Human Thrombopoietin Receptor Research Articles

Efficacy and safety of hetrombopag in the treatment of recombinant human thrombopoietin–resistant thrombocytopenia after allogeneic hematopoietic stem cell transplantation

BackgroundThrombocytopenia after allogeneic hematopoietic cell transplantation is a challenging clinical problem. Recombinant human thrombopoietin (rhTPO) and thrombopoietin receptor agonists are increasingly used in posttransplant thrombocytopenia. However, the use of hetrombopag in patients with posttransplant thrombocytopenia, especially in patients with resistance to rhTPO, has not yet been reported. ObjectivesThe present study aimed to investigate the efficacy and safety of hetrombopag in patients with rhTPO-resistant posttransplant thrombocytopenia. MethodsThis retrospective study included 21 patients with rhTPO-resistant posttransplant thrombocytopenia who received hetrombopag from August 2021 to July 2022. The primary endpoint was the overall response rate, including partial response and complete response (CR). We also evaluated the predictors of hetrombopag efficacy and adverse events. ResultsThe overall response rate to hetrombopag was 81%, and the CR rate was 62%. The median time from hetrombopag initiation to response and CR were 16 and 31 days, respectively. Decreased megakaryocytes in bone marrow negatively correlated with CR to hetrombopag (P = .027). All the patients tolerated hetrombopag well without any significant increase in adverse events. At the last follow-up, 71% of responders had discontinued hetrombopag and sustained their best response. ConclusionOur results suggested that hetrombopag is an effective treatment option to promote platelet recovery in patients with posttransplant thrombocytopenia, even in patients resistant to rhTPO.

Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma

Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, one of the possible adverse reactions from the immune system is immunogenicity: the production of anti-drug antibodies to the medicinal product, including neutralising antibodies, which may affect the efficacy and safety profile of the medicinal product.Aim. Validate the procedure for determining neutralising antibodies to romiplostim in human plasma for further clinical studies of immunogenicity.Materials and methods. The study used rabbit polyclonal antibodies to romiplostim, Nplate® produced by Amgen Europe as a standard sample; a placebo produced by LLC "GEROPHARM", a cell line 32D-hTPOR clone 63 with stable expression of human TPO receptor and a chemiluminescence assay kit CellTiter-Glo® Luminescent Cell Viability Assay produced by Promega to assess specificity. The experiment was carried out on cell line 32D-hTPOR clone 63, which was seeded on the first day and the neutralizing antibody concentrations were titrated with a constant concentration of romiplostim, then the chemiluminescence was detected on the second day. Statistical processing of the results was carried out using Prism 9 software.Result and discussion. The specificity of the procedure was demonstrated; at maximum concentration, the medicinal product differs from placebo by 309 times in the residual level of cell viability. The linearity of the procedure in terms of the coefficient determination is 0.9969. The precision of the procedure was determined: the repeatability was 1–9 %, the intermediate precision was 3–18 %. The coefficient of variation in selectivity of the procedure was 22 %. For the accuracy parameter, the values for recovery/spike were determined as 90–101 %. It was proven that there was no matrix effect.Conclusion. It can be stated that the procedure is linear, specific, highly precise, correct, selective and with a proven absence of matrix effect, which allows it to be used to determine the immunogenicity of romiplostim medicinal products in clinical studies.

Short-Term Efficacy of Combination Therapy with Recombinant Human Thrombopoietin (rh-TPO) and Thrombopoietin Receptor Agonists (TPO-Ra) in Patients with Severe Immune Thrombocytopenia

Introduction: Bleeding manifestations in patients with immune thrombocytopenia (ITP) range from mild skin bruises to lifethreatening intracranial hemorrhage. When the risk of bleeding is becoming high or life-threatening bleeding events occurs, we often use a combination of therapies to rapidly raise platelets to the safe range, including platelet transfusions, high-dose glucocorticoid, and intravenous immunoglobulins. Recombinant human thrombopoietin (rh-TPO) and thrombopoietin receptor agonists (TPO-Ra) are two kinds of thrombopoietic drugs commonly used in China. In patients with severe ITP refractory to glucocorticoids, we lack experience with the use of rh-TPO and TPO-Ra in this emergency situation. Patients and methods： We included ITP patients with platelets <10*10 9/L and bleeding symptoms requiring treatment, who were refractory to glucocorticoids and used thrombopoietic drugs. The bleeding score was calculated with reference to the Chinese guidelines for adult ITP. The patients were divided into three groups according to the medication used: rh-TPO group(rh-TPO 30000IU Qd), TPO-Ra group(Eltrombopag 25mg-75mg/d or Avatrombopag 2.5mg-7.5mg/d or Hetrombopagolamine 20mg-60mg/d), and rh-TPO+TPO-Ra group(combination of two drugs at the same dosage). The platelet count should be rechecked every 1-2 days during the treatment. Platelet transfusion and hemostatic drugs can be used as needed. According to the standardized and international criteria, and taking into account the characteristics of this study, platelet counts were classified as short-term Complete Response(sCR), short-term Response(sR), and short-term No Response (sNR), based on the highest value of platelet counts from the start of treatment to the time of hospital discharge, short-term Overall Response(sOR)=sCR+sR. The three groups were compared the effective time(defined as the time to first sCR/sR after treatment) , peak platelet count after treatment, time to peak platelet count after treatment and other short-term efficacy indicators. Results Nineteen patients were include in this study, with 68.4% (13/19) was female. The median age was 58 years [range 17-79]. 15.8%(3/19) were newly diagnosed with ITP, 10.5%(2/19) had persistent ITP, and 73.7%(14/19) had chronic ITP. All patients were treated with glucocorticoids prior to this treatment, but they had a poor response. The median platelet count was 3*10 9/L [range 1-9] and the median bleeding score was 4 [range 2-9] before treatment. The common bleeding sites were skin ecchymosis and oral mucous membrane hemorrhage in 100%(19/19)and 47.3%(9/19), respectively. Severe bleeding symptoms were the manifestation in 3 cases: including 2 heavy menstrual flow and 1 hemoptysis and gastrointestinal. 100%(19/19) achieved a sOR and 31.6%(6/19) achieved a sCR. The median bleeding score was 0[0-2] after treatment and none had active bleeding symptoms. Platelet transfusion and hemostatic drugs were administered to all patients. Further details are provided in Table 1. Among 19 patients, 7(36.8%) used rh-TPO alone, 6(31.6%) TPO-Ra alone and 6(31.6%) used rh-TPO+TPO-Ra. There was no statistically significant difference in Clinical characteristics, including ages, gender, platelet counts and bleeding score among the three groups of patients before treatment (P=0.727, 0.594, 0.719, 0.299). In terms of treatment efficacy, we were unable to perform a statistical analysis of sOR rates because all three groups reached 100%. The sCR and the peak platelet count after treatment of rhTPO+TPO-Ra group was higher than the other two groups, 28.6%(2/7), 16.7%(1/6) and 50%(3/6) of patients achieved sCR respectively, the peak platelet counts after treatment in the three groups were 62*10 9/L, 72*10 9/L, 79.5*10 9/L respectively in this treatment, but there was no statistically significant difference (P=0.425，0.924). In addition, the effective time and time to peak platelet count after treatment of the three group are also no statistically significant difference(p=0.692,0.505). Further details are provided in Table 2. Conclusion This retrospective study suggests that the patients with severe ITP who were refractory to glucocorticoids, the use of rh-TPO and TPO-Ra may rapidly increase to safe ranges, but the combination is not necessary.

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation

AbstractThe thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F–mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F–driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F–positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F–specific activation.

Platelet Response to Lusutrombopag, a Thrombopoietin Receptor Agonist, in Patients with Chronic Liver Disease and Thrombocytopenia Undergoing Non-Emergency Invasive Procedures: Results from a Phase 3 Randomized, Double-Blind, Placebo-Controlled Study

Thrombocytopenia is a common complication of chronic liver disease (CLD), which may be caused by multiple factors including decreased production of thrombopoietin (TPO). The presence of thrombocytopenia may complicate the conduct of routine invasive procedures that are commonly performed in CLD patients, leading to delayed or cancelled procedures. Lusutrombopag (formerly S-888711) is an orally available, small molecule agonist of human TPO receptor. Here we report the platelet response data from a confirmatory global phase 3, randomized, double-blind, placebo-controlled study (L-PLUS 2 study) evaluating lusutrombopag for the treatment of thrombocytopenia in patients with CLD who were undergoing non-emergency invasive procedures.Key eligibility criteria included 18 years of age or older, platelet count < 50 × 109/L at baseline, Child-Pugh Class A or Class B, and expecting to undergo a non-emergency invasive procedure. Patients with existing or history of portal vein thrombosis (PVT), or with past or present thrombosis or prothrombotic conditions were excluded. Patients were randomized 1:1 to receive oral lusutrombopag 3 mg once daily or placebo for up to 7 days. Stopping criteria based upon platelet count measurement on days 5, 6 and 7 were used to prevent platelet overshoot. Invasive procedures were performed between Day 9 and Day 14. Platelet transfusion was mandated by the study protocol if a patient's platelet count prior to the invasive procedure was < 50×109/L. The primary efficacy endpoint was proportion of patients who require no platelet transfusion prior to the primary invasive procedure and no rescue therapy for bleeding from randomization to 7 days after the primary procedure. Safety assessment included adverse events, physical exams, laboratory tests and imaging for detection of PVT.A total of 215 patients were randomized, constituting the ITT population (lusutrombopag 108, placebo 107). Baseline patient characteristics were similar between the two treatment groups. The most common invasive procedures included endoscopic variceal ligation (28%), endoscopy with or without polypectomy/biopsy (27%), dental extraction (11%), and transarterial chemoembolization (9%). The study met its primary efficacy endpoint: 64.8% of patients in lusutrombopag group required no platelet transfusion prior to their procedures and no rescue therapy for bleeding up to 7 day after the procedure, compared to 29.0% of patients in the placebo group (p<0.0001). The proportion of patients who met the definition of platelet responder (platelet count ≥ 50×109/L and an increase from baseline of ≥ 20×109/L at least once during the study) was 64.8% in lusutrombopag group and 13.1% in placebo group (p<0.0001). Figure 1 shows the median platelet count over the study period for both treatment groups. Among patients in the lusutrombopag group who did not receive platelet transfusion, the mean peak platelet count was 86.9×109/L (mean increase from baseline 47.1×109/L) which occurred on average around Day 12, and the median duration of platelet count maintained at ≥ 50×109/L was 19 days. Treatment-emergent adverse events (TEAEs) were reported for 47.7% of patients in lusutrombopag group and 48.6% in placebo group, with serious TEAEs at 6.5% in each treatment group. Three PVT events were observed, 1 in lusutrombopag group and 2 in placebo group; all were incomplete occlusion considered not clinically significant and all events resolved with therapy. Bleeding-related TEAEs occurred in 3 patients of lusutrombopag group and 6 patients in placebo group.In conclusion, oral lusutrombopag 3 mg once daily demonstrated superiority over placebo as treatment for thrombocytopenia in patients with CLD undergoing invasive procedures in this global phase 3 study, meeting all pre-specified primary and secondary endpoints. Lusutrombopag was well tolerated, with a safety profile similar to that of placebo. [Display omitted] DisclosuresAfdhal:Shionogi: Consultancy; Merck: Consultancy; Ligand: Consultancy; Jansen: Consultancy; Gilead: Consultancy; Echosens: Consultancy; Spring Bank Pharmaceuticals: Employment, Equity Ownership; Allurion: Equity Ownership; TRIO Healthcare: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Duggal:Shionogi: Consultancy. Ochiai:Shionogi: Employment. Motomiya:Shionogi: Employment. Kano:Shionogi: Employment. Nagata:Shionogi: Employment. Peck-Radosavljevic:Shionogi: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria; Lilly: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Jansen: Consultancy, Honoraria; MSD: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Takeda: Research Funding.

TET2 Inhibitory Effects of Eltrombopag Contribute Its Hematopoietic Activity

Severe aplastic anemia (sAA) is bone marrow (BM) failure syndrome characterized by immune mediated BM hypoplasia and pancytopenia usually treated with immunosuppressive therapy (IST) or allogeneic BM transplant. Despite the successes of IST in treating acquired, idiopathic sAA, most of the available treatment options only convert severe into non-severe forms of disease. Recently, eltrombopag (Epag), a small molecule thrombopoietin receptor (TPOR) agonist has been introduced into the routine treatment of de-novo and refractory AA with robust clinical response. Epag treatment showed remarkable tri-lineage responses in AA, suggesting an effect on hematopoietic stem and progenitor cells (HSPCs) amplification. The use of hematopoietic growth factors in AA that increases HSPCs has been associated with justified fears of facilitating AA evolution to MDS. Similarly, Epag has parallel effects on the amplification of HSPCs and there has been recent signals that long term treatment of Epag may increase the MDS risk. Using an in-house developed highly stable cell free high throughput screening of bioactive small molecule chemical library, we identified Epag as one of the most potent inhibitor of TET2. TET2 is the most abundant DNA dioxygenase in HSPCs. The loss-of-function mutations of TET2 frequently occurs in myeloid neoplasia. Here using in vitro and in vivo model system we report that effect of Epag, in part, is mediated by its ability to inhibit TET-dioxygenase activity. We demonstrate that Epag directly binds to TET2 in the presence of Fe2+ and inhibits its dioxygenase function in cell free system. In cell culture model we observed that nearly 40% of Epag partitioned into to nucleus suggesting its potential TET2 inhibitory role in cells. To test if TPOR signaling activation by Epag has any impact on TET activity we engineered murine cell lines BaF3 and 32D by introducing human TPOR. Irrespective of the status of TPOR-JAK-STAT pathway activation, as reflected in STAT5 phosphorylation, Epag demonstrated a robust TET-inhibition activity. However, in similar experiments peptide agonist of TPOR or another small molecule TPOR agonist Avotrombopag did not have any TET inhibitory effects. Consistent with these observations, Epag but not TPO or Avotrombopag inhibited TET activity in murine BM. Thus, Epag mediated TET2 inhibition in these cells are specific and independent of TPOR signaling. Consistent with previous reports, we observed that Epag treatment can significantly expand murine HSPCs even in the absence of TPOR signaling activation. The hematopoietic activity of Epag in murine system is dependent on its ability to inhibit TET2 as confirmed by the lack of Epag effects on Tet2-/- HSPCs. We further confirmed that TET-inhibition by Epag is central for its ability to expand HSPCs in murine model under in vivo settings using BM transplant experiments. We transplanted a mixture of cells derived from Tet2+/+ Pep Boy (CD45.1, 95%) and Tet2-/- C57BL/6 (CD45.2, 5%) into lethally irradiated recipient PepBoy mice. Epag treatment of mice reconstituted with the mixture of BM cells increases the fraction of Tet2+/+ BM cells only. Interestingly, Epag mimic loss of Tet2 in vivo as observed in the expansion of myeloid compartments of Tet2+/+ fractions reflected in CD11b-CD11c+, CD11b+CD11c-, CD11b+CD11c-Ly6C+Ly6G- (monocytes) and CD11b+CD11c-Ly6ClowLy6G+ (neutrophils). We did not observe any significant change in B220+, CD4+ or CD8+ lymphoid populations. On the contrary, we observed a growth restrictive effect of Epag treatment on Tet2-/- myeloid cells. Consistent with our hypothesis that Epag inhibits TET-dioxygenase activity, we observed hypermethylation in the mononuclear cells derived AA patients (n= 16) after Epag treatment. This was further confirmed in ex vivo Epag treatment of BM cells derived from healthy donor. Epag treatment significant increase clonogenic expansion of normal BM cells but not TET2 mutant myeloid cells. In summary, here we demonstrate that Epag is a potent inhibitor of TET-dioxygenase and its biological consequences are independent of TPOR-JAK-STAT activation. Epag treatment mimics TET2 loss of function in AA cells, however it restricts clonal expansion of TET2 mutant myeloid cells. Disclosures Patel: Alexion: Other: educational speaker. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491

AbstractMutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning

AbstractThe single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F− essential thrombocythemia and primary myelofibrosis patients, respectively, have “canonical” MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other “noncanonical” MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

Pharmacological characterization of hetrombopag, a novel orally active human thrombopoietin receptor agonist

Nonpeptide thrombopoietin receptor (TPOR/MPL) agonists, such as eltrombopag, have been used to treat thrombocytopenia of various aetiologies. Here, we investigated the pharmacological properties of hetrombopag, a new orally active small‐molecule TPOR agonist, in preclinical models. Hetrombopag specifically stimulated proliferation and/or differentiation of human TPOR‐expressing cells, including 32D‐MPL and human hematopoietic stem cells, with low nanomolar EC 50 values through stimulation of STAT, PI3K and ERK signalling pathways. Notably, hetrombopag effectively up‐regulated G1‐phase–related proteins, including p‐RB, Cyclin D1 and CDK4/6, normalized progression of the cell cycle, and prevented apoptosis by modulating BCL‐XL/BAK expression in 32D‐MPL cells. Moreover, hetrombopag and TPO acted additively in stimulating TPOR‐dependent signalling, promoting cell viability, and preventing apoptosis. Orally administered hetrombopag specifically promoted the viability and growth of 32D‐MPL cells in hollow fibres implanted into nude mice with much higher potency than that of the well‐known TPOR agonist, eltrombopag, in association with activation of TPOR‐dependent signal transduction in vivo. Taken together, our findings indicate that, given its favourable pharmacological characteristics, hetrombopag may represent a new, orally active, small‐molecule TPOR agonist for patients with thrombocytopenia.

Affinity maturation of an TpoR targeting antibody in full-length IgG form for enhanced agonist activity.

It has been observed that converting scFv formatted antibodies to full-length IgG often associates with loss of affinity. We aim to address this issue in this paper by establishing an integrated affinity maturation method applying yeast display technology platform. To demonstrate that, we employed a human thrombopoietin receptor targeting antibody named 3D9 which was identified previously from a combinational antibody library in scFv-Fc fusion protein form. We have observed that significant potency loss happened when 3D9 was transformed to full-length IgG form. In this study, we tested whether the potency of the full-length IgG can be improved by affinity maturation of 3D9 using a modified Fab yeast display platform. An efficient CDR3 targeted mutagenesis strategy was designed for Fab library with pre-designed CDR diversity. Next generation sequencing was also used for evaluation of the enrichment process and investigation of sequence-function relationship of the antibody. A variant with improved affinity and higher potency was identified. The study demonstrates that the strategy we used here are efficient for optimizing affinity and activity of full-length IgGs.

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His(499) near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His(499) is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His(499) regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.

Lusutrombopag: First Global Approval.

Lusutrombopag (Mulpleta®) is an orally bioavailable, small molecule thrombopoietin (TPO) receptor agonist being developed by Shionogi for chronic liver disease (CLD) patients with thrombocytopenia prior to elective invasive surgery. Lusutrombopag acts selectively on the human TPO receptor and activates signal transduction pathways that promote the proliferation and differentiation of bone marrow cells into megakaryocytes, thereby increasing platelet levels. In September 2015, lusutrombopag received its first global approval in Japan for the improvement of CLD-associated thrombocytopenia in patients scheduled to undergo elective invasive procedures. This article summarizes the milestones in the development of lusutrombopag leading to this first approval.

The treatment for primary immune thrombocytopenia with romiplostim in adult and paediatric patients: use experience at a Spanish university hospital

Primary immune thrombocytopenia (ITP) is characterized by accelerated platelet destruction, as well as suboptimal platelet production. Thrombopoietin (TPO) receptor agonists bind to and activate human TPO receptor, and have been shown to increase platelet counts. In this study, we assessed the effectiveness and safety of long-term administration of TPO agonist romiplostim in adult and paediatric patients. This is a retrospective observational study that included every ITP patient (adults and children) who received romiplostim since its inclusion in our institutional formulary. Data on patients' demographics, romiplostim doses, platelet counts, use of rescue medication and concurrent therapies were collected. Outcomes for effectiveness evaluation were proportion of patients who achieved a platelet response (platelet count >50×10(9) per litre and double the platelet count at baseline on any scheduled visit, excluding counts obtained within 8weeks after receipt of rescue medications), proportion of patients who achieved a durable response (platelet responses during 6 or more weeks of the last 8weeks of treatment), proportion of patients needing rescue medication, proportion of patients able to stop or reduce concurrent treatment and mean number of weekly platelet responses. Safety was assessed on the basis of the incidence of adverse events documented on the patients' medical records. This study enrolled ten adults and four paediatric patients. None of the paediatric patients and one adult patient had been splenectomized (contraindicated in the other adults). In the adult population, eight achieved a response at least once during treatment, and 1 achieved a durable response. Four patients needed rescue medication (mostly intravenous immunoglobulins). Three patients were able to stop concurrent ITP therapies, and the mean number of weekly platelet responses was 6. All four paediatric patients achieved a response at least once during treatment, and three achieved durable responses. Three patients needed rescue medication. The only patient who was receiving concurrent ITP medication was able to stop it, and the mean number of weekly platelet responses was 25. No serious adverse events were registered during treatment in either population. The effectiveness of romiplostim was variable with few adult patients achieving a durable response. Our paediatric patients responded better with most achieving a durable response. The treatment was safe for both groups of patients. Studies should be conducted to identify patients more likely to benefit from this treatment.

Novel Small-Molecule Human G-CSF Receptor Agonist Stimulates Neutrophil Counts In Monkeys and Displays Anti-Proliferative Effects On Tumor Cells Mediated By a Reduction Of Intracellular Iron

Previously we reported on the discovery of a series of novel, non-peptidyl, small molecules that selectively activate the human granulocyte colony stimulating factor receptor (hG-CSFR) in a manner distinct from G-CSF and similar to the mechanism of small-molecule human thrombopoietin receptor (hTPOR) agonists [Marschke KB, et al. ASH 53rd Annual Meeting Abstracts, Blood 2011 118(21):1453, Abstract 3391]. The lead compound, LG7455, activates STAT5 and STAT3 signal transduction and induces G-CSF-dependent hematopoietic cell growth, but not TPO-dependent or erythropoietin-dependent cell growth. LG7455 promotes the differentiation of human bone marrow cells into granulocytes, significantly increasing expression of the granulocyte-specific marker CD15 (FUT4). The G-CSF-like effect of LG7455 requires the expression of human or monkey G-CSFR, and a histidine in the transmembrane domain is needed for activity, similar to what has been found for small-molecule hTPOR agonists, such as eltrombopag. LG7455 increases the binding of GCSF in a manner consistent with allosteric receptor modulation. To test the ability of LG7455 to stimulate neutrophil counts in vivo, cynomolgus monkeys were dosed intravenously (IV) 3 times per day (TID) to increase LG7455 exposure. Increases in peripheral blood neutrophils were observed in male cynomolgus monkeys with LG7455 (2 mg/kg/dose, TID). Because of the potential for LG7455 to interact with metal ions, their role in the activity of LG7455 was investigated. The ability of LG7455 to activate luciferase expression in cells transiently transfected with an hG-CSFR expression vector and a STAT5-responsive luciferase reporter was dependent on the addition of zinc (II) ion (up to 30 µM) to the cell culture media. The addition of Zn(II) also significantly enhanced the efficacy (63% vs. 23% relative to rhG-CSF) and potency ( EC50 of 28 nM vs. 300 nM) by which LG7455 stimulated the growth of UT-7 cells made dependent on G-CSF by stable transfection of hGCSFR (UTP-hGCSFR). LG7455 did not activate luciferase expression or stimulate growth of cells incubated in the presence of additional iron (III), copper (II), magnesium (II) or calcium (II) ions. Similar to the antitumor effect of the well-described iron chelator salicylaldehyde isonicotinyl hydrazine (SIH), LG7455 inhibited the growth of a selection of human tumor cell lines, including the myeloid leukemia cell lines HL-60 and Kasumi-1, the lung carcinoma cell line A549, the embryonic kidney cell line HEK293, and the hepatocellular carcinoma cell line HepG2 (IC50's of 5.0, 12.1, 6.1, 1.4 and 1.0 µM, respectively). SIH did not stimulate but instead inhibited the growth of UTP-hGCSFR cells. The anti-proliferative effect of LG7455 on HL-60 cells was accompanied by an increase in apoptosis as determined by Annexin V/propidium iodide staining and FACS analysis. The addition of excess Fe(III) to the tumor cells was able to overcome the anti-proliferative effect of LG7455 in a concentration-dependent manner, indicating that a reduction in intracellular iron plays a role in the effect. The addition of Zn(II) and Cu(II) were less efficient at blocking the anti-proliferative effect of LG7455 than Fe(III). Addition of Mg(II) or Ca(II) had no effect. LG7455 increased the fluorescence of HL-60 cells loaded with calcein-AM, a fluorescent dye quenched when bound to iron, further supporting that a reduction of free intracellular iron occurs in cells treated with LG7455. Lastly, as has been shown with other iron chelators with anti-proliferative activity, LG7455 increased intracellular reactive oxygen species (ROS) formation in HL-60 cells loaded with the cell-permeant ROS indicator, CM-H2-DCFDA, and evaluated by flow cytometry. Further optimization of the LG7455 series may provide a novel, orally-available adjuvant anticancer therapy that is also a supportive care agent to treat neutropenia in patients receiving bone-marrow suppressive treatments. Disclosures:Marschke:Ligand Pharmaceuticals Incorporated: Employment. Vajda:Ligand Pharmaceuticals Incorporated: Employment. Zhi:Ligand Pharmaceuticals Incorporated: Employment.

Tryptophan at the transmembrane–cytosolic junction modulates thrombopoietin receptor dimerization and activation

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

Discovery of Non-Peptidyl Small-Molecule Human GCSF Receptor Agonists for the Potential Treatment of Neutropenia,

Abstract 3391Granulocyte colony stimulating factor (GCSF) is the essential cytokine for the regulation of neutrophilic granulocytes. Binding of GCSF to its receptor (GCSFR) triggers receptor dimerization, leading to activation of JAK1 and JAK2, phosphorylation of GCSFR, STAT3, STAT5, and Ras/mitogen-activated protein kinase (MAPK), and results in proliferation and differentiation of granulocytic cells. Recombinant human GCSF (rhGCSF) is used successfully to alleviate chemotherapy-induced neutropenia, neutropenia associated with hematopoietic stem cell transplantation, and severe chronic neutropenia. A small molecule oral GCSFR agonist may offer a safer and more convenient alternative to the current injectable rhGCSF therapy. Whereas a previous effort to identify small-molecule mimetics of GCSF found SB-247464 that selectively activated the murine GCSFR, no small-molecule human GCSF mimetics have been developed. Recently, we have discovered a series of novel non-peptidyl small molecules that selectively activate human GCSFR (hGCSFR) function, and may provide a significant innovation in the treatment of neutropenia. In cells transiently transfected with an hGCSFR expression vector and a STAT3-responsive luciferase reporter, a lead compound, LG7455, activates luciferase expression with an efficacy of 50% relative to rhGCSF, and potency (EC50) of 100 nM. LG7455 also activates luciferase expression in cells transfected with hGCSFR and a STAT5-responsive luciferase reporter (65%, 40 nM EC50). The activity of LG7455 is dependent on the expression of hGCSFR, and LG7455 is not active in luciferase assays when human thrombopoietin receptor (hTPOR) or erythropoietin receptor (hEPOR) is expressed. In UT-7 cells made responsive to GCSF by stable transfection of hGCSFR (UTP-hGCSFR), LG7455 stimulated cell growth and increased the phosphorylation of STAT3 and STAT5. LG7455 did not increase growth of TPO- or EPO-responsive UT-7 cells. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), LG7455, increased the percentage of cells positive for the granulocyte-specific marker CD15 (FUT4). The effect of LG7455 in BM-HCs was additive to the effect of rhGCSF. LG7455 is active in luciferase assays with expressed cynomolgus monkey GCSFR, but not mouse, guinea pig or rabbit GCSFR. Similar to what has been demonstrated for small-molecule human TPOR agonists such as eltrombopag, the activity of LG7455 is dependent on a specific residue in the hGCSFR transmembrane domain. When histidine 627 (His-627) in hGCSFR is changed to asparagine present at a similar location in the mouse GCSFR (Asp-602), unlike rhGCSF, LG7455 is no longer active. LG7455 is active, however, on mouse GCSFR with Asp-602 replaced by His. In radioligand-binding experiments using UTP-hGCSFR cells, LG7455 did not displace [125I]rhGCSF, however binding of [125I]rhGCSF was augmented in a concentration dependent manner consistent with allosteric receptor modulation. These data demonstrate that LG7455 is a novel small-molecule selective hGCSFR agonist that activates the receptor in a manner distinct from GCSF and similar to the mechanism of small-molecule hTPOR agonists. Further optimization of the LG7455 chemical series should provide orally-available molecules to treat neutropenia with improved safety and convenience compared to current injectable rhGCSF. Disclosures:Marschke:Ligand Pharmaceuticals: Employment. Rungta:Ligand Pharmaceuticals: Employment. Slavin:Ligand Pharmaceuticals: Employment. Sanders:Ligand Pharmaceuticals: Employment. Roach:Ligand Pharmaceuticals: Employment. Pickens:Ligand Pharmaceuticals: Employment. Shen:Ligand Pharmaceuticals: Employment. van Oeveren:Ligand Pharmaceuticals: Employment. Hong:Ligand Pharmaceuticals: Employment. Sun:Ligand Pharmaceuticals: Employment. Bissonnette:Ligand Pharmaceuticals: Employment. Syka:Ligand Pharmaceuticals: Employment. Zhi:Ligand Pharmaceuticals: Employment.

Metabolism and Disposition of Eltrombopag, an Oral, Nonpeptide Thrombopoietin Receptor Agonist, in Healthy Human Subjects

The metabolism and disposition of eltrombopag, the first-in-class small molecule human thrombopoietin receptor agonist, were studied in six healthy men after a single oral administration of a solution dose of [(14)C]eltrombopag (75 mg, 100 μCi). Eltrombopag was well tolerated. The drug was quickly absorbed and was the predominant circulating component in plasma (accounting for 63% of the total plasma radioactivity). A mono-oxygenation metabolite (M1) and acyl glucuronides (M2) of eltrombopag were minor circulating components. The predominant route of elimination of radioactivity was fecal (58.9%). Feces contained approximately 20% of dose as glutathione-related conjugates (M5, M6, and M7) and another 20% as unchanged eltrombopag. The glutathione conjugates were probably detoxification products of a p-imine methide intermediate formed by metabolism of M1, which arises through cytochrome P450-dependent processes. Low levels of covalently bound drug-related intermediates to plasma proteins, which could result from the reaction of the imine methide or acyl glucuronide conjugates with proteins, were detected. The bound material contributes to the longer plasma elimination half-life of radioactivity. Renal elimination of conjugates of hydrazine cleavage metabolites (mostly as M3 and M4) accounted for 31% of the radiodose, with no unchanged eltrombopag detected in urine.

Pharmacological profile of AS1670542, a novel orally-active human thrombopoietin receptor agonist

Eltrombopag, an orally-active small molecule thrombopoietin (TPO) receptor agonist, was used for the first time in 2008 to treat patients with chronic idiopathic thrombocytopenic purpura. Here, we investigated the pharmacological effect of a new orally-active small molecule TPO receptor agonist which may be effective in treating these patients. 50% effective concentration values for cell proliferation with AS1670542 or eltrombopag were 1.9 and 13 nM, respectively, while those for megakaryocyte colony formation from human cord blood CD34 + cells with AS1670542 or eltrombopag were 260 and 950 nM, respectively. On Day 14 after the start of administration, AS1670542 significantly increased the number of human platelets in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice with transplanted human hematopoietic stem cells at 0.3 ( P < 0.05); in contrast, while administration of eltrombopag also increased the numbers of these platelets at 30 mg/kg/day ( P = 0.058), no statistical significance was noted in the increase. Here, we identified AS1670542, a novel orally-active TPO receptor agonist which mimics the biological activity of TPO and may demonstrate greater in vitro and in vivo pharmacologically efficacy than eltrombopag.