The Effect of Eltrombopag on the Expression of Platelet Collagen Receptor GPVI in Pediatric ITP.

Using eltronmobag as an alternative pathway, which depend on stimulation of thrombocyte synthesis, in chronic and persistent immune thrombocytopenia may be more promising treatment than the classic type, not only by increasing platelets count but also through enhancing of the platelets activation and upregulation of GPVI expression on platelet surface.

This study will include 40 pediatric patients with chronic or persistent ITP, recruited from the Hematology clinic of pediatric hospital Ain Shams University, aiming to investigate the efficacy, \& the safety and tolerability of eltrombopag therapy for children as well as the ability of eltrombopag to enhance the platelet activation through the up-regulation of glycoprotein VI (GPVI) receptor expression in comparison with other lines of treatment.

Patients will be divided into 2 groups : Group 1 on eltrombopag ; Group 2 : receiving other lines of therapy .

All Patients will be subjected to :

1. bleeding score assessment ( baseline \&every month ) and Health related quality of life based on Kids' ITP Tools (KIT) questionnaires( baseline and week 24 )
2. Baseline and at week 24 bone marrow examination with reticulin stain
3. clinical examination every 2 weeks and complete blood counts.
4. Assessment of soluble form of glycoprotein VI using sandwich enzyme-linked immunosorbent assay (ELISA) as well as assessment of platelet activation by GPVI using flowcytometry (Gardiner, etal.,2010 ) at baseline and at the end of 6 months treatment period

Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia, defined as a peripheral blood platelet count less than 100 ×109/l and the absence of any obvious initiating and/ or underlying cause of the thrombocytopenia. (Buchanan, 1989).

Novel thrombopoiesis-stimulating agents have been developed increasing platelet production by overdriving the system and thereby correcting the thrombocytopenia (Cines etal., 2002). Because these peptides bear no structural resemblance to thrombopoietin agonist(TPO) but still bind and activate the TPO receptor, these compounds are called TPO receptor agonists (TRAs). (Meletis etal.,2010).

Newly formed young platelets are larger and express higher levels of glycoprotein Ibα (GPIbα), which decreases as platelets age. For the purpose of quantitative platelet analysis, GPIbα of the GPIb-IX-V complex and GPVI are of particular interest because these receptors are essentially platelet specific, are critical for initiation of thrombus formation at arterial shear rates, and are implicated in wider platelet functions beyond hemostasis and thrombosis, as well as platelet aging and clearance. (Gardiner etal., 2014).

GP VI is thought to be the major signaling receptor involved in platelet activation on exposed collagen. Following GP VI interactions with collagen, platelets initiate strong activation and release the content of alpha and dense granules.( Yun etal.,2016) GPVI expression was upregulated in megakaryocytes after TPO stimulation (Holmes etal., 2002). Eltrombopag could upregulate GPVI expression in ITP patients, thereby enhancing their platelet adhesion capacity leading to reduce bleeding symptoms (Chiou etal.,2015).

The Aim is to :

To investigate the efficacy, \& the safety and tolerability of eltrombopag therapy for children with persistent and chronic immune thrombocytopenia as well as the ability of eltrombopag to enhance the platelet activation through the up-regulation of glycoprotein VI (GPVI) receptor expression in comparison with other lines of treatment.

Study methods :

This study will include 40 pediatric patients with chronic or persistent ITP, age ≥ 1 to ≤ 18 years, recruited from the Hematology clinic of pediatric hospital Ain Shams University

* Baseline bleeding score assessment (ITP-BAT (v1.0) score) and Health related quality of life based on Kids' ITP Tools (KIT) questionnaires will be done.
* Patients will be divided into 2 groups:

  1. Group 1 (eltrombopag arm n=20 patients): Patients who showed no response (platelet count ≤ 20x109/L) initially for 3 months or relapse after 6 months after at least one prior ITP therapy. patients will receive a total daily dose of eltrombopag of (25-50mg/d). Dose adjustments may be made based on platelets count with an increment of 25mg once per day at 2 weeks intervals (Maximum dose: 75 mg orally once a day). If platelet counts reached more than 200 × 109 per L, the dose will be decreased by 25 mg once per day at 2 weeks intervals. If platelet counts increased to more than 400 × 109 per L, treatment will be interrupted until platelet counts decreases to less than 150 × 109 per L then the dose will be resumed at the next lower dose.

     Patients, who responded poorly to eltrombopag in 6 months or developed adverse effects, will be asked to discontinue the medication. Those who responded will be followed for further 6 month period.
  2. Group 2 (n=20 patients) Patients who are currently receiving other lines of treatment (steroids, IVIG, azathioprine, and rituximab).
* All patients will be followed clinically every 2 weeks throughout the period of the study for safety and efficacy, and complete blood counts.
* Baseline and at week 24 bone marrow examination with reticulin stain will be done
* Baseline and every 3 months liver and kidney function tests. Throughout the study periods, patients with increased serum alanine transaminase (ALT) ≥ 3 times upper limit normal (ULN) with a total bilirubin ≥2 times ULN or those with increased serum ALT ≥ 5 times ULN will be withdrawn, as well as patients with increased serum creatinine above ULN.
* Adverse events, episodes of bleeding and bleeding score will be collected and evaluated every month throughout the study.
* Health related quality of life based on Kids' ITP Tools (KIT) questionnaires will be repeated at the end of study period.
* Assessment of soluble form of glycoprotein VI using sandwich enzyme-linked immunosorbent assay (ELISA) as well as assessment of platelet activation by GPVI using flowcytometry (Gardiner, etal.,2010 ) at baseline and at the end of 6 months treatment period

Statistical Analysis : Analysis of data will be done using Statistical Program for Social Science version 21 (SPSS Inc., Chicago, IL, USA).

Eligibility Criteria:
Inclusion Criteria:

* Age ≥ 1 to ≤ 18 years at enrollment. Patient should be able to swallow tablet form. Weight should be ≥ 12kg.
* Diagnosis of persistent (3-\<12 months duration) or chronic cases (≥12 months duration) with day 1 platelet count ≤ 20x10\^9.
* Patients have a bleeding score grade 3 for skin and/or grade 2 or higher for mucosal domains and/or higher than grade 1 for organ domain at the baseline visit or the worst bleeding incident episode in the patient's medical reports in the last 3 months prior screening, using ITP-BAT (v1.0) score.
* Normal Kidney function tests and liver function tests.

Exclusion Criteria:

* Acute thrombocytopenic purpura patients or Other causes of thrombocytopenia. Patients with Evans syndrome
* Hypertension, cardiovascular disease, diabetes, hepatitis C virus (HCV), HIV, hepatitis B surface antigen(HBsAg) seropositive status.
* Baseline bone marrow biopsy with evident fibrosis (reticulin stain grade 2 or more)
* Patients who have previously received eltrombopag.