Use of telomerase inhibitors for treatment of myeloproliferative disorders and myeloproliferative neoplasms

Abstract

To provide new treatments for myeloproliferative disorders or neoplasms and myelodysplastic syndrome which target neoplastic progenitor cells responsible for malignant phenotypes of the diseases in individuals who are resistant to, or has experienced adverse events as a result of taking, commonly prescribed front-line therapies for the disorders.SOLUTION: The invention provides methods for using telomerase inhibitor compounds to treat or mitigate symptoms associated with myeloproliferative neoplasms such as essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), acute myelogenous leukemia (AML) by targeting neoplastic progenitor cells characteristic of the diseases.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 734,941, filed on December 7, 2012; U.S. Provisional Patent Application No. 61 / 799,069, filed on March 15, 2013; U.S. Patent Application No. 13 / 841,711, filed on March 15, 2013; and U.S. Provisional Patent Application No. 61 / 900,347, filed on November 5, 2013, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] The present invention relates to methods of using telomerase inhibitor compounds for treating or preventing symptoms associated with myeloproliferative diseases or myeloproliferative tumors such as essential thrombocythemia (ET).

Background Art

[0003] Hematological malignancies are forms of cancer that begin in cells of the blood - forming tissues such as the bone marrow or in cells of the immune system. Examples of blood cancers include acute and chronic leukemias, lymphomas, multiple myeloma, and myelodysplastic syndromes.

[0004] Myeloproliferative neoplasms or MPNs are blood tumors that arise from neoplastic hematopoietic bone marrow progenitor cells such as erythroid, platelet, and granulocyte progenitor cells. The proliferation of tumor progenitor cells results in the overproduction of any combination of white blood cells, red blood cells, and / or platelets, depending on the disease. These overproduced cells can also be abnormal and cause further clinical complications. There are various types of chronic myeloproliferative disorders. Included in the MPN disease spectrum are essential thrombocythemia (ET), polycythemia vera (PV), and chronic myeloid leukemia (CML), myelofibrosis (MF), chronic neutrophilic leukemia, chronic eosinophilic leukemia, and acute myeloid leukemia (AML). Myelodysplastic syndromes (MDS) are a collection of symptoms that include cancers of the blood and bone marrow. Myelodysplastic syndromes (MDS) include diseases such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with single-lineage dysplasia, and chronic myelomonocytic leukemia (CMML).

[0005] Essential thrombocythemia Circulating blood platelets are anucleate yet retain the ability to biosynthesize small amounts of megakaryocyte-derived mRNA and fully functional proteins (Gnatenko et al., Blood 101, 2285-2293 (2003)). Essential thrombocythemia (ET) is a subtype of myeloproliferative disorder characterized by increased neoplastic proliferation of megakaryocytes, elevated numbers of circulating platelets, and thrombotic and hemorrhagic events to be considered, often neurological symptoms (Nimer, Blood 93, 415-416 (1999)). ET is seen with equal frequency in men and women, but the additional peak incidence of women at 30 years of age can account for the clearly higher disease prevalence in women after this age. The molecular basis of ET remains established, but has historically been considered a "clonal" disorder (El-Kassar et al., Blood 89, 128 (1997); "Evidence that ET is a clonal disorder with origin in a multipotent stem cell" PJ Fialkow, Blood 1981 58:916-919). In addition to the obvious exaggeration of platelet volume in subsets of ET platelets, the cells remain morphologically indistinguishable from their normal counterparts. There are currently no functional or diagnostic tests available for ET, and it remains diagnosed by the exclusion of other possible blood disorders. Incidence estimates of 2-3 per 100,000 per year are not inconsistent with other types of leukemia, but the prevalence is at least 10-fold higher due to the low mortality associated with ET.

[0006] Current treatments for ET mainly focus on the prevention of thrombotic / hemorrhagic events and necessarily involve the non-specific reduction of blood platelet levels. However, none of these existing treatments specifically target the tumor progenitor cells that drive the malignancy underlying the condition. For example, the treatment of ET with cytotoxic chemotherapy reduces tumor cells but leaves residual progenitor cells. This results in the generation of new tumor cells from the progenitor cells, leading to the continuation of the condition. In addition, many individuals with ET develop resistance to front-line treatments such as hydroxyurea or discontinue the use of these drugs altogether due to adverse side effects.

[0007] Polycythemia vera Patients with polycythemia vera (PV) exhibit increased erythropoiesis. Treatment is aimed at reducing the excess number of erythrocytes. PV can progress in stages in its later stages similar to primary myelofibrosis with cytopenia and myelosuppression and fibrosis. A genetic mutation in the Janus kinase 2 gene (JAK2) on chromosome 9, which causes increased proliferation and survival of hematopoietic precursors in vitro, has been identified in the majority of patients with PV. Patients with PV have an increased risk of cardiovascular and thrombotic events and transformation to acute myeloid leukemia or primary myelofibrosis. Treatment of PV includes intermittent phlebotomy to maintain a hematocrit of less than 45% in men and less than 40% in women. Other possible treatments include hydroxyurea, interferon-alpha, and low-dose aspirin (includee).

[0008] Myelofibrosis Myelofibrosis or MF, or primary myelofibrosis, is a myeloproliferative neoplasm in the same disease spectrum as ET. Patients with MF often have the JAK2 V617F mutation in their bone marrow. Occasionally, ET progresses to MF. JAK2 inhibition is currently considered the standard treatment for MF in countries where the Janus kinase inhibitor, ruxolitinib (Jakafi®), is approved. There is no evidence that JAK2 inhibitors such as Jakafi® selectively inhibit the proliferation of leukemic clones that are the cause of the disease, and thus they may not be "disease-modifying".

[0009] Acute myeloid leukemia Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells. AML is the most common acute leukemia affecting adults. Patients with AML have a rapid proliferation of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Replacing normal bone marrow and white blood cells reduces red blood cells, platelets, and normal white blood cells. Symptoms of AML include fatigue, shortness of breath, easy bruising and bleeding, and an increased risk of infection. Like acute leukemia, AML progresses rapidly and generally leads to death within weeks or months if left untreated. Standard treatment for AML is treatment using chemotherapy aimed at inducing remission, and patients may undergo a hematopoietic stem cell transplant.

[0010] Myelodysplastic syndrome Myelodysplastic syndrome (MDS) is a collection of symptoms including cancers of the blood and bone marrow. Myelodysplastic syndrome (MDS) includes diseases such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with single lineage dysplasia, and chronic myelomonocytic leukemia. Immature blood stem cells (blasts) do not become healthy red blood cells, white blood cells, or platelets. Blasts die soon after moving into the bone marrow or the blood. This reduces the space for forming healthy white blood cells, red blood cells, and / or platelets in the bone marrow.

[0011] Myelodysplastic syndrome (MDS) is a collection of hematological medical symptoms that necessarily includes ineffective hematopoiesis of the myeloid class of blood cells. Patients with MDS often develop severe anemia and require frequent blood transfusions. Due to low or dysfunctional platelets and neutrophils, the risks of bleeding and infection also occur, respectively. If the disease worsens, patients may develop cytopenia (low blood cell count) caused by progressive bone marrow failure. The disease may also transform into acute myeloid leukemia (AML). If the overall proportion of bone marrow blasts rises above a certain cut-off (20% for WHO and 30% for FAB), it is said that transformation into acute myeloid leukemia (AML) has occurred.

[0012] Therefore, there is a need for novel treatments for myeloproliferative diseases or tumors, such as ET, PV, MF, CML, and AML, and myelodysplastic syndromes, that target pre-tumor cells that are the cause of the malignant phenotypes of the disease, particularly in individuals who are resistant to or have experienced adverse events as a result of front-line treatments commonly prescribed for this disease.

[0013] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles) are referenced. The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0015] The invention provided herein discloses methods for using telomerase inhibitor compounds to treat and alleviate symptoms associated with myeloproliferative tumors such as essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), and acute myeloid leukemia (AML), particularly by targeting pre-tumor cells specific to these diseases. The invention provided herein also discloses methods for using telomerase inhibitor compounds to treat and alleviate symptoms associated with myelodysplastic syndromes (MDS) such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with monosomy dysplasia, and chronic myelomonocytic leukemia, particularly by targeting pre-tumor cells that cause an abnormally high number of cell production specific to these diseases.

[0016] Accordingly, in one aspect, the present specification provides a method for reducing at least one symptom associated with a myeloproliferative neoplasm in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with the myeloproliferative neoplasm. In some embodiments, the symptoms include headache, dizziness or fainting, chest pain, weakness, syncope, changes in vision, numbness or tingling in the limbs, flushing, throbbing or burning pain in the limbs (erythromelalgia), splenomegaly, epistaxis, purpura, bleeding from the mouth or gums, bloody stools, or stroke. In some embodiments, the myeloproliferative neoplasm is, for example, essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), and acute myeloid leukemia (AML). In some embodiments of any of the embodiments herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any of the embodiments herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any of the embodiments herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any of the embodiments herein, the oligonucleotide further comprises a lipid moiety attached to the 5’ and / or 3’ terminus of the oligonucleotide. In some embodiments of any of the embodiments herein, the lipid moiety is attached to the 5’ and / or 3’ terminus of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any of the embodiments herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any of the embodiments herein, the telomerase inhibitor is imetelstat. In some embodiments of any of the embodiments herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient.In some embodiments of any embodiment of the present specification, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intraocular administration. In some embodiments of any embodiment of the present specification, administration of a therapeutically effective amount of a telomerase inhibitor includes contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment of the present specification, an effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment of the present specification, an effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, an effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, an effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, an effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the individual has a gain-of-function mutation V617F in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of the telomerase inhibitor is of the individual's JAK2. Reduce the percentage of V617F gene mutations (allelic burden). In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits CFU-mega. In some embodiments, inhibition of CFU-Mega is independent of the decrease in JAK2 gene mutations. In some embodiments, the individual is resistant or intolerant to existing telomerase inhibitor-based therapies. In some embodiments, the individual is human.

[0017] Accordingly, in one aspect, provided herein is a method for reducing at least one symptom associated with essential thrombocythemia in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with essential thrombocythemia. In some embodiments, the symptoms include headache, dizziness or fainting, chest pain, fatigue, syncope, changes in vision, numbness or tingling in the extremities, flushing, throbbing or burning pain in the extremities (erythromelalgia), splenomegaly, epistaxis, purpura, bleeding from the mouth or gums, bloody stools, or stroke. In some embodiments of any of the embodiments herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is from 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any of the embodiments herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any of the embodiments herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any of the embodiments herein, the oligonucleotide further comprises a lipid moiety attached to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of any of the embodiments herein, the lipid moiety is attached to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any of the embodiments herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any of the embodiments herein, the telomerase inhibitor is imetelstat. In some embodiments of any of the embodiments herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any of the embodiments herein, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intraocular administration.In some embodiments of any embodiment of the present specification, administration of a therapeutically effective amount of a telomerase inhibitor comprises contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the individual has a gain-of-function mutation V617F in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutation. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits CFU-mega. In some embodiments, inhibition of CFU-Mega is independent of the decrease in JAK2 gene mutations. In some embodiments, the individual is resistant or intolerant to existing non-telomerase inhibitor-based therapies. In some embodiments, the existing non-telomerase inhibitor-based therapy is hydroxyurea, anagrelide, or interferon α-2B. In some embodiments, the individual is human.

[0018] In another aspect, provided herein is a method for reducing the proliferation of tumor progenitor cells in an individual diagnosed with or suspected of having a myeloproliferative neoplasm or myelodysplastic syndrome, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces the proliferation of the individual's tumor progenitor cells. In some embodiments, the myeloproliferative neoplasm is, for example, essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), and acute myeloid leukemia (AML). In some embodiments, for ET, the reduction in the proliferation of tumor progenitor cells results in a platelet count of less than about 600×10 3 / μL in the blood of the individual. In some embodiments, the reduction in the proliferation of tumor progenitor cells results in a platelet count of less than 400×10 3 / μL in the blood of the individual. In some embodiments of any of the embodiments herein, the individual does not experience thromboembolism. In some embodiments of any of the embodiments herein, the reduction in the proliferation of tumor cells results in a platelet count of less than about 400×10 3 / μL in the blood of the individual within 2 months or less after initiation of administration of the telomerase inhibitor. In some embodiments of any of the embodiments herein, the reduction in the proliferation of tumor cells results in a platelet count of less than about 400×10 3 / μL in the blood of the individual within 1 month or less after initiation of administration of the telomerase inhibitor. In some embodiments, the individual is resistant or intolerant to existing non-telomerase inhibitor-based therapies. In some embodiments, for example, for MF, the reduction in the proliferation of tumor progenitor cells results in a platelet count of greater than about 100×10 9 / L in the blood of the individual. In some embodiments, for example, for MF, the reduction in the proliferation of tumor progenitor cells results in a modified hemoglobin value of at least 90 g / L, or 100 g / L or 110 g / L or 120 g / L. In some embodiments, for example, for MF, the reduction in the proliferation of tumor progenitor cells results in at least 1.0×10 9 / L or at least 2.0×10 9It becomes the modified absolute neutrophil count of / L. In some embodiments of any embodiment herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any embodiment herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any embodiment herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any embodiment herein, the oligonucleotide further comprises a lipid moiety attached to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of any embodiment herein, the lipid moiety is attached to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any embodiment herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any embodiment herein, the telomerase inhibitor is imetelstat. In some embodiments of any embodiment herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any embodiment herein, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intraocular administration. In some embodiments of any embodiment herein, administration of a therapeutically effective amount of the telomerase inhibitor comprises contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment herein, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg.In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the individual has a gain-of-function mutation V617F in the Janus kinase 2 (JAK2) gene. In some embodiments, the administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutation. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor inhibits CFU-mega. In some embodiments, the inhibition of CFU-Mega is independent of the decrease in the JAK2 gene mutation. In some embodiments, the individual is human.

[0019] In another aspect, the present specification provides a method for reducing the proliferation of pre-tumor cells in an individual diagnosed or suspected of having essential thrombocythemia, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein the administration of the telomerase inhibitor reduces the proliferation of the individual's pre-tumor cells. In some embodiments, the reduction in the proliferation of pre-tumor cells results in a platelet count of less than about 600×10 3 / μL in the blood of the individual. In some embodiments, the reduction in the proliferation of pre-tumor cells results in a platelet count of less than about 400×10 3 / μL in the blood of the individual. In some embodiments of any embodiment of the present specification, the individual does not experience thromboembolism. In some embodiments of any embodiment of the present specification, the reduction in the proliferation of tumor cells results in a platelet count of less than about 400×10 3results in a platelet count of less than / μL. In some embodiments of any of the embodiments herein, the decrease in tumor cell growth results in, within or less than 1 month after the start of administration of the telomerase inhibitor, approximately 400×10 in the blood of the individual 3results in a platelet count of less than / μL. In some embodiments, the individual is resistant or intolerant to existing non - telomerase inhibitor - based therapies. In some embodiments, the existing non - telomerase inhibitor - based therapies are hydroxyurea, anagrelide, or interferon α - 2B. In some embodiments of any of the embodiments herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 - 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any of the embodiments herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any of the embodiments herein, the oligonucleotide comprises multiple N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any of the embodiments herein, the oligonucleotide further comprises a lipid moiety that binds to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of any of the embodiments herein, the lipid moiety binds to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any of the embodiments herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any of the embodiments herein, the telomerase inhibitor is imetelstat. In some embodiments of any of the embodiments herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any of the embodiments herein, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhaled, or intraocular administration. In some embodiments of any of the embodiments herein, administration of a therapeutically effective amount of the telomerase inhibitor comprises contacting one or more pre - neoplastic cells with the telomerase inhibitor.In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the individual has a gain-of-function mutation V617F in the Janus kinase 2 (JAK2) gene. In some embodiments, the administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutation. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor inhibits CFU-mega. In some embodiments, the inhibition of CFU-Mega is independent of the decrease in the JAK2 gene mutation. In some embodiments, the individual is human.

[0020] In another aspect, provided herein is a method for maintaining the platelet count in the blood of an individual diagnosed or suspected of having essential thrombocythemia at less than about 400×10 3 / μL, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor maintains the platelet count of the individual at less than about 400×10 3 / μL. In some aspects, the telomerase inhibitor is administered no more than once every two weeks. In other aspects, the telomerase inhibitor maintains the platelet count in the individual's blood at from about 150×10 3 / μL to about 400×10 3It is administered to maintain between / μL. In some embodiments of any embodiment herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any embodiment herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any embodiment herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any embodiment herein, the oligonucleotide further comprises a lipid moiety that binds to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of any embodiment herein, the lipid moiety binds to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any embodiment herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any embodiment herein, the telomerase inhibitor is imetelstat. In some embodiments of any embodiment herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any embodiment herein, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intraocular administration. In some embodiments of any embodiment herein, administration of a therapeutically effective amount of the telomerase inhibitor comprises contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment herein, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg.In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the individual has a gain-of-function mutation V617F in the Janus kinase 2 (JAK2) gene. In some embodiments, the administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutation. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments of any embodiment of the present specification, the administration of the telomerase inhibitor inhibits CFU-mega. In some embodiments, the inhibition of CFU-Mega is independent of the decrease in the JAK2 gene mutation. In some embodiments, the individual is resistant or intolerant to existing non-telomerase inhibitor-based therapies. In some embodiments, the existing non-telomerase inhibitor-based therapy is hydroxyurea, anagrelide, or interferon α-2B. In some embodiments, the individual is human.

[0021] Accordingly, in one aspect, the present specification provides a method for reducing at least one symptom associated with polycythemia vera (PV) in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with polycythemia vera. In some embodiments, the symptoms include headache, dizziness or fainting, chest pain, weakness, syncope, vision changes, limb numbness or tingling, shortness of breath, weakness or fatigue, splenomegaly, epistaxis, purpura, bleeding from the mouth or gums, or bloody stools. In some embodiments of some embodiments of any of the embodiments herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of some embodiments of any of the embodiments herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of some embodiments of any of the embodiments herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of some embodiments of any of the embodiments herein, the oligonucleotide further comprises a lipid moiety attached to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of some embodiments of any of the embodiments herein, the lipid moiety is attached to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of some embodiments of any of the embodiments herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of some embodiments of any of the embodiments herein, the telomerase inhibitor is imetelstat. In some embodiments of some embodiments of any of the embodiments herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of some embodiments of any of the embodiments of the specification, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intraocular administration.In some embodiments of any embodiment of the present specification, administration of a therapeutically effective amount of a telomerase inhibitor comprises contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits the proliferation of erythroblasts. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits erythroid colony-forming units. In some embodiments of any embodiment of the present specification, the individual has a gain-of-function mutation of V617F in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutation. In some embodiments, the individual is resistant or intolerant to an existing non-telomerase inhibitor-based therapy. In some embodiments, the individual is human.

[0022] Accordingly, in one aspect, the present specification provides a method for reducing at least one symptom associated with myelofibrosis in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with myelofibrosis. In some embodiments, the symptoms include splenomegaly and splenic pain, early satiety, anemia, bone pain, fatigue, fever, night sweats, weight loss, weakness, syncope, epistaxis, purpura, bleeding from the mouth or gums, bloody stools, or stroke. In some embodiments of any of the embodiments of the present specification, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10-20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any of the embodiments of the present specification, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any of the embodiments of the present specification, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any of the embodiments of the present specification, the oligonucleotide further comprises a lipid moiety attached to the 5’ and / or 3’ terminus of the oligonucleotide. In some embodiments of any of the embodiments of the present specification, the lipid moiety is attached to the 5’ and / or 3’ terminus of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any of the embodiments of the present specification, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any of the embodiments of the present specification, the telomerase inhibitor is imetelstat. In some embodiments of any of the embodiments of the present specification, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any of the embodiments of the specification, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhaled, or intraocular administration.In some embodiments of any of the embodiments of this specification, administration of a therapeutically effective amount of a telomerase inhibitor comprises contacting one or more pre - cancerous cells with the telomerase inhibitor. In some embodiments of any of the embodiments of this specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any of the embodiments of this specification, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of this specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of this specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of this specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any of the embodiments of this specification, administration of the telomerase inhibitor does not inhibit cytokine - dependent megakaryocyte proliferation. In some embodiments of any of the embodiments of this specification, the individual has a gain - of - function V617F mutation in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutations. In some embodiments of any of the embodiments of this specification, administration of the telomerase inhibitor inhibits cytokine - independent megakaryocyte proliferation. In some embodiments of any of the embodiments of this specification, administration of the telomerase inhibitor inhibits CFU - mega. In some embodiments, inhibition of CFU - Mega is independent of the decrease in JAK2 gene mutations. In some embodiments, the individual is resistant or intolerant to existing non - telomerase inhibitor - based therapies. In some embodiments, the individual is human.

[0023] In another aspect, a method for reducing myelofibrosis in an individual diagnosed or suspected of having a myeloproliferative neoplasm or myelodysplastic syndrome is provided herein, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces the individual's myelofibrosis. In another aspect, about 100×10 9Provided herein are methods in patients with MF for maintaining platelet counts above / L, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor increases the platelet count. In another aspect, provided herein are methods in patients with MF for maintaining hemoglobin levels at least at 90 g / L, or 100 g / L or 110 g / L or 120 g / L, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor increases the hemoglobin level. In another aspect, provided herein are methods in patients with MF for maintaining an absolute neutrophil count at least at 1.0×10 9 / L or at least 2.0×10 9 / L, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor increases the neutrophil count. In some aspects, the telomerase inhibitor is administered no more than once every two weeks. In other aspects, the telomerase inhibitor increases the platelet count in the individual's blood from about 150×10 3 / μL to about 400×10 3It is administered to maintain between / μL. In some embodiments of any embodiment herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any embodiment herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any embodiment herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any embodiment herein, the oligonucleotide further comprises a lipid moiety that binds to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of any embodiment herein, the lipid moiety binds to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any embodiment herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any embodiment herein, the telomerase inhibitor is imetelstat. In some embodiments of any embodiment herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any embodiment herein, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intravitreal administration. In some embodiments of any embodiment herein, administration of a therapeutically effective amount of the telomerase inhibitor comprises contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment herein, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg.In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg.

[0024] Accordingly, in one aspect, the present specification provides a method for reducing at least one symptom associated with acute myeloid leukemia in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with acute myeloid leukemia. In some embodiments, the symptoms include splenomegaly and splenic pain, anemia, bone pain, fatigue, fever, night sweats, weight loss, weakness, syncope, epistaxis, purpura, bleeding from the mouth or gums, bloody stools, or stroke. In some embodiments of any of the embodiments herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any of the embodiments herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any of the embodiments herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any of the embodiments herein, the oligonucleotide further comprises a lipid moiety attached to the 5’ and / or 3’ end of the oligonucleotide. In some embodiments of any of the embodiments herein, the lipid moiety is attached to the 5’ and / or 3’ end of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any of the embodiments herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any of the embodiments herein, the telomerase inhibitor is imetelstat. In some embodiments of any of the embodiments herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. In some embodiments of any of the embodiments herein, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhaled, or intraocular administration.In some embodiments of any embodiment of this specification, administration of a therapeutically effective amount of a telomerase inhibitor comprises contacting one or more pre - tumor cells with the telomerase inhibitor. In some embodiments of any embodiment of this specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment of this specification, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of this specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of this specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of this specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of this specification, administration of the telomerase inhibitor does not inhibit cytokine - dependent megakaryocyte proliferation. In some embodiments of this specification, the individual has a gain - of - function V617F mutation in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of the telomerase inhibitor reduces the proportion of the individual's JAK2 V617F gene mutation. In some embodiments of any embodiment of this specification, administration of the telomerase inhibitor inhibits cytokine - independent megakaryocyte proliferation. In some embodiments of any embodiment of this specification, administration of the telomerase inhibitor inhibits CFU - mega. In some embodiments, the inhibition of CFU - Mega is independent of the decrease in the JAK2 gene mutation. In some embodiments, the individual is resistant or intolerant to an existing therapy based on a non - telomerase inhibitor. In some embodiments, the individual is human.

[0025] Accordingly, in one aspect, provided herein is a method for reducing at least one symptom associated with myelodysplastic syndromes, such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with single lineage dysplasia, and chronic myelomonocytic leukemia, in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with myelodysplastic syndromes. In some embodiments, the symptoms include shortness of breath, fatigue, weakness, syncope, epistaxis, purpura, bleeding from the mouth or gums, bloody stools, petechiae, or stroke. In some embodiments of any of the embodiments herein, the telomerase inhibitor comprises an oligonucleotide. In some embodiments, the oligonucleotide is complementary to the RNA component of telomerase. In some embodiments, the oligonucleotide is 10 to 20 base pairs in length. In some embodiments, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In some embodiments of any of the embodiments herein, the oligonucleotide comprises at least one N3’→P5’ phosphorothioamidate nucleoside internucleotide linkage. In some embodiments of any of the embodiments herein, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. In some embodiments of any of the embodiments herein, the oligonucleotide further comprises a lipid moiety that binds to the 5’ and / or 3’ terminus of the oligonucleotide. In some embodiments of any of the embodiments herein, the lipid moiety is bound to the 5’ and / or 3’ terminus of the oligonucleotide via a linker. In some embodiments, the linker is a glycerol or aminoglycerol linker. In some embodiments of any of the embodiments herein, the lipid moiety is a palmitoyl (C16) moiety. In some embodiments of any of the embodiments herein, the telomerase inhibitor is imetelstat. In some embodiments of any of the embodiments herein, the telomerase inhibitor is administered with a pharmaceutically acceptable excipient.In some embodiments of any embodiment of the specification, the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhaled, or intraocular administration. In some embodiments of any embodiment of the present specification, administration of a therapeutically effective amount of the telomerase inhibitor includes contacting one or more pre-tumor cells with the telomerase inhibitor. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In some embodiments of any embodiment of the present specification, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments of the present specification, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments of any embodiment of the present specification, administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments, the individual is resistant or intolerant to existing non-telomerase inhibitor-based therapies. In some embodiments, the individual is human. The present invention also provides, for example, the following items. (Item 1) A method for reducing at least one symptom associated with a myeloproliferative neoplasm or myelodysplastic syndrome in an individual in need thereof, comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with a myeloproliferative neoplasm or myelodysplastic syndrome. (Item 2) The method according to item 1, wherein the myeloproliferative neoplasm is selected from the group consisting of essential thrombocythemia (ET), polycythemia vera (PV), chronic myeloid leukemia (CML), myelofibrosis (MF), chronic neutrophilic leukemia, chronic eosinophilic leukemia, and acute myeloid leukemia (AML). (Item 3) The method according to item 2, wherein the symptoms include headache, dizziness or fainting, chest pain, weakness, loss of consciousness, changes in vision, numbness or tingling in the limbs, flushing, throbbing or burning pain in the limbs (erythromelalgia), splenomegaly, epistaxis, purpura, bleeding from the mouth or gums, bleeding from the mouth or gums, bloody stools, or stroke. (Item 4) The method according to item 2, wherein the myeloproliferative neoplasm (MPN) is essential thrombocythemia (ET) or polycythemia vera (PV). (Item 5) The method according to item 2, wherein the myeloproliferative neoplasm (MPN) is myelofibrosis (MF). (Item 6) The method according to item 2, wherein the myeloproliferative neoplasm (MPN) is acute myeloid leukemia (AML). (Item 7) The method according to item 1, wherein the myelodysplastic syndrome is selected from the group consisting of refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with single lineage dysplasia, and chronic myelomonocytic leukemia (CMML). (Item 8) The method according to item 7, wherein the myelodysplastic syndrome (MDS) is chronic myelomonocytic leukemia (CMML). (Item 9) A method for reducing the proliferation of tumor progenitor cells in an individual diagnosed or suspected of having a myeloproliferative neoplasm or myelodysplastic syndrome, comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces the proliferation of the individual's tumor progenitor cells. (Item 10) The method according to item 9, wherein the myeloproliferative neoplasm is selected from the group consisting of essential thrombocythemia (ET), polycythemia vera (PV), chronic myeloid leukemia (CML), myelofibrosis (MF), chronic neutrophilic leukemia, chronic eosinophilic leukemia, and acute myeloid leukemia (AML). (Item 11) The method according to item 10, wherein the myeloproliferative neoplasm (MPN) is essential thrombocythemia (ET) or polycythemia vera (PV). (Item 12) The method according to item 10, wherein the myeloproliferative neoplasm (MPN) is myelofibrosis (MF). (Item 13) The method according to item 10, wherein the myeloproliferative neoplasm (MPN) is acute myeloid leukemia (AML). (Item 14) The method according to item 9, wherein the myelodysplastic syndrome is selected from the group consisting of refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with unilineage dysplasia, and chronic myelomonocytic leukemia (CMML). (Item 15) A decrease in the proliferation of tumor progenitor cells results in a platelet count of less than about 600×10 3 / μL in the blood of the individual, the method according to item 11. (Item 16) The method according to item 9, wherein the individual is resistant or intolerant to an existing treatment based on a telomerase inhibitor. (Item 17) A method for maintaining a platelet count of less than about 400×10 3 / μL in the blood of an individual diagnosed or suspected of having essential thrombocythemia, comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor maintains the platelet count of the individual at less than about 400×10 3 / μL, the method. (Item 18) The method according to item 17, wherein the telomerase inhibitor is administered no more than once every two weeks. (Item 19) A method for reducing myelofibrosis in an individual diagnosed or suspected of having a myeloproliferative neoplasm or myelodysplastic syndrome, comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces myelofibrosis in the individual, the method. (Item 20) The method according to item 9, wherein the telomerase inhibitor comprises an oligonucleotide. (Item 21) The method according to item 13, wherein the oligonucleotide is complementary to the RNA component of telomerase. (Item 22) The method according to item 14, wherein the oligonucleotide has a length of 10-20 base pairs. (Item 23) The method according to item 15, wherein the oligonucleotide contains the sequence TAGGGTTAGACAA. (Item 24) The method according to item 20, wherein the oligonucleotide contains at least one N3’→P5’ phosphorothioamidate nucleoside internucleoside linkage. (Item 25) The method according to item 24, wherein the oligonucleotide contains a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleoside linkages. (Item 26) The method according to item 20, wherein the oligonucleotide further contains a lipid moiety that binds to the 5’ and / or 3’ end of the oligonucleotide. (Item 27) The method according to item 26, wherein the lipid moiety binds to the 5’ and / or 3’ end of the oligonucleotide via a linker. (Item 28) The method according to item 27, wherein the linker is a glycerol or aminoglycerol linker. (Item 29) The method according to item 27, wherein the lipid moiety is a palmitoyl (C16) moiety. (Item 30) The method according to any one of items 9, wherein the telomerase inhibitor is imetelstat. (Item 31) The method according to item 9, wherein the telomerase inhibitor is administered with a pharmaceutically acceptable excipient. (Item 32) The method according to item 9, wherein the telomerase inhibitor is formulated for oral, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, intranasal, inhalation, or intraocular administration. (Item 33) The method according to item 9, wherein the administration of the therapeutically effective amount of the telomerase inhibitor comprises contacting one or more tumor progenitor cells with the telomerase inhibitor. (Item 34) The method according to item 30, wherein the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. (Item 35) The method according to item 30, wherein the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. (Item 36) The method according to item 9, wherein the administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. (Item 37) The method according to item 9, wherein the individual has a gain-of-function mutation V617F in the Janus kinase 2 (JAK2) gene. (Item 38) The method according to item 37, wherein the administration of the telomerase inhibitor reduces the proportion of the JAK2 V617F gene mutation in the individual. (Item 39) The method according to item 9, wherein the administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. (Item 40) The method according to item 9, wherein the administration of the telomerase inhibitor inhibits CFU-mega. (Item 41) The method according to item 40, wherein the inhibition of CFU-Mega is independent of the decrease in the JAK2 gene mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0027] The present invention particularly provides a method for reducing the proliferation of tumor progenitor cells and alleviating the symptoms of an individual. The invention provided herein particularly relates to a method for treating and alleviating the symptoms associated with myeloproliferative neoplasms (MPNs) such as essential thrombocythemia (ET), polycythemia vera, myelofibrosis, and acute myeloid leukemia by targeting tumor progenitor cells specific to these diseases, using a telomerase inhibitor compound. The invention provided herein also particularly relates to a method for treating and alleviating the symptoms associated with myelodysplastic syndromes (MDS) such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with unilineage dysplasia, and chronic myelomonocytic leukemia by targeting tumor progenitor cells that cause an abnormally high number of cells specific to these diseases, using a telomerase inhibitor compound. The inventors have made the surprising discovery that telomerase inhibitors (such as imetelstat) can effectively reduce the circulating blood platelet levels in individuals with MPN and MDS. Additionally, this decrease in platelet levels is seen independently of the common ET-associated mutation in the Janus kinase 2 gene (JAK2) (seen in approximately 50% of ET cases) and is effective in individuals previously resistant to treatment with hydroxyurea, a common front-line treatment for ET. The present invention also provides a method for using a telomerase inhibitor (e.g., imetelstat) to maintain platelet counts within a relatively normal range in the blood of individuals diagnosed or suspected of having ET. Without being bound by theory, unlike other common treatments for MPN and MDS, the telomerase inhibitor compounds used in the methods of the present invention are specifically thought to inhibit tumor progenitor cells that drive the malignancy that is the cause of this condition.

[0028] I. General Technology Unless otherwise indicated, the practice of the present invention employs conventional techniques in nucleic acid chemistry, molecular biology, microbiology, cell biology, biochemistry, and immunology, which are well known to those skilled in the art. Such techniques are described in detail in the literature such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, Third Edition (Sambrook and Russel, 2001), which are collectively referred to herein as "Sambrook"; Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987, including supplements up to 2001); PCR: The Polymerase Chain Reaction, (edited by Mullis et al., 1994). Nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques described, for example, in Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; Komberg and Baker, DNA Replication, Second Edition. (Freeman, San Francisco, 1992); Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlmann and Peyman, Chemical Reviews, 90:543-584, 1990.

[0029] II. Definitions The term "nucleoside" means a moiety having the general structure represented below, where B represents a nucleobase and the 2'-carbon may be substituted as described below. When incorporated into an oligomer or polymer, the 3'-carbon is further bonded to an oxygen or nitrogen atom. [Chemical formula]

[0030] This structure includes 2'-deoxy and 2'-hydroxyl (i.e., deoxyribose and ribose) forms, and analogs. Although less common, the 5'-NH group can be replaced by a 5'-oxygen. "Analogs" include, with respect to nucleosides, synthetic nucleosides having a modified nucleobase moiety (see the definition of "nucleobase" below) and / or a modified sugar moiety such as 2'-fluoro sugar, and further analogs. Such analogs are generally designed to affect binding properties, e.g., stability, specificity, or others. The term nucleoside includes natural nucleosides and analogs including 2'-deoxy and 2'-hydroxyl forms as described, for example, in Komberg and Baker, DNA Replication, 2nd edition (Freeman, San Francisco, 1992). "Analogs" include, with respect to nucleosides, synthetic nucleosides having a modified nucleobase moiety (see the definition of "nucleobase" below) and / or a modified sugar moiety as extensively described, for example, by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., stability, properties, or others, as disclosed by Uhlmann and Peyman, Chemical Reviews 90:543 - 584, 1990). Oligonucleotides containing such nucleosides, and those generally containing synthetic nuclease-resistant internucleoside linkages, may themselves be referred to as "analogs".

[0031] "Polynucleotide" or "oligonucleotide" means a polymer or oligomer of ribose and / or deoxyribose nucleoside subunits having from about 2 to about 200 contiguous subunits. The nucleoside subunits can be linked by various subunit - subunit linkages including, but not limited to, phosphodiester, phosphotriester, methylphosphonate, P3’→N5’ phosphoramidate, N3’→P5’ phosphoramidate, N3→P5’ thiophosphoramidate, and phosphorothioate linkages. The term also includes polymers or oligomers having modifications to the sugar (e.g., 2’ substitution), base (see definition of "nucleoside" above), and 3’ and 5’ termini known to those of skill in the art. In embodiments where the oligonucleotide moiety contains multiple subunit - subunit linkages, each linkage may be formed using the same chemistry or a mixture of linkage chemistries may be used. When an oligonucleotide is represented by a sequence of letters such as "ATGUCCTG", it will be understood that the nucleotides are in the 5’→3’ order from left to right. The representation of the base sequence of an oligonucleotide in this manner does not imply the use of any particular type of subunit between the nucleosides in the oligonucleotide.

[0032] "Nucleic acid base" includes (i) the natural DNA and RNA nucleic acid bases (uracil, thymine, adenine, guanine, and cytosine), (ii) modified nucleic acid bases or nucleic acid base analogs (e.g., 5 - methylcytosine, 5 - bromouracil, or inosine), and (iii) nucleic acid base analogs. Nucleic acid base analogs are compounds whose molecular structure mimics that of the common DNA or RNA bases.

[0033] The term "lipid" is used herein broadly to include substances that are soluble in organic solvents but poorly soluble, if at all, in water. The term lipid includes, but is not limited to, hydrocarbons, oils, fats (such as fatty acids and glycerides), sterols, steroids, and derivative forms of these compounds. In some embodiments, the lipid is a fatty acid and its derivatives, a hydrocarbon and its derivatives, and a sterol such as cholesterol. Fatty acids typically contain an even number of carbon atoms (usually 12 to 24 carbons) in a straight chain, may be saturated or unsaturated, and can contain or be modified to contain various substituents. For the sake of simplicity, the term "fatty acid" also includes fatty acid derivatives such as fats or esters. In some embodiments, the term "lipid" also includes amphiphilic compounds containing both a lipid and a hydrophilic moiety.

[0034] A "telomerase inhibitor" is a compound that can reduce or inhibit the activity of the telomerase reverse transcriptase in mammalian cells. Such inhibitors can be small molecule compounds as described herein, or hTR template inhibitors that include oligonucleotides as described herein. In one aspect, the telomerase inhibitor is Imetelstat or Imetelstat sodium. In another aspect, the telomerase inhibitor is GRN163L.

[0035] An "hTR template inhibitor" is a compound that blocks the template region of the RNA component of human telomerase, thereby inhibiting the activity of the enzyme. The inhibitor is typically an oligonucleotide that can hybridize to this region. In some embodiments, the oligonucleotide contains a sequence effective to hybridize to a more specific portion of this region having the sequence 5'-CUAACCCUAAC-3'.

[0036] If the growth of cells in the presence of a compound is less than that observed in the absence of the compound, the compound is said to "inhibit cell growth". That is, cell growth is slowed or stopped in the presence of the compound. Inhibition of cancer cell growth may be demonstrated, for example, by a decrease in the number of cells or the rate of cell expansion, a decrease in tumor volume or tumor growth rate, or an increase in the survival rate of the subject being treated.

[0037] An oligonucleotide having "nuclease-resistant binding" means a subunit binding in which the backbone is substantially resistant to nuclease cleavage, either in a non-hybridized or hybridized form, by common extracellular and intracellular nucleases in the body. That is, the oligonucleotide shows little or no nuclease cleavage under normal nuclease conditions in the body where the oligonucleotide is exposed. The N3’→P5’ phosphoramidate (NP) or N3’→P5’ thiophosphoramidate (NPS) bonds described below are nuclease-resistant.

[0038] An "individual" can be a mammal, such as any common laboratory model organism. Mammals include, but are not limited to, humans and non-human primates, livestock, sport animals, pets, mice, rats, and other rodents. In some embodiments, the individual is a human.

[0039] An "effective amount" or "therapeutically effective amount" or "clinically effective amount" means the amount of a therapeutic compound that, when administered to a mammalian subject, either as a single dose or as part of a series of doses, such as a telomerase inhibitor, is effective to produce the desired therapeutic effect.

[0040] As used herein, "tumor cell" means a cell that exhibits relatively autonomous growth, and thus they exhibit an abnormal growth phenotype characterized by a profound loss of control of cell proliferation. Tumor cells include cells that may be actively replicating or in a temporarily non-replicating quiescent state (G1 or G0), and similarly, tumor cells may include cells having a fully differentiated phenotype, a poorly differentiated phenotype, or a mixture of both types of cells. Thus, not all tumor cells are necessarily replicating cells at a given point in time. "Tumor cell" encompasses cells of benign tumors and cells of malignant tumors.

[0041] As used herein, "preneoplastic cell" means a cell of a cell population having the ability to become neoplastic.

[0042] As used herein, the terms "tumor" or "tumorous" or "neoplasm" mean abnormal new cell growth. Unlike hyperplasia, neoplastic growth persists in the absence of an initiating stimulus.

[0043] As used herein, the singular form also includes plural references unless otherwise indicated.

[0044] The aspects and embodiments of the invention described herein are to be understood to include "comprising", "consisting of", and "consisting essentially of" aspects and embodiments.

[0045] Every maximum numerical limitation recited throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly recited herein. Every minimum numerical limitation recited throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly recited herein. Every numerical range recited throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly recited herein.

[0046] III. Telomerase inhibitor compounds Telomerase is a ribonucleoprotein that catalyzes the addition of telomeric repeat sequences (in humans, having the sequence 5'-TTAGGG-3') to the ends of chromosomes. See, for example, Blackburn, 1992, Ann. Rev. Biochem. 61:113-129. The enzyme is expressed in the majority of cancer cells but not in mature somatic cells. Loss of telomeric DNA can play a role in the induction of cellular senescence. Harley, 1991, Mutation Research 256:271-282. Cells from various cancers, including cancers of the skin, connective tissue, adipose, breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervous system (CNS), retina, and hematological tumors (such as myeloma, leukemia, and lymphoma), have been shown to be telomerase positive. Targeting telomerase can avoid many of the deleterious side effects associated with chemotherapy regimens that indiscriminately target dividing cells and can be effective in providing a highly discriminatory treatment between malignant and normal cells.

[0047] To date, inhibitors of telomerase that have been identified include oligonucleotides (e.g., oligonucleotides having nuclease-resistant binding) as well as small molecule compounds. Further information regarding telomerase inhibitor compounds can be found in U.S. Patent No. 7,998,938, the disclosure of which is incorporated herein by reference in its entirety.

[0048] A. Small Molecule Compounds Small molecule inhibitors of telomerase include, for example, BRACO19 (see Mol. Pharmacol. 61(5):1154 - 62, 2002); DODC (diethyloxadicarbocyanine), and telomestatin. These compounds can act as G4 stabilizers that promote the formation of inactive G4 structures in the RNA component of telomerase. Other small molecule inhibitors of telomerase include BIBR1532 (2-[(E)-3-naphthalen-2-ylbut-2-enoylamino]benzoic acid) (see also Ward & Autexier, Mol. Pharmacol. 68:779 - 786, 2005 and J. Biol. Chem. 277(18):15566 - 72, 2002); AZT and other nucleoside analogs such as ddG and ara-G (see, for example, U.S. Pat. Nos. 5,695,932 and 6,368,789), and certain thiopyridine, benzothiophene, and pyrido[b]thiophene derivatives described in U.S. Pat. Nos. 5,767,278, 5,770,613, 5,863,936, 5,656,638, and 5,760,062, the disclosures of which are incorporated herein by reference. Another example includes 3-chlorobenzothiophene-2-carboxy-2'-[(2,5-dichlorophenylamino)thia]hydrazine described in U.S. Pat. No. 5,760,062 (incorporated herein by reference).

[0049] B. Oligonucleotide-based telomerase inhibitors: sequences and compositions Genes encoding both the protein and RNA components of human telomerase have been cloned and sequenced (see U.S. Patent Nos. 6,261,836 and 5,583,016, respectively, both of which are incorporated herein by reference). Oligonucleotides can be targeted to the mRNA encoding the telomerase protein component (the human form of which is known as human telomerase reverse transcriptase, or hTERT) or the RNA component of the telomerase holoenzyme (the human form of which is known as human telomerase RNA, or hTR).

[0050] The nucleotide sequence of the RNA component of human telomerase (hTR) is shown by the sequence (SEQ ID NO:1) described below in the 5'→3' direction. The sequence is shown using standard abbreviations for ribonucleotides. Those skilled in the art will recognize that the sequence also represents the sequence of cDNA in which uridine (U) is replaced by thymidine (T) and ribonucleotides are replaced by deoxyribonucleotides. The template sequence of the RNA component is located within the region defined by nucleotides 46-56 (5'-CUAACCCUAAC-3'), which is complementary to the telomere sequence composed of 1, 2, 3 telomere repeat units. The template region functions to identify the sequence of telomere repeats that telomerase adds to the ends of chromosomes and is essential for the activity of the telomerase enzyme (see, for example, Chen et al., Cell 100:503-514, 2000; Kim et al., Proc. Natl. Acad. Sci. USA 98(14):7982-7987, 2001). The designation of antisense, ribozyme or small interfering RNA (siRNA) agents to inhibit or cause the destruction of mRNA is well known (see, for example, Lebedeva, I., et al. Annual Review of (see Pharmacology and Toxicology, Vol. 41:403-419, April 2001; Macejak, D., et al., Journal of Virology, Vol. 73(9):7745-7751, September 1999, and Zeng, Y., et al., PNAS Vol. 100(17):9779-9784, August 19, 2003), such agents may be designated to target hTERT mRNA, thereby inhibiting the production of hTERT protein in target cells such as cancer cells (see, e.g., U.S. Patent Nos. 6,444,650 and 6,331,399).

[0051] Oligonucleotides that target hTR (i.e., the RNA component of the enzyme) act as inhibitors of telomerase enzyme activity by blocking or interfering with the interaction between hTR and hTERT protein, which is an interaction required for telomerase function (see, e.g., Villeponteau et al., U.S. Patent No. 6,548,298).

[0052] The preferred target region of hTR is the template region, spanning nucleotides 30 to 67 of SEQ ID NO:1 (GGG UUG CGG AGG GUG GGC CUG GGA GGG GUG GUG GCC AUU UUU UGU CUA ACC CUA ACU GAG AAG GGC GUU AGG CGC CGU GCU UUU GCU CCC CGC GCG CUG UUU UUC UCG CUG ACU UUC AGC GGG CGG AAA AGC UCG GCC UGC CGC CUU CCA CCG UUC AUC UAG AGC AAA CAA AAA AUG UCA GCC UGC UGG CCC GUU CGC CUC CCG GGG ACC UGC GGC GGG UCG GCC UGC CCA GCC CCC GAA CCC CGC CUG GAG CCG CGG UCG GCC CGG GGC UUC UCC GGA GGC ACC CAC UGC CAC CGC GAA GAG UUG GGC UCU GUC AGC CGC GGG UCU UCU CGG GGG CGA GGG CGA GGU UCA CCG UUC AGG CGC AGG AAG AGG AAC GGA GCG AGU CCC GCC GCG GCG CGA UUC CUG AGC UGU GGG ACG UGC ACC CAG GAC UCG GGC UCA CAC AUG CAG UUC GCU UUC CUG UUG GUG GGG GGA ACG CCA UCG UGC GCA UCC GUC ACC CCC UCG GCC GGC AGU GGG GGC UUG UGA ACC CCC AAA ACC UGA CUG ACU GGG CCA GUG UGC U). Oligonucleotides targeting this region are referred to herein as "hTR template inhibitors" (see, for example, Herbert et al., Oncogene 21(4):638-42 (2002)). Preferably, such oligonucleotides contain sequences that are complementary or near-complementary to several portions of an 11-nucleotide region having the sequence 5'-CUA ACC CUA AC-3' (SEQ ID NO:23).

[0053] Another preferred target region is the region spanning nucleotides 137-179 of hTR (see Pruzan et al., Nucl. Acids Research, 30:559-568, 2002). Within this region, the sequence spanning 141-153 is a preferred target. PCT Publication WO98 / 28442 describes the use of oligonucleotides at least 7 nucleotides in length for inhibiting telomerase, and the oligonucleotides are designated to be complementary to accessible portions of hTR sequences outside the template region, including nucleotides 137-196, 290-319, and 350-380 of hTR. Preferred hTR target sequences are described below and are identified by SEQ ID NOs: 2-22.

[0054] The region of the therapeutic oligonucleotide targeting the hTR sequence is preferably exactly complementary to the corresponding hTR sequence. In certain instances, mismatches may be tolerated, although it is expected that this will reduce the specificity and activity of the resulting oligonucleotide complex. In certain embodiments, the base sequence of the oligonucleotide is therefore selected to include at least a 5-nucleotide sequence that is exactly complementary to the hTR target, and enhanced telomerase inhibition can be obtained when using complementary sequences of increased length, such as at least 8, at least 10, at least 12, at least 13, at least 15 nucleotides, etc., that are exactly complementary to the hTR target. In other embodiments, the sequence of the oligonucleotide includes at least a 5- to 20-nucleotide, at least 8- to 20-nucleotide, at least 10- to 20-nucleotide, or at least 10- to 15-nucleotide sequence that is exactly complementary to the hTR target sequence.

[0055] Optimal telomerase inhibitory activity can be obtained when the full length of the oligonucleotide is selected such that it is complementary to the hTR target sequence. However, the full length of the oligonucleotide need not be completely complementary to the target sequence, and the oligonucleotide sequence may include regions that are not complementary to the target sequence. Such regions may be added, for example, to confer other properties to the compound, such as sequences that facilitate purification. Alternatively, the oligonucleotide may include multiple repeats of a sequence complementary to the hTR target sequence.

[0056] When the oligonucleotide includes regions that are not complementary to the target sequence, such regions are generally located at one or both of the 5' or 3' termini. Exemplary sequences targeting human telomerase RNA (hTR) include the following. [Table A]

[0057] The internucleoside linkages of the oligonucleotide may include any available oligonucleotide chemistry, such as phosphodiester, phosphotriester, methylphosphonate, P3’→N5’ phosphoramidate, N3’→P5’ phosphoramidate, N3’→P5’ thiophosphoramidate, and phosphorothioate. Generally, although not necessarily, all internucleoside linkages within the oligonucleotide will be of the same type, but the oligonucleotide components may be synthesized using a mixture of different linkages.

[0058] In some embodiments, the oligonucleotide has at least one N3’→P5’ phosphoramidate (NP) or N3’→P5’ thiophosphoramidate (NPS) linkage, which linkage has the structure: 3’-(-NH--P(=O)(--XR)--O-)-5’ where X is O or S and R is selected from the group consisting of hydrogen, alkyl, and aryl, and when XR is OH or SH, may be represented by its pharmaceutically acceptable salt. In other embodiments, the oligonucleotide includes all NPs or, in some embodiments, all NPS linkages.

[0059] In one embodiment, the sequence for the hTR template inhibitor oligonucleotide is a sequence complementary to nucleotides 42 - 54 of SEQ ID NO:1 described above. This sequence (TAGGGTTAGACAA; SEQ ID NO:12) and the oligonucleotide having N3’→P5’ phosphorothioamidate (NPS) linkages are designated herein as GRN163. See, for example, Asai et al., Cancer Research 63:3931 - 3939 (2003) and Gryaznov et al., Nucleosides Nucleotides Nucleic Acids 22(5 - 8):577 - 81 (2003).

[0060] The oligonucleotide GRN163, when administered alone, has shown inhibitory activity in vitro in cell cultures, including squamous carcinoma, breast epithelium, renal carcinoma, renal adenocarcinoma, pancreas, brain, colon, prostate, leukemia, lymphoma, myeloma, epidermis, cervix, ovary and liver cancer cells.

[0061] The oligonucleotide GRN163 has also been tested and shown to be therapeutically effective in various animal tumor models, including both small cell and non - small cell ovarian and lung cancers (see, for example, U.S. Patent No. 7,998,938, the disclosure of which is incorporated herein by reference).

[0062] C. Lipid - oligonucleotide complexes In some embodiments, the telomerase inhibitors based on the oligonucleotides disclosed herein include at least one covalently attached lipid group (see U.S. Publication No. 2005 / 0113325, which is incorporated herein by reference). This modification provides superior cellular uptake properties such that equivalent biological effects can be obtained using a smaller amount of the complexed oligonucleotide compared to the unmodified form. When applied to the human therapeutic setting, this can translate to a reduced toxicity risk and cost savings.

[0063] The lipid group L is generally an aliphatic hydrocarbon or fatty acid, including derivatives of hydrocarbons and fatty acids. Examples include saturated straight-chain compounds having 14 to 20 carbons such as myristic (tetradecanoic) acid, palmitic (hexadecanoic) acid, and stearic (octadecanoic) acid, and their corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane, and octadecane. Examples of other suitable lipid groups that may be employed include sterols such as cholesterol, and substituted fatty acids and hydrocarbons, particularly polyfluorinated forms of these groups. The scope of the lipid group L includes derivatives such as amines, amides, esters, and carbamate derivatives. The type of derivative is often determined by the mode of attachment to the oligonucleotide as exemplified below. [Chemical formula] (wherein R is (CH2) 14 CH3 (palmitoyl)). This compound is designated herein as GRN163L (imetelstat).

[0064] In one exemplary structure, the lipid moiety is palmitoylamide (derived from palmitic acid) and complexes with the 5'-thiophosphate group of the NPS-linked oligonucleotide through an aminoglycerol linker. The NPS oligonucleotide having the sequence shown for GRN163 and complexed in this manner (shown below) is designated herein as GRN163L (Imetelstat). In a second exemplary structure, the lipid as palmitoylamide complexes through the terminal 3'-amino group of the NPS oligonucleotide.

[0065] D. Pharmaceutical Compositions In some aspects of the invention, when employed as a medicament, the telomerase inhibitor compounds disclosed herein can be formulated with a pharmaceutically acceptable excipient or carrier and formulated into a pharmaceutical composition.

[0066] When employed as a pharmaceutical, the telomerase inhibitor compounds can be administered in the form of a pharmaceutical composition. These compounds can be administered by various routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective in both injectable and oral compositions. Such compositions are prepared by methods well known in the pharmaceutical art and contain at least one active compound. When employed as an oral composition, the telomerase inhibitor compounds disclosed herein are protected from acid digestion in the stomach by a pharmaceutically acceptable protecting agent.

[0067] The present invention also includes pharmaceutical compositions containing, as an active ingredient, a telomerase inhibitor compound associated with one or more pharmaceutically acceptable excipients or carriers. When preparing the compositions of the present invention, the active ingredient is usually mixed with, diluted by, or encapsulated within such excipients or carriers and can take the form of capsules, sachets, paper, or other containers. When the excipient or carrier functions as a diluent, it can be an individual, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can take the form of, for example, tablets, pills, powders, troches, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or within a liquid medium), and ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0068] In the preparation of the formulations, it may be necessary to grind the active lyophilized compound to provide an appropriate particle size before combining it with other ingredients. If the active compound is substantially insoluble, it is generally ground to a particle size of less than 200 mesh. If the active compound is substantially water-soluble, the particle size is usually adjusted, for example, to about 40 mesh, by grinding to provide a substantially uniform distribution within the formulation.

[0069] It may be convenient or desirable to prepare, purify, and / or handle the corresponding salts of the active compound, such as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts are described in Berge et al., 1977, "Pharmaceutically Acceptable Salts", J. Pharm. Sci., Vol. 66, pp. 1-19. For example, if the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO - ), the salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, Na + . Examples of suitable organic cations include ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R + , NH2R2 + , NHR3 + , NR4 + ), but are not limited thereto.

[0070] Some examples of suitable excipients or carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations may further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl benzoate and propyl hydroxybenzoate; sweetening agents; and flavoring agents. The compositions of the present invention can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a patient by employing procedures known in the art.

[0071] The composition can be formulated into unit dosage forms containing an active ingredient, where each dosage is from about 5 mg to about 100 mg or more, for example, any range between these values including from about 1 mg to about 5 mg, from 1 mg to about 10 mg, from about 1 mg to about 20 mg, from about 1 mg to about 30 mg, from about 1 mg to about 40 mg, from about 1 mg to about 50 mg, from about 1 mg to about 60 mg, from about 1 mg to about 70 mg, from about 1 mg to about 80 mg, or from about 1 mg to about 90 mg. The term "unit dosage form" means a physically discrete unit suitable as a unit dose for an individual, and each unit contains a predetermined amount of the active substance calculated to produce the desired therapeutic effect together with a suitable pharmaceutical excipient or carrier.

[0072] Telomerase inhibitor compounds are effective over a wide dosage range and are usually administered in a therapeutically effective amount. However, it will be understood that the actual amount of telomerase inhibitor compound administered will be determined by the physician in light of relevant circumstances including the condition to be treated, the selected route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, etc.

[0073] For the preparation of solid compositions such as tablets, the telomerase inhibitor compound, the main active ingredient, is mixed with a pharmaceutical excipient or carrier to form a preformulation composition containing a homogeneous mixture of the compounds of the present invention. When referring to these preformulation compositions as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition such that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules.

[0074] The tablets or pills of the present invention can be coated or formulated in order to provide a dosage form that gives the advantage of a sustained action and to protect the telomerase inhibitor compound from acid hydrolysis in the stomach. For example, the tablets or pills can contain an internal administration component and an external administration component in the form of a wrapper in which the latter covers the former. The two components can be separated by an enteric layer that functions to resist disintegration in the stomach and allows the internal component to pass through intact or delay release into the duodenum. Various materials can be used for such an enteric layer or coating, and such materials include a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0075] The liquid forms in which the novel compositions of the present invention can be incorporated for oral or injectable administration include aqueous solutions, syrups with suitable flavors, aqueous or oily suspensions, and flavored emulsions, including edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0076] Compositions for inhalation or gas injection include solutions and suspensions, and powders, in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof. The liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described above. The compositions can be administered via the oral or nasal respiratory route for local or systemic action. Compositions in pharmaceutically acceptable solvents can be nebulized using an inert gas. The nebulized solution can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing apparatus. Solution, suspension, or powder compositions can also be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0077] IV. Methods of the Present Invention The telomerase inhibitor compounds (such as pharmaceutical compositions) provided herein are useful for modulating a disease state. In some embodiments, the cell proliferative disease is related to an increase in the expression or activity of telomerase or cell proliferation (precursor cells associated with abnormal production of platelets in essential thrombocythemia (ET)) or both.

[0078] In some aspects, provided herein are methods for reducing at least one symptom associated with MPN in an individual in need thereof. In some aspects, provided herein are methods for reducing at least one symptom associated with MDS in an individual in need thereof. Also provided herein are methods for reducing the proliferation of precursor cells of a patient having MPN or MDS, and methods for maintaining normal levels of platelet concentration and / or red blood cell concentration and / or white blood cell concentration in an individual diagnosed or suspected of having MPN or MDS.

[0079] Myeloproliferative neoplasms, or MPNs, are blood cancers that arise from malignant hematopoietic bone marrow precursor cells, such as precursor cells of red blood cells, platelets, and granulocytes. The proliferation of malignant precursor cells results in the overproduction of any combination of white blood cells, red blood cells, and / or platelets, depending on the disease. These overproduced cells can also be abnormal and can lead to further clinical complications. There are various types of chronic myeloproliferative diseases. Those included in the MPN disease spectrum include essential thrombocythemia (ET), polycythemia vera (PV), and chronic myelogenous leukemia (CML), myelofibrosis (MF), chronic neutrophilic leukemia, chronic eosinophilic leukemia, and acute myelomgenous leukemia (AML).

[0080] Myelodysplastic syndromes (MDS) are a group of conditions that include cancers of the blood and bone marrow. Myelodysplastic syndromes (MDS) include diseases such as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage dysplasia, refractory cytopenia with unilineage dysplasia, and chronic myelomonocytic leukemia. Immature blood stem cells (blasts) do not become healthy red blood cells, white blood cells, or platelets. Blasts die soon after moving into the bone marrow or the blood. This reduces the space available to form healthy white blood cells, red blood cells, and / or platelets in the bone marrow.

[0081] A. Essential thrombocythemia Megakaryocytes are bone marrow cells responsible for the production of platelets necessary for normal blood clotting. Megakaryocytes usually account for 1 in 10,000 bone marrow cells but can increase in number nearly 10-fold during certain disease processes.

[0082] Megakaryocytes are derived from hematopoietic stem cell precursors in the bone marrow. Once a cell has completed differentiation and become a mature megakaryocyte, it begins the process of producing platelets. Many cytokines are suspected of playing a role in stimulating megakaryocytes to produce platelets, but it is thrombopoietin, a cytokine, that induces megakaryocytes to form small proplatelet processes. Platelets are retained within these intracellular membranes in the cytoplasm of megakaryocytes. Each of these proplatelet processes can give rise to 2000 - 5000 new platelets upon disintegration. Overall, 2 / 3 of these newly produced platelets remain in the circulation and 1 / 3 are sequestered by the spleen.

[0083] Essential thrombocythemia (ET) is a chronic disorder associated with an increase or abnormal production of blood platelets. Platelet formation in ET occurs in a cytokine-independent manner, and megakaryocytes produce platelets in an uncontrolled way. Since platelets are involved in blood clotting, abnormal production can lead to inappropriate blood clot formation or bleeding, increasing the risk of gastrointestinal bleeding, heart attack, and stroke.

[0084] In many cases, many patients with ET are asymptomatic. Generally, it is diagnosed after the platelet count is found to be high as part of a regular health check-up. When symptoms of ET are present, they can include fatigue or be involved in small or large vascular disorders or bleeding. Small vascular disorders (often considered primary vasomotor) can cause headaches, visual disturbances or silent migraines (migraines without headache symptoms), dizziness or fainting, coldness or blueness of the fingers or toes, or burning, redness, and pain in the hands and feet (www.mpnresearchfoundation.org / Essential-Thrombocythemia). Thrombotic complications can cause strokes, transient ischemic attacks (TIAs), heart attacks, deep vein thrombosis or pulmonary embolism (blood clots in the lungs) and can be quite serious. Bleeding can manifest as easy bruising, nosebleeds, heavy periods, gastrointestinal bleeding or blood in the urine (www.mpnresearchfoundation.org / Essential-Thrombocythemia). A small number of people with ET can later develop acute leukemia or myelofibrosis, both of which can be life-threatening. Acute myeloid leukemia is a type of rapidly progressing blood and bone marrow cancer. Myelofibrosis is a progressive bone marrow disease that causes bone marrow scarring, severe anemia, and enlargement of the liver and spleen.

[0085] According to the World Health Organization, the diagnosis of ET requires criteria A1 to A4 (A1) Persistently 450×10 9A platelet count exceeding 450×10⁹ / L; (A2) the bone marrow showing an increase in the number of large, mature megakaryocytes and no significant increase in left shift of granulopoiesis or erythropoiesis; (A3) not meeting the WHO criteria for polycythaemia vera, primary myelofibrosis, chronic myeloid leukaemia, myelodysplastic syndromes, or other myeloid tumours; and (A4) having no acquired mutations or clonal markers for thrombocytosis or reactive causes (Swedlow et al., (2008) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Lyon, IARC Press). When diagnosing ET, some clinicians use the criteria of the British Committee for Standards in Haematology (published in 2010), which are similar to the 2008 WHO criteria but differ in some important respects (Beer et al., (2010) Blood 117(5):1472-1482).

[0086] Tests that can be performed to diagnose ET include: (1) blood tests to rule out other causes of thrombocytosis, including tests for markers of iron deficiency and inflammation (other mimicking haematological diseases are also excluded); (2) tests for JAK2 gene mutations (occurring in approximately 50% of cases) or MPL (occurring in up to approximately 5% of cases); (3) bone marrow biopsy to look for the classical features of ET, including an increase in platelet precursors. Further information regarding the diagnosis of ET can be found in US Patent Application Publication No. 2006 / 0166221, which is incorporated herein by reference.

[0087] ET is usually treated through modification of cardiovascular risk factors, antiplatelet therapy, and cytoreductive therapy (Beer et al., Blood 117(5):1472-1482; hereafter (Beer et al., 2010)). With regard to cardiovascular risk factors, patients are screened for the presence of hypertension, diabetes, smoking, hypercholesterolemia, and obesity, and are treated when instructed according to appropriate guidelines for these conditions (Beer et al., 2010). Antiplatelet therapy includes, but is not limited to, aspirin and antiplatelet agents such as clopidogrel, unless contraindicated. ET patients may be stratified based on thrombotic risk, with high-risk patients being over 60 years of age, having had a previous thrombotic event, or having a platelet count greater than 1500×10 9 / L. These high-risk patients are likely to benefit from cytoreductive therapy (Beer et al., 2010).

[0088] Although the risk of leukemic transformation may increase when ET patients are treated with hydroxycarbamide (hydroxyurea), it remains front-line treatment for most patients who require treatment (Beer et al., 2010). Other treatments include, but are not limited to, interferon, anagrelide, pipobroman, busulfan, and irradiation with radioactive phosphorus.

[0089] Current drug therapies for ET are not curative and there is little evidence suggesting a survival benefit. None of these current strategies address or directly target any of the malignant clonal cells that cause the disease, disease progression, or the symptoms patients suffer from that affect quality of life. The goal of current therapies is to prevent thrombohemorrhagic complications. A major advance in understanding the etiology of ET was the description in 2005 of the JAK2 somatic mutation (V617F), present in 50-60% of ET patients (James et al., (2005) Nature 434:1144-1148; Kralovics et al., (2005) N Engl J Med 352:1779-90; Baxter et al., (2005) Lancet 365:1054-61; Levine et al., (2005) Cancer Cell 7:387-97). In addition to the presence and amount of the JAK2 / V617F mutation, baseline leukocytosis has recently been recognized as a new disease-related risk factor for ET (Ziakas PD. (2008) Haematologica 93:1412-1414; Carobbio et al., (2007) Blood 109:2310-2313). Evidence also suggests that leukocytosis may have importance as a prognostic factor and may be a cause of vascular events (Barbui et al., (2009) Blood 114:759-63).

[0090] B. Polycythemia vera Patients with polycythemia vera (PV) are marked by increased erythropoiesis. Treatment is directed at reducing the excessive number of red blood cells. PV can progress to a stage in the later course similar to that of primary myelofibrosis with cytopenia and hypoplasia and fibrosis of the bone marrow. A Janus kinase 2 gene (JAK2) gene mutation on chromosome 9 that causes increased proliferation and survival of hematopoietic progenitor cells in vitro has been identified in most patients with PV. Patients with PV have an increased risk of cardiovascular and thrombotic events and transformation to acute myeloid leukemia or primary myelofibrosis. Treatment for PV includes intermittent phlebotomy to maintain a hematocrit level of less than 45% in men and less than 40% in women. Other possible treatments include hydroxyurea, interferon-α, and low-dose aspirin (includee).

[0091] C. Myelofibrosis Myelofibrosis or MF is a myeloproliferative neoplasm in the same disease spectrum as ET. Patients with MF often have the JAK2 V617F mutation in their bone marrow. Occasionally, ET progresses to MF. JAK2 inhibition is currently considered the standard treatment for MF in countries where the Janus kinase inhibitor, ruxolitinib (Jakafi®), is approved. There is no evidence that JAK2 inhibitors, such as Jakafi®, selectively inhibit the proliferation of the leukemic clone that is the cause of the disease, and thus they may not be "disease modifying".

[0092] D. Acute Myeloid Leukemia Acute myeloid leukemia (AML) is a cancer of the myeloid blood cells. AML is the most common acute leukemia affecting adults. Patients with AML have a rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Replacing normal bone marrow and white blood cells reduces red blood cells, platelets, and normal white blood cells. Symptoms of AML include fatigue, shortness of breath, easy bruising and bleeding, and an increased risk of infection. Like acute leukemia, AML progresses rapidly and will generally lead to death within a few weeks or months if left untreated. Standard treatment for AML is treatment using chemotherapy aimed at inducing remission, and patients may undergo a hematopoietic stem cell transplant.

[0093] E. Myelodysplastic syndromes Myelodysplastic syndromes (MDS) are a collection of symptoms including cancers of the blood and bone marrow. Immature blood stem cells (blasts) do not become healthy red blood cells, white blood cells, or platelets. Blasts die shortly after moving into the bone marrow or the blood. This reduces the room for healthy white blood cells, red blood cells, and / or platelets to form in the bone marrow.

[0094] Myelodysplastic syndromes (MDS) are a collection of hematological medical conditions that necessarily include ineffective hematopoiesis of myeloid-class blood cells. Patients with MDS often develop severe anemia and require frequent blood transfusions. The disease may worsen, and patients may develop cytopenia (low blood cell counts) caused by progressive bone marrow failure. The disease may also transform into acute myeloid leukemia (AML). Transformation into acute myeloid leukemia (AML) is said to have occurred if the overall percentage of bone marrow blasts rises above a certain cut-off (20% for WHO and 30% for FAB).

[0095] F. Method for treating MPN or MDS using a telomerase inhibitor Provided herein is a method for reducing the proliferation of pre - tumor cells and alleviating related symptoms in an individual diagnosed with or suspected of having MPN or MDS through administration of a telomerase inhibitor (such as any telomerase inhibitor disclosed herein).

[0096] The method can be carried out in an adjuvant setting. An "adjuvant setting" means a clinical context in which an individual has a history of a proliferative disease and typically (but not necessarily) responds to treatment methods including, but not limited to, surgery (such as surgical resection), radiation therapy, and chemotherapy. However, due to their history of proliferative disease, these individuals are considered at risk of disease occurrence. Treatment or administration in an "adjuvant setting" means treatment in a subsequent manner. The degree of risk (i.e., when an individual in an adjuvant setting is considered "high - risk" or "low - risk") depends on several factors, generally usually the degree of the disease at the time of first treatment.

[0097] The method provided herein can also be carried out in a "neoadjuvant setting". That is, the method can be performed before primary / definitive treatment. In some embodiments, the individual has been previously treated. In some embodiments, the individual has not been previously treated. In some embodiments, the treatment is a first - line therapy.

[0098] 1. A method for alleviating symptoms of myeloproliferative neoplasms and myelodysplastic syndromes In some aspects, the invention relates to methods for inhibiting symptoms or conditions (disabilities, dysfunctions) associated with myeloproliferative neoplasms, as described in detail above. By itself, it is not required that all effects of the condition be completely prevented or reversed, but the effects of the presently disclosed methods are intended to reach a significant therapeutic benefit for the patient. The therapeutic benefit is not necessarily a complete prevention or treatment of a particular condition that itself necessarily results from a myeloproliferative neoplasm, but rather includes results such as reducing or preventing symptoms caused by a cell proliferative disorder, reducing or preventing the occurrence of such symptoms (quantitatively or qualitatively), reducing the severity or physiological impact of such symptoms, and / or enhancing the recovery of an individual after experiencing myeloproliferative neoplasm symptoms.

[0099] In some aspects, the invention relates to methods for inhibiting symptoms or conditions (disabilities, dysfunctions) associated with myelodysplastic syndromes (MDS), as described in detail above. By itself, it is not required that all effects of the condition be completely prevented or reversed, but the effects of the presently disclosed methods are intended to reach a significant therapeutic benefit for the patient. The therapeutic benefit is not necessarily a complete prevention or treatment of a particular condition that itself necessarily results from a myelodysplastic syndrome, but rather includes results such as reducing or preventing symptoms caused by a cell proliferative disorder, reducing or preventing the occurrence of such symptoms (quantitatively or qualitatively), reducing the severity or physiological impact of such symptoms, and / or enhancing the recovery of an individual after experiencing myelodysplastic syndrome symptoms.

[0100] As used herein, the phrase "alleviating at least one symptom associated with" a disease, disorder, or condition (such as MPN or MDS) indicates reversing, inhibiting the progression of, or preventing the disease or condition to which such term applies, or reversing, inhibiting the progression of, or preventing one or more symptoms of the disease or condition to which such term applies. Specifically, the compositions of the present invention (such as any telomerase inhibitor compound disclosed herein) can, when administered to an individual, treat or prevent one or more symptoms or conditions associated with MPN or MDS, and / or reduce or alleviate the symptoms or conditions associated with this disease. Protecting an individual from the effects or symptoms caused by MPN or MDS itself includes both preventing or reducing the occurrence and / or severity of the effects of the disease, and treating patients in whom the effects of the disease have already occurred or are beginning to occur. The beneficial effects can be readily evaluated by those skilled in the art and / or trained clinicians treating the patients. Preferably, patients treated using the methods of the present invention have a positive or beneficial difference in the severity or incidence of at least one clinical or biological score, value, or measure used to evaluate such patients, compared to patients who have not received treatment.

[0101] Accordingly, in some embodiments, provided herein is a method for reducing at least one symptom associated with MPN or MDS in an individual in need thereof, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces at least one symptom associated with MPN or MDS. In some embodiments, the symptoms include headache, dizziness or lightheadedness, chest pain, fatigue, fainting, changes in vision, numbness or tingling in the extremities, flushing, throbbing or burning pain in the extremities (erythromelalgia), splenomegaly, nosebleeds, purpura, bleeding from the mouth or gums, bloody stools, heart attack (myocardial infarction) or stroke. In some embodiments, the telomerase inhibitor can comprise an oligonucleotide that can be complementary to the RNA component of telomerase and can optionally be of a length between 10 and 20 base pairs. In one embodiment, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In other embodiments, the oligonucleotide comprises multiple N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. The oligonucleotide can optionally complex to a lipid moiety at either its 5’ or 3’ terminus via a linker (such as a glycerol or aminoglycerol linker). In some embodiments, the lipid moiety is a palmitoyl (C16) moiety. In yet another embodiment, the telomerase inhibitor is imetelstat. In some embodiments, administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In other embodiments, administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments, administration of the telomerase inhibitor inhibits CFU-mega. In yet other embodiments, inhibition of CFU-Mega is independent of a decrease in the JAK2 V617F gene mutation. In some embodiments, the individual can be resistant or intolerant to existing non-telomerase inhibitor-based therapies (including but not limited to hydroxyurea, anagrelide, or interferon α-2B). In other embodiments, the individual is human.

[0102] In some embodiments, the effective amount of the telomerase inhibitor administered to the patient is from 7.5 mg / kg to 9.3 mg / kg. In other embodiments, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In another embodiment, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor includes any one of at least about 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 10.1 mg / kg, 10.2 mg / kg, 10.3 mg / kg, 10.4 mg / kg, 10.5 mg / kg, 10.6 mg / kg, 10.7 mg / kg, 10.8 mg / kg, 10.9 mg / kg, 11 mg / kg, 11.1 mg / kg, 11.2 mg / kg, 11.3 mg / kg, 11.4 mg / kg, 11.5 mg / kg, 11.6 mg / kg, 11.7 mg / kg, 11.8 mg / kg, 11.9 mg / kg, 12 mg / kg, 12.1 mg / kg, 12.2 mg / kg, 12.3 mg / kg, 12.4 mg / kg, 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg, or 13 mg / kg.In some embodiments, the effective amount of the telomerase inhibitor administered to the subject is not 9.4 mg / kg.

[0103] In some aspects, a subject diagnosed or suspected of having MPN has a gain-of-function V617F mutation in the Janus kinase 2 (JAK2) gene. There are many methods for determining whether a subject has this mutation and for measuring the gene mutation, which are well known in the art (see, e.g., U.S. Patent Application Nos. 2009 / 0162849, 2007 / 0224598, and 2009 / 0162849, the disclosures of each of which are incorporated herein by reference). In some embodiments, administration of the telomerase inhibitor reduces the percentage of the subject's JAK2 V617F gene mutation.

[0104] 2. Method for reducing tumor cell proliferation In another aspect, the present disclosure provides a method for reducing the proliferation of tumor progenitor cells in an individual diagnosed with or suspected of having essential thrombocythemia, the method comprising administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor reduces the proliferation of the individual's tumor progenitor cells. In some embodiments, the telomerase inhibitor can comprise an oligonucleotide that can be complementary to the RNA component of telomerase and can optionally be between 10 and 20 base pairs in length. In one embodiment, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In other embodiments, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. The oligonucleotide can optionally complex to a lipid moiety at either its 5’ or 3’ terminus via a linker (such as a glycerol or aminoglycerol linker). In some embodiments, the lipid moiety is a palmitoyl (C16) moiety. In yet another embodiment, the telomerase inhibitor is imetelstat. In some embodiments, administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In other embodiments, administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments, administration of the telomerase inhibitor inhibits CFU-mega. In yet other embodiments, inhibition of CFU-Mega is independent of a decrease in the JAK2 V617F gene mutation. In some embodiments, the individual can be resistant or intolerant to existing non-telomerase inhibitor-based therapies (including but not limited to hydroxyurea, anagrelide, or interferon α-2B). In other embodiments, the individual is human.

[0105] In some aspects, the reduction in the proliferation of tumor progenitor cells is in the individual's blood and includes, among other values, about 600×10 3 / μL, 575×10 3 / μL, 550×10 3 / μL, 525×10 3 / μL, 500×10 3 / μL, 475×10 3 / μL, 450×10 3 / μL, 425×10 3 / μL, 400×10 3 / μL, 375×10 3 / μL, 350×10 3 / μL×10 3 / μL, 325×10 3 / μL, 300×10 3 / μL, 275×10 3 / μL, 250×10 3 / μL, 225×10 3 / μL, 200×10 3 / μL, 175×10 3 / μL, or less than 150×10 3 / μL results in a platelet count that also includes values between these numerical values that are less than any of these. In other embodiments, the decrease in tumor cell proliferation results in a decrease in the platelet count (e.g., any of the platelet counts described above) in the blood of an individual within about 24 weeks, 23 weeks, 22 weeks, 21 weeks, 20 weeks, 19 weeks, 18 weeks, 17 weeks, 16 weeks, 15 weeks, 14 weeks, 13 weeks, 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, or 2 weeks or less after the start of telomerase inhibitor administration.

[0106] In some embodiments, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In other embodiments, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In another embodiment, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg.In some embodiments, an effective amount of a telomerase inhibitor comprises at least about 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 10.1 mg / kg, 10.2 mg / kg, 10.3 mg / kg, 10.4 mg / kg, 10.5 mg / kg, 10.6 mg / kg, 10.7 mg / kg, 10.8 mg / kg, 10.9 mg / kg, 11 mg / kg, 11.1 mg / kg, 11.2 mg / kg, 11.3 mg / kg, 11.4 mg / kg, 11.5 mg / kg, 11.6 mg / kg, 11.7 mg / kg, 11.8 mg / kg, 11.9 mg / kg, 12 mg / kg, 12.1 mg / kg, 12.2 mg / kg, 12.3 mg / kg, 12.4 mg / kg, 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg, or 13 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor administered to an individual is not 9.4 mg / kg.

[0107] In some aspects, an individual diagnosed or suspected of having ET has a gain-of-function V617F mutation in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of the telomerase inhibitor reduces the percentage of the individual's JAK2 V617F gene mutation.

[0108] 3. A method for maintaining normal levels of circulating platelets In other aspects, in the blood of an individual diagnosed or suspected of having essential thrombocythemia, the platelet count is about 400×10 3Methods are provided herein for maintaining below about 400×10 3 / μL, which comprises administering to the individual a clinically effective amount of a telomerase inhibitor, wherein administration of the telomerase inhibitor maintains the platelet count of the individual below about 400×10

[0109] In some aspects, administration of a telomerase inhibitor (such as any telomerase inhibitor described herein) maintains the platelet count at a physiologically normal level. In some embodiments, administration of the telomerase inhibitor in the individual's blood comprises, among others, about 600×10 3 / μL, 575×10 3 / μL, 550×10 3 / μL, 525×10 3 In some embodiments, the telomerase inhibitor can be complementary to the RNA component of telomerase and can be, in some cases, an oligonucleotide that can be between 10 and 20 base pairs in length. In one embodiment, the oligonucleotide comprises the sequence TAGGGTTAGACAA. In other embodiments, the oligonucleotide comprises a plurality of N3’→P5’ phosphorothioamidate nucleoside internucleotide linkages. The oligonucleotide can optionally complex with a lipid moiety at either its 5’ or 3’ end via a linker (such as a glycerol or aminoglycerol linker). In some embodiments, the lipid moiety is a palmitoyl (C16) moiety. In yet another embodiment, the telomerase inhibitor is imetelstat. In some embodiments, administration of the telomerase inhibitor does not inhibit cytokine-dependent megakaryocyte proliferation. In other embodiments, administration of the telomerase inhibitor inhibits cytokine-independent megakaryocyte proliferation. In some embodiments, administration of the telomerase inhibitor inhibits CFU-mega. In yet other embodiments, inhibition of CFU-Mega is independent of a decrease in the JAK2 V617F gene mutation. In some embodiments, the individual can be resistant or intolerant to existing non-telomerase inhibitor-based therapies (including, but not limited to, hydroxyurea, anagrelide, or interferon α-2B). In other embodiments, the individual is human. / μL, 500×10 3 / μL, 475×10 3 / μL, 450×10 3 / μL, 425×10 3 / μL, 400×10 3 / μL, 375×10 3 / μL, 350×10 3 / μL×10 3 / μL, 325×10 3 / μL, 300×10 3 / μL, 275×10 3 / μL, 250×10 3 / μL, 225×10 3 / μL, 200×10 3 / μL, 175×10 3 / μL, or less than 150×10 3 / μL, maintaining platelet counts that also include values between these numbers and less than any of these values. In other embodiments, administration of a telomerase inhibitor maintains platelet counts in the blood of an individual at about 100 - 400×10 3 / μL, 150 - 200×10 3 / μL, 150 - 250×10 3 / μL, 150 - 300×10 3 / μL, 150 - 350×10 3 / μL, 150 - 400×10 3 / μL, 200 - 250×10 3 / μL, 200 - 300×10 3 / μL, 200 - 350×10 3 / μL, 200 - 400×10 3 / μL, 250 - 300×10 3 / μL, 250 - 350×10 3 / μL, 250 - 400×10 3 / μL, 300 - 350×10 3 / μL, 300 - 400×10 3 / μL, or between 350 and 400×10 3 / μL, maintaining platelet counts between any of these values.

[0110] In yet other embodiments, to maintain the platelet count at a physiologically normal level, administration of a telomerase inhibitor is required once or less per day, every other day, every three days, once a week, every eleven days, every other week, every three weeks, once a month, every six weeks, every other month, or a longer period, including the periods included therein.

[0111] In some embodiments, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.3 mg / kg. In other embodiments, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In another embodiment, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor includes any one of at least about 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 10.1 mg / kg, 10.2 mg / kg, 10.3 mg / kg, 10.4 mg / kg, 10.5 mg / kg, 10.6 mg / kg, 10.7 mg / kg, 10.8 mg / kg, 10.9 mg / kg, 11 mg / kg, 11.1 mg / kg, 11.2 mg / kg, 11.3 mg / kg, 11.4 mg / kg, 11.5 mg / kg, 11.6 mg / kg, 11.7 mg / kg, 11.8 mg / kg, 11.9 mg / kg, 12 mg / kg, 12.1 mg / kg, 12.2 mg / kg, 12.3 mg / kg, 12.4 mg / kg, 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg, or 13 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor administered to an individual is not 9.4 mg / kg.

[0112] In some embodiments, an individual diagnosed with or suspected of having ET has a gain-of-function V617F mutation in the Janus kinase 2 (JAK2) gene. In some embodiments, administration of a telomerase inhibitor reduces the percentage of the individual's JAK2 V617F gene mutations.

[0113] G. Administration of Telomerase Inhibitor In some embodiments, a telomerase inhibitor (such as any telomerase inhibitor disclosed herein) is administered in the form of an injection. The injection can contain the compound in combination with an aqueous injectable excipient or carrier. Non-limiting examples of suitable aqueous injectable excipients or carriers are well known to those of skill in the art, and those, as well as methods of formulating the formulations, can be found in standard references such as Alfonso AR: Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton Pennsylvania, 1985. Suitable aqueous injectable excipients or carriers optionally contain solubilizing agents such as 10% mannitol or other sugars, 10% glycine, or other amino acids, and include water, saline, aqueous dextrose solutions, and others. The composition can be injected subcutaneously, intraperitoneally, or intravenously.

[0114] In some embodiments, intravenous administration is used, which can be a continuous intravenous infusion over a period of several minutes to 1 hour or more, such as about 15 minutes. The dosage can vary widely depending on the type of telomerase inhibitor, the size of the unit dose, the type of excipient or carrier, and other factors well known to those of skill in the art. The remainder contains the excipient(s) or carrier, and the telomerase inhibitor can, for example, be included in an amount from about 0.001% to about 10% (w / w), from about 0.01% to about 1%, from about 0.1% to about 0.8%, or any range therein.

[0115] For oral administration, the telomerase inhibitor can be in the form of tablets or capsules prepared by conventional means, for example, together with pharmaceutically acceptable excipients or carriers such as binders, fillers, lubricants, disintegrants, or wetting agents. Liquid preparations for oral administration can be in the form of, for example, solutions, syrups, or suspensions, or can be provided as dry products to be constituted with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl paraben or propyl paraben or sorbic acid). The preparations can also contain suitable buffering salts, flavorings, and coloring agents.

[0116] In some embodiments, the telomerase inhibitor can be administered by inhalation through an aerosol spray or nebulizer that can contain a suitable propellant such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or combinations thereof. In one non-limiting example, dosage units for pressurized aerosols can be delivered through a metered valve. In other embodiments, gelatin capsules and cartridges can be used, for example, in an inhaler and can be formulated to contain a powdered mixture of the compound with a suitable powder base such as, for example, starch or lactose.

[0117] In some embodiments, the amount of telomerase inhibitor administered to an individual is included in any of the following ranges: about 0.5 to about 5 mg, about 5 to about 10 mg, about 10 to about 15 mg, about 15 to about 20 mg, about 20 to about 25 mg, about 20 to about 50 mg, about 25 to about 50 mg, about 50 to about 75 mg, about 50 to about 100 mg, about 75 to about 100 mg, about 100 to about 125 mg, about 125 to about 150 mg, about 150 to about 175 mg, about 175 to about 200 mg, about 200 to about 225 mg, about 225 to about 250 mg, about 250 to about 300 mg, about 300 to about 350 mg, about 350 to about 400 mg, about 400 to about 450 mg, or about 450 to about 500 mg. In some embodiments, the amount of telomerase inhibitor in an effective amount administered to an individual (e.g., in unit dosage form) is in the range of about 5 mg to about 500 mg, such as about 30 mg to about 300 mg or about 50 mg to about 200 mg. In some embodiments, the concentration of the telomerase inhibitor administered to an individual is, for example, any of about 0.1 to about 200 mg / ml, about 0.1 to about 180 mg / ml, about 0.1 to about 160 mg / ml, about 0.1 to about 140 mg / ml, about 0.1 to about 120 mg / ml, about 0.1 to about 100 mg / ml, about 0.1 to about 80 mg / ml, about 0.1 to about 60 mg / ml, about 0.1 to about 40 mg / ml, about 0.1 to about 20 mg / ml, about 0.1 to about 10 mg / ml, about 2 to about 40 mg / ml, about 4 to about 35 mg / ml, about 6 to about 30 mg / ml, about 8 to about 25 mg / ml, about 10 to about 20 mg / ml, about 12 to about 15 mg / ml, or any of about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2 mg / ml, 2.1 mg / ml, 2.2 mg / ml, 2.3 mg / ml, 2.4 mg / ml, or 2.5 mg / ml, and is diluted (about 0.1 mg / ml) or concentrated (about 180 mg / ml).In some embodiments, the concentration of the telomerase inhibitor is at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 1.3 mg / ml, 1.5 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 33.3 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml, 160 mg / ml, 170 mg / ml, 180 mg / ml, 190 mg / ml, 200 mg / ml, 210 mg / ml, 220 mg / ml, 230 mg / ml, 240 mg / ml, or 250 mg / ml.

[0118] An exemplary effective amount of the telomerase inhibitor administered to an individual is at least about 25 mg / m 2 、 30 mg / m 2 、 50 mg / m 2 、 60 mg / m 2 、 75 mg / m 2 、 80 mg / m 2 、 90 mg / m 2 、 100 mg / m 2 、 120 mg / m 2 、 125 mg / m 2 、 150 mg / m 2 、 160 mg / m 2 、 175 mg / m 2 、 180 mg / m 2 、 200 mg / m 2 、 210 mg / m2 , 220 mg / m 2 , 250 mg / m 2 , 260 mg / m 2 , 300 mg / m 2 , 350 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 540 mg / m 2 , 750 mg / m 2 , 1000 mg / m 2 , or 1080 mg / m 2 and includes but is not limited to any of these. In various embodiments, the amount of telomerase inhibitor administered to an individual is about 350 mg / m 2 , 300 mg / m 2 , 250 mg / m 2 , 200 mg / m 2 , 150 mg / m 2 , 120 mg / m 2 , 100 mg / m 2 , 90 mg / m 2 , 50 mg / m 2 , or 30 mg / m 2 and includes a telomerase inhibitor less than any of these. In some embodiments, the amount of telomerase inhibitor per administration is 25 mg / m 2 , 22 mg / m 2 , 20 mg / m 2 , 18 mg / m 2 , 15 mg / m 2 , 14 mg / m 2 , 13 mg / m 2 , 12 mg / m 2 , 11 mg / m 2 , 10 mg / m 2 , 9 mg / m 2 , 8 mg / m 2 , 7 mg / m 2 , 6 mg / m 2 , 5 mg / m 2 , 4 mg / m 2 , 3 mg / m 2 , 2 mg / m 2 , or 1 mg / m 2 and is less than any of these. In some embodiments, the effective amount of telomerase inhibitor administered to an individual is included in any of the following ranges. From about 1 to about 5 mg / m2 , from about 5 to about 10 mg / m 2 , from about 10 to about 25 mg / m 2 , from about 25 to about 50 mg / m 2 , from about 50 to about 75 mg / m 2 , from about 75 to about 100 mg / m 2 , from about 100 to about 125 mg / m 2 , from about 125 to about 150 mg / m 2 , from about 150 to about 175 mg / m 2 , from about 175 to about 200 mg / m 2 , from about 200 to about 225 mg / m 2 , from about 225 to about 250 mg / m 2 , from about 250 to about 300 mg / m 2 , from about 300 to about 350 mg / m 2 , or from about 350 to about 400 mg / m 2 . In some embodiments, the effective amount of telomerase inhibitor administered to the individual is from about 20 to about 300 mg / m 2 , from about 50 to about 250 mg / m 2 , from about 100 to about 150 mg / m 2 , about 120 mg / m 2 , about 130 mg / m 2 , or about 140 mg / m 2 , or about 260 mg / m 2 , such as from about 5 to about 300 mg / m 2 .

[0119] In some embodiments of any of the above aspects, the effective amount of the telomerase inhibitor administered to the individual comprises any of at least about 1 mg / kg, 2.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 6.5 mg / kg, 7.5 mg / kg, 9.4 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In various embodiments, the effective amount of the telomerase inhibitor administered to the individual comprises a telomerase inhibitor of less than any of about 350 mg / kg, 300 mg / kg, 250 mg / kg, 200 mg / kg, 150 mg / kg, 100 mg / kg, 50 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 7.5 mg / kg, 6.5 mg / kg, 5 mg / kg, 3.5 mg / kg, 2.5 mg / kg, or 1 mg / kg.In other embodiments of any of the above aspects, the effective amount of the telomerase inhibitor administered to the individual is at least about 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 10.1 mg / kg, 10.2 mg / kg, 10.3 mg / kg, 10.4 mg / kg, 10.5 mg / kg, 10.6 mg / kg, 10.7 mg / kg, 10.8 mg / kg, 10.9 mg / kg, 11 mg / kg, 11.1 mg / kg, 11.2 mg / kg, 11.3 mg / kg, 11.4 mg / kg, 11.5 mg / kg, 11.6 mg / kg, 11.7 mg / kg, 11.8 mg / kg, 11.9 mg / kg, 12 mg / kg, 12.1 mg / kg, 12.2 mg / kg, 12.3 mg / kg, 12.4 mg / kg, 12.5 mg / kg, 12.6 mg / kg, 12.7 mg / kg, 12.8 mg / kg, 12.9 mg / kg, or 13 mg / kg. In some embodiments, the effective amount of the telomerase inhibitor administered to the individual is not 9.4 mg / kg. In other embodiments, the effective amount of the telomerase inhibitor administered to the individual is from 7.5 mg / kg to 9.3 mg / kg. In other embodiments, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 11.7 mg / kg. In still other embodiments, the effective amount of the telomerase inhibitor is from 9.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 6.5 mg / kg to 11.7 mg / kg. In some embodiments herein, the effective amount of the telomerase inhibitor is from 7.5 mg / kg to 9.4 mg / kg.

[0120] Exemplary dosing frequencies for pharmaceutical compositions (such as pharmaceutical compositions containing any telomerase inhibitor disclosed herein) include, but are not limited to, once daily; once every other day; twice a week; three times a week; once a week without a break; once a week for 3 out of 4 weeks; once every 3 weeks; once every 2 weeks; once a week for 2 out of 3 weeks. In some embodiments, the pharmaceutical composition is administered about once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 8 weeks. In some embodiments, the composition is administered at least about once, twice, three times, four times, five times, six times, or seven times (i.e., once daily) a week, or either three times a day or twice a day. In some embodiments, the interval between each administration is less than about 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between each administration is longer than about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there are no interruptions in the dosing schedule. In some embodiments, the interval between each administration is about 1 week or less.

[0121] In other aspects, a pharmaceutical composition (such as a pharmaceutical composition containing any telomerase inhibitor disclosed herein) is administered to maintain the platelet count in an individual diagnosed or suspected of having essential thrombocythemia between about 150×10 3 / μL and 400×10 3 / μL. Under these conditions, the interval between each administration can be once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks or more. In some embodiments, the interval for administration of the telomerase inhibitor can be reduced over time if the individual's platelet count remains below 400×10 3 / μL in the individual's blood. In some aspects, there is provided a method for determining the dosing frequency of a telomerase inhibitor for the treatment of ET, comprising a) measuring the individual's platelet count by any method known in the art, and b) administering the telomerase inhibitor when the individual's platelet count exceeds 400×10 3 / μL.

[0122] The administration of a pharmaceutical composition (such as a pharmaceutical composition containing any telomerase inhibitor disclosed herein) can be extended over a long period, for example, from about 1 month to about 7 years (such as during maintenance therapy). In some embodiments, the composition is administered for any period of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In other embodiments, the composition is administered for the remainder of the individual's life.

Examples

[0123] Example 1: Preparation and Lipid Complexation of Oligonucleotide N3’→P5’ Phosphoramidate (NP) or N3’→P5’ Thiophosphoramidate (NPS) This example shows a method for synthesizing lipid complex oligonucleotide N3’→P5’ phosphoramidate (NP) or N3’→P5’ thiophosphoramidate (NPS).

[0124] Materials and Methods Starting Compounds These compounds may be prepared, for example, as described in McCurdy et al., Tetrahedron Letters 38:207-210 (1997) or Pongracz & Gryaznov, Tetrahedron Letters 49:7661-7664 (1999). The starting 3’-aminonucleoside monomers may be prepared as described in Nelson et al., J. Org. Chem. 62:7278-7287 (1997) or by the method described in Gryaznov et al., U.S. Patent Application Publication No. 2006 / 0009636.

[0125] Lipid Conjugation Depending on the nature of the linkage selected, various synthetic approaches can be used to complex the lipid moiety L to the oligonucleotide. See, for example, Mishra et al., Biochim. et Biophys. Acta 1264:229-237 (1995), Shea et al., Nucleic Acids Res. 18:3777-3783 (1995), or Rump et al., Bioconj. Chem. 9:341-349 (1995). Generally, complexation is achieved through the use of suitable functional groups at the oligonucleotide terminus. For example, the 3'-amino group present at the 3' terminus of NP and NPS oligonucleotides can be reacted with carboxylic acids, acid chlorides, acid anhydrides, activated esters using a suitable coupling catalyst to form amide bonds. Thiol groups are also suitable as functional groups (see Kupihar et al., Bioorg. Med. Chem. 9:1241-1247 (2001)). Various amino- and thiol-functionalized modifiers of different chain lengths are commercially available for oligonucleotide synthesis.

[0126] Specific approaches for attaching lipid groups to the termini of NP or NPS oligonucleotides include those described in U.S. Patent Application Publication No. 2005 / 0113325, which is hereby incorporated by reference in its entirety. In addition to the amide bonds described above, for example, lipids may also be attached to the oligonucleotide chain using phosphoramidite derivatives of the lipid to form phosphoramidate or thiophosphoramidate linkages that link the lipid and the oligonucleotide. The free 3'-amino of a fully protected support-bound oligonucleotide can also react with a suitable lipid aldehyde and then be reduced with sodium cyanoborohydride, which generates an amino bond.

[0127] For attachment of lipids to the 5’ end, the oligonucleotide can be synthesized using a modified, lipid-containing solid support, as also described in US Patent Application Publication No. 2005 / 0113325. The reaction of 3’-amino-1,2-propanediol with a fatty acyl chloride (RC(O)Cl), followed by dimethoxytritylation of the primary alcohol and succinylation of the secondary alcohol, provides an intermediate that is then linked to the solid support via the free succinyl carboxyl group. Examples of modified supports are shown below, where S represents a long-chain alkylamine CPG support and R represents a lipid.

Chemical formula

[0128] Following this procedure, the oligonucleotide is synthesized in the 5’ to 3’ direction, starting from deprotection of the ODMT group and phosphitylation, as described, for example, by Pongracz & Gryaznov (1999). This is effective, after cleavage from the solid support, for generating, for example, the following structure.

Chemical formula

[0129] The above structure is designated herein as GRN163L (Imetelstat or Imetelstat sodium) when R is (CH2) 14 CH3 (palmitoyl).

[0130] FlashPlate (trademark) assay This assay was essentially performed as described by Asai et al., Cancer Research 63:3931-3939 (2003). Briefly, this assay detects and / or measures telomerase activity by measuring the addition of TTAGGG telomere repeat sequences to a biotinylated telomerase substrate primer. The biotinylated product is captured on a streptavidin-coated microtiter plate, and an oligonucleotide probe labeled with 33P and complementary to 3.5 telomere repeats is used to measure the telomerase product. Unbound probe is removed by washing, and the amount of probe annealed to the captured telomerase product is quantified by scintillation counting.

[0131] Example 2: Imetelstat inhibits the spontaneous proliferation of CFU-Meg from patients with essential thrombocythemia and myelofibrosis in vitro, but not from healthy individuals. This example demonstrates the dose-dependent suppression of colony-forming units of megakaryocytes (CFU-Mega) by imetelstat in patients with essential thrombocythemia or myelofibrosis, independent of the JAKV617F mutation status or cytoreductive therapy, and shows the specificity of imetelstat for malignant megakaryocytic cells.

[0132] Materials and Methods To measure the effect of imetelstat on megakaryocyte proliferation and differentiation, the following method was used. (1) Cord blood (CB) cells were enriched for CD34-positive expressing cells using a negative cell separation device; (2) the cells were incubated with imetelstat (1-15 μM) in StemSpan® SFEM, a serum-free liquid medium containing a cytokine formulation designated for the development of megakaryocyte progenitor cells; (3) cord blood cells were cultured for a total of 17 days; (4) the cells were counted and evaluated at various time points for the differentiation marker (CD41) by flow cytometry and for telomerase activity by the TRAP assay.

[0133] To measure the CFU-Mega dose-response curve, mononuclear cells (MNCs) from 3 healthy individuals, 11 ET patients, and 1 patient with myelofibrosis (MF) (determined using WHO 2009 criteria) were isolated from peripheral blood, suspended in IMDM or plated into collagen ± cytokines (TPO, IL3, IL6, SCF, EPO), and treated with 0, 0.1, 1, and 10 μM of imetelstat or mismatch controls, and incubated at 37°C for several hours (cell suspension) or 10 - 12 days (collagen + 5% CO2). Megakaryocytes were stained and the number of CFU-Mega was scored. Dose-response analysis utilized a four-parameter log-logistic model for Log 10 (colony number). Telomerase activity was measured in MNCs by the TRAP assay.

[0134] Results Figures 1A and 1B show that imetelstat does not inhibit megakaryocyte proliferation or differentiation in healthy donors.

[0135] Table 1 shows spontaneous proliferation of CFU-Mega and inhibition by imetelstat. [Table 1]

[0136] Table 2 shows cytokine-stimulated proliferation of CFU-Mega and lack of inhibition by imetelstat. [Table 2]

[0137] Figure 7 shows that imetelstat inhibits megakaryocyte proliferation or differentiation in patients with myelofibrosis.

[0138] The dose-response curve in Figure 2 and the results in Figure 7 indicate that imetelstat decreases the proliferation of tumor precursors. CFU-Mega from peripheral blood indicates that imetelstat inhibits the proliferation of neoplastic (spontaneous) megakaryocytes from patients with ET and MF, but does not inhibit the proliferation of normal (cytokine-dependent) megakaryocytes from healthy individuals. This dose-dependent suppression of CFU-Mega formation by imetelstat in patients with ET is independent of the JAK V617F mutation status or cytoreductive therapy. Example 3: Phase II study (Phase II Imetelstat ET Study) to evaluate the activity of Imetelstat (GRN163L) in patients with essential thrombocythemia who require cytoreduction and who have been refractory or intolerant to existing therapies or who have refused standard therapy

[0139] This example demonstrates that imetelstat rapidly induces and maintains substantial hematological and molecular genetic responses in patients with essential thrombocythemia (ET) who are refractory or intolerant to existing therapies. Materials and Methods

[0140] Clinical Trial Plan Patients with ET who had been refractory or intolerant to at least one of the existing therapies (or who had refused standard therapy) and who required cytoreduction were initiated on Imetelstat at a dose of 7.5 - 11.7 mg / kg as a 2-hour intravenous infusion twice weekly and titrated to a platelet response. Once a platelet count of 250 - 300 × 10 3 / μL was achieved, maintenance dosing with imetelstat was then initiated with dose increases or decreases based on platelet response and toxicity, with the goal of decreasing the dosing frequency in the maintenance phase.

[0141] The selection criteria for ET-specific patients were: (1) a confirmed diagnosis of ET according to the criteria of the World Health Organization (WHO); (2) patients suffering from ET who required cytoreduction and had failed or were intolerant to at least one of the existing therapies (or had refused standard treatment). The clinical examination criteria (within 14 days of the first administration of the clinical trial drug) were: (1) platelets exceeding 600,000 / μL; (2) ANC of 1500 / μL or more; (3) hemoglobin of 10 g / dL or more.

[0142] General criteria for all patients were: (1) being willing and able to sign an informed consent form; (2) male or female aged 18 years or older; (3) an ECOG performance status of 0-2. The clinical examination criteria for all patients (within 14 days of the first administration of the clinical trial drug) were: (1) INR (or PT) and aPTT less than 1.5 times the upper limit of normal values (ULN); (2) serum creatinine of 2 mg / dL or less; (3) serum bilirubin less than 2.0 mg / dL (for patients with Gilbert syndrome, serum bilirubin less than 3 times the ULN); (4) AST (SGOT) and ALT (SGPT) less than 2.5 times the ULN; (5) alkaline phosphatase less than 2.5 ULN; (6) any clinically significant toxicity from previous cancer treatment and / or major surgery had to have recovered to grade 0-1 before the start of the clinical trial treatment.

[0143] Patients who met any of the following criteria were excluded from screening and clinical trial registration: (1) women who were pregnant or lactating; (2) those who had previously received a stem cell transplant; (3) those who had received investigational treatment within 4 weeks prior to the first clinical trial drug administration; (4) (a) uncontrolled congestive heart failure (CHF); (b) the need for antiarrhythmic treatment for ventricular arrhythmia; (c) clinically significant severe conductive hearing loss as determined by the clinical trial responsible physician; (d) ongoing angina pectoris requiring treatment; (e) cardiovascular diseases of New York Heart Association (NYHA) class II, III, or IV; (f) known to be serologically positive for human immunodeficiency virus (HIV); (g) medical symptoms of severe complications as determined by the clinical trial responsible physician, including active or chronic relapsing bleeding, clinically relevant ongoing infection, cirrhosis, and chronic obstructive or restrictive lung disease; or (h) any other severe, acute, or chronic medical or psychiatric condition, abnormal clinical test values, or difficulties that, in accordance with the protocol requirements, could increase the risk associated with participating in the clinical trial or receiving the clinical trial drug or could interfere with the interpretation of the clinical trial results and that, in the judgment of the clinical trial responsible physician, would make the patient unsuitable for the clinical trial, including clinically significant cardiovascular diseases or conditions.

[0144] The primary evaluation item was the best overall hematological response rate (RR) (complete remission (CR) + partial remission (PR)). The time frame was from the first administration (day 1 of cycle 1) to the end of the clinical trial (12 months after the last participant was administered).

[0145] The secondary evaluation items were the measurement of the duration of hematological response, the measurement of molecular genetic response (JAK2 V617F / MPL W515 mt patients), and the purpose was to test safety and tolerability by monitoring multiple patients with hematological toxicity, non-heme Grade 3 and 4 adverse events (AEs), and hemorrhagic events. The time frame was from the first administration (day 1 of cycle 1) to the end of the clinical trial (12 months after the last participant was administered). The spontaneous proliferation of CFU-Mega (only at the selected sites) was for exploratory purposes.

[0146] Table 3 describes the definition of response for this clinical trial. The European LeukemiaNet response criteria were applied from Barosi et al., Blood (2009). Heme response was counted as the most recent at 4 weeks. [Table 3]

[0147] Patient basic information is provided in Table 4 below. [Table 4]

[0148] Results Figure 3 shows that 100% overall hematological remission was achieved in all 14 patients with ET who had been treatment failures or intolerant to prior therapies. Complete remission was achieved in 13 out of 14 (92.9%) patients and partial remission in 1 out of 14 (7.1%) patients. All patients who achieved hematological CR are continuing treatment. Data on the time until the platelet count ≤ 400 × 10 3 / μL (shown as diamonds for each patient) first occurred had a median of 3.1 weeks (range 2.1 to 23.1 weeks), while the time to complete remission had a median of 6.1 weeks (range 5.1 to 14.1 weeks) (Figure 3).

[0149] Data on dosing frequency for the 13 patients who achieved hematological complete remission and initiated maintenance therapy are provided in Table 5 below. Maintenance dosing frequency generally decreased over time (ranging from once weekly to every 7 weeks), and the majority of patients (84.6% or 11 / 14) received imetelstat once every 2 weeks or less frequently (based on the median). 85.7% (6 / 7) of patients eligible to continue treatment were still on maintenance therapy after 1 year. [Table 5]

[0150] As shown in Figure 4A, the percentage of %JAK2 V617F gene mutations decreased over time in all patients. On the other hand, Figure 4B shows that molecular genetic response (PR) was achieved within the range of 3 to 6 months in 6 / 7 (85.7%) of the patients tested for JAK2 V617 (JAPK2 V617F).

[0151] Table 6 shows the results regarding the exploratory evaluation item (CFU-Mega). In the two patients tested, a decrease in spontaneous proliferation of ex-vivo CFU-Mega (93% and 96% decrease from baseline, respectively) was demonstrated, confirming the existing ex-vivo data.

Table 6

[0152] Figure 5 shows that the spontaneous proliferation of CFU-Mega is not correlated with the decrease in JAK2 gene mutations in one patient (Patient No. 4).

[0153] The data indicate that imetelstat has a relatively selective inhibitory effect on the proliferation of neoplastic clones that drive myeloproliferative neoplasms (MPNs) such as essential thrombocythemia and may modify the biological characteristics underlying the disease.

[0154] Table 7 shows clinically significant frequent non-hematological adverse events.

Table 7

[0155]

Table 8

[0156] Example 4: Open-label pilot trial of the efficacy and safety of Imetelstat (GRN163L) in patients with DIPSSplus intermediate-2 risk or high-risk primary myelofibrosis (PMF), myelofibrosis transformed from polycythemia vera (post-PV MF), or myelofibrosis transformed from essential thrombocythemia (post-ET MF) Materials and methods Clinical trial plan Patients with DIPSSplus intermediate-2 risk or high-risk primary myelofibrosis (PMF), myelofibrosis transformed from polycythemia vera (post-PV MF), or myelofibrosis transformed from essential thrombocythemia (post-ET MF) who were not active on standard therapy were initiated on 9.4 mg / kg of Imetelstat given as a 2-hour intravenous infusion once every 21 days (cohort A). Alternatively, patients were administered once weekly for 3 weeks followed by infusions (9.4 mg / kg) every 21 days for 2 hours (cohort B). Patients may be treated for up to 9 cycles. Patients may continue treatment beyond 9 cycles.

[0157] The selection criteria for PMF-specific patients were: (1) a confirmed diagnosis of ET by World Health Organization (WHO) criteria; (2) the presence of megakaryocyte proliferation and dysplasia with reticulin and / or collagen fibrosis, or (4) not meeting the WHO criteria for CML, PV, MDS, or other myeloid neoplasms, or (5) no evidence of reactive myelofibrosis.

[0158] The selection criteria for patients specific to post-PV MF were defined as follows: (1) a confirmed diagnosis of PV according to the World Health Organization (WHO) criteria; (2) a grade of bone marrow fibrosis of 2-3 (on a 0-3 scale) or grade 3-4 (on a 0-4 scale); and (3) either (a) a persistent loss of the need for anemia or bleeding in the absence of cytoreductive therapy, or (b) a leukoerythroblastic peripheral blood picture, or (c) an increase in splenomegaly defined as an increase in a palpable spleen of 5 cm or more or the appearance of a new palpable spleen, or (d) the onset of two or more of three systemic symptoms (weight loss of 10% or more within 6 months, night sweats, fever of unknown origin (37.5 °C or higher)).

[0159] The selection criteria for patients specific to post-ET MF were defined as follows: (1) a confirmed diagnosis of ET according to the World Health Organization (WHO) criteria; (2) a grade of bone marrow fibrosis of 2-3 (on a 0-3 scale) or grade 3-4 (on a 0-4 scale); and (3) either (a) anemia and a reduction of 2 g / dL or more from the baseline hemoglobin level, or (b) a leukoerythroblastic peripheral blood picture, or (c) an increase in splenomegaly defined as an increase in a palpable spleen of 5 cm or more or the appearance of a new palpable spleen, or (d) an increase in castate dehydrogenase, or (e) the onset of two or more of three systemic symptoms (weight loss of 10% or more within 6 months, night sweats, fever of unknown origin (37.5 °C or higher)).

[0160] The general criteria for all patients were as follows: (1) the willingness and ability to sign an informed consent form; (2) male or female aged 18 years or older; and (3) an ECOG performance status of 0-2. The examination criteria for all patients (within 14 days of the first administration of the clinical trial drug) were as follows: (1) AST (SGOT) and ALT (SGPT) ≤ 2.5 × ULN; (2) creatinine ≤ 3 mg / dL; (3) absolute neutrophil count ≥ 1000 / μL; (4) platelet count ≥ 50,000 / μL; and (5) the absence of active treatment with systemic anticoagulation and baseline PT and aPTT not exceeding 1.5 times the ULN.

[0161] Any patient who met any of the following criteria was excluded from screening and clinical study enrollment: (1) Women who were pregnant or lactating; (2) Those who had received any of chemotherapy, immunomodulatory drug therapy, immunosuppressive therapy, corticosteroids, prednisone 10 mg / day or equivalent, growth factor treatment, or JAK inhibitor therapy within 14 days prior to enrollment; (4) Subjects with other ongoing malignant lesions; (5) Those known to be positive for HIV; (6) Any unresolved toxicity greater than or equal to grade 2 from previous anticancer therapy; (6) Incomplete recovery from any previous surgery; (7) The presence of acute ongoing infections requiring antibiotics; (8) Uncontrolled comorbidities or any concurrent conditions that put the patient's safety or protocol compliance at risk.

[0162] The primary evaluation item was the best overall response rate (RR) (clinical improvement (CI) or complete remission (CR) or partial remission (PR)). The time frame was from the first administration (day 1 of cycle 1) to the first 9 cycles of treatment.

[0163] The purpose of the secondary evaluation items was to measure (a) adverse events, (b) splenic response: defined as either a minimum 50% reduction in palpable splenomegaly of the spleen that was at least 10 cm at baseline or a palpable spleen that was 5 cm or more at baseline, and (c) transfusion independence: defined as a history of at least 2 units of red blood cell transfusions in the most recent month for hemoglobin values less than 85 g / L where transfusion dependence was not associated with clinically obvious bleeding. The time frame was from the first administration (day 1 of cycle 1) to the end of the clinical trial. The exploratory purposes were (a) histological evaluation of the reversal of myelofibrosis to a lower grade and (b) a subset of patients with baseline leukocytosis and thrombocytosis who achieved at least a 50% reduction in their numbers at the end of cycles 3, 6, and 9.

[0164] Table 9 describes the efficacy definitions for this clinical trial. The consensus criteria of the International Working Group (IWG) for treatment efficacy in myelofibrosis with myeloid metaplasia were used.

Table 9

[0165] Results Clinical utility was observed in the patients enrolled in this clinical trial. Thirty-three patients accrued. The first 18 were enrolled and followed for a minimum of 3 months, and the rest were discontinued. There were 11 patients in cohort A and 7 patients in cohort B; 44% had PMF, 33% had post-PV MF, and 22% had post-ET MF. The median age was 68 years, and the baseline risk was high at 56% and intermediate-2 risk at 44%. Seven patients were transfusion-dependent. The median spleen size was 13 cm, and 11 patients had constitutional symptoms. The karyotype was abnormal in 7 patients, and 89% were JAK2-mutated. Fifteen patients (83%) had been previously treated, 7 with JAK inhibitors and 3 with pomalidomide.

[0166] Toxicity At a median follow-up of 3.2 months, 16 patients (89%) continued treatment; the 2 discontinuation cases were due to unrelated death and disease progression. In cohort A, there were no grade 4 treatment-related adverse events, and grade 3 events were limited to thrombocytopenia in 27% and anemia in 9%. In cohort B, 2 patients (29%) experienced grade 4 thrombocytopenia, and grade 3 events were limited to thrombocytopenia, neutropenia, and anemia in 1 patient each. Dose reduction was required for grade 3 or 4 myelosuppression in only 2 patients (11%).

[0167] Efficacy The overall response rate was 44%. This included 5 patients (28%) who met the BM and peripheral blood morphologic criteria for complete remission (CR) (n = 4) or partial remission (PR) (n = 1), and 3 patients who had resolution of drug-induced grade 1 thrombocytopenia during validation of clinical improvement and response duration. Four (22%) CR patients experienced reversal of bone marrow (BM) fibrosis and recovery of normal megakaryocyte morphology. Two CR patients were transfusion-dependent at baseline but became transfusion-independent. Complete molecular genetic responses were demonstrated in 2 CR patients. One had 10% JAK2V617F and the other had 50% JAK2V617F. Among the 13 patients who developed leukocytosis, 10 (77%) normalized the count or had a reduction of more than 50%. Eleven (61%) patients had complete or partial resolution of leukemoid reaction.

[0168]

Table 10

[0169] Example 5: Imetelstat inhibits the spontaneous proliferation of CD34+ cells from patients with acute myeloid leukemia in vitro but not from healthy individuals. This example demonstrates the dose-dependent inhibition of CD34+ cells by imetelstat in patients with acute myeloid leukemia and suggests the specificity of imetelstat for malignant CD34+ cells.

[0170] Materials and Methods The following methods were used to measure the efficacy of imetelstat. (1) In colony formation assays and liquid cultures, bone marrow cells were incubated with imetelstat (0.1 - 10 μM) for a total of 14 days, and the cells were counted and evaluated at various time points.

[0171] To measure the CFU dose-response curve, bone marrow cells from four healthy individuals or five AML patients were plated and treated with 0, 0.1, 1, and 10 μM of imetelstat or mismatch control isolated from peripheral blood. CFU-GM (colony-forming unit - granulocyte, macrophage) and BFU-E (burst-forming unit - erythroblast) were stained and the numbers of CFU-GM and BFU-E were scored.

[0172] Results Imetelstat did not reduce CFU from the bone marrow of healthy donors in the 14-day CFU assay.

[0173] A decrease in CFU of bone marrow cells from AML patients was observed in treatment with imetelstat in the 14-day CFU assay.

[0174] In the 14-day liquid culture assay, Imtelstat reduced cell proliferation from the bone marrow cells of newly diagnosed AML patients.

[0175] Imetelstat reduced the proliferation of CD34-positive cells derived from the bone marrow cells of AML patients, but not from those of normal patients. Figure 6 represents the rate of cell proliferation in cultures after in-vitro treatment with Imetelstat of CD34-positive cells obtained from healthy donors and CD34-positive cells from AML patients on days 5, 7, and 9.

[0176] Examples are intended to be merely illustrative of the present invention and, therefore, one should not think of limiting the present invention in any way. Embodiments that explain and will also elaborate on the above-described aspects and embodiments of the present invention are provided. The foregoing examples and detailed description are provided for illustration and are not limiting. All publications, patent applications, and patents cited herein are hereby incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are hereby expressly incorporated by reference for the purpose of describing and disclosing the compositions and methods that may be used in connection with the present invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims in light of the teachings of the present invention.

Claims

【Claim 1】 The composition described in the specification.

Images