scholarly journals Pharmacokinetic and Pharmacodynamic Properties of Romiplostim

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1463-1463
Author(s):  
Karen Arkam ◽  
Sameer Doshi ◽  
Bing-Bing Yang
Keyword(s):  
Healthy Volunteers ◽  
Healthy Subjects ◽  
Immune Thrombocytopenia ◽  
Receptor Complex ◽  
Platelet Response ◽  
Employment Equity ◽  
Platelet Production ◽  
Platelet Counts ◽  
Equity Ownership ◽  
Chronic Immune Thrombocytopenia

Abstract Background: Chronic Immune thrombocytopenia (ITP) is characterized by low platelet counts, resulting from increased platelet destruction and inadequate platelet production. Romiplostim is a 59 kDa peptibody which binds to and activates the thrombopoietin (TPO) receptor on platelet precursors in the bone marrow, and increases platelet counts. This analysis integrates the pharmacokinetic (PK) and pharmacodynamic (PD) properties of romiplostim in animals, healthy volunteers and patients with ITP, and describes its intricate PK-PD inter-relationship. Methods and Results: In healthy subjects, over a wide range of doses examined, the PK and PD (platelet response) of romiplostim were dependent on both the dose administered and the baseline platelet counts. Following SC administration, platelet counts increased in a dose-dependent fashion after 4 to 9 days, peaking at 12 to 16 days (Wang Clin Pharmacol Ther. 2004;76:628-38). When romiplostim binds to the TPO receptor on megakaryocytes and platelets, the peptibody-receptor complex is internalized and degraded inside the cells. Therefore, as platelet counts increase, a higher number of free receptors are available to clear romiplostim (Wang AAPS J. 2010;12:729-40). Results from rodent studies suggest that as the dose increases, the TPO receptors become saturated and the contribution of the kidney to clearance increases. Additionally, proteolysis plays a role in the clearance of romiplostim; however, the cytochrome P450 enzymes are not involved in protein catabolism (Wang Pharm Res. 2011;28:1931-8), hence there are no known drug-drug interactions or dietary restrictions (Nplate Prescribing Information 2014). Following SC administration, serum concentrations of romiplostim were markedly lower, however, platelet response was similar after the same dose of intravenous (IV) and SC administration (Wang Clin Pharmacol Ther. 2004;76:628-38). This suggests that the PD response is driven by the length of time that the romiplostim concentrations remained above a threshold rather than by the magnitude of concentrations achieved. This effect was verified in a mechanistic PK-PD modeling study in animals (Krzyzanski Pharm Res. 2013;30:655-69). In patients with ITP receiving SC romiplostim at a dose of 1 mcg/kg, the peak platelet response was achieved at 18 days (range 8 to 43; Bussel N Engl J Med. 2006;355:1672-81). Pharmacodynamic model analysis showed that compared with healthy subjects, patients with ITP had a shorter platelet life span and a decreased rate of production of progenitor cells, but no major difference in the time to maturation of megakaryocytes. The PD response in this modeling analysis was not notably affected by age, body weight, sex, and race (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). The frequency of once-weekly dosing was selected because once every 2 weeks dosing was determined to be inadequate to achieve and maintain platelet counts in the therapeutic range (Bussel N Engl J Med. 2006;355:1672-81). A mechanistic PK-PD model based on data from the healthy subjects further suggested that weekly dosing resulted in a sustained platelet response while dosing less frequently resulted in high fluctuation of platelet counts (Wang AAPS J. 2010;12:729-40). Large inter- and intra-individual variability in the PD response was observed at a given dose; therefore, dose adjustments should be made based on a patient's platelet counts, using a titrated dosing scheme to prevent having platelet counts over 400 x 109/L (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). Conclusion: Romiplostim is a peptibody that binds and activates the TPO receptor, and consequently increases platelet production in individuals with chronic ITP. The peptibody-receptor complex is internalized and degraded inside the cells, without involvement of the liver. Romiplostim's PD response is driven by the length of time that its concentrations remained above a threshold rather than by the magnitude of concentrations achieved. Moreover, weekly dosing has demonstrated a sustained platelet response while less frequent dosing resulted in fluctuating platelet counts. Disclosures Arkam: Amgen Inc.: Employment, Equity Ownership. Off Label Use: Romiplostim is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. This abstract also describes PK data from healthy volunteers.. Doshi:Amgen Inc.: Employment, Equity Ownership. Yang:Amgen Inc.: Employment, Equity Ownership.

Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3279-3279 ◽  
Author(s):  
Ann Janssens ◽  
Michael D. Tarantino ◽  
Robert Bird ◽  
Maria Gabriella Mazzucconi ◽  
Ralph Vincent V. Boccia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Platelet Response ◽  
Employment Equity ◽  
Advisory Committees ◽  
Platelet Production ◽  
Platelet Counts ◽  
Equity Ownership

Abstract Abstract 3279 Background: ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts. Adverse Event Subject Incidence Platelet Response Disclosures: Janssens: Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

Novel Protein Agonist of Thrombopoiesis Acts Via a Physiocrine Pathway Distinct From That of Thrombopoietin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2376-2376
Author(s):  
Minh-Ha T Do ◽  
Wei Zhang ◽  
Kyle Chiang ◽  
Chi-Fang Wu ◽  
Chulho Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Employment Equity ◽  
Advisory Committees ◽  
Platelet Production ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Naturally Occurring ◽  
Equity Ownership

Abstract Abstract 2376 Thrombopoietin (TPO) is recognized as the main regulator of platelet production, yet its genetic ablation in mice does not completely obliterate thrombopoiesis, suggesting that alternate pathways could lead to platelet formation. We recently identified a naturally-occurring protein that acts as a potent agonist of platelet production by a mechanism distinct from that of TPO. This protein belongs to a novel class of human extracellular signaling proteins called physiocrines that are generated from tRNA synthetases by alternative splicing or proteolysis. Physiocrines interact with several classes of receptors through unique mechanisms to modulate cellular differentiation and tissue homeostasis in normal and pathological processes. The newly identified thrombopoietic physiocrine, termed ATYR0030, is an engineered version of a naturally-occurring physiocrine derived from the tyrosyl tRNA synthetase (YRS). In vivo, systemic administration of ATYR0030 or YRS physiocrine to rats led to an increase in platelets counts comparable to that seen with TPO treatment, but with a greater effect in animals with low baseline platelet levels. When injected into normal animals preselected for low platelet counts, ATYR0030 treatment resulted in an increase in platelets up to, but not beyond, normal levels (Figure 1), suggesting a role in platelet homeostasis and differentiating its effects from the known activity of TPO. Intravenous administration of ATYR0030 also accelerated recovery of platelet counts in carboplatin-treated rats, indicating a possible role in bone marrow reconstitution after chemical insult. Consistent with homeostatic properties, no toxicity was seen in a repeat-dose 28-day non-GLP safety study in rats dosed up to 100-fold above the efficacious range. Histopathology assessment revealed no tissue abnormalities, no increase in bone marrow reticulin and no hyperplasia of myeloid precursors. Clinical chemistry and hematology parameters were in the normal range with a modest increase in platelet counts, as anticipated in animals with normal platelet levels. Our in vitro data suggest that ATYR0030 may play a role in megakaryopoiesis by facilitating cell migration and adhesion to the vasculature. In contrast to TPO, ATYR0030 does not directly signal through the TPO receptor and does not activate the JAK/STAT pathway but rather appears to engage specific G-protein coupled receptors. In vitro, ATYR0030 does not stimulate proliferation of cultured M07e human megakaryoblasts or primary bone marrow cells isolated from AML patients (Figure 2). The parent synthetase is present in human platelets and is secreted in response to platelet activation, perhaps providing a feedback mechanism to stimulate the release of new platelets. In an effort to link the biological activity of ATYR0030 and the role that the parent synthetase plays in human physiology, we have begun to analyze samples from patients with abnormal platelets counts to determine circulating levels of the parent synthetase. The unique thrombopoietic activity of ATYR0030 may lead to an orthogonal approach to restoring normal platelet levels in thrombocytopenic patients who currently have limited treatment options. For example, in the myelodysplastic syndrome population, TPO-receptor agonists carry a risk of stimulating blast proliferation and accelerating disease progression to acute myeloid leukemia (AML). The distinct proliferation profile of ATYR0030 may translate into important safety benefits by reducing the risk of progression to AML. In addition, the potential role of ATYR0030 in regulating platelet homeostasis may provide a greater safety margin in the normalization of platelet levels, thereby also limiting the risk of thrombosis. Leveraging the therapeutic potential of this thrombopoietic physiocrine may lead to the development of a novel treatment option with a favorable safety profile. Disclosures: Do: aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Zhang:aTyr Pharma: Employment, Equity Ownership. Chiang:aTyr Pharma: Employment, Equity Ownership. Wu:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Park:aTyr Pharma: Equity Ownership. Yang:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kunkel:aTyr Pharma: Consultancy, Stock Ownership. Ashlock:aTyr Pharma: Employment, Equity Ownership. Mendlein:aTyr Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Belani:Atyr Pahrma: Consultancy, Equity Ownership, Patents & Royalties. Vasserot:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Watkins:aTyr Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Longitudinal Prospective Study Evaluating the Effects of Eltrombopag Treatment On Bone Marrow in Patients with Chronic Immune Thrombocytopenia: Interim Analysis At 1 Year.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2195-2195 ◽  
Author(s):  
Russell K. Brynes ◽  
Attilio Orazi ◽  
Raymond S.M. Wong ◽  
Kalpana Bakshi ◽  
Christine K Bailey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Trabecular Bone ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Employment Equity ◽  
Advisory Committees ◽  
Study Results ◽  
Equity Ownership ◽  
Chronic Immune Thrombocytopenia

Abstract Abstract 2195 Introduction: Eltrombopag (epag), a thrombopoietin receptor agonist (TPO-RA), increases platelet counts in patients with chronic immune thrombocytopenia (cITP). TPO-RAs have been associated with varying degrees of increases in bone marrow reticulin (Brynes 2011; Ghanima 2011). Due to lack of pretreatment evaluations, the incidence and clinical significance of these findings have not been established. Inconsistencies in specimen preparation, staining, and analysis across institutions further confound conclusions. The purpose of this 2-year (y) study (NCT01098487) is to assess for bone marrow fibrosis (reticulin and/or collagen) in patients treated with epag for cITP. Baseline and 1y findings are presented. Methods: Bone marrow biopsies are being collected at baseline (before treatment with epag) and at 1 and 2y of treatment. Specimens are centrally processed and stained for reticulin (silver) and collagen (trichrome) and undergo central independent pathology review of cellularity; megakaryocyte, erythroid, and myeloid quantity and appearance; trabecular bone quality; reticulin grade; and presence of collagen (European Consensus scale-MF; Thiele 2005). Results: Baseline and 1y (10–14 months) data are available for 101 patients. Median age is 42y (18–78); 70 patients are female; 50% are Caucasian/European, 22% are East Asian, and 29% are Central South Asian. Median time since ITP diagnosis is 4.2y (0.2–45.7). All patients had received prior ITP therapy, and 8 patients had received prior TPO-RA treatment (epag [7], romiplostim [1]), the last dose ≥6 months before enrollment. At baseline, 91 patients had reticulin grade 0 (MF-0), 10 MF-1, and 0 MF≥2. At 1y, 59 patients had MF-0, 38 MF-1, 3 MF-2, and 1 MF-3 (Figure). Compared with baseline, there was no change at 1y in MF grade in 61 patients, a decrease by 1 grade in 3, an increase by 1 grade in 35, and an increase in 2 or 3 grades in 1 patient each (Table). Three patients had collagen at 1y (1 patient each with MF-1, MF-2, and MF-3). None of the 4 patients with MF≥2 had adverse events or hematologic abnormalities considered related to impaired bone marrow function, and none withdrew due to bone marrow findings. Among the 8 patients with prior TPO-RA treatment, all had baseline reticulin of MF-0 and none had collagen; at 1y, 6 remained MF-0, 1 was MF-1, and 1 MF-3 (collagen demonstrated). Cellularity was normal in 83% and 80% of patients at baseline and 1y, respectively. Other than normalization of erythroid lineage numbers, no changes occurred in marrow cellular composition. In 3 of 4 patients with MF≥2, cellularity was increased at 1y. Trabecular bone thinning was found at baseline in 28 patients (the majority with prior steroid use) and 51 patients at 1y. Discussion: 10% of patients had MF-1 at baseline. After 1y of treatment, no increase or a mild increase in reticulin was observed in 63% and 35% of patients. No patient with MF≥2 (n=4) had clinical signs or symptoms indicative of bone marrow dysfunction and none withdrew from the study. Results were similar to those reported for EXTEND, an eltrombopag extension study (median treatment duration >2 years; Brynes 2011). Conclusion: These data suggest that treatment with epag is generally not associated with clinically relevant increases in bone marrow reticulin or collagen. The potential association of TPO-RAs and increased bone marrow reticulin needs further study. Disclosures: Brynes: GlaxoSmithKline: Research Funding. Orazi:GlaxoSmithKline: Research Funding. Wong:Roche: Research Funding; MSD: Research Funding; Johnson & Johnson: Research Funding; Bayer: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biogen-Idec: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; GlaxoSmithKline: Research Funding; Bristol-Myers Squibb: Research Funding. Bakshi:GlaxoSmithKline: Employment, Equity Ownership. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Brainsky:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties.

Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Multiple Doses of AG-519, an Allosteric Activator of Pyruvate Kinase-R, in Healthy Subjects

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1264-1264
Author(s):  
Ann J Barbier ◽  
Susan Bodie ◽  
Gary Connor ◽  
Elizabeth Merica ◽  
Charles Kung ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Healthy Volunteers ◽  
Pyruvate Kinase ◽  
Small Molecule ◽  
Healthy Subjects ◽  
Employment Equity ◽  
Multiple Doses ◽  
Equity Ownership ◽  
Dose Dependent ◽  
2,3 Dpg

Abstract BACKGROUND Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by deficiency of the glycolytic enzyme red cell PK (PK-R) due to mutations in the PKLR gene. PK catalyzes the last enzymatic step in the glycolytic pathway and is the main source of adenosine triphosphate (ATP) production in red blood cells. PKLR mutations lead to defective proteins that are hypothesized to reduce ATP levels in red cells, leading to hemolysis. Small molecule allosteric activation of PK-R resulting in increases in ATP and decreases in 2,3-diphosphoglycerate (2,3-DPG) in healthy volunteers has been observed with an earlier molecule, AG-348, the first small molecule PK-R activator to enter clinical trials (Yang et al. EHA 2015, S138). AG-519 is the second small molecule PK-R activator to enter clinical trials. AG-519 is a potent, highly selective and orally bioavailable PK-R activator devoid of the aromatase inhibitory effects that were observed with AG-348. AIMS AG-519 is currently being tested in a randomized, double-blind, phase 1 study in healthy volunteers (NCT02630927), with the objective of identifying a safe and pharmacodynamically active dose and schedule to support potential ongoing development in patients with PK deficiency. Here we report the first 4 cohorts of the multiple ascending dose (MAD) phase of this study. The single ascending dose (SAD) phase of the study and the first two cohorts of the MAD phase of the study have been reported previously (Barbier et al. EHA 2016, P752). METHODS Healthymen and women (non-childbearing potential) aged 18-60 years who provided informed consent were eligible. The MAD phase of the study consisted of 5 dose cohorts. The dose levels administered were determined during interim data reviews of each completed MAD cohort, as well as data from completed SAD cohorts. At each dose level, 8 subjects were enrolled and randomized to receive AG-519 (n=6) or placebo (n=2) twice daily (BID; approximately every 12 hours) for 14 days. Safety assessments included adverse events (AEs), vital signs, electrocardiogram and clinical laboratory parameters. Serial blood samples were drawn to measure plasma concentrations of AG-519 and whole blood concentrations of 2,3-DPG and ATP for pharmacokinetic and pharmacodynamic (PD) assessments. RESULTS Data are available for 32 subjects enrolled across 4 dose cohorts in the MAD phase of the study: 8 subjects each in cohort 1 (125 mg BID), cohort 2 (375 mg BID), cohort 3 (25 mg BID), and cohort 4 (300 mg BID). Blinded safety reviews indicated that multiple doses up to 375 mg have been well tolerated with no serious AEs or dose-limiting toxicities reported to date. One case of probable drug-induced Grade 2 thrombocytopenia was previously reported in 1 subject in the 375 mg cohort; the event was rapidly reversible with no clinical sequelae. The protocol was amended to require daily monitoring of platelets in subsequent cohorts and no other subjects have developed thrombocytopenia during treatment. The preliminary analysis of free testosterone and estradiol confirmed the absence of aromatase inhibitory activity. AG-519 steady-state was reached the third day after the first dose based on trough concentration values. The clearance of AG-519 after multiple doses was similar to that observed after single doses in the SAD cohorts. Dose-dependent increases in ATP in blood (Figure 1) and decreases in 2,3-DPG in blood correlated with dose-dependent increases in exposure of AG-519, with a peak effect at or below 375 mg BID. ATP response at 25 mg appears to be greater than 50% of maximal response. Results from the fifth MAD cohort, which evaluated the PD results with 10 mg BID, will be presented. ATP = adenosine triphosphate; BID = twice daily CONCLUSION AG-519 is well tolerated in healthy subjects at doses ranging from 25 mg to 375 mg BID for 14 days. The robust dose-dependent changes in ATP and 2,3-DPG concentrations in blood from healthy volunteers are consistent with increased activity of PK-R, the expected PD effect of AG-519. These data support the hypothesis that AG-519 may be able to enhance glycolytic activity in red cells of patients with PK deficiency to address the underlying cause of the disease. Figure 1 Change of ATP concentration in blood from baseline Figure 1. Change of ATP concentration in blood from baseline Disclosures Barbier: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bodie:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Connor:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Le:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yuan:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bowden:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Cohen:Agios Pharmaceuticals, Inc.: Consultancy.

EXTEND Study Update: Safety and Efficacy of Eltrombopag In Adults with Chronic Immune Thrombocytopenia (ITP) From June 2006 to February 2010

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 67-67 ◽  
Author(s):  
Mansoor N. Saleh ◽  
Gregory Cheng ◽  
James B. Bussel ◽  
Huiping Sun ◽  
Bhabita Mayer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Thromboembolic Events ◽  
Bone Marrow Biopsies ◽  
Baseline Platelet Count ◽  
Safety And Efficacy ◽  
Platelet Counts ◽  
Equity Ownership ◽  
Chronic Immune Thrombocytopenia

Abstract Abstract 67 Background: Eltrombopag is an oral thrombopoietin receptor agonist approved for treatment of chronic immune thrombocytopenia (ITP) in the US and other countries. In 6-week and 6-month placebo-controlled trials, eltrombopag safely increased platelet counts and reduced bleeding symptoms in patients with previously treated chronic ITP. The safety and efficacy of eltrombopag treatment are being evaluated in EXTEND, an ongoing open-label, extension study in ITP patients who completed a previous eltrombopag study. Methods: Enrolled patients had previously received eltrombopag or placebo in one of the following studies: two 6-week studies (773A and B), RAISE (6-month), or REPEAT (intermittent treatment). In EXTEND, specific goals include: 1) identification of a dose of eltrombopag that increases platelet counts (≥100,000/μ L) to support reduction of concomitant ITP medications (if taken); 2) identification of minimally effective doses of eltrombopag and concomitant ITP medication to maintain platelet counts ≥50,000/μ L; and 3) evaluation of the safety and efficacy of eltrombopag. Patients who completed at least 2 years of therapy and transitioned off study due to commercial availability of eltrombopag were considered to have completed the study. Results: Of 299 patients enrolled, 8% (23) completed the study, 41% (122) withdrew, and 52% (154) remain on study. The main reasons for withdrawal were adverse events (AEs, 11%), patient decision (11%), and lack of efficacy (10%). At baseline, platelet counts were ≤15, >15–<30, 30–50, and >50,000/μ L in 43%, 27%, 17%, and 13% of patients, respectively; 38% of patients were splenectomized; 33% were receiving concomitant ITP medication at baseline, and 53% had received ≥3 previous ITP therapies. 249, 210, 138, and 24 patients had been taking eltrombopag for ≥26, 52, 104, and 156 weeks, respectively, with a median duration of exposure of 100 weeks at the time of data analysis. The proportion of patients achieving a platelet count ≥50,000/μ L was similar regardless of the following baseline characteristics: splenectomy (84%) vs no splenectomy (89%); use of ITP medication (88%) vs no use of ITP medication (87%); and baseline platelet count <30,000/μ L (83%) vs 30–50,000/μ L (98%) vs >50,000/μ L (95%). Overall, 87% (261/299) of patients achieved a platelet count ≥50,000/μ L on treatment; 37 of these had a baseline platelet count of ≥50,000/μ L. Median platelet counts increased to ≥50,000/μ L by week 2 and remained consistently ≥50,000/μ L through week 164. The incidence of any bleeding symptoms (WHO grades 1–4) declined from 56% at baseline to 16% and 20% at weeks 52 and 104, respectively. Clinically significant bleeding (WHO grades 2–4) was reduced from 16% (47/299) at baseline to 3% (2/77) and 7% (3/41) at weeks 52 and 104, respectively. AEs and SAEs occurred in 88% (262) and 26% (79) of patients, respectively. The most frequent AEs were headache (26%), nasopharyngitis (23%), and upper respiratory tract infection (21%). AEs led to withdrawal of 13% (38) of patients, 9% (27) of which were due to SAEs. Twenty-one thromboembolic events (TEE) have been reported in 5% (16) of patients; the incidence rate is 3.17/100 patient years (95% CI [1.81, 5.15]). The most common TEEs were DVT (8) and MI (4). No association has been observed with elevated platelet counts, as only 3/16 patients experienced the TEE closest to their maximum platelet count achieved on study. Hepatobiliary laboratory abnormalities were reported in 29 patients (10%). All were reversible; the majority while on therapy. Of 299 patients enrolled, 6 (2%) have been withdrawn due to a hepatobiliary AE. After examining bone marrow biopsies from >150 patients treated with eltrombopag for >1 year, no clinically relevant increase in reticulin fiber deposition has been observed. Conclusions: Eltrombopag was effective in increasing and maintaining platelet counts ≥50,000/μ L and reducing bleeding symptoms. Eltrombopag has an overall positive risk/benefit assessment and was well tolerated during treatment of patients with chronic ITP even with exposures of more than 3 years. Bone marrow biopsies will continue to be assessed. Hepatobiliary laboratory abnormalities and thromboembolic events are risks that need to be monitored. Disclosures: Saleh: GlaxoSmithKline, Novartis, Imcoline, Celgene: Honoraria, Speakers Bureau. Cheng:GlaxoSmithKline: Consultancy, Honoraria, Speakers Bureau. Bussel:GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mayer:GlaxoSmithKline: Employment, Equity Ownership. Bailey:GlaxoSmithKline: Employment. Brainsky:GlaxoSmithKline: Employment.

scholarly journals Absence of Plasma Gastric Biomarker Elevations with Chronic Dosing of Avatrombopag, a Novel Oral Thrombopoietin Receptor Agonist, in Patients with Chronic Immune Thrombocytopenia in Phase 3 Trials

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3740-3740 ◽  
Author(s):  
James B. Bussel ◽  
Lee F. Allen ◽  
Wei Tian ◽  
David J. Kuter
Keyword(s):  
Acid Production ◽  
Research Funding ◽  
Receptor Agonist ◽  
Immune Thrombocytopenia ◽  
Employment Equity ◽  
Phase 3 ◽  
Thrombopoietin Receptor ◽  
Thrombopoietin Receptor Agonist ◽  
Equity Ownership ◽  
Chronic Immune Thrombocytopenia

Abstract Background: Avatrombopag (AVA) is an oral, thrombopoietin receptor agonist approved by FDA for the treatment of thrombocytopenia in patients with chronic liver disease scheduled to undergo a procedure. AVA is also being developed for other indications including chronic immune thrombocytopenia (cITP) (Bussel et al, Blood, 2014; Wojciech et al, Br J Haematol, in press 2018). In repeated-dose studies in mice, rats and cynomolgus monkeys, there were AVA dose, concentration, and duration-dependent histologic changes in gastric fundic glands that reversed by 4 weeks after dosing was stopped. This was associated with decreased gastric acid production, hypergastrinemia, and increased intragastric pH in all species. The NOAEL in 2-year toxicology studies was defined at exposures 4 to 21-fold the recommended human AVA dose. Hypergastrinemia and parietal cell hyperplasia have also been reported with proton pump inhibitors (PPIs) and H2-receptor antagonists (H2RA). These findings may be secondary to suppression of gastric acid production, and, in rodents, may lead to gastric carcinoids. Findings of hypergastrinemia-related gastric hyperplasia in rodents have a low risk/relevance to humans. To further evaluate the preclinical findings and screen for atrophic gastritis, gastric biomarkers (gastrin, gastrin-17, and pepsinogen I/II [PG-I/PG-II]) were measured in 2 Phase 3 trials with chronic AVA dosing. These multicenter, randomized, controlled Phase 3 trials were conducted in patients with cITP, and assessed clinical safety and efficacy of AVA versus placebo (PBO) (Study 1- NCT01438840) or eltrombopag (ELT) (Study 2- NCT01433978); Study 2 was terminated early due to enrollment challenges. Methods: Both Phase 3 trials enrolled adults (≥18 years) with chronic ITP (Baseline platelet count (PC) <30x109/L), who were randomized 2:1 to receive either 20 mg/day AVA or PBO (Study 1) or 1:1 to 20 mg/day AVA or ELT (50 mg/day) (Study 2) orally with food for 26 weeks. Individual patient AVA (5-40 mg/day) or ELT (25-75 mg/day) dose titration was permitted. Patients with Baseline fasting gastrin-17 levels greater than upper limit of normal (ULN) were excluded from the study, as were patients taking PPIs or H2RAs if not on a stable dose or if gastrin-17 levels were >1.5 times the ULN. Safety analyses included adverse event (AE) monitoring and laboratory testing, including fasting gastric biomarkers. Patients who developed 2 consecutive fasting gastrin-17 levels >2.5 times the ULN or low serum PG-I levels or low PG-I/PG-II ratio (with low fasting gastrin-17 levels) were to be discontinued from the trials. Patients with elevations >5 times the ULN were to undergo endoscopy to assess the gastric mucosa. Results: A total of 64 patients were treated with AVA across the two trials (Study 1 n=47; Study 2 n=17); the majority were white (92.2%) and female (64.1%), with a mean age of 46.6 years. The median duration of exposure to AVA was 35.1 weeks, with a mean daily dose of 24.6 mg; 75% of subjects received AVA doses between 5 and 40 mg for ≥20 weeks. AVA was shown to be superior to PBO in the median cumulative number of weeks of platelet response (PC ≥50×109/L in the absence of rescue therapy) in Study 1 (12.4 vs. 0.0 weeks, respectively; p <0.0001). Mean Baseline PCs in Study 1 were similar in AVA- and PBO-treated subjects (14.1 and 12.7x109/L, respectively), and peaked at 205.4 and 11.6x109/L on Week 2; mean Baseline PCs were lower in Study 2 in AVA- compared to ELT-treated subjects (13.0 and 18.2x109/L, respectively), and increased to 184.8 and 91.6x109/L at Week 2. Of the AVA-treated patients, no significant changes in the mean fasting gastrin, gastrin-17, PG-I, PG-II, or the PG-I/PG-II ratio were observed from Baseline over the course of the trial (Figure 1). In addition, no patients met the gastric biomarker change criteria for discontinuation or to undergo endoscopy. Common AEs in Study 1 included headache, contusion and upper respiratory tract infections, with 4 cases of thromboembolic events. Conclusions: In Phase 3 trials, AVA was shown to be effective and well tolerated in the treatment of thrombocytopenia in patients with cITP, with most patients demonstrating substantial platelet increases. In particular, treatment of cITP with AVA was not associated with an increase in gastric biomarkers including gastrin, gastrin-17 or pepsinogen and, therefore, no signal of gastric toxicity was identified with chronic AVA dosing of up to 26 weeks. Disclosures Bussel: Prophylix: Consultancy, Research Funding; Protalex: Consultancy; Amgen Inc.: Consultancy, Research Funding; Uptodate: Honoraria; Rigel: Consultancy, Research Funding; Momenta: Consultancy; Novartis: Consultancy, Research Funding. Allen:Dova Pharmaceuticals: Employment, Equity Ownership. Tian:Dova Pharmaceuticals: Employment, Equity Ownership. Kuter:Pfizer: Consultancy; ONO: Consultancy; Amgen Inc.: Consultancy; Novartis: Consultancy; Syntimmune: Consultancy; Protalex: Research Funding; Rigel: Consultancy, Research Funding; Argenx: Consultancy; Dova Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Consultancy, Research Funding; BMS: Research Funding; Principia: Research Funding.

Evaluation of Romiplostim In a Randomized Placebo-Controlled Phase 3 Study of a Japanese Population with Chronic Immune Thrombocytopenia (ITP).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3704-3704
Author(s):  
Koji Miyazaki ◽  
Yukari Shirasugi ◽  
Kiyoshi Ando ◽  
Yoshiaki Tomiyama ◽  
Shinichiro Okamoto ◽  
...  
Keyword(s):  
Platelet Count ◽  
Placebo Group ◽  
Adverse Events ◽  
Target Range ◽  
Platelet Response ◽  
Employment Equity ◽  
Phase 3 ◽  
Platelet Counts ◽  
Equity Ownership ◽  
H Pylori

Abstract Abstract 3704 Introduction: Chronic immune thrombocytopenia (ITP) is an autoimmune disorder characterized by both increased platelet destruction and decreased platelet production. Romiplostim increases platelet counts by binding and activating the thrombopoietin receptor. Romiplostim has been approved for the treatment of adult ITP in the United States, Europe, Canada, and Australia. This study evaluated dosing, efficacy, and safety in a Japanese population of adults with ITP. Methods: This phase 3 study was placebo-controlled, double-blind, and randomized 2:1 (romiplostim:placebo). Patients were eligible for the study if they were Japanese patients with ITP diagnosed at least 6 months before the initial screening, were aged ≥ 20 years, and were H pylori negative or had received at least 1 treatment for H pylori eradication. Patients were stratified by splenectomy status (yes or no). After a 3-week evaluation period, patients were treated for 12 weeks with a weekly subcutaneous injection of either romiplostim or placebo. The starting dose was 3 mcg/kg with dose adjustments to a maximum of 10 mcg/kg to achieve a platelet count within the target range of ≥ 50 to ≤ 200 × 109/L. Patients were monitored posttreatment until their platelet count dropped to ≤ 50 × 109/L or for a maximum of 12 weeks. The primary endpoint was number of weeks with platelet response (platelet count ≥ 50 × 109/L). Results: Thirty-four patients enrolled (22 romiplostim, 12 placebo), 24 (71%) were female, the median (range) age was 55 (44 - 64) years, and the median (range) baseline platelet count was 19 (3 – 32) × 109/L. Patients had received a median of 4 (1 – 19) prior ITP therapies, and 15 (44%) had previously undergone a splenectomy; 23 (68%) patients were receiving concurrent ITP therapy at baseline. All patients completed the study. Romiplostim demonstrated superiority to placebo on weekly platelet response, incidence of increase in platelet count ≥ 20 × 109/L from baseline, change from baseline in mean of last 4 platelet counts during week 2 to week 13, and number of weeks with platelet counts within the target range (Table 1), and 16 (73%) romiplostim patients had platelet counts ≥ 200 × 109/L. Twenty-one (95%) patients in the romiplostim group had a platelet response with a median time of 1 week until first response. Results for weekly platelet response were comparable irrespective of splenectomy status and baseline concurrent ITP therapy. Two (17%) patients in the placebo group achieved a platelet response. In romiplostim-treated patients, posttreatment platelet counts remained > 50 × 109/L for 8 weeks in one and for 12 weeks in another. The mean weekly dose of romiplostim for the study was 2.6 mcg/kg compared with the dose range of 3–4 mcg/kg in prior phase 3 studies (Kuter et al. Lancet. 2008;371:395–403). There was a low incidence of rescue medication use in the study (Table 1). The adverse events profile was comparable to that seen in non-Japanese studies. Patients in both treatment groups experienced similar proportions of adverse events (91% romiplostim, 92% placebo). Adverse events with > 10% higher frequency in the romiplostim group than placebo group) were (romiplostim, placebo) nasopharyngitis (41%, 17%), headache (32%, 17%), peripheral edema (18%, 0%), back pain (14%, 0%), and pain in extremity (14%, 0%). Significant (≥ grade 3) bleeding events occurred in 1 patient in the romiplostim group (subarachnoid hemorrhage) and 1 patient in the placebo group (subarachnoid hemorrhage, cerebral hemorrhage, and gastrointestinal hemorrhage). There were no adverse events of bone marrow reticulin, thrombosis, or detection of neutralizing antibodies. Conclusion: Romiplostim significantly increased and maintained platelet counts and was well-tolerated in a Japanese ITP population. Disclosures: Tomiyama: Kyowa Hakko Kirin Co.: Speakers Bureau; GlaxoSmithKline: Speakers Bureau. Kurokawa:Novartis: Consultancy; Shionogi & Co., Ltd.: Consultancy. Wei:Amgen Inc.: Employment, Equity Ownership. Lizambri:Amgen Inc.: Employment, Equity Ownership.

scholarly journals Long Term Discontinuation of Eltrombopag after Remission in Primary Immune Thrombocytopenia: 8-Year Follow-up Data from 15 Spanish Centers

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2352-2352
Author(s):  
Tomas Jose Gonzalez-Lopez ◽  
Fernando Fernandez-Fuertes ◽  
Maria Cristina Pascual Izquierdo ◽  
Isabel Caparros ◽  
Silvia Bernat ◽  
...  
Keyword(s):  
Platelet Count ◽  
Median Time ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Previous Treatment ◽  
Complete Response ◽  
Platelet Response ◽  
Platelet Counts

Background: Successful discontinuation of eltrombopag in certain immune thrombocytopenia (ITP) patients after complete response has already been demonstrated. However, the frequency of this phenomenon and type of candidate patients are still matter of discussion. Moreover, possibility of long term discontinuation responses is not clearly established. Methods: Here we retrospectively evaluated our whole cohort of 508 adult patients (aged 18 years or more) with primary ITP treated with eltrombopag included in the Spanish Eltrombopag Registry with a focus on the patients who achieved a durable (at least six months) platelet response after stopping eltrombopag. Successful discontinuation of eltrombopag (SDOE) was defined as those patients who reached remission and maintained platelet counts ≥ 50x109/l for at least 6 months in absence of eltrombopag or any rescue therapies administered. Long term discontinuation of eltrombopag (LTDOE) was defined as those patients who reached remission and maintained platelet counts ≥ 50x109/l for at least 36 months in the absence of eltrombopag or any rescue therapies administered. The study was approved by the Hospital Universitario de Burgos Ethics Committee and fulfilled Helsinki declaration standards. Results: While 37.4% of our patients relapsed of ITP with subsequent platelet count drop sometime during first six months of discontinuation of eltrombopag, a total of 74 patients (14.6%) were able to achieve SDOE. The median age of SDOE patients was 62 [range, 47-79] years. There were 47 women and 27 men. According to the standard definition, patients were allocated to newly diagnosed (n=17), persistent (n=15) and chronic (n=42) ITP groups. The median time from diagnosis to eltrombopag initiation was 31 [range, 4-104] months. The median number of previous therapies was 2 [range, 1-2], including splenectomy (14%), rituximab (18%) and romiplostim (12%). As expected, all patients but 1 achieved a complete response (platelet count ≥100 x 109/L) prior to eltrombopag discontinuation The median duration of eltrombopag treatment was 7 [range, 2-19] months. Reasons for eltrombopag discontinuation were: persistent response despite a reduction in dose over time (n=43), platelet count >400x109/L (n=16), aspartate aminotransferase elevation (n=5), diarrhea (n=4), thrombosis (n=3), patient's request (n=2) and other reasons (n=1). Analysis of these SDOE discontinued patients show that with a median follow-up of 55 [range, 29-79] months, 38 patients (51.3%) maintained treatment-free response 36 months after stopping eltrombopag with no need of additional ITP therapies (median time of eltrombopag discontinuation was 70 [range, 50-77] months).This condition is what we define now as LTDOE. Nevertheless, 36 patients relapsed beyond 6 months but before 36 months of eltrombopag discontinuation (median time of eltrombopag discontinuation was 10 [range,7 -22] months). Characteristics of LTDOE population were a median time since ITP diagnosis of 32 [range, 5-88] months with 15/38 patients having ITP <1 year. 9 patients (24%) were male and their median age was 50 [range, 37-64] years. They had received a median of only two previous treatment lines [range: 1-2 lines]. The median platelet count before starting eltrombopag was 19 x 109/L [range, 8-40]. Meanwhile, platelet count before eltrombopag stop was 218 x 109/L [range, 123-356]. The main characteristics (age, gender, duration of ITP, prior ITP lines, platelet count before starting eltrombopag, duration of eltrombopag treatment, and platelet count before eltrombopag withdrawal) of the 38 patients with LTDOE were compared with those of the SDOE cohort who did not achieve a LTDOE. Unfortunately, no predictive factors of LTDOE could be identified. Conclusion: Durable platelet response following eltrombopag cessation may be observed in only 15% of primary ITP patients treated with this drug. On the contrary, half of patients who achieve a sustained response after eltrombopag withdrawal will get a long term discontinuation. However, we are lacking predictor factors for successful and long-term discontinuation of eltrombopag in primary ITP. Disclosures Gonzalez-Lopez: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Pascual Izquierdo:Novartis: Consultancy; Sanofi: Consultancy. Sánchez-González:Amgen: Consultancy, Speakers Bureau; Gilead: Speakers Bureau; Navartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Jarque:Takeda: Consultancy, Speakers Bureau; Shire: Consultancy, Speakers Bureau; Shionogi: Consultancy, Speakers Bureau; Servier: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Grifols: Consultancy; Gilead: Consultancy, Speakers Bureau; CellTrion: Consultancy; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Abbie: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau.

Erythropoietin Stimulates Thrombopoiesis in the Absence of C-Mpl Signaling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2451-2451
Author(s):  
Norma E Fox ◽  
Rose Chen ◽  
Ian Hitchcock ◽  
Kenneth Kaushansky ◽  
Amy Geddis
Keyword(s):  
Mouse Model ◽  
Stem Cell Factor ◽  
Statistical Significance ◽  
Null Mouse ◽  
Platelet Response ◽  
Platelet Production ◽  
Platelet Counts ◽  
Severe Reduction ◽  
Stimulation Of

Abstract Thrombopoietin (TPO) is essential for normal megakaryopoiesis, and mice and humans lacking the TPO receptor c-Mpl have significantly impaired platelet production. However, in the c-Mpl-null mouse model platelet counts, while reduced to ~10% of normal, are not zero, suggesting that another cytokine is able to support some degree of residual thrombopoiesis. We and others have reported that elimination or severe reduction of stem cell factor, G-CSF, IL-3, IL-6 or IL-11 does not eliminate residual thrombopoiesis. Because megakaryocytes (MKs) and erythrocytes are derived from a common progenitor, we asked if erythropoietin (EPO) can stimulate thrombopoiesis in c-Mpl-null mice. We administered 90 u recombinant EPO or vehicle by subcutaneous injection every 3 days to c-Mpl-null or WT control mice and measured baseline and weekly platelet counts. In three independent experiments, at 2 weeks platelet counts in c-Mpl-null mice receiving EPO were significantly higher that at baseline (5–7 mice per group, average of mean platelet counts 425,000/mm3 vs. 285,000/mm3, p=0.0015). There was a trend towards higher platelet counts in WT mice receiving EPO but this did not reach statistical significance. No difference in platelet counts was observed in mice injected with vehicle. In one experiment c-Mpl-null or WT mice were injected with EPO for 4 weeks and the platelet response in the c-Mpl-null animals was sustained for the duration of the experiment. Western blotting showed that murine MKs express the EPO receptor. To determine if EPO stimulates MK production directly we stimulated WT murine MKs in vitro with either 6 u/ml EPO, 100 ng/ml rhTPO or both and monitored activation of ERK and STAT5 signaling by immunoblotting. Stimulation of MKs with EPO resulted in phosphorylation of ERK and STAT5 (15- and 14-fold above baseline, respectively), compared to TPO (97- and 75-fold above baseline). Stimulation with EPO and TPO together had an additive effect (phospho-ERK increased 121-fold and phospho-STAT5 increased 100-fold). To determine if EPO acts primarily on early or late MKs, we harvested bone marrow from c-Mpl-null mice after 2 weeks of treatment with EPO or vehicle and measured CFU-MK frequency and MK ploidy. Although there was a small increase in the frequency of CFU-MK in mice treated with EPO compared to vehicle, these differences were not significant (n=3, p=0.7), possibly due to the difficulty in assaying CFU-MK in vitro without TPO. In addition, EPO did not significantly enhance MK ploidy in c-Mpl-null mice, although MKs in the 32N and greater peaks were slightly more numerous. Therefore, we conclude that EPO can augment platelet production in the absence of c-Mpl signaling, although it is not yet clear if EPO primarily acts on early or late cells. Additional experiments are underway to determine if ablating EPO receptors in a TPO-null mouse model will eliminate residual thrombopoiesis. These findings may have clinical relevance for treating patients with congenital amegakaryocytic thrombocytopenia and other causes of thrombocytopenia in which c-Mpl signaling is impaired.

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