Thrombopoietin (TPO) is produced in the liver and regulates the proliferation and differentiation of hematopoietic stem cells and megakaryocytes, called pancytopenic factors. TPO activates downstream signaling pathways through its receptor c-Mpl (also called MPL) to promote cell survival and proliferation. Because TPO is important for hematopoiesis, alterations in its receptors and hormones lead to diseases: e.g. congenital and acquired thrombocythemia, thrombocytopenia. dysregulation of TPO expression or altered function of c-Mpl can lead to aplastic anemia (AA).
Recombinant TPO (rhTPO), not only drives megakaryocyte differentiation, but also promotes the proliferation of megakaryocyte progenitors. In vitro experiments showed that rhTPO significantly increased the number and size of megakaryocyte colony-forming units (CFU-MK). In the absence of TPO, CFU-MK formation was completely terminated. in the mid-1990s, with the understanding of the structure and function of TPO, platelet thrombopoietin began to be used in the treatment of human diseases. the first generation of TPO drugs included rhTPO and polyethylene glycolylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF).
rhTPO is a glycoprotein molecule expressed in mammalian cells and modified by intact glycosylation, and is homologous to endogenous TPO. When administered intravenously to cancer patients, rhTPO causes a rise in platelets after 5 d, peaking at 10-14 d. PEG- rHuMGDF is expressed by E. coli and has one more methionine at the amino terminus than the full-length TPO molecule, with only 165 amino acids at the amino terminus of the TPO molecule. When injected subcutaneously, PEG- rHuMGDF has similar effects to rhTPO.
All trials involving first-generation TPO drugs were stopped because of the cross-reactivity between the recombinant drug and endogenous TPO and thus its ability to cause antigenic reactions, leading to the development of thrombocytopenia in 13 of 538 healthy volunteers who received PEG-rHuMGDF [1].
Cwirla et al [2] identified a peptide containing 14 amino acids that binds cCMpl with high affinity. because it is not homologous to TPO, it does not elicit an immune response. Four of these linear peptides were covalently fused to the Fc fragment of the human IgG1 heavy chain to increase its longevity and activity in circulation [3]. This “peptidome”, named romistatin, binds to the c-Mpl receptor and successfully stimulates the same downstream signaling pathway by mimicking rhTPO.
Subcutaneous administration of romisetin increased platelet counts in 80% of patients with immune thrombocytopenic purpura (ITP). Long-term studies have shown that the drug is well tolerated and shows no signs of antigenic reactions. The U.S. Food and Drug Administration (FDA) approved romiplostim for refractory ITP in 2008.
Another TPO analogue, etrepipate, was synthesized by large-scale screening of non-peptide substances and is an oral version of a TPO analogue. Unlike peptide analogs, etriboplat binds non-competitively to TPO to cCMpl. It interacts with the transmembrane structural domain that activates the active receptor of cCMpl [5]. In clinical trials ITP patients receiving itrapropa had increased platelet counts despite a significantly lower increase in platelets than those on romiplostim. In 2008 the FDA also approved itrapropa as a second-line treatment for patients with ITP.
I. Application in the treatment of aplastic anemia AA
Thrombocytopenia is one of the major causes of death in patients with aplastic anemia (AA). Thrombocytopenia is caused by alterations in the number and function of hematopoietic stem and progenitor cells resulting in impaired megakaryocyte production and insufficient production of mature platelets. Recent studies have shown that TPO analogs are effective in the treatment of bone marrow failure syndrome.
Although TPO levels are high in bone marrow failure syndrome, supraphysiological pharmacological levels of TPO analogs may be able to treat bone marrow failure. Previous studies have shown poor results with other growth factors such as erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF), perhaps because these factors act on biased mature myeloid progenitor cells, whereas TPO is able to act on more primitive progenitor cells and hematopoietic stem cells (HSC), supporting its role in the treatment of bone marrow failure.
Romistatin activates signaling through the JAK-STAT and MAPK pathways. In a non-randomized phase II trial, 25 patients with immunosuppression-tolerant severe AA and thrombocytopenia were treated with romipristin. Prior to romipristin, all patients required platelet transfusions and were treated with a median duration of 2 (1 to 4) courses of immunosuppressive therapy (IST). The initial dose of romiplostim was 50 mg per day and was increased at a standard rate of 25 mg every 2 weeks when the platelet count remained below 20×109/L.
After 12 to 16 weeks of treatment 44% (11/25) of patients were effective and all three lines were able to show a hematopoietic response. Most effective patients were able to come off platelet transfusion (9/11) and platelet count was eventually able to increase to a mean of 44 x 109/L. Six patients (6/11) had a significant increase in the red lineage and nine patients (9/11) had a neutrophil response. These clinical results support the hypothesis that TPO analogs can directly enhance trilineage hematopoiesis.
In a recent clinical trial of patients with severe AA who were refractory to immunosuppression treated with etriboplat, results showed that patients who responded well to etriboplat were able to sustain improvement and that a subset of patients were eventually able to have significant increases in neutrophils, platelets and red blood cells. Patients with a hematologic response were followed for more than 8 months and were found to have normalized their bone marrow picture and peripheral blood.
Previous studies of patients with ITP treated with TPO analogs found that platelet agonists may cause myelofibrosis. However, in the current study of AA, there is no evidence that TPO analogs cause the development of myelofibrosis in AA patients. These patients were monitored for drug response and toxicity using a series of bone marrow examinations, including pre- and post-treatment bone marrow biopsies, which were performed every 6 months for more than 30 months.
In patients with ITP, those treated with TPO analogs had ultra-high levels of megakaryocytes, which were used to compensate for platelet destruction, and myelofibrosis was likely associated with the release of fibrogenic factors from megakaryocytes. Compared to healthy controls, serum concentrations of TPO were significantly higher in the treated patients, but this number did not change over time. Immunophenotyping of leukocyte telomere length and T-cell subsets in effective and ineffective patients did not differ significantly before, after or during treatment with romiplostim [8].
II. Application in myelodysplastic syndromes (MDS)
Clinical studies have shown that romiplostim stimulates the growth of megakaryocytes in MDS patients and potentially reduces the number of malignant clonal cells, which provides a theoretical basis for experiments with TPO analogs. TPO levels are also increased in MDS, but only mildly so compared to AA. Romipristin has been used in patients with low-risk MDS and thrombocytopenia. This study reported a decrease in bleeding events and a decrease in the frequency of platelet transfusions in 46% of patients.
Two cases (5%) progressed to acute myeloid leukemia (AML), and four cases (9%) had increased primitive cell counts, which subsequently decreased when romiplostim was discontinued. In a subsequent double-blind randomized trial with low-risk, intermediate-risk-1 patients with placebo as the control group and romisetin as the experimental group, there were 219 cases, with a 2:1 ratio of experimental to control group, romisetin group (147): placebo group (72). Twelve cases progressed to AML, 10 in the treatment group and 2 in the control group, yielding a risk ratio (HR) of 2.51 for patients with romiplostim.
The number of primitive granulocytes in peripheral blood increased up to 10% in 28 cases, 25 of which were in the treatment group. Eight cases were diagnosed with AML, and this trial was terminated early because more patients in the treatment group progressed to AML. The results of the increased number of primitive cells and the high risk of conversion to AML in these MDS patients were added to the prescribing information for romiplostim.
However, in the control group, more than 2 cases that developed AML were not analyzed earlier; the chance of progression to leukemia was 8.9% with romipristine and 8.5% in the control group, and the HR for romipristine to prompt progression to AML was 1.2. Of the 22 cases that progressed to leukemia, 12 were refractory anemia with primocytosis-I (RAEB-I).
Common treatment options for MDS include demethylation and ranadolamide. These regimens are effective in half of the patients, but are associated with significant drug toxicity and common hematocrit. TPO analogs have been combined with these regimens for low-risk MDS with the expectation of reducing the complications of platelet reduction. Clinical studies have been initiated with the combination of romipristin and the desmethyl drug decitabine. Patients with low-risk or intermediate-risk-1 MDS are randomized 2:1 to either the romisetin or placebo group.
Clinically significant events (CSTEs) of thrombocytopenia were used as study endpoints. The results of the study showed a trend toward higher platelet counts, less frequent platelet transfusions, and fewer bleeding events in patients with low-risk or intermediate-risk-1 MDS treated with romiplostim. In the phase 2 study of MDS patients at low and intermediate risk receiving azacitidine in combination with romisetin. 40 patients were randomly assigned to romisetin 500 μg, 750 μg, and placebo in 4 courses of azacitidine, administered subcutaneously once weekly. The evaluation endpoints were CSTEs and the number of platelet transfusions.
The results showed no statistically significant difference in CSTEs. However, on day 1 of the 3 sessions – when platelet counts were lowest – the mean number of platelets was increased in the experimental group when compared to the placebo group. In a phase II multicenter open study, 28 patients with thrombocytopenia and low-risk MDS were given romiplostim 750 μg subcutaneously once a week or once every 2 weeks or intravenously once every week for 8 weeks.
The safety and efficacy of romiplostim suggest that 750 μg administered subcutaneously once weekly is an appropriate starting dose for future clinical studies in MDS and thrombocytopenia [20]. In a phase I pilot study exploring the safe dose of etrepipate, this study required that patients be primed with MDS, have a platelet count << span="">75×109/L, and be able to receive azacitidine. In 3 cycles of azacitidine therapy, the combination was administered with etriboplat and the drug dose was gradually increased. A total of 12 patients with a median age of 74 years were treated.
Serious adverse effects included infection, lower extremity deep vein thrombosis, and transient ischemic attack. Four cases achieved complete remission or bone marrow remission. Despite the administration of azacitidine, platelet counts increased or remained stable in 9 cases. There was no increase in primitive cell count, disease progression, or myelofibrosis as reported in previous drug studies.
Thrombocytopenia occurred in 44% to 74% of patients with low-risk MDS treated with ranadolide, which led to reduction of ranadolide or discontinuation of therapy. Combination therapy with TPO analogs can reduce the degree of thrombocytopenia, maintain adequate doses and regimens, and achieve better outcomes. The phase II multicenter, placebo-controlled randomized trial randomized 39 patients with low- or intermediate-risk-1 MDS to the placebo, romiplostim 500 μg, and 750 μg groups, and these patients received 4 concurrent courses of ranadolide.
In the romipristin group, treatment efficiency was higher and blood product transfusion rates were lower. Because of the small number of cases, no dose response was observed for romiplostim. 50% of patients in the placebo group, 36% in the romiplostim 500 μg group, and 15% in the romiplostim 750 μg group required a reduction in the ranadolide dose. The results confirmed that romipristine reduced the risk of ranadomide dose reduction or discontinuation.
In another study, the combination of ranadomide and etrapolpa in patients with AML and MDS showed that the combination significantly inhibited the proliferation of malignant clones in most patients with primary MDS and AML. Furthermore, in primary MDS, etriboplat was able to reduce the anti-megakaryocytic and platelet-reducing side effects of ranadomide. These results provide a preclinical rationale for the combination of MDS and AML.
An ongoing Italian multicenter clinical trial has yielded promising results in patients with low- and intermediate-risk-1 MDS with IPSS and platelets less than 30 x 109/L. Romilastine dosing was started at 50 mg and increased every 2 weeks up to 300 mg.
At a median follow-up of 6 months, defined by platelets greater than 100×109/L and no bleeding events, 5 of 10 patients were in complete remission and 1 was in partial remission. Only one patient in the placebo group was in remission, which was considered “unstable remission”. No patient in the treatment group progressed to progressive MDS (intermediate risk-2 to high risk according to WHO criteria), while one patient in the placebo group progressed.
III. Outlook
The new generation of thrombopoietin analogs can act on hematopoietic stem cells and megakaryocytes to promote the proliferation and differentiation of myeloid cells, including megakaryocytes, without the antigenicity of rhTPO, opening up new ideas for the treatment of bone marrow failure disorders. Previous clinical trials have confirmed its efficacy in bone marrow failure disorders including AA and MDS. However, the current study is mainly as an adjuvant drug and second-line treatment option, which still needs to be validated by large sample clinical trials. In addition, its mechanism of action on hematopoietic stem and progenitor cells needs to be further investigated.