days spent in the hospital was seen in the senicapoc treatment group (18·6 vs. 15·1 d, respectively, P = 0·030). However, no differences were seen between treatment groups for other health economic parameters, such as the number of subjects transfused, number of transfusions per subject, or number of units transfused per subject.

Senicapoc plasma concentrations

Blood samples were collected from c. 60 patients receiving senicapoc. Mean senicapoc plasma concentrations obtained during the treatment phase were 137 ng/ml for all patients, 127 ng/ml for patients in the HC cohort, and 149 ng/ml for patients in the non-HC cohort. No relevant differences in plasma concentrations were seen between patients in the HC and non-HC cohorts.


Similar proportions of patients in the senicapoc (87%) and placebo groups (83%) experienced at least one treatment-emergent adverse event not related to SCD during the treatment and follow-up periods. Table V summarizes the events occurring with a frequency of at least 5% in either the senicapoc or placebo groups. Events that occurred more frequently in the senicapoc versus placebo group (>5% differential) were nausea (16% vs. 10%) and urinary tract infections (14% vs. 8%).

Table V.   Treatment-emergent adverse events* with an incidence of at least 5% in either treatment group, safety population.
 All subjects
Senicapoc N = 146Placebo N = 143
N (%)
  1. *non-SCPC related.

At least 1 event127 (87)119 (83)
Nausea23 (16)14 (10)
Urinary tract Infection21 (14)12 (8)
Headache20 (14)23 (16)
Arthralgia13 (9)7 (5)
Upper respiratory tract Infection12 (8)20 (14)
Vomiting12 (8)11 (8)
Pyrexia12 (8)15 (10)
Pneumonia11 (8)13 (9)
Back pain11 (8)10 (7)
Pain in extremity11 (8)9 (6)
Nasopharyngitis10 (7)11 (8)
Cough10 (7)6 (4)
Constipation9 (6)13 (9)
Fatigue9 (6)8 (6)
Hypokalaaemia7 (5)3 (2)
Haematuria7 (5)1 (<1)
Diarrhoea5 (3)12 (8)
Abdominal pain5 (3)7 (5)
Pharyngolaryngeal pain4 (3)10 (7)
Pruritus4 (3)7 (5)
Drug hypersensitivity2 (1)7 (5)

Nine patients (6%) in the placebo group and 12 patients (8%) in the senicapoc group dropped out of the study because of an adverse event. Sickle cell crisis-related events were the most common reason for discontinuation (one patient in the placebo and four patients in the senicapoc groups). Four patients (two each in the placebo and senicapoc groups) discontinued due to pregnancy and two (one each in the placebo and senicapoc groups) due to cerebral or cerebellar infarction. No other event resulted in more than one discontinuation.

Eighty-six patients (43/143, 30%, in the placebo group and 43/146, 29%, in the senicapoc group) experienced at least one serious adverse event other than sickle cell crisis. Pneumonia, catheter-related infections, anaemia, fever, and asthma, were the most common serious events in both treatment groups and accounted for 81% (70/86) of the events, collectively. Two deaths considered unrelated to study treatment occurred in the placebo group during follow-up assessments.

There were no clinically relevant changes in vital signs or physical examination findings between groups, and no differences between treatment groups or cohorts for any ECG parameter, including heart rate, PR interval, QRS duration, or QTc interval.

By the pre-defined limits for clinically important laboratory studies, abnormal results that were more frequent among the senicapoc group compared to the placebo recipients included elevations in: alkaline phosphatase [eight (5%) of 146 vs. two (1%) of 143], gamma-glutamyl transferase (GGT) [45 (31%) vs. 29 (20%)], haemoglobin [5 (3%) vs. 1 (<1%)] and platelets [8 (6%) vs. 6 (4%)], as shown in Table VI. The mechanism of the elevation in GGT is unknown, but was not associated with an increase in alanine transaminase (ALT) or aspartate transaminase (AST) values. The greatest mean change in GGT was 24 u/l and was reversible upon discontinuation of senicapoc. The majority of subjects in both treatment groups with clinically important GGT elevations began with Screening and/or Baseline values above the reference range. The greatest mean changes in non-fasting glucose were 1·09 mmol/l. Clinically important changes in laboratory values that were more frequent among placebo compared to the senicapoc recipients included elevated creatinine [four (3%) vs. one (<1%)], total bilirubin [four (3%) vs. two (1%)] and LDH [21 (15%) vs. 10 (7%)]. The lower frequencies of clinically important changes in total bilirubin and LDH are consistent with the postulated reduction in RBC haemolysis expected with senicapoc.

Table VI.   Clinically relevant changes in laboratory values, safety population.
  1. AST, aspartate transaminase; ALT, alanine transaminase; BUN, blood urea nitrogen; GGT, gamma-glutamyl transferase; LDH, lactate dehydrogenase; CPK, creatine phosphokinase; WBC, white blood cell count.

Laboratory parameterNumber of subjects with clinically important laboratory value
N = 146
N (%)
N = 143
N (%)
 Low: ≤2·5; mmol/l1 (<1)0
 High: >6 mmol/l2 (1)4 (3)
 High: ≥180 μmol/l1 (<1)4 (3)
 High: ≥175 u/l4 (3)3 (2)
 High: ≥125 u/l6 (4)6 (4)
Alkaline phosphatase
 High: ≥350 u/l8 (5)2 (1)
 High: ≥12·5 mmol/l1 (<1)2 (1)
Total bilirubin
 High: ≥205 μmol/l2 (1)4 (3)
 High: ≥100 u/l (female)45 (31)29 (20)
 ≥150 u/l (male)
 High: ≥750 u/l10 (7)21 (15)
 High: ≥850 u/l (female)6 (4)5 (4)
    ≥975 u/l (male)
 High: ≥130 g/l5 (3)1 (<1)
 Low: ≤2·5 × 109/l2 (1)0
 High: >30 × 109/l01 (<1)
Platelet count
 High: >1000 × 109/l8 (6)6 (4)


Gardos channel inhibition by senicapoc in patients with SCD has been shown to decrease haemolysis, the number of dense erythrocytes, and the number and percentage of reticulocytes in circulation (Brugnara et al, 1993; De Franceschi et al, 1994; Ataga et al, 2008). However, the hypothesis that these changes would lead to improvements in vaso-occlusive complications through the reduction in HbS polymerization, red cell sickling and red cell adhesion was untested. This multi-centre clinical study was undertaken to test this hypothesis and collect long-term data on the safety of senicapoc in this patient population.

Consistent with the results seen in a previous clinical study, patients in the senicapoc group of this study had improvements in anaemia and haemolysis (Ataga et al, 2008). The numbers of dense erythrocytes also decreased, indicating that cell hydration was apparently improved during senicapoc treatment. The current study demonstrated both the durability of the haematological and biochemical improvements over 52 weeks of senicapoc administration and attenuation of improvements upon cessation of treatment. These results also indicate that senicapoc had its intended effect, that of preventing dehydration of red cells, presumably by inhibition of the Gardos channel.

The observed improvements in haematology parameters following senicapoc administration, however, were not accompanied by a significant reduction in painful crises nor were there improvements in other secondary efficacy endpoints. In fact, in a subset analysis, crisis rates were greater in the senicapoc treatment group in certain strata (non-HC and having a history of 2–4 crises in the previous year, or HbSS patients) although these were secondary analyses and based on smaller numbers of patients without statistical correction for multiple analyses. The apparent increase in pain crises in these patients was unexpected because senicapoc appeared to be successful in producing improvements in their haematological parameters.

The reasons for the apparent increase in painful events in certain subpopulations of those receiving senicapoc are not obvious and are probably complex. Vaso-occlusive events in SCD are believed to be influenced by multiple factors, including leucocyte adhesion molecules, inflammatory factors, endothelial cell interactions, haemorheology and erythrocyte heterogeneity (Okpala et al, 2002; Kaul, 2008). An increase in blood viscosity resulting from the senicapoc-associated increase in haemoglobin could have paradoxically resulted in the observed increase in painful events. Indeed, increased rates of pain crisis were associated with high haematocrit levels in the Cooperative Study of Sickle Cell Disease, a natural history study of SCD in the United States (Platt et al, 1991). However, post-hoc analyses showed no statistically significant correlation between total haemoglobin or change from baseline haemoglobin and the crisis rate experienced by patients receiving senicapoc in this study.

Alternatively, there is evidence that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contribute to the processing of nociceptive signals in the spinal cord (Schmidtko et al, 2009). Multiple animal studies show that inhibition of NO or cGMP synthesis can considerably reduce both inflammatory and neuropathic pain (Schmidtko et al, 2009). The decreased haemolysis observed following treatment with senicapoc quite likely increases the availability of NO, which in turn may have contributed to the increased pain experienced by these patients. This is consistent with the recent report of increased pain crises following treatment of patients with SCD-associated pulmonary hypertension with the phosphodiesterase-5 inhibitor, sildenafil (Machado et al, 2009).

Chronic treatment with senicapoc was overall well tolerated. At the dose tested, only nausea appeared to be reasonably associated with senicapoc, but it did not appear to be a significant cause of drug discontinuation. No dose-limiting toxicity was observed and discontinuations for adverse events occurred with similar frequencies in the placebo and senicapoc groups. Small increases in GGT levels that occurred were not associated with liver toxicity, and normalized upon cessation of senicapoc treatment. The mechanism of the elevation in GGT is unknown, but was not associated with an increase in ALT or AST levels. These changes in GGT are, therefore, unlikely to be of clinical significance. Similarly, the changes in blood glucose over the course of the study were erratic, based on non-fasting sampling, and unlikely to be of clinical significance.

Given the long half-life of senicapoc (12–14 d), the dosing regimen consisted of loading and maintenance doses designed to ensure that subjects rapidly achieved steady-state senicapoc plasma concentrations and maintained concentrations to maximize pharmacological effects. Senicapoc plasma concentrations measured at various times during the study demonstrated that the regimen achieved these goals because senicapoc concentrations exceeded those that were measured in the Phase II study (i.e., 100 ng/ml) in which nearly complete blockade of the Gardos channel was achieved (Ataga et al, 2008). The increase in average concentration may be due to the improved bioavailability of the senicapoc oral formulation, which was modified following the phase II study.

In summary, senicapoc did not improve the rate of SCPCs in patients with SCD, despite significant improvements in laboratory parameters. Further study is needed to understand if and how improvements in haematological characteristics induced by senicapoc can modulate other clinical outcomes in SCD patients.


This study was funded by Icagen, Inc., Research Triangle Park, NC. Statistical computations and analyses were performed by Stacy Woodard, Ph.D., who was employed by Quintiles Transnational Corporation. Patrice Ferriola, Ph.D. provided assistance in preparation and editing of the manuscript and was supported by Icagen, Inc. All authors had access to the to the primary clinical trial data. All investigators and clinical site support personnel are listed in the Appendix I.

Authors contribution

K.I.A., J.H.H. and J.W.S. participated in the study design. K.I.A., M.V., S.K.B. and J.W.S. participated in the writing and review of the manuscript. All listed authors, with the exception of J.W.S. and J.H.H., were significant clinical contributors to the trial. J.H.H. and J.W.S. were involved in all aspects of trial conduct including analysis of the data.

Conflict of interest disclosure

J.H.H. and J.W.S. are (were) employed by Icagen, Inc. and own equity in Icagen. All remaining authors declare no competing financial interest other than research funding for the study.


Appendix I

Clinical investigators and support personnel by study site

Nik Abdul-Rachid, MD
Jonathan Bernstein, MD
Ronald Kline, MD
Arlene Bayreder, RN, PNP
Jennifer Buitrago, RN, PNP
Comprehensive Cancer Centers of Nevada, Las Vegas, NV, USA
Patricia Adams-Graves, MD
Marion Dugdale, MD
University of Tennessee Medical Group Memphis, TN, USA
Kenneth Ataga, MD
Eugene P. Orringer, MD
Susan K. Jones, RN
Dell H. Strayhorn, RN, MPH, FNP-C
University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Moji Asogbade, MD
Swee-Lay Thein, MD
Kings College Hospital, London, England
Samir Ballas, MD
Monique McRay
Margaret Lusardi, RN, BA
Thomas Jefferson University, Philadelphia, PA, USA
Jerry Barbosa, MD
Nanette Grana, MD
Claudia Lukas, PA-C
Irmel Ayala, MD
Teri Vrchoticky, PharmD
Karol Kerr, MD
Lele Aung, MD
Pediatric Physicians Services St. Petersburg, FL, USA
Rita Bellevue, ND
Jean D’Augustine, MD
New York Methodist Hospital, Brooklyn, NY, USA
Lennette Nejamin, MD
Gwendolyn I. Swinson, RN
Barbara McMillion, ANP
Montefiore Medical Center, Bronx, NY, USA
Joel Bessman, MD
Robert P. Whitehead, MD
Kausar Suleman, MD
Maurice Willis, MD
Linda Valencia-Stephens, RN
Phyllis Crawford, RN, BSN,
San Juana C. Hirsch, RN
University of Texas Medical Branch at Galveston, Galveston, TX, USA
Carolyn Bigelow, MD
Vincent Herrin, MD
Robert Darryl Hamilton, MD
University of Mississippi Medical Center, Jackson, MS, USA
Morey Blinder, MD
Joshua Field, MD
Washington University School of Medicine, St. Louis, MO, USA
Timothy Carlos, MD
Robert Redner, MD
Oakland Veterans Affairs Medical Center, Oakland, CA, USA
Kenneth Charles, MD
Terence A. Seemungal, MD
Niall Leelah, MD
Titilayo A. Ajayi, MD
The General Hospital Trinidad, Port of Spain, Trinidad
James CHingos, MD
Dat C Pham, MD
Ru-Amir Walker, MD
Fauzia N. Rana, MD
Joseph Mignone, MD
Augusto E. Villegas, MD
University of Florida Health Science Center at Jacksonville, Jacksonville, FL, USA
Alice Cohen, MD
Maya Sha, MD
Elois Rogers-Phillips, MD
Indu Sabnani, MD
Newark Beth Israel Medical Center, Newark, NJ, USA
Julia Cruz, MD
David Leedy, PAC
Fraqncis O’Brien, MD
Jennifer MacLean, RN
Wake Forest University Health Sciences, Wake Forest, NC, USA
Charles Daeschner, MD
Mauro Grossi, MD
Pediatric Hematology/Oncology East Carolina University School of Medicine, Greenville, NC, USA
Carlton Dampier, MD
Lori Luck, MD
Nkechi Mba, MD
Maureen Meier, RN, CCRC,
Maureen Rockstein, RN, CCRC
Michele Cahill, RN
Amy Moore, RN, BSN
St. Christopher’s Hospital for Children, Philadelphia, PA, USA
Laura DeCastro, MD
Melissa D. Hall, NP
Jude C. Jonassaint, RN
Sherri Zimmerman, MD
Charles Greenberg, MD
Daphne Friedman, MD
Jacob Laubach, MD
Nicole Whitlatch, MD
John R. Pawloski, MD
Carey Anders, MD
Mark Lanasa, MD, PhD
Jeanne Palmer, MD
Yosuf Zafar, MD
Karen Haith, GNP
Duke University Health System, Durham, NC, USA
Maria Stella Figueiredo, MD, PhD
Faustine Moreira Neto
Rita de Cássia Rosario Cavalheiro, MD
Perla Vicari, MD
Universidade Federal de São Paulo, São Paulo –SP, Brazil
Francis Flug, MD
Burton Appel, MD
Steven Diamond, MD
John Gregory, MD
Steven Halpern, MD
Paul Harlow, MD
Michael B. Harris, MD
Cindy S. Steele, MD
Bruce Terrin, MD
Tomorrow’s Children’s Institute, Hackensack, NJ, USA
Frédéric Galacteros, MD
Habibi Anoosha, MD
Balleyguier Marie-Odette, MD
Bachir Dora, MD
Hôpital Henri Mondsor, Creteil, France
Kamar Godder, MD
Scott Gullquist MD
Nancy Lynn Dunn, MD
Gita Vasers Massey, MD
Joseph Laver, MD
Asadullah Khan, MD
Elizabeth Hall, FNP,
Ann Mauck, FNP
Debra Shockey, CPNP
Robbie Casper, FNP-C
Bernadette Temple, PNP
Anne Mauck, FNP
Edward Clifton Russell, MD
Thomas Brunner, FNP
Virginia Commonwealth University, Richmond, VA, USA
Anne Greist, MD
Amy Shapiro, MD
Randall Riley, MD
Kimberly Hummel, PA
Asmar Muhammad, PA
Lorrie Miller-Rice, MD
Rekha Parameswaran, MD
Indiana Hemophilia & Thrombosis Center, Indianapolis, IN, USA
Edouard Guillaume, MD
Eric Jaffe, MD
Claudia Wilson, MD
Interfaith Medical Center, Brooklyn, NY, USA
Ward Hagar, MD
Kathryn Stewart
Christie Hoehner, RN
Sandie Edwards, CCRP
Lori Appel Styles, MD
Children’s Hospital & Research Center at Oakland, Oakland, CA, USA
Nathan Hagston, MD
Nehal Parikh, MD
Arnold Altman, MD
Eileen Gillan, MD
Katherine Steven, APRN
Emily Peluso, APRN
Connecticut Children’s Medical Center, Hartford, CT, USA
Jonathon Harrison, MD
Parvin Saidi, MD
Claire Philipp, MD
Shelonitada Rose, MD
Lisa Michaels, MD
Richard Drachtman, MD
University of Medicine and Dentistry of New Jersey, New Brunswick, NJ, USA
Kathryn Hassel, MD
Rachelle Nuss, MD
University of Colorado School of Medicine, Denver, CO, USA
Johnson Haynes, MD
Ardie Pack-Mabien, MSN, CRNP
University of South Alabama, Mobile, AL, USA
Atif Hussein, MD
Lanetta Bronte, MD
James Cohen, MD
Raja Mudad, MD
Maria Restrepo, MD
Stephanie Bankston, ARNP
Robert Glasser, MD
Mark Steven Lewis, MD
Robert Cano, MD
Sandra Franco, MD
Daren Grosman, MD
Anna Abraham, MD
Gabriel Domenech, MD
Alejandra T. Perez, MD
Memorial Cancer Institute, Hollywood, FL, USA
Sherron Jackson, MD
Mary Ellen Cavalier, MD
Debbie Disco, RN, CPNP
Jessica Peterson, RN
Elizabeth Rackoff, RN
Shayla Bergman, MD
Shannon Johnson, RN
Ellen Debenham, RN
Medical University of South Carolina, Charleston, SC, USA
Kajed Jeroudi, MD
Margaret Ann Springer, MD
Lora Todd, RN
Pat F. Bass III, MD
Louisiana State University Health Sciences Center, Shreveport, LA, USA
Clinton Joiner, MD
Vinod Balsa, MD
Karen Kalinyak, MD
Ralph Gruppo, MD
Punam Malik, MD
Joseph Palumbo, MD
Theodosia Kalfa, MD, PhD
Denise Lagory, RPH
Tammy Nordheim, RN
Vernocia Johnson, BAGS
Aemita Terry, RN, BSN,
Darice Wilson, RN, BSN
Matthew Hodgson, MA
Patricia Boyd, RN
Lana Hackworth, RN
Patrick Kelly, MD
Franklin Smith, MD
Andrea Norman, RN, BSN
Donyiella Godfrey
Edward Chan, MD
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Harinder Juneja, MD
David Ellent, MD
Deborah Brown, MD
Kenneth K. Wu, MD, PhD
Dipak Ghelani, MD
The University of Texas Medcical School, Houston, TX, USA
Adetola Kassim, MD
Laura Winslow, NP
David Nilson, RN
Steve Klintworth, RN
The Vanderbilt Clinic, Nashville, TN, USA
Charles Knupp, MD
Darla Liles, MD
East Carolina University at Brody School of Medicine, Greenville, NC, USA
Lakshmanan Krishnamurti, MD
Sriya W.C. Gunawardena, MD
Beth A. Windsor, MSN, CRNP,
Allison A. Sakara, MSN, CRNP
Charyl L. Lindsey, MSN, CRNP, Study Coordinator
Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA
Abdullah Kutlar, MD
Leigh Wells, MSN
Lisa Daitch, PA-C
Kathleen McKie, MD
Virgil McKie, MD
Kavita Natarajan, MD
Medical College of Georgia, Augusta, GA, USA
Sophie Lanzkron, MD
Mandy David, PA-C
Yvette Roane, PA-C
John’s Hopkins University Medical Center, Baltimore, MD, USA
Clarisse Lopes de Castro Lobo, MD
Patricia Gomes Moura, MD
Elizabeth da Costa Ribeiro Cerqueira, MD
Ana Maria Mach Queiroz, MD
Ana Paula de Almeida Queiroz, PharmD
Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti – HEMORIO, Frio de Janeiro – RJ, Brazil
Thomas Loew, MD
Jorge DiPaola, MD
Frederick Goldman, MD
M. Sue O’Dorisio, MD PhD
Mohamed Radhi, MD
Raymond Tannous, MD
Rajeev Vibhakar, MD
Raymond Hohl, MD
Myrl Holida, PA-C
Sharron McMillan, RN
University of Iowa Hospitals and Clinics
Iowa City, Iowa, USA
Ronald McCaffrey, MD
W. Hallowell Churchill, MD
Maureen M. Okam, MD
Elyse Mandell APRN, BC
Manuel Souza APRN, BC
Jean M. Connors, MD
Brigham and Women’s Hospital, Boston, MA, USA
Scott Miller, MD
Kathy Rey, PAC
Sreedhar P. Rao, MD
SUNY-Brooklyn, Brooklyn, NY, USA
Reginald Moore, MD
Anne-Marie Langevin, MD
Shannon Carpenter, MD
Howard Britton, MD
Shafqat Shah, MD
The Universwity of Texas Health Science Center at San Antonio, San Antonio, TX, USA
Brigitta Mueller, MD
Donald H. Mahoney, MD
Kenneth McClain, MD
Gladstone, Airewele, MD
Rosalind Bryant, NPN-LPA
Texas Children’s Hospital, Houston, TX, USA
Onyinye Onyekwere, MD Christopher DeGannes, MD
Oswaldo Castro, MD
Howard University Hospital, Washington, DC, USA
Athena Pefkarou, MD
Enrique Alberto Escalon, MD
Roberto Warman, MD
Ziad Ahmad Khatib, MD
John Allen Fort, MD
Miami Children’s Hospital, Miami, FL, USA
Marvin Reid, MD
Colette A. Cunningham-Myrie, MD
Vanessa Cumming, MD
Monika Parshad-Asnani, MD
Jennifer Knight-Madden, MD
Wendy Madden
Norma Lewis
Tropical Medicine Research Institute, University of the West Indies, Mona, Kingston 7, Jamaica
Suzanne Saccente, MD
David Becton, MD
Kimo C. Stein, MD
Robert L. Saylors, MD
Arkansas Children’s Hospital, Little Rock, AR, USA
Sharada Samaik, MD
Wanda J. Whitten-Shurney, MD
Meera Chitlur, MD
Kristin Strother, RN, CNP
Ordella G. Henderson, RN, CNP
Children’s Hospital of Michigan, Detroit, MI, USA
Yogen Saunthararajah, MD
Richard Labotka, MD
Robert Molokie, MD
Santosh Saraf, MD
Seema Sidhwani, MD
Michael Gowhari, MD
University of Illinois at Chicago, Chicago, IL, USA
Charles Scher, MD
Marc Jeffrey Kahn, MD
Charles Stanley Hemenway, MD
Marta Kligerman Rozans, MD
Marshall A. Schorin, MD
Tulane University Health Sciences Center, New Orleans, LA, USA
Gary Schilelr, MD
Mary Carol Territo, MD
Ronald Paquette, MD
UCLA Division of Hematology-Oncology, Los Angeles, CA, USA
Lucia Mariano da Rocha Silla, MD
Laura Maria Fogliatto, MD
Joao Ricardo Friedrisch, MD
Denise Silveira Lehugteur, MD
Hospital das Clinicals, Porto Alegre - RS, Brazil
Wally Smith, MD
Imoigele Aiusiku, MD
Larry White, CCRC
Medical College of Virginia/Virginia Commonwealth University, Richmond, VA, USA
Kim Smith-Whitley, MD
Janet Kwiatkowski, MD
Kwaku Ohene-Frempong, MD
Children’s Hospital of Philadelphia, Philadelphia, PA, USA
William Solomon, MD
Peter Gillette, MD
Sarah Egan, MD
Boriana Kamenova, MD
SUNY Downsgtate Medical Center, Brooklyn, NY, USA
Luther St. James III, MD
Carol Tanner-St. James, MD
Loray Blair-Britt, MD
Charles C. Nesbitt, RN, ARNP
Rataf Shafik Iskander, PA
Nancy Mather, ARNP-C
Twin Lakes Family Practice, Holly Hill, FL, USA
Lori Styles, MD
Kathryn Stewart
Mary GTruskier, PNP
Sandie Edwards, CCRP
Robert Ward Hagar, MD
Oakland Children’s Hospital & Research Center, Oakland, CA, USA
Uma Subramanian–Srinivsan, MDUniversity of IllinoisChicago, IL, USA
Paul Swerdlow, MD
Jesus Ortega, MD
Lisabette Littsey, MSN, RN-C
Arthur R. Jett, PA
Hudson-Weber Cancer Research Center, etroit, MI, USA
Cameron Tebbi, MD
Tuyng Wynn, MD
Dana Obzut, MD
Hans-Cristoph Rossbach, MD
St. Joseph’s Children’s Hospital of Tampa, Tampa, FL, USA
Paul Telfer, MD
Banu Kaya, MD
Royal London Hospital, London E1 1BB
Mark Uddon, MDBen Taub General Hospital, Houston, TX, USA
Trib Vats, MD
Cynthia E. Gonzales, MD
Lawrence S. Frankel, MD
Backus Children’s Hospital, Savannah, GA, USA
Anthony Villella, MD
Jeffrey L. Blumer, PhD, MD
Brian W. Berman MD
Joseph Gibbons, MD
Eloise Lemon, RN
University Hospitals Case Medical Center, Cleveland, OH, USA
Kusum Viswanathan, MD
Donna Boruchov, MD
Maged Khalil, MD
Madhumati R. Kalavar, MD
Seema Naik, MD
Theresa Liu Dumlao, MD
Alka Arora, MD
Brookdale University Hospital and Medical Center, Brooklyn, NY, USA
Rajasekharan Warrier, MD
Renee Gardner, MD
Lolie Yu, MD
Maria Velez, MD
Tammuella Singleton, MD
Louisiana State University Health Sciences Center, New Orleans, LA, USA
Steven Wolff, MD
Lateesa Posey, APRN, BC
Meharry Medical College, Nashville, TN, USA
Gerald Woods, MD
Kathleen Neville, MD
Susan Sarcone, RN
Kristin Stegenga, RN
Julie Routhieaux, RN
Greta Roath
Children’s Mercy Hospital, Kansas City, MO, USA
Louis Wright, MD
Sumeet Bhatia, MBBS, MD
Adult Primary Care Associates, Group Practice, Indianapolis, IN, USA
Theodore Wun, MDUniversity of California, Davis Cancer Center, Sacramento, CA, USA
Zahida Yasin, MD
Jane Pruemer, PharmD
Joseph Palascak, MD
Amit Gupta, MD
John Pancoast, MD
Zeina Naheleh, MD
Abdul-Rahman Jazieh, MD
Barrett Cancer Center, Cincinnati, OH, USA
Anaadriana Zakarija, MD
David Green, MD
Claude Herman, PA
Gerald Soff, MD
Northwestern University Feinberg School of Medicine, Chicago, IL, USA