These authors contributed equally to this work.
Phase I study of eptifibatide in patients with sickle cell anaemia
Version of Record online: 3 OCT 2007
British Journal of Haematology
Volume 139, Issue 4, pages 612–620, November 2007
How to Cite
Lee, S. P., Ataga, K. I., Zayed, M., Manganello, J. M., Orringer, E. P., Phillips, D. R. and Parise, L. V. (2007), Phase I study of eptifibatide in patients with sickle cell anaemia. British Journal of Haematology, 139: 612–620. doi: 10.1111/j.1365-2141.2007.06787.x
- Issue online: 8 OCT 2007
- Version of Record online: 3 OCT 2007
- Received 11 May 2007; accepted for publication 18 June 2007
- sickle cell anaemia;
- CD40 ligand;
The αIIbβ3 antagonist eptifibatide is an effective treatment for patients with acute coronary syndromes (ACS). Platelet reactivity and CD40 ligand (CD40L) may play a role in the pathophysiology of sickle cell anaemia (SCA) similar to that in ACS, suggesting that inhibition of platelet aggregation and CD40L release by eptifibatide may benefit patients with SCA. Following eptifibatide infusion, safety and pharmacodynamic data were obtained from four SCA patients in their non-crisis, steady states. Eptifibatide was well tolerated, with no adverse changes in the haematological, biochemical or coagulation parameters studied. Eptifibatide did not increase plasma levels of platelet factor 4 or beta-thromboglobulin, P-selectin exposure or platelet:leucocyte aggregate formation. Moreover, decreases in platelet aggregation and soluble CD40L (sCD40L) levels achieved in SCA patients were comparable to those observed in the treatment of ACS. Finally, indicators of inflammation, macrophage inflammatory protein-1α, tumour necrosis factor-α and myoglobin were reduced following eptifibatide infusion, while vasodilation correlatives, matrix metalloproteinases (MMP-2 and MMP-9) and leptin were increased. Based on these phase I results, eptifibatide may benefit SCA patients by inhibiting platelet aggregation, decreasing sCD40L levels and favourably altering plasma levels of inflammatory mediators.
Sickle cell anaemia (SCA) is characterized by a hypercoagulable state of multifactorial aetiology (Ataga & Orringer, 2003). In these patients, vaso-occlusive phenomena of both red blood cells (RBC) and leucocytes (Hebbel et al, 2004) are compounded by ischaemia-reperfusion injury (Solovey et al, 2004), elevated leucocyte counts, abnormal activation of granulocytes and monocytes (Belcher et al, 2000; Platt, 2000), and increased levels of multiple inflammatory mediators (Hebbel et al, 2004). As a result, SCA is increasingly referred to as a chronic inflammatory disease. Concurrently, activated platelets are thought to contribute to the pathophysiology of SCA via P-selectin exposure (Matsui et al, 2001), endothelial activation (Marcondes et al, 2000) and aggregate formation (Palabrica et al, 1992). Platelet-released factors increase RBC adhesivity (Brittain et al, 2001), promote coagulation (Dale et al, 2002) and increase vasoconstriction (Frishman & Grewall, 2000). Furthermore, activated platelets expose and release the tumour necrosis factor (TNF) family member, CD40 ligand (CD40L) (Henn et al, 1998), which is capable of mediating a broad range of immune and inflammatory responses (Schonbeck & Libby, 2001). As SCA is characterized by both chronic inflammation (Platt, 2000) and hypercoagulability (Berney et al, 1992), platelet CD40L may contribute to the pathophysiology of SCA as it does in other vascular diseases (Schonbeck et al, 2000). Our previous findings, that elevated levels of platelet-derived soluble CD40L (sCD40L) in plasma contribute to the upregulation of tissue factor (TF), endothelial cell expression of adhesion molecules and B-cell proliferation in patients with SCA (Lee et al, 2006), strongly suggests that the inhibition of platelet activity and sCD40L release may be an effective treatment for certain SCA-related complications.
The integrin, αIIbβ3 is critical for platelet aggregation, adhesion, granule secretion and platelet-induced procoagulant activity (Phillips & Agin, 1977). Blockade of αIIbβ3 inhibits thrombotic vascular occlusion (Savage et al, 1996), prothrombin activation (Byzova & Plow, 1997) and platelet release of inflammatory CD40L (Nannizzi-Alaimo et al, 2003). Eptifibatide (Integrilin®), an antiplatelet agent, was shown to effectively treat acute coronary syndromes (ACS) by αIIbβ3 inhibition (Tcheng, 1997). By blocking αIIbβ3 and decreasing the release of platelet CD40L (Nannizzi-Alaimo et al, 2003), eptifibatide may be beneficial in the treatment of certain SCA-related complications by reducing the pro-inflammatory, adhesive, and hypercoaguable states present in these patients. The phase I study described here was performed to assess the safety, ex vivo platelet function and inflammatory profile following the administration of eptifibatide to SCA patients.
Approval for this study was obtained from the Committee on the Protection of the Rights of Human Subjects at the University of North Carolina-Chapel Hill, with informed consent provided according to the Declaration of Helsinki. Study participants were eligible if they met the following criteria: (i) adults (age 18–50 years) with a confirmed diagnosis of SCA; (ii) had no history of acute vaso-occlusive events requiring hospitalization during the preceding 6 weeks; (iii) had clinically acceptable values for haematology, chemistry, urinalysis and electrocardiogram for a patient with SCA; (iv) had a negative pregnancy test, if female; (v) had a clinically acceptable physical examination; (vi) had no evidence of infiltrates on a chest x-ray; and (vii) weighed ≤100 kg. Patients were excluded from participating in the study if: (i) they were pregnant or breastfeeding; (ii) had laboratory values that indicated major organ dysfunction (e.g. serum creatinine >176·8 μmol/l, aspartate transaminase and/or alanine transaminase >3 times normal); (iii) had received a RBC transfusion within the previous 3 months; (iv) had a history of clinically significant active cardiovascular, neurological, endocrine, hepatic or renal disorder; (v) had uncontrolled hypertension; (vi) had a previous hemorrhagic stroke; (vii) had a history of recent illicit drug or alcohol abuse; (viii) had been exposed to any investigational drug within the preceding 6 weeks; (ix) were on chronic anticoagulation therapy; and (x) were on aspirin, non-steroidal anti-inflammatory drugs or other anti-platelet medications.
Four patients were enrolled onto this open-label phase I study. All of the patients were male, and they were studied while in their non-crisis, steady states. The median age was 31·5 years (range: 20–34 years). Patients 1, 3 and 4 were on hydroxycarbamide (also known as hydroxyurea) at the time of evaluation.
To evaluate the safety and pharmacodynamics of eptifibatide, the study design was divided into four phases: a Screening Phase; a Treatment Phase; a Post-Treatment Phase; and a Follow-up Phase. Eligible patients were admitted to the General Clinical Research Center (GCRC) 1 d prior to study drug administration. On the day of administration of the study drug, two baseline samples for platelet aggregation and sCD40L were obtained using a 21-gauge needle. Beginning immediately thereafter, each patient received two 180 μg/kg boluses of eptifibatide 10 min apart, followed immediately by a continuous infusion at 2 μg/kg/min for 6 h. Throughout this treatment phase, safety assessments were obtained by monitoring vital signs, clinical laboratory test results (complete blood counts, prothrombin time/activated partial thromboplastin time (PT/PTT), renal and liver function tests), and any observed or reported adverse events. The post-treatment phase began immediately after the eptifibatide infusion was completed and lasted for 24 h. ‘Post’ refers to measurements taken immediately after the 6 h infusion. Seven days after completion of the study drug infusion, each patient returned as an outpatient for the follow-up phase, which involved a thorough history and physical examination, a detailed assessment for adverse events, clinical laboratory safety tests, and final plasma samples for pharmacodynamic analyses.
The pharmacodynamics of eptifibatide in this patient population were analysed by performing platelet aggregation studies on platelet-rich plasma samples taken prior to the study drug infusion, immediately following discontinuation of the drug, 24-h after commencement of the infusion and at the 7-d postinfusion follow-up. For all platelet aggregation studies, the blood samples were drawn into 1·2 mmol d-phenylalanyl-l-prolyl-l-arginyl chloromethyl ketone, rested for 15 min at 37°C, then centrifuged at 200 g for 15 min before the platelets were removed for use. Aggregation was then measured in platelet-rich plasma by an optical aggregometer (Chrono-Log, Havertown, PA, USA) using autologous platelet-poor plasma as a reference and either adenosine diphosphate (ADP) or thrombin receptor activating peptide (TRAP) (SFLLRN) as agonists.
CD40L, beta-thromboglobulin and platelet factor 4 measurement.
Peripheral blood samples were collected by venepuncture from the antecubital vein via a 21-gauge needle into tubes containing 0·13 M sodium citrate anticoagulant. To separate plasma from blood cells, samples were centrifuged at 200 g. Prostaglandin (PGI2, 1 U/ml) was added to plasma before preparing platelet-poor plasma by removing platelets following centrifugation at 750 g, and microparticles following centrifugation at 16 000 g. Each centrifugation was 15 min in duration and was preceded by a 15–30 min rest of platelets at 37°C. According to the manufacturer's recommendations (see below), a fibrin clot was formed by the addition of 1U thrombin/ml to platelet-free plasma. The resulting defibrinated plasma was stored at −80°C until further analysis. Soluble CD40L, beta-thromboglobulin (βTG) and platelet factor (PF4) levels were measured in thawed defibrinated plasma with enzyme-linked immunosorbent assay kits (Alexis Biochemicals, San Diego, CA, USA and American Diagnostica, Greenwich, CT, USA respectively). All the assays relied on antibody capture techniques and horseradish peroxidase conjugates. Reactions were developed using a tetramethylbenzidine developing solution.
Plasma levels of inflammatory cytokines were measured using luminex multi-analyte profiling (MAP) technology (Oliver et al, 1998). Briefly, dual lasers identified specific fluorescent microspheres and enabled quantification of the target analyte in human plasma samples. The assays were performed by Rules Based Medicine (Houston, TX, USA). Measurements were obtained at baseline and immediately following the discontinuation of the study drug.
Platelet exposure of P-selectin and CD40L was measured by whole blood flow cytometry as described previously (Michelson, 2000). Briefly, 5 μl of whole blood sample obtained from 0·13 mol/l sodium citrate anticoagulated blood was incubated with either fluorescein isothiocyanate-conjugated anti-CD62P and phycoerythrin (PE)-conjugated anti-CD40L or appropriate fluorophore- and isotype-matched control antibodies (BD PharMingen, San Diego, CA, USA). Samples were incubated at room temperature for 30 min, fixed with 0·7% paraformaldehyde, and read by a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Platelets were selected based on characteristic forward- and side-scatter profiles.
The mean differences and corresponding 95% confidence intervals in platelet aggregation were analysed by one-way analysis of variance (anova), with Bonferroni's multiple comparison post-test using GraphPad Prism 4 for Windows, GraphPad Software (San Diego, CA, USA) (Fig 1A–D). P-values <0·05 were considered significant.
Eptifibatide was well tolerated by the study subjects. None of the patients experienced any acute pain episodes or other side effects that were thought to be secondary to the administration of eptifibatide. One subject complained of a sore throat and a non-productive cough on the day 7 follow-up visit that was thought to be because of a viral upper respiratory tract infection. There were no bleeding manifestations or clinically meaningful changes in any of the safety laboratory studies (haematological, biochemical or coagulation parameters) that were evaluated before, during and following eptifibatide infusion (Table I).
|Patient||WBC (×109/l)||Haemoglobin (g/l)||Platelets (×109/l)||PT/INR (s)||PTT (s)||Na (mmol/l)||K (mmol/l)||BUN (mmol/l)||Creatinine (μmol/l)||Bilirubin (μmol/l)||AST (U/l)||ALT (U/l)|
Eptifibatide inhibits platelet aggregation in SCA patients
The inhibition of ex vivo platelet aggregation by eptifibatide was examined in all four patients. Platelet response to ADP and TRAP was measured at baseline, postinfusion (immediately following eptifibatide infusion), 24-h, and 7-d posteptifibatide infusion. Eptifibatide significantly inhibited platelet aggregation in all of the patients (Fig 1). ADP-induced platelet aggregation was inhibited by approximately 90% immediately following eptifibatide infusion (6-h after the start of infusion, Fig 1A). Aggregation induced by varying concentrations of TRAP was inhibited by approximately 46–67% (Fig 1B–D). Aggregation returned to normal at the 24-h time point for all agonists, indicating that eptifibatide is of short duration in these patients as in other patient populations.
Platelet reactivity and platelet:leucocyte aggregates in SCA plasma are unaffected by eptifibatide infusion
The platelet activation markers, βTG and PF4, were measured to determine if eptifibatide occupation of αIIbβ3 resulted in ‘outside-in’ signalling that increased platelet granule release in study subjects. Neither βTG nor PF4 were increased following eptifibatide infusion (Fig 2A). Furthermore, surface expression of platelet P-selectin remained unchanged in the subjects following the infusion of eptifibatide (Fig 2B). Blood cells simultaneously positive for the leucocyte marker CD45 and the platelet marker glycoprotein IX were analysed by whole blood flow cytometry to determine the amount of platelet:leucocyte aggregates in SCA patient samples. The high degree of platelet:leucocyte aggregates found in the blood of the study patients was not increased following infusion of eptifibatide, remaining nearly constant for all time points measured (Fig 2C). These results suggest that the already high levels of platelet activation and platelet:leucocyte aggregates that exist in SCA are not further increased following eptifibatide infusion.
Elevated CD40L in SCA plasma is reduced by eptifibatide infusion
Immediately following eptifibatide infusion, sCD40L levels were decreased in three of the four patients studied compared with baseline values, but remained unchanged for one patient (Fig 3). On average, sCD40L levels were reduced by approximately 35% in those patients whose plasma sCD40L were decreased following eptifibatide infusion (Fig 3B).
Profile of inflammatory indicators in SCA plasma is altered by eptifibatide infusion
Plasma from the study subjects was assayed for expression of indicators of inflammation before and after eptifibatide infusion. Myoglobin, a marker of muscle damage, was reduced by an average of 35%, macrophage inflammatory protein-1α (MIP-1α) expression was reduced by 37%, and TNFα was reduced by 32% (Fig 4A). Conversely, eptifibatide infusion increased levels of the matrix metalloproteinases, MMP-2 and MMP-9, by an average of 34% and 81% respectively (Fig 4B). The adipokine, leptin was similarly increased by an average of 70% in plasma from study patients following eptifibatide treatment (Fig 4C).
Sickle cell anaemia is a chronic inflammatory state (Hebbel et al, 2004). Patients with SCA and related hemoglobinopathies exhibit increased thrombin generation, abnormal activation of fibrinolysis, increased platelet activation and decreased levels of anticoagulant proteins (Ataga & Orringer, 2003). These changes, combined with increased TF procoagulant activity and TF-expressing endothelial cells and microparticles (Key et al, 1998; Shet et al, 2003), contribute to the development of a hypercoagulable state in these patients. The aetiology of this hypercoagulable state is multifactorial, with contributions from an abnormal RBC membrane phospholipid asymmetry (Kuypers et al, 1996; Setty et al, 2000), adherence of sickle RBCs to vascular endothelium (Hebbel et al, 2004), and ischaemia-reperfusion injury (Solovey et al, 2004).
Platelet activation (Papadimitriou et al, 1993) and the subsequent release of sCD40L characterizes the vascular pathogenesis of SCA just as it does in ACS (Lee et al, 1999). sCD40L levels are increased 30-fold in SCA (Lee et al, 2006) compared with the less than onefold elevation in ACS (Heeschen et al, 2003). Antagonism of the platelet integrin, αIIbβ3, by eptifibatide is safe and effective for the treatment of ACS (Curran & Keating, 2005), with patients benefiting from the inhibition of platelet aggregation and sCD40L release (Nannizzi-Alaimo et al, 2003; Furman et al, 2004; Welt et al, 2004). Platelet antagonism and the subsequent inhibition of sCD40L release is also beneficial to patients with coronary artery disease, as shown using aspirin with the thienopyridine and P2Y12 receptor antagonist, Clopidogrel (Azar et al, 2006; Yip et al, 2006). As higher levels of sCD40L are predictive of vascular inflammation and future cardiovascular events (Schonbeck et al, 2001; Prasad et al, 2003; Nylaende et al, 2006), treatments that decrease levels of sCD40L may reduce such vascular inflammation and adverse clinical events observed in patients with SCA.
Platelet granule release, P-selectin exposure and platelet:leucocyte aggregation have been reported to occur both in vitro and in vivo following αIIbβ3 inhibition by eptifibatide (Scholz et al, 2002; Keating et al, 2006). However, these results were not reproduced in other studies of αIIbβ3 antagonists (Massberg et al, 2003; Furman et al, 2005). Despite these conflicting data on platelet activation following αIIbβ3 inhibition, treatment of ACS patients with eptifibatide results in improved clinical outcomes (Tcheng et al, 1995; Tcheng, 1997). In the current study, eptifibatide was well tolerated by SCA patients. No adverse events were reported that were attributed to eptifibatide and there were no adverse changes in any of the measured haematological, biochemical or coagulation parameters. Of particular importance, no patients experienced acute pain episodes during the course of the study, an adverse event that might arise with increased platelet reactivity and platelet:leucocyte aggregate formation. Furthermore, the patients did not exhibit increased platelet reactivity following treatment with eptifibatide, as βTG and PF4 levels, as well as P-selectin exposure and platelet:leucocyte aggregates remained relatively constant for all time points studied. In addition, all of the patients exhibited the expected inhibition of platelet aggregation immediately following the eptifibatide infusion with no evidence of undesirable platelet activity.
The dramatic, reversible inhibition of platelet aggregation achieved in SCA patients as a result of eptifibatide antagonism of αIIbβ3 demonstrates potentially beneficial pharmacodynamics of the drug in this patient population. Despite conflicting reports regarding platelet survival, SCA patients in their non-crisis steady state appear to exhibit increased platelet aggregation, a finding perhaps due to the increased number of larger, and presumably younger circulating platelets, and increased plasma levels of platelet agonists such as thrombin, ADP and epinephrine (Ataga & Orringer, 2003). Although pharmacodynamic studies of eptifibatide have largely focused on its short-acting, reversible decrease in platelet aggregation, benefits to ACS patients receiving eptifibatide are significant up to a year following the treatment (O'Shea et al, 2002; Puma et al, 2006). Therefore, comparable short-term pharmacodynamic activity in SCA patients may similarly have lasting benefits.
We have also demonstrated that sCD40L was reduced by eptifibatide infusion in three of the four patients. Reductions averaged 35% in these three patients, suggesting that eptifibatide may lower sCD40L levels in the majority of SCA patients. In addition to its effects on platelet aggregation and sCD40L release, eptifibatide has multiple effects that may provide therapeutic benefit. It disrupts platelet aggregates in vitro (Moser et al, 2003); inhibits platelet-derived cytokine RANTES (regulated upon activation, normal t-cell expressed and secreted) release ex vivo and in vitro (Nannizzi-Alaimo et al, 2003; Welt et al, 2004); inhibits interleukin-1 receptor antagonist levels ex vivo (Aggarwal et al, 2003); and improves endothelium-dependent and nitric oxide-mediated vasodilation in patients with coronary artery disease (Heitzer et al, 2003). With its multiple effects, it is highly conceivable that SCA patients may achieve lasting benefit from eptifibatide treatment comparable to those obtained in patients with ACS (Tcheng et al, 1995; Tcheng, 1997). The reversible effect of eptifibatide on platelet aggregation, as well as its effects on sCD40L levels and endothelium-dependent and nitric oxide-mediated vasodilation, have the potential to be particularly beneficial in the treatment of SCA patients during acute pain episodes, a period characterized by further increases in sCD40L levels (Lee et al, 2006).
Finally, infusion of eptifibatide resulted in reduced levels of the potent inflammatory cytokines MIP-1α and TNFα as well as reduced myoglobin, a marker of muscle injury. Simultaneously, eptifibatide infusion resulted in an upregulation of the vasodilators MMP-2, MMP-9 and the adipokine leptin. While we cannot predict the clinical relevance of these eptifibatide-induced alterations to SCA plasma, it is intriguing that these changes favour a less inflammatory profile and point to potential therapeutic benefits for SCA patients.
In conclusion, this study suggests that eptifibatide is safe in SCA patients. The inhibition of αIIbβ3-mediated platelet aggregation in SCA was comparable to that observed in ACS. Additionally, eptifibatide treatment resulted in decreased sCD40L levels and appeared to promote a favourable alteration of cytokine expression in SCA patients. The emerging role of platelets as mediators of both inflammation and activated coagulation suggest that antiplatelet therapies, such as eptifibatide, may hold promise for the treatment of SCA by inhibiting platelet aggregation and sCD40L-mediated inflammatory and prothrombotic changes. Further studies are warranted to establish the safety and efficacy of eptifibatide as a treatment modality for SCA.
The authors thank Susan Jones, RN and the UNC Comprehensive Sickle Cell Center; and the UNC General Clinical Research Center. This work was supported by grants from American Heart Association: 01502457N (LVP) and NIH: HL67440 (LVP), HL070769 (LVP and EPO), HL69768 (SPL), RR17059 (KIA) and RR00046 (KIA, EPO).
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