WO2010029158A1 - Aplidine in the treatment of chronic myeloproliferative disorders - Google Patents

Aplidine in the treatment of chronic myeloproliferative disorders Download PDF

Info

Publication number
WO2010029158A1
WO2010029158A1 PCT/EP2009/061820 EP2009061820W WO2010029158A1 WO 2010029158 A1 WO2010029158 A1 WO 2010029158A1 EP 2009061820 W EP2009061820 W EP 2009061820W WO 2010029158 A1 WO2010029158 A1 WO 2010029158A1
Authority
WO
WIPO (PCT)
Prior art keywords
aplidine
chronic myeloproliferative
myeloproliferative disorder
mice
treatment
Prior art date
Application number
PCT/EP2009/061820
Other languages
French (fr)
Inventor
Maria Verrucci
Anna Rita Migliaccio
Alessandro M. Vannucchi
José María JIMENO DOÑAQUE
Original Assignee
Pharma Mar, S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharma Mar, S.A. filed Critical Pharma Mar, S.A.
Publication of WO2010029158A1 publication Critical patent/WO2010029158A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/15—Depsipeptides; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use of Aplidine and analogues thereof in the treatment of chronic myeloproliferative disorders, especially those wherein there is an accumulation of abnormal megakaryocytes.
  • the present invention relates to the treatment of polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis, post-PV myelofibrosis and post-ET myelofibrosis.
  • Chronic myeloproliferative disorders also referred as myeloproliferative neoplasms (MPN)
  • MPN myeloproliferative neoplasms
  • CEL/ HES chronic eosinophilic leukaemia/ hypereosinophilic syndrome
  • WHO World Health Organization
  • CMPDs share a common stem cell- derived clonal heritage and their phenotypic diversity is attributed to different configurations of abnormal signal transduction, resulting from a spectrum of mutations affecting protein tyrosine kinases or related molecules.
  • histology-based classification and diagnostic criteria for these disorders can be refined by employing molecular disease markers.
  • the bone marrow criteria defined by the WHO classification of myeloproliferative disorders of 2001 are based on characteristic increase and clustering of morphologically abnormal enlarged megakaryocytes as a pathognomonic clue to describe three distinct phenotypic entities: essential thrombocythemia (ET), polycythemia vera (PV) and prefibrotic, early fibrotic, and fibrotic primary myelofibrosis (PMF) .
  • EERT essential thrombocythemia
  • PV polycythemia vera
  • PMF fibrotic primary myelofibrosis
  • bone marrow histopathology allows expert pathologists to differentiate between the three prefibrotic myeloproliferative disorders.
  • PV and ET have marked increases of red blood cell and platelet production, respectively. Treatment is directed at reducing the excessive numbers of blood cells. Both PV and ET can develop a spent phase late in their courses that resembles PMF with cytopenias, marrow hypoplasia and fibrosis.
  • the World Health Organization classification system has recently revised its diagnostic criteria for PV, ET, and PMF (2008 WHO diagnostic criteria) to include JAK2 and MPL mutations as clonal markers (A.Tefferi, 2008, American Journal of Hematology, 83(6), 491-7; and A.Tefferi and JW Vardiman, 2008, Leukemia, 22, 14-22).
  • Primary myelofibrosis (also known as myelofibrosis, chronic idiopathic myelofibrosis, agnogenic myeloid metaplasia and myoelosclerosis with myeloid metaplasia) is caused by the growth and proliferation of an abnormal bone marrow hematopoietic stem cell (CD34+), resulting in the replacement of the bone marrow with fibrous connective tissue.
  • myelofibrosis also known as myelofibrosis, chronic idiopathic myelofibrosis, agnogenic myeloid metaplasia and myoelosclerosis with myeloid metaplasia
  • CD34+ abnormal bone marrow hematopoietic stem cell
  • Bone marrow fibrosis 'myelofibrosis' is the most recognized and best characterized feature of primary myelofibrosis (PMF).
  • PMF primary myelofibrosis
  • other conditions might be accompanied by bone marrow fibrosis, including other myeloid disorders, lymphoid disorders, metastatic cancer, autoimmune diseases, and inflammatory or infectious conditions.
  • PMF is, in the majority of cases, associated with reticulin and/ or collagen myelofibrosis and it can therefore be considered as part of the primary process.
  • overt collagen bone marrow fibrosis in conditions other than PMF is chronologically a 'secondary' process.
  • PV polycythemia vera
  • ET essential thrombocythemia
  • PMF Primary myelofibrosis
  • MK megakaryocytes
  • TGF- ⁇ transforming growth factor
  • bFGF basic fibroblast growth factor
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • the first one was developed by transplanting recipient mice with bone marrow cells genetically engineered to overexpress the thrombopoietin (TPO) gene, the main physiologic factor that stimulates megakaryocytopoiesis (Yan XQ et al., 1996, Blood, 88: 402-9) .
  • TPO thrombopoietin
  • the second animal model of PMF has been identified in mice genetically impaired for their expression of the transcription factor GATA- 1 , the so-called GATA- 1 low mice (Vannucchi et al., 2004, Pathobiologie, 52, 275-279).
  • mice recapitulate several aspects of the disease, e.g. myelofibrosis has a quite slow evolution during the lifespan of the mice, making it more suitable for targeted drug studies.
  • Prognosis Overall median survival in PMF is approximately 5 years but it varies substantially among patients based on the presence or absence of well-defined prognostic determinants (Cervantes F. et al., 1997, Br J Haematol, 97, 635-640).
  • prognostic determinants Classically, the most important indicators of adverse prognosis have been: the presence of anemia, advanced age, hypercatabolic symptoms, leukocytosis or leukopenia, circulating blasts and high-risk cytogenetic abnormalities (+8, 12p-). More recent studies have shown that JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in PMF (Barosi G.
  • Thalidomide inhibits TNF- ⁇ and TNF- ⁇ , interleukin (IL)- l ⁇ , IL-6, IL- 12, and GM-CSF and stimulates T-lymphocyte proliferation.
  • IL interleukin
  • Clinical improvements mainly related to anemia or thrombocytopenia, have been disclosed.
  • Imatinib A specific inhibitor of AbI, PDGFR, c-Kit and Arg tyrosine kinases which has become the standard therapy in chronic myelogenous leukaemia.
  • imatinib had only modest activity, although most of the patients experienced decreased splenomegaly and transiently improved hematopoiesis (Cortes et al., 2003, Cancer, 97, 2760-2766).
  • PTK787/ZK 222584 An oral inhibitor of the VEGF receptor VEGFR- I and VEGFR-2 tyrosine kinases (FIi- I and FIk- 1 /KDR) as well as the PDGFR and c-Kit which has shown modest activity in patients with PMF (Giles et al., 2007, Leukemia research, 31 (7), 891 -897).
  • VEGFR-2 A synthetic inhibitor of VEGFR-2, c-Kit, and Flt-3, which was used in phase II trials in patients with chronic myeloproliferative disorders on the premise of their antiangiogenic activity, but overall clinical activity was minimal (Giles et a!., 2003, Cancer, 97, 1920- 1928).
  • - SU6668 is a potent antiangiogenic inhibitor of receptor tyrosine kinases including those of VEGFR, FOFR, and PDGFR and c-Kit. It has been hypothesised that 8U6668 may be effective in PMF based upon its inhibitory target profile (Hasselbach et al., 2003, Medical hypotheses, 61 (2), 244-247).
  • Raf- 1 a member of the Raf/mitogen-activated protein kinase/ extracellular-signal -regulated kinase (ERK) kinase (MEK) / ERK signaling pathway
  • ERK extracellular-signal -regulated kinase
  • MEK extracellular-signal -regulated kinase
  • ERK signaling pathway a member of the Raf/mitogen-activated protein kinase/ extracellular-signal -regulated kinase (ERK) kinase (MEK) / ERK signaling pathway
  • ERK extracellular-signal -regulated kinase
  • MEK extracellular-signal -regulated kinase
  • c-Kit receptor tyrosine kinases
  • Aplidine (dehydrodidemnin B) is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans, and is the subject of WO 91/04985. It is related to compounds known as didemnins, Aplidine has the following structure:
  • the problem to be solved by the present invention is to provide a therapy that is useful in the treatment of chronic myeloproliferative disorders, especially those wherein there is an accumulation of abnormal megakaryocytes.
  • this invention is directed to pharmaceutical compositions, kits and methods for the treatment of chronic myeloproliferative disorders using Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and to the use of Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof in the manufacture of medicaments for the treatment of chronic myeloproliferative disorders.
  • the invention provides for the use of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of chronic myeloproliferative disorders.
  • the invention provides Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic myeloproliferative disorders.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, to be used in the treatment of chronic myeloproliferative disorders.
  • the present invention additionally provides a method for treating any mammal, notably a human, affected by a chronic myeloproliferative disorder, comprising administering to the affected individual a therapeutically effective amount of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof.
  • a medical kit for administering Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof comprising printed instructions for administering it according to the uses and methods of treatment set forth herein, and a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.
  • the invention refers to a method for treating a human subject having a chronic myeloproliferative disorder, said method comprising: a) obtaining a nucleic acid sample from said human subject; b) determining the presence of the JAK2V617F mutation; and c) treating with Aplidine the JAK2V617F positive subjects.
  • the invention relates to a method for determining an increased likelihood of pharmacological effectiveness of treatment by Aplidine in a human subject diagnosed with a chronic myeloproliferative disorder comprising obtaining a nucleic acid sample from said human subject and determining the presence of the JAK2V617F mutation, wherein the identification of the JAK2V617F mutation indicates an increased likelihood of pharmacological effectiveness by Aplidine.
  • Figure 1 Platelet count in younger GATA- l low mice (9 month old) injected with one or two courses of Aplidine (60 ⁇ g/kg/day, i.p.) and/ or vehicle.
  • FIG. 1 Platelet count in older GATA- l low mice (18 month old) injected with one course of Aplidine (60 mg/kg/day, ip) or vehicle.
  • Figure 5 Bone marrow cellularity in younger GATA- l low mice (9 month old) injected with one or two courses of Aplidine (60 ⁇ g /kg/ day, ip) and/ or vehicle.
  • Figure 6 Bone marrow cellularity in older GATA- l low mice (18 month old) injected with one course of Aplidine (60 ⁇ g/kg/day, ip) or vehicle.
  • Figure 7 Bone marrow megakaryocyte number in younger GATA- l low mice (9 month old) injected with one or two courses of Aplidine (60 ⁇ g/kg/day, ip) and/ or vehicle.
  • FIG. 9 Platelet count in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Platelet count was monitored at days 7 and 14 of each course of treatment. At the beginning of the assay the baseline levels of wild type and GATA- l low were 0.8 x 10 6 and 0.1 x 10 6 platelets/ ⁇ L, respectively, and at the end of the assay these baseline levels were 0.75 x 10 6 and 0.2 x 10 6 platelets/ ⁇ L.
  • FIG. 10 Hematocrit level in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Hematocrit level was monitored at days 7 and 14 of each course of treatment. At the beginning of the assay the baseline levels of wild type and GATA- l low were 45% and 38%, respectively, and at the end of the assay these baseline levels were 48% and 43%.
  • FIG. 11 Bone marrow cellularity in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively. At the beginning of the assay the baseline levels of wild type and GATA- l low 16 x 10 6 and 6 x 10 6 cells/ femur, respectively and at the end of the assay these baseline levels were 16 x 10 6 and 4 x 10 6 cells/ femur.
  • FIG. 12 Bone marrow megakaryocyte number in 8- 10 month- old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
  • Figure 13 Bone marrow fiber deposition in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
  • Figure 14 Bone marrow microvessel density (angiogenesis) in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
  • FIG. 15 Bone marrow TGF- ⁇ mRNA levels in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
  • 2- ⁇ Ct is the amount of target mRNA normalized to the beta-2- microglobulin gene (housekeeping gene).
  • FIG. 16 Bone marrow VEGF mRNA levels in 8- 10 month-old GATA- l low mice injected with two or four courses of Aplidine ( 100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
  • 2- ⁇ Ct is the amount of target mRNA normalized to the beta-2- microglobulin gene (housekeeping gene).
  • FIG. 1 GATAl expression in megakaryocytes and frequency of megakaryocytes in sections immunostained for GATAl and counterstained by hamatoxilyn-eosin, in bone marrow from 8- 10 month-old GATA- l low mice injected with four courses of Aplidine (100 ⁇ g/kg/day x 5d, q 2 Id, i.p.) or vehicle.
  • Figure 18 Effects of Aplidine on the clonogenic activity and the proliferation of cell lines; values shown are IC50 (mean+SD of at least 3 individual experiments).
  • Figure 19 Effects of Aplidine on the clonogenic activity of progenitor cells from MPN patients; values shown are IC50 (mean+SD).
  • Figure 20 Effects of Aplidine on the proportion of BFU-E colonies harboring the JAK2V617F mutation obtained from JAK2V617F-mutant PMF patients.
  • ⁇ values represent the percentage (%) of colonies harboring the JAK2V617F mutation (V617F) or not (wt) of the total number of colonies genotyped (from a minimum of 20 to 50).
  • FIG. 21 Effects of Aplidine on the differentiation of progenitor cells (CD34 + ) from PMF patients towards the megakaryocytic lineage (CD61 + ). Values are presented as the Mean+SD of five independent experiments.
  • FIG. 22 Algorithm of treatment for primary myelofibrosis (PMF).
  • PMF primary myelofibrosis
  • CMPDs chronic myeloproliferative disorders
  • MK abnormal megakaryocytes
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • treatment refers to the act of treating as “treating” is defined immediately above.
  • Aplidine is a cyclic depsipeptide with the following structure:
  • Aplidine is intended here to cover any pharmaceutically acceptable salt, solvate, hydrate, prodrug, or any other compound which upon administration to the patient is capable of providing (directly or indirectly) the compound as described herein.
  • the preparation of salts, solvates, hydrates, and prodrugs can be carried out by methods known in the art.
  • salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two.
  • nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.
  • acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate.
  • mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate
  • organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate.
  • alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N, N- dialkylenethanolamine, triethanolamine and basic aminoacids salts.
  • prodrug is used in its broadest sense and encompasses those derivatives that are converted in vivo (ie. metabolized) to Aplidine.
  • the prodrug can be hydro lyzed, oxidized, or otherwise react under biological conditions to provide Aplidine.
  • prodrugs include, but are not limited to, derivatives and metabolites of Aplidine that include biohydrolyzable moeities such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
  • Prodrugs can tipically be prepared using well-known methods, such as those described by Burger “Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and “Design and Applications of Prodrugs” (H. Bundgaard ed., 1985, Harwood Academic Publishers).
  • any drug referred to herein may be in crystalline form either as free compound or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention.
  • Methods of solvation are generally known within the art.
  • Suitable Aplidine analogues include those compounds disclosed in WO 02/02596, WO 01 /76616, and WO 2004/84812. We incorporate by specific reference the content of these patent applications.
  • the term "Aplidine analogue” is intended here also to cover any pharmaceutically acceptable salt, solvate, hydrate, prodrug, or any other compound which upon administration to the patient is capable of providing (directly or indirectly) an "Aplidine analogue" as described above.
  • Aplidine and Aplidine analogues for use in accordance with the present invention may be prepared following any of the synthetic processes disclosed in WO 02/02596, WO 01 /76616, and WO 2004/84812, which are incorporated herein by reference. In particular, we include by reference the compounds defined in the claims of those patent applications.
  • compositions comprising Aplidine, an Aplidine analogue, or a pharmaceutically acceptable salt thereof, may be formulated according to the chosen route of administration.
  • the pharmaceutical composition of the invention can be administrated by any suitable route, including but not limited to oral, rectal, transdermal, ophthalmic, nasal, topical, vaginal or parenteral.
  • the pharmaceutical composition is formulated in order to be suitable for parenteral administration to a subject, e.g., a human being, preferably by intravenous, intramuscular, intraperitoneal or subcutaneous administration.
  • suitable formulations for parenteral administration are solutions, suspensions, emulsions, lyophilized compositions and the like.
  • the administration of the pharmaceutical composition of the invention to a mammal in need thereof can be carried out by conventional means.
  • the administration of the pharmaceutical composition of the invention is by intravenous administration and includes an intravenous delivery through standard devices, e.g., a standard peripheral intravenous catheter, a central venous catheter, or a pulmonary artery catheter, etc.
  • standard devices e.g., a standard peripheral intravenous catheter, a central venous catheter, or a pulmonary artery catheter, etc.
  • the pharmaceutical composition of the invention may be administrated using the appropriate equipments, apparatus, and devices which are known by the skilled person in art.
  • Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof may be supplied and stored as a sterile lyophilized product, comprising the active ingredient and excipients in a formulation adequate for therapeutic use.
  • a formulation comprising the active ingredient and mannitol is preferred.
  • pharmaceutical compositions of Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof is given in WO 99/42125, which is incorporated herein by reference in its entirety.
  • Administration of Aplidine, Aplidine analogues, a n d pharmaceutical compositions comprising the same is preferably by intravenous infusion.
  • the infusing step is typically repeated on a cyclic basis, which may be repeated as appropriate over for instance 1 to 20 cycles.
  • the cycle includes a phase of infusing the drug, and usually also a phase of not infusing the drug.
  • the cycle is worked out in weeks, and thus the cycle normally comprises one or more weeks of a drug infusion phase, and one or more weeks of non-infusion to complete the cycle.
  • a cycle of 3 or 4 weeks is preferred, but alternatively it can be from 2 to 6 weeks.
  • the infusion phase can itself be a single administration in each cycle of say 1 to 72 hours, more usually of about 1 , 3 or 24 hours; or an infusion on a daily basis in the infusion phase of the cycle for preferably 1 to 5 hours, especially about 1 or 3 hours; or an infusion every week or every two weeks in the infusion phase of the cycle for preferably 1 to 24 hours, especially about 1 , 3 or 24 hours.
  • the dose will be selected according to the dosing schedule, having regard to the existing data on Dose Limiting Toxicity, on which see for example the above mentioned WO 01 /35974 patent specification and the Phase I studies cited in the background of the invention. These documents are also incorporated herein in full by specific reference.
  • schedules and dosages are for example:
  • Schedule and dosages b, c) and d) are the most preferred to be used in the present invention. It is particularly preferred the dosing and administration regime defined in b), i.e. the administration of aplidine at about 5 mg/m 2 body surface area, as a 3-hour intravenous infusion every two weeks in a four- week cycle treatment schedule.
  • the correct dosage of the compound may change according to the particular formulation, the mode of application, and the particular situs, patient and disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Dose delays and/ or dose reductions and schedule adjustments are performed as needed depending on individual patient condition and tolerance of treatments.
  • Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof may be used with other drugs to provide a combination therapy in the treatment of chronic myeloproliferative disorders.
  • the other drug may form part of the same composition, or be provided as a separate composition for administration at the same time or at different time.
  • the present invention provides a method for treating any mammal, notably a human, affected by a chronic myeloproliferative disorders, comprising administering to the affected individual a therapeutically effective amount of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof.
  • said chronic myeloproliferative disorder is a BCR-ABL negative myeloproliferative disorder.
  • the BCR-ABL fusion gene is a specific chromosomal abnormality that is due to a reciprocal translocation between chromosomes 9 and 22.
  • the ABL gene from chromosome 9 joins to the BCR gene on chromosome 22, to form the BCR-ABL fusion gene.
  • the changed chromosome 22 with the fusion gene on it is called the Philadelphia chromosome.
  • PV Polycythemia vera
  • ET essential thrombocythemia
  • PMF primary myelofibrosis
  • said chronic myeloproliferative disorder is characterised by an increase in the proliferation of abnormal megakaryocytes.
  • the three classic BCR-ABL negative myeloproliferative disorders (PV, ET and PM) are characterized by an increase in the proliferation and some degree of clustering of abnormal megakaryocytes.
  • the methods of the invention are used to treat PV, ET and PMF.
  • the chronic myeloproliferative disorder is primary myelofibrosis (PMF).
  • PMF can show a variable degree of bone marrow fibrosis "myelofibrosis".
  • myelofibrosis the disease can be classified in pre-fibrotic, early fibrotic or fibrotic PMF.
  • Assessment of bone marrow fibrosis includes basic parameters such as cellularity and fiber content (Thiele et al., 2005, Haematologica, 90(8), 1128-32).
  • the methods of the invention are used for treating early stage primary myelofibrosis (pre-fibrotic or early fibrotic PMF) wherein fibrosis is not yet fully established.
  • the chronic myeloproliferative disorder is polycythemia vera (PV). In a further preferred embodiment, the chronic myeloproliferative disorder is essential thrombocythemia (ET).
  • IWG-MRT International Working Group for Myelofibrosis Research and Treatment
  • the methods of the invention are used for treating polycythemia vera (PV) having progressed to post-PV myelofibrosis and essential thrombocythemia (ET) which has progressed to post-ET myelofibrosis.
  • PV polycythemia vera
  • ET essential thrombocythemia
  • the invention further provides the use of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a chronic myeloproliferative disorder.
  • the invention provides Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, for use in the treatment of a chronic myeloproliferative disorder.
  • the present invention is also directed to a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, to be used in the treatment of a chronic myeloproliferative disorder.
  • a medical kit for administering Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof comprising printed instructions for administering it according to the uses and methods of treatment set forth herein, and a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  • the Aplidine, Aplidine analogue or pharmaceutically acceptable salt thereof for the methods, uses, pharmaceutical compositions and kits of the present invention is Aplidine.
  • Example 4 the effects of Aplidine on progenitor cells (CD34+) obtained from patients suffering of a chronic myeloproliferative disorder, particularly PV or PMF, wherein said patients were JAK2V617F mutated (5 PV patients and 4 or the 5 PMF).
  • the mammal preferably a human, suffering from a chronic myeloproliferative disorder is harbouring the JAK2V617F mutation.
  • the chronic myeloproliferative disorder is primary myelofibrosis (PMF).
  • V617F mutation of the Janus kinase (JAK) 2 gene has been associated with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PM) (A.Tefferi, 2008, American Journal of Hematology, 83(6), 491-7; A.Tefferi and JW Vardiman, 2008, Leukemia, 22, 14-22; and Baxter EJ et al., 2005, Lancet., 365 (9464): 1054- 1061).
  • PV polycythemia vera
  • ET essential thrombocythemia
  • PM primary myelofibrosis
  • This mutation has provided a molecular explanation for the disregulated hematopoiesis typical of these disorders, a diagnostic test that distinguishes them from other types of myeloproliferative disorders, and an opportunity to develop targeted therapy that could potentially avoid the toxicities associated with the conventional chemotherapeutic agents currently employed in their treatment.
  • the invention refers to a method for treating a human subject having a chronic myeloproliferative disorder, said method comprising: a) obtaining a nucleic acid sample from said human subject; b) determining the presence of the JAK2V617F mutation; and c) treating with Aplidine the JAK2V617F positive subjects.
  • the invention relates to a method for determining an increased likelihood of pharmacological effectiveness of treatment by Aplidine in a human subject diagnosed with a chronic myeloproliferative disorder comprising obtaining a nucleic acid sample from said human subject and determining the presence of the JAK2V617F mutation, wherein the identification of the JAK2V617F mutation indicates an increased likelihood of pharmacological effectiveness by Aplidine.
  • the JAK2V617F mutation is present in the Janus kinase 2 (JAK2), a cytoplasmic protein-tyrosine kinase that catalyzes the transfer of the gamma-phosphate group of adenosine triphosphate to the hydroxyl groups of specific tyrosine residues in signal transduction molecules. It can be an homozygous or heterozygous mutation. A detailed description of this mutation, literature references for it and how to determine its presence is given in Tefferi et al, Mayo Clinic Proceedings 2005, July 205 80(7) 947-958.
  • the methods of the invention can be carried out in any type of sample from the patient, such as a biopsy sample, tissue, cell or fluid
  • peripheral blood cells or bone marrow samples will be examined.
  • JAK2V617F mutation detection In order to carry out the JAK2V617F mutation detection, several approaches are available such as PCR detection (Baxter EJ et al., 2005, Lancet., 365 (9464): 1054-1061), RNAase cleavage (Kambas et al., 2009, Eur. J. Haematol., 83(3), 215-219) or single-nucleotide polymorphism array (Knoops L. et al., 2009, Cancer Genet Cytogenet, 192(2), 102-4).
  • detection of the JAK2V617F mutation is performed by PCR, as illustrated in Example 4.
  • mice Male wild-type and GATA l low mice were generated in the animal facility of Istituto Superiore di Sanita as described (Vannucchi et al., 2000, Blood, 95, 2559-2568). Two GATAl low (neodHS) mice (one female and one male of mixed C57 B1/6-SV 129 background) were crossed with CD l mice (Charles River, Calco, Italy) to generate a GATAl low colony, according to standard genetic protocols (Martelli F et al., 2005, Blood, 106, 4102- 13), that was kept at the animal facilities of the Istituto Superiore di Sanita.
  • mice were all genotyped by polymerase chain reaction (PCR) at birth for the absence of the deleted GATAl sequences (negative genotyping) and for the presence of the neo cassette (positive genotyping). Littermates whose genotype contained the GATAl region and did not contain the neo cassette were considered to be wild-type and used as negative controls. All the experiments were performed with sex- and age-matched mice under protocols approved by the institutional animal care committee. The mutants are also available from Jackson Laboratories (Bar Harbor, ME; JAX@Mice DATAbase- STOCK Gatal ⁇ tm2Sho>).
  • mice develop myelofibrosis while aging, they were divided into 3 age classes: disease-free (1-6 months old), early myelofibrotic (8- 12 months old, when presence of the disease is detectable only in bone marrow), and myelofibrotic ( 15 months old to natural death, when the complete clinical picture of the human disease is manifested).
  • Example 1 Activity of Aplidine (60 ug/kg/d x 9d, ip) in the GATA-I low mice myelofibrosis model
  • mice were individually weighted. Mice were then injected intraperitoneally (i.p.) with an Aplidine solution using a syringe with a needle of 26GA (Becton Dickinson S. A., Madrid, Spain) for 9 consecutive days. A vehicle or control group was treated in parallel with vehicle alone. The volume of the Aplidine solution injected in each animal was adjusted on the basis of the weight so that each animal received precisely 60 ⁇ g of Aplidine /kg/ day. After every Aplidine injection, mice were also injected, under the skin with 200 ⁇ L of 0.9% sodium chloride solution using again a syringe with a needle 26GA.
  • 26GA Becton Dickinson S. A., Madrid, Spain
  • mice were sacrificed at days 10 and 16 for parameters evaluation (platelet count, hematocrit, bone marrow cellularity, etc). At least three mice were treated for each time point. Several animals were left recover for 4 weeks and then treated with a second cycle of Aplidine (60 ⁇ g/kg/day) or vehicle for 9 consecutive days (days 38-46); they were sacrificed 7 days after the last injection (day 53) and then parameters were analyzed. Body weight was monitored at sequential time points after treatment.
  • Hematologic values platelet count and hematocrit.
  • blood was collected from the retro-orbital plexus for evaluation of hematological parameters (hematocrit and platelet counts). Blood was collected with heparin- coated pipettes. The volume of blood collected from each animal was 800-1000 ⁇ L. 400-500 ⁇ L of blood were centrifuged at 3000 rpm for 15 minutes to separate plasma from blood cells. Plasma was recovered and kept frozen till its further analysis.
  • the number of platelets was evaluated diluting 10 ⁇ L of whole blood in 90 ⁇ L of Stromatolytic agent for blood platelet counts (Stromatol ® ,
  • Platelets were recognized on the basis of the color and morphologic/ size characteristics and counted with an inverted microscope (Axiostar plus, ZEISS spa, Arese, MI, Italy).
  • Hematocrit levels were evaluated by transferring 20-40 ⁇ L of whole blood into microhematocrit tubes that were centrifuged with a hematocrit-specific microcentrifugette 4203 (ALC International srl, Cologno Monzese MI, Italy), at 12.000 rpm for 10 minutes. After centrifugation, each micro hematocrit tube was measured with a decimeter to calculate the ratio between the volume of the tube occupied by red cells and that occupied by serum. The ratio between the two volumes was then expressed in percent. - Bone marrow cellularity
  • the number of cells in the marrow was calculated by diluting the marrow cells obtained by flushing the whole femur cavity with 10 mL of Iscove's Modified Dulbecco's Medium (IMDM, Gibco-BRL, Gaithsburg, MD, USA).
  • IMDM Iscove's Modified Dulbecco's Medium
  • the femur was held with sterile tweezers inside a Petri plate (60x15 mm) filled with 5 mL of medium to cut the extremities of the bone with a sterile surgical scissor.
  • a needle (21GxI 1 / 2 0.45 x 13 mm) of a 5 mL syringe was inserted in one extreme of the bone cavity and the medium (IMDM) flushed into the femur cavity 5-6 times until all the marrow cells were cleaned from the cavity.
  • the resulting cell suspension was filtered with a 100 ⁇ m Nylon Cell Strainer (BD Falcon, Erenbodegen, Belgium) and transferred in a 50 mL Falcon tube.
  • Total cells/femur number of cells/ quadrant x dilution factor x 10 4 x mL of cell solution.
  • HBSS Hank's balanced salt solution
  • Section area was calculated by visually tracing perimeter of the area.
  • Bone marrow cellularity is typically reduced in GATA- l low mice as the consequence of fibrosis.
  • a first course of Aplidine induced a moderate increase in bone marrow cellularity which was more evident 7 days after treatment cessation (day 16) and even more at day 53 in the animals receiving vehicle in the second course.
  • a second Aplidine course increased bone marrow cellularity to almost normal levels (levels in control wild type mice). The re was a normalization of total femur cellularity at the end of Aplidine treatment.
  • Bone marrow megakaryocyte number There was an increase in bone marrow megakaryocyte number 7 days after treatment cessation (day 16) in the group of younger mice ( Figure 7).
  • Example 2 Activity of Aplidine ( 100 ⁇ g/kg/d x 5d, q 21 d, ip) in the GATA-I low mice myelofibrosis model
  • mice 8- 10 month old GATA- l low mice were used in this experiment. These young mice received either two or four cycles of treatment. Age-matched sham injected GATA- l low and wild-type controls were used.
  • mice were individually weighted. Mice (10 animals) were then injected intraperitoneally (i.p.) with an Aplidine solution using a syringe with a needle of 26GA (Becton Dickinson S. A., Madrid, Spain) for 5 consecutive days, every 3 weeks. A vehicle or control group composed of 6 mice was treated in parallel with vehicle alone. The volume of the Aplidine solution injected in each animal was adjusted on the basis of the weight so that each animal received precisely 100 ⁇ g of Aplidine/kg/day. After every Aplidine injection, mice were also injected, under the skin with 200 ⁇ L of 0.9% sodium chloride solution using again a syringe with a needle 26GA.
  • 26GA Becton Dickinson S. A., Madrid, Spain
  • mice were sacrificed (5 animals in the Aplidine group and 3 animals in the vehicle group) and liver, spleen, tibias and femurs were surgically removed. The liver and spleen were weighted. Half of the liver and spleen was used for histological analysis (formalin 10% v/v).
  • the remaining animals were treated with two additional cycles of Aplidine (100 ⁇ g/kg/day) for 5 consecutive days; and at days 49, 56, 70 and 78 body weigh and blood parameters were monitorized and they were sacrificed 14 days after the end of the fourth cycle (day 79) and then parameters were analyzed.
  • Bones were decalcified with acidified EDTA, pH 7.2 for 3-5 days depending on bone thickness and consistency, according to standard procedures (Formigli L. et at, J Oral Pathol Med. 1995 May; 24(5) :216- 20; F Martelli et at, Blood, 15 December 2005, Vol. 106, No. 13, pp. 4102-41 13). Consecutive 2.5- to 3- ⁇ M sections were stained with Gomori-silver (MicroStain MicroKit; Diopath, Bologna, Italy).
  • MVD microvessel density
  • Anti-Mouse CD34 Purified (Clone MEC14.7) (rat IgG2a), Cedarlane Laboratories, dilution 1 :200).
  • Antibody dilution and blocking of unspecific binding sites was performed with Ultra V- Block, code TA-060-UB, Thermo Scientific. Secondary labelled antibody incubation and stain development was performed using SuperPicTure Kit HRP Broad Spectrum, Histo-Line Laboratories, dilution 1 : 1 (Petrosyan K. et al., 2002, J Histotechnology, 25(4), 247- 250).
  • MVD was calculated as the mean number of stained vessels per 40Ox high power field, calculating the mean of five random chosen areas. Counting was performed by two separate investigators in a blinded fashion.
  • Section of 4-6 ⁇ m were cut from paraffin embedded bone marrow samples and, after deparaffination, incubated with the anti-GATA- 1 monoclonal antibody (from Santa Cruz Biotechnology, Santa Cruz, CA) and the immunostaining developed with the Ultraystain polyvalent HRP Immunostaining kit (Ylem, Rome, Italy), as described by the manufacturer.
  • the images were also used to calculate the frequency of morphologically recognizable megakaryocytes per mm 2 , the percentage of megakaryocytes that reacted with the Gatal antibody and the average intensity of the Gatal staining in the GatalP° s megakaryocytes. In each case, it was analyzed at least three field per mouse per a total of three untreated and three Aplidine treated Gatal low mice. The intensity of the Gatal immunostaining was determined with the MethaMorph program and expressed in pixels/cell. Values are expressed as mean ( ⁇ SD) of these independent measurements and data obtained in untreated and Aplidine-treated mice compared by analysis of variance Anova with the Origin 6.1 program for Windows XP.
  • Bone marrow cellularity was analyzed in mice that were sacrificed after 2 or 4 courses of Aplidine treatment. Two courses of Aplidine were not able to induce an increase in bone marrow cellularity; however, 4 courses of Aplidine increased bone marrow cellularity to almost normal levels (Figure 1 1).
  • TGF-beta and VEGF expression associated with reduced microvessel density, would suggest a possible activity of the drug in human PMF where levels of these two cytokines are abnormally increased.
  • the frequency of megakaryocytes that do not react with the Gatal antibody is slightly increased by Aplidine- treatment.
  • the intensity of the immunostaining is greater in those megakaryocytes expressing Gatal in Aplidine-treated mice than in the vehicle control group. It is hypothetised that Gatal P° S megakaryocytes from Aplidine-treated mice progress in maturation more efficiently than those from control mice, which is associated with the increase in platelets counts, and are therefore selectively depleted from the marrow.
  • UKEl cells were cultured in Iscove modified Dulbecco medium (IMDM) (Lonza, Ltd, Basel Switzerland) and HEL, SET2 and K562 cells were cultured in RPMI 1640 medium (Lonza, Ltd, Basel Switzerland).
  • IMDM Iscove modified Dulbecco medium
  • HEL HEL
  • SET2 and K562 cells were cultured in RPMI 1640 medium (Lonza, Ltd, Basel Switzerland).
  • the antiproliferative activity of Aplidine was also studied in murine cells overexpressing the wild-type (Ba/F3-wt) or V617F mutant allele (Ba/ F3 V617F).
  • Ba/ F3 cells were donated by Dr. R. Skoda (Basel, Switzerland), (Kralovics R. et al., 2005, N Engl J Med., 352 (17): 1779- 1790).
  • Agar clonogenic assay The agar colony assay explored the capacity of Aplidine to prevent clonogenic proliferation of the cell.
  • Clonogenic assay for continuous cell lines was performed in 0.3% soft agar in Dulbecco's medium (Lonza, Ltd, Basel Switzerland) in the absence of growth factor, except in case of wild-type Ba/ F3 cells that were incubated in the presence of murine IL- 3 (50 pg/mL) (Miltenyi Biotech; Gladbach, Germany). Cells were plated at 10 3 /mL in 35-mm dishes containing different final concentrations of Aplidine (from 0.01 to 10 nM), or no addition (control dishes) and allowed to incubated at 37°C in 5% CO2 atmosphere.
  • Colonies were enumerated after 14 days using inverted microscopy (Nikon Eclipse TSlOO, Germany).
  • the IC50 value i.e., the concentration of Aplidine at which the number of colonies was reduced of 50% compared to control dishes, was calculated using plotted data.
  • a short-term (48-hr) proliferation assay in liquid cultures was performed.
  • IxIO 3 cells were plated into the wells of micro titer plates in 100 ⁇ L volume of RPMI 1640 (Lonza, Ltd, Basel Switzerland) with different concentrations of the drug.
  • the tetrazolium salt WST- I (Roche Diagn . GmbH , Mannheim, Germany) was added to each well and incubated for 4 hr at 37°C in 5% CO2 atmosphere.
  • the colored product was measured by spectrophotometry at 450 nm with reference wavelength at 650 nm.
  • the IC50 i.e., the drug concentration at which 50% inhibition of cell proliferation was observed, was determined using the Origin v7.0 software.
  • Example 4 Effects of Aplidine on the clonogenic activity and differentiation of progenitor cells (CD34 + ) from MPN patients
  • CD34 + cells were obtained by immunomagnetic selection (Miltenyi Biotech; Gladbach, Germany) according to manufacturer's instructions modified as described previously (Vannucchi AM et al., 2005, Am J Pathol., 167 (3):849-858). Purity of the isolated CD34 + cell population was evaluated by flow cytometry (FACScan, Becton Dickinson) after labeling with PE-HPCA2 anti-CD34 monoclonal antibody (Becton Dickinson).
  • Routine patient genotyping for the JAK2V617F mutation was performed on peripheral blood (PB) granulocytes separated by differential centrifugation over a Ficoll-Paque gradient (Lympholyte, Cederlane, Canada); contaminating red cells were removed by hypotonic lysis, and the cell pellet was processed for DNA purification using the QIAmp DNA blood Kit (Qiagen, GmbH, Germany). DNA was quantified with the NanoDrop technology (Wilmington, DE, USA) . Genomic analysis for JAK2V617F mutation was performed by an allele-specific (ASO) PCR using 75 ng granulocyte DNA, exactly as described by Baxter et al.
  • ASO allele-specific
  • Agar clonogenic assay The effects of Aplidine on the growth of BFU-E, CFU-GM and CFU-Mk from MPN patients were evaluated. All 5 PV patients and 4 of the 5 PMF patients analyzed were JAK2V617F mutated.
  • Mononuclear cells from MPN patients or control subjects were plated at 10 5 /mL for the growth of BFU-E and CFU-GM in 35-mm-diameter dishes in Methylcellulose (MethoCult, StemCell Technologies, Vancouver, Canada), without Aplidine or in the presence of different concentrations of Aplidine (0 to 10 nM), and in the presence of the following cytokines (all from Miltenyi Biotech; Gladbach, Germany): SCF 50ng/mL, IL-3 IOng/mL, IL-6 IOng/mL, GM-CSF IOng/mL, G-CSF IOng/mL and EPO 3U/mL.
  • cytokines all from Miltenyi Biotech; Gladbach, Germany
  • CD34 + progenitor cells were plated at 5xlO 4 /mL in a 24-well plate in MegaCult-c Collagen and Medium with lipids (StemCell Technologies, Vancouver, Canada), in presence of the following cytokines (all from Miltenyi Biotech; Gladbach, Germany): Thrombopoietin 50ng/mL, IL-3 10ng/mL, IL-6 10ng/mL. Cultures were incubated at 37°C in a humidified 5% CO2 atmosphere, and colonies were enumerated at day 14 according to standard criteria.
  • CD34+ cells purified from PMF patients and control subjects were plated at 10 5 /mL in 500 ⁇ L volume in a 48-well plate in serum-free medium (SYN-H medium; AbCys Synergie, Paris, France) supplemented with the following recombinant cytokines (all from Miltenyi Biotech; Gladbach, Germany): SCF 5 (ng/mL), IL-3 (2 ng/mL), 11-6 (1 ng/mL), IL- I l (40 ng/mL) and TPO (50 ng/mL) for the first seven days of culture.
  • serum-free medium SYN-H medium
  • IL-3 2 ng/mL
  • 11-6 1 ng/mL
  • IL- I l 40 ng/mL
  • TPO 50 ng/mL
  • CD61 + Mks were recovered from the culture, labeled with CD41-PE and CD61-FITC (both from Becton Dickinson) and analyzed by flow cytometry using a FACSan (Becton Dickinson). As shown in Figure 21, it was found that the number of CD61 + cells was no different between cultures containing or not Aplidine, overall suggesting that the drug mainly affected early proliferation of Mk progenitors rather than influencing their differentiation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The use of Aplidine and analogues thereof in the treatment of chronic myeloproliferative disorders, especially those wherein there is an accumulation of abnormal megakaryocytes, is provided.

Description

APLIDINE IN THE TREATMENT OF CHRONIC MYELOPROLIFERATIVE DISORDERS
FIELD OF THE INVENTION
The present invention relates to the use of Aplidine and analogues thereof in the treatment of chronic myeloproliferative disorders, especially those wherein there is an accumulation of abnormal megakaryocytes. In particular, the present invention relates to the treatment of polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis, post-PV myelofibrosis and post-ET myelofibrosis.
BACKGROUND OF THE INVENTION
Chronic Myeloproliferative disorders
Chronic myeloproliferative disorders (CMPD), also referred as myeloproliferative neoplasms (MPN), comprise the four classic myeloproliferative disorders (chronic myelogenous leukemia, primary myelofibrosis, polycythemia vera and essential thrombocythemia) together with chronic neutrophilic leukemia, chronic eosinophilic leukaemia/ hypereosinophilic syndrome (CEL/ HES) and "CMPD unclassificable", according to the World Health Organization (WHO) classification of 2001. All of these disorders involve dysregulation at the multipotent hematopoietic stem cell (CD34+), with one or more of the following shared features:
• Overproduction of one or several blood elements with dominance of a transformed clone.
• Hypercellular marrow/ marrow fibrosis. • Cytogenetic abnormalities.
• Thrombotic and/or hemorrhagic diatheses. • Extramedullary hematopoiesis (liver/ spleen).
• Transformation to acute leukemia.
• Overlapping clinical features.
It is now well established that CMPDs share a common stem cell- derived clonal heritage and their phenotypic diversity is attributed to different configurations of abnormal signal transduction, resulting from a spectrum of mutations affecting protein tyrosine kinases or related molecules. In principle, therefore, histology-based classification and diagnostic criteria for these disorders can be refined by employing molecular disease markers.
The bone marrow criteria defined by the WHO classification of myeloproliferative disorders of 2001 are based on characteristic increase and clustering of morphologically abnormal enlarged megakaryocytes as a pathognomonic clue to describe three distinct phenotypic entities: essential thrombocythemia (ET), polycythemia vera (PV) and prefibrotic, early fibrotic, and fibrotic primary myelofibrosis (PMF) . Furthermore, bone marrow histopathology allows expert pathologists to differentiate between the three prefibrotic myeloproliferative disorders.
E T , P V a n d P M F a r e t h e c l a s s i c B C R-ABL negative myeloproliferative disorders (MPDs). Patients with PV and ET have marked increases of red blood cell and platelet production, respectively. Treatment is directed at reducing the excessive numbers of blood cells. Both PV and ET can develop a spent phase late in their courses that resembles PMF with cytopenias, marrow hypoplasia and fibrosis. Today, a multimodal diagnostic approach combining cytomorphology, cytogenetics and biologically relevant clinical markers is needed for ET, PV and PMF differential diagnose (Michiels et al., 2007, Leukemia Research, 31 (8): 1031-8). The recent discovery of JAK2 and/ or MPL mutations in PV, ET and PMF has had a major impact on how these disorders are now diagnosed and treated. Thus, the World Health Organization classification system has recently revised its diagnostic criteria for PV, ET, and PMF (2008 WHO diagnostic criteria) to include JAK2 and MPL mutations as clonal markers (A.Tefferi, 2008, American Journal of Hematology, 83(6), 491-7; and A.Tefferi and JW Vardiman, 2008, Leukemia, 22, 14-22).
Primary myelofibrosis
Primary myelofibrosis (also known as myelofibrosis, chronic idiopathic myelofibrosis, agnogenic myeloid metaplasia and myoelosclerosis with myeloid metaplasia) is caused by the growth and proliferation of an abnormal bone marrow hematopoietic stem cell (CD34+), resulting in the replacement of the bone marrow with fibrous connective tissue.
Bone marrow fibrosis 'myelofibrosis' is the most recognized and best characterized feature of primary myelofibrosis (PMF). However, other conditions might be accompanied by bone marrow fibrosis, including other myeloid disorders, lymphoid disorders, metastatic cancer, autoimmune diseases, and inflammatory or infectious conditions. PMF is, in the majority of cases, associated with reticulin and/ or collagen myelofibrosis and it can therefore be considered as part of the primary process. Whereas the development of overt collagen bone marrow fibrosis in conditions other than PMF is chronologically a 'secondary' process. Particularly, it has been described that patients with either polycythemia vera (PV) or essential thrombocythemia (ET) can experience fibrotic disease transformation, being referred as post- PV myelofibrosis and post-ET myelofibrosis, respectively.
Pathophysiology Primary myelofibrosis (PMF) is a complex myeloproliferative disorder associated with abnormalities of megakaryocytic proliferation and maturation (dismegakaryopoiesis) which result in prominent accumulation of abnormal megakaryocytes (MK) in the bone marrow and increased release of several cytokines in the bone marrow microenvironment; variable degree of bone marrow fibrosis, osteosclerosis and angiogenesis (neovascularization) ; presence of immature myeloid and erythroid cells, and tear-drop erythrocytes in the peripheral blood; and extramedullary hematopoiesis. Constitutive mobilization of CD34+ hematopoietic progenitor cells from the bone marrow to the peripheral blood distinguishes patients with PMF from other chronic myeloproliferative disorders, and may be related to the severity of the disease and the risk of leukemic transformation. Both fibrogenesis and angiogenesis are considered to develop consequent to the intramedullary release of various growth-promoting factors from rapidly proliferating and displastic megakaryocytes. Among these growth factors are transforming growth factor (TGF-β), basic fibroblast growth factor (bFGF) , platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). The risk of pre-fibrotic PMF to transform into early fibrotic PMF and subsequent fibrotic PMF with extramedullary hematopoiesis has been observed to be dependent on the degree of hypercellularity and the degree of maturation defects of megakaryopoiesis.
Two animal models of PMF are currently available and have provided important insights into the pathogenic mechanisms of myelofibrosis with special regard to the role of megakaryocytes. The first one was developed by transplanting recipient mice with bone marrow cells genetically engineered to overexpress the thrombopoietin (TPO) gene, the main physiologic factor that stimulates megakaryocytopoiesis (Yan XQ et al., 1996, Blood, 88: 402-9) . The second animal model of PMF has been identified in mice genetically impaired for their expression of the transcription factor GATA- 1 , the so-called GATA- 1 low mice (Vannucchi et al., 2004, Pathobiologie, 52, 275-279).
The GATA- I low murine model of PMF has been described (Vannuchi AM et al., 2002, Blood, 100, 1 123-32 ) and further characterized in subsequent studies (Vannucchi AM et al., 2005, Blood,
105, 3493-3501 ; Martelli F et al., 2005, Blood, 106, 4102- 13; Migliaccio et al., 2008, Exp Hematol, 36, 158- 171) as a suitable animal model for the human disease. In contrast to the TPO-overexpressing murine model, the GATA- I low mice recapitulate several aspects of the disease, e.g. myelofibrosis has a quite slow evolution during the lifespan of the mice, making it more suitable for targeted drug studies.
Information from both experimental PMF in mice and cellular and plasma/bone marrow cytokine studies in the human disease strongly suggests the role of cytokines, arising from megakaryocytes and other cells within the abnormal clone, in mediating the bone marrow stromal reaction characteristic of PMF. Most recently, approximately 50% and
5% of the patients with PMF were shown to display gain-of-function mutations involving JAK2V617F and MPLW515K/L, respectively.
Diagnosis
The diagnostic criteria of the World Health Organisation (WHO) for PV, ET and PMF were revised in 2008. Bone marrow histology assessment remains the gold standard criterion for diagnosis and staging of PV, ET and PMF and its differentiation from primary or secondary erythrocytosis, reactive thrombocytosis and thrombocythemias associated with atypical MPD, myelodysplastic syndromes, and chronic myeloid leukemia. Clinical, molecular and pathological criteria are used to solidify a differential diagnosis of PV, ET and PMF. In Table 2 of Tefferi et al. (A.Tefferi and JW Vardiman, 2008, Leukemia, 22, 14-22) the 2008 WHO diagnostic criteria for PV, ET and PMF are shown. Diagnosis of PMF requires meeting all three major criteria and two minor criteria.
Prognosis Overall median survival in PMF is approximately 5 years but it varies substantially among patients based on the presence or absence of well-defined prognostic determinants (Cervantes F. et al., 1997, Br J Haematol, 97, 635-640). Classically, the most important indicators of adverse prognosis have been: the presence of anemia, advanced age, hypercatabolic symptoms, leukocytosis or leukopenia, circulating blasts and high-risk cytogenetic abnormalities (+8, 12p-). More recent studies have shown that JAK2V617F mutational status predicts progression to large splenomegaly and leukemic transformation in PMF (Barosi G. et al., 2007, Blood, 1 10(12), 4030-6). Accordingly, patients will be categorized into a low-risk, a high-risk or intermediate-risk group. Median survival may exceed 10 years in low-risk disease but may be less than 2 years in high-risk disease, most patients dying from infections, haemorrhages or evolution to acute leukaemia.
Treatment
Unfortunately, no therapeutic approach has been efficacious at reducing overall mortality, and therefore therapy for PMF is mainly supportive and is aimed at improving quality of life through palliation of symptoms and control of peripheral blood counts. In Figure 22, an algorithm of treatment for PMF is shown, wherein a suggested treatment to be followed is indicated according to the risk group and the list of drugs which may be used to palliate each of the main PMF symptoms (Arana-Yi C. et al., 2006, Oncologist, 1 1 , 929-943). Cumulative evidence regarding the importance of bone marrow stromal proliferation and neovascularization in PMF and the implication of profibrotic and angiogenic cytokines in this process, such as bFGF, TGF-β, VEGF and PDGF have driven to novel therapeutic strategies mainly focused on the development of agents targeted to the aforementioned factors and other signal transduction inhibitors. Current investigational approaches in PMF include: angiogenesis inhibitors (e.g. thalidomide), tyrosine kynase inhibitors (e.g. imatinib mesylate), farnesyl transferase inhibitors (e.g. Rl 15777), etanercept (a potent anti-TNFα inhibitor) and others; in addition to the potential role of autologous and allogeneic stem cell transplantation (SCT) approaches (Hennessy et al., 2005, Cancer, 103, 32-43 and Arana-Yi C. et al., 2006, Oncologist, 1 1 , 929-943).
Experience with thalidomide has been reported in PMF based on its antiangiogenic and immunomodulatory properties. Thalidomide inhibits TNF-α and TNF-β, interleukin (IL)- l β, IL-6, IL- 12, and GM-CSF and stimulates T-lymphocyte proliferation. Clinical improvements, mainly related to anemia or thrombocytopenia, have been disclosed.
In addition, several receptor kinase inhibitors have been described as anti-angiogenic agents and are currently under investigation for PMF treatment:
- Imatinib: A specific inhibitor of AbI, PDGFR, c-Kit and Arg tyrosine kinases which has become the standard therapy in chronic myelogenous leukaemia. In several clinical trials in patients with PMF, imatinib had only modest activity, although most of the patients experienced decreased splenomegaly and transiently improved hematopoiesis (Cortes et al., 2003, Cancer, 97, 2760-2766). - Vatalanib (PTK787/ZK 222584): An oral inhibitor of the VEGF receptor VEGFR- I and VEGFR-2 tyrosine kinases (FIi- I and FIk- 1 /KDR) as well as the PDGFR and c-Kit which has shown modest activity in patients with PMF (Giles et al., 2007, Leukemia research, 31 (7), 891 -897).
- SU5416: A synthetic inhibitor of VEGFR-2, c-Kit, and Flt-3, which was used in phase II trials in patients with chronic myeloproliferative disorders on the premise of their antiangiogenic activity, but overall clinical activity was minimal (Giles et a!., 2003, Cancer, 97, 1920- 1928).
- SU6668 is a potent antiangiogenic inhibitor of receptor tyrosine kinases including those of VEGFR, FOFR, and PDGFR and c-Kit. It has been hypothesised that 8U6668 may be effective in PMF based upon its inhibitory target profile (Hasselbach et al., 2003, Medical hypotheses, 61 (2), 244-247).
- Sorafenib (BAY 43-9006) is a potent inhibitor of Raf- 1 (a member of the Raf/mitogen-activated protein kinase/ extracellular-signal -regulated kinase (ERK) kinase (MEK) / ERK signaling pathway) and several receptor tyrosine kinases involved in neovascularization, including VEGFR-2, VEGFR-3, PDGFR-β, Flt-3, and c-Kit. However, its in vitro activity in primary cells isolated from patients with PMF appears to be minimal (Smith et al., 2005, Blood, 106, abstract 4943).
Despite the above mentioned efforts, nowadays, allogenic hematopoietic stem cell transplantation is considered the only available therapy for patients with PMF, with potential to eliminate bone marrow fibrosis and possibly cure patients. Nonetheless, the use of fully myeloablative conditioning regimes has been associated with high morbidity and mortality. Thus, there is no established and effective treatment for PMF, let alone for post-PV/ET myelofibrosis. Therefore, the development of novel therapies and agents to treat patients suffering from these diseases is urgently needed.
Aplidine
Aplidine (dehydrodidemnin B) is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans, and is the subject of WO 91/04985. It is related to compounds known as didemnins, Aplidine has the following structure:
Figure imgf000010_0001
More information on Aplidine and analogues thereof, their uses, formulations and synthesis can be found in patent applications WO 91/04985, WO 99/42125, WO 01/35974, WO 01/76616, WO 2004/084812, WO 02/30441, WO 02/02596, WO 03/33013, WO 2004/080477, WO 2004/080421, WO 2007/ 101235, WO 2008/080956, PCT/GB2008/050331 , US 61/034,870, and US 60/981,463. We incorporate by specific reference the content of each of these patent application texts. In contrast to the lack of bone marrow toxicity, Aplidine has been shown, in both animal preclinical studies and human clinical Phase I studies to have cytotoxic potential against a broad spectrum of tumor types including leukemia and lymphoma. See for example: Faircloth, G. et al.: "Dehydrodidemnin B (DDB) a new marine derived anticancer agent with activity against experimental tumour models", 9th NCI-EORTC Symp. New Drugs Cancer Ther. (March 12- 15, Amsterdam) 1996, Abst 1 1 1 ;
Faircloth, G. et al.: "Preclinical characterization of Aplidine, a new marine anticancer depsipeptide", Proc. Amer. Assoc. Cancer Res. 1997, 38: Abst 692;
Depenbrock H, Peter R, Faircloth GT, Manzanares I, Jimeno J, Hanauske AR.: "In vitro activity of Aplidine, a new marine-derived anticancer compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells" Br. J. Cancer, 1998; 78: 739-744;
Faircloth G, Grant W, Nam S, Jimeno J, Manzanares I, Rinehart K. : "Schedule-dependency of Aplidine, a marine depsipeptide with antitumor activity", Proc. Am. Assoc. Cancer Res. 1999; 40: 394;
Broggini M, Marchini S, D'Incalci M, Taraboletti G, Giavazzi R, Faircloth G, Jimeno J.: "Aplidine blocks VEGF secretion and VEGF/ VEGF-Rl autocrine loop in a human leukemic cell line", Clin. Cancer Res. 2000; 6 (suppl): 4509;
Erba E, Bassano L, Di Liberti G, Muradore I, Chiorino G, Ubezio P, Vignati S, Codegoni A, Desiderio MA, Faircloth G, Jimeno J and D'Incalci M.: "Cell cycle phase perturbations and apoptosis in tumour cells induced by Aplidine", Br. J. Cancer 2002; 86: 1510- 1517;
Paz-Ares L, Anthony A, Pronk L, Twelves C, Alonso S, Cortes-
Funes H, Celli N, Gomez C, Lopez-Lazaro L, Guzman C, Jimeno J, Kaye
S. : "Phase I clinical and pharmacokinetic study of Aplidine, a new marine didemnin, administered as 24-hour infusion weekly" CUn.
Cancer Res. 2000; 6 (suppl): 4509; Raymond E, Ady-Vago N, Baudin E, Ribrag V, Faivre S, Lecot F, Wright T, Lopez Lazaro L, Guzman C, Jimeno J, Ducreux M, Le Chevalier T, Armand JP. : "A phase I and pharmacokinetic study of Aplidine given as a 24-hour continuous infusion every other week in patients with solid tumor and lymphoma", CZm. Cancer Res. 2000; 6 (suppl): 4510;
Maroun J, Belanger K, Seymour L, Soulieres D, Charpentier D, Goel R, Stewart D, Tomiak E, Jimeno J, Matthews S. :"Phase I study of Aplidine in a 5 day bolus q 3 weeks in patients with solid tumors and lymphomas", CZm. Cancer Res. 2000; 6 (suppl): 4509;
Izquierdo MA, Bowman A, Martinez M, Cicchella B, Jimeno J, Guzman C, Germa J, Smyth J. : "Phase I trial of Aplidine given as a 1 hour intravenous weekly infusion in patients with advanced solid tumors and lymphoma", Clin. Cancer Res. 2000; 6 (suppl): 4509. Ciruelos, E; Twelves, C; Dominguez,MJ; Mckay, H; Castellano, D;
Ruiz, A; Lόpez-Lazaro, L; Celli, N; Paz-Ares, L. : "Phase I Clinical and Pharmacokinetic study of the marine compound Aplidine administered as a 3 hour infusion every 2 weeks", ASCO 38th Annual Meeting; May 18-21 , 2002.
The mechanism by which Aplidine induces cell growth inhibition and apoptosis was investigated in the human leukaemia cell line MOLT- 4 by Broggini et al. (Broggini M et al., 2003, Leukemia, 17, 52-59). The obtained data demonstrated that Aplidine inhibits the growth and induces apoptosis in MOLT-4 cells through inhibition of VEGF secretion which blocks the VEGF/ VEGFR- I loop, suggesting a possible effect on tumor angiogenesis.
Consequently, a study was designed to investigate the antiangiogenic effect of Aplidine . In the chick embryo allantoic membrane (CAM) assay, Aplidine in vivo inhibited spontaneous angiogenesis which was elicited by exogenous VEGF and FGF-2, and induced by VEGF overexpressing 1A9 ovarian carcinoma cells. At concentrations achievable in the plasma of patients, Aplidine in vitro inhibited endothelial cell functions related to angiogenesis. Finally, Aplidine prevented the formation of capillary-like structures by endothelial cells. These findings were indicative that Aplidine has antiangiogenic activity in vivo and inhibits endothelial cell functional responses to angiogenic stimuli in vitro (Taraboletti G. et al. , 2004, British Journal of Cancer, 90, 2418-2424).
Since at the present there is no established and effective treatment for chronic myeloproliferative disorders, especially for those wherein there is an accumulation of abnormal megakaryocytes, there is an urgent need for providing a therapy useful in the treatment of said diseases. The problem to be solved by the present invention is to provide a therapy that is useful in the treatment of chronic myeloproliferative disorders, especially those wherein there is an accumulation of abnormal megakaryocytes.
SUMMARY OF THE INVENTION
We have established that Aplidine and analogues thereof effectively reverse or slow the progression of chronic myeloproliferative disorders, especially those wherein there is an accumulation of abnormal megakaryocytes, and therefore it can be successfully used in the treatment of these diseases.
Thus, this invention is directed to pharmaceutical compositions, kits and methods for the treatment of chronic myeloproliferative disorders using Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and to the use of Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof in the manufacture of medicaments for the treatment of chronic myeloproliferative disorders. In accordance with one aspect, the invention provides for the use of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of chronic myeloproliferative disorders.
In a further aspect, the invention provides Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic myeloproliferative disorders.
In another aspect, the present invention is also directed to a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, to be used in the treatment of chronic myeloproliferative disorders.
The present invention additionally provides a method for treating any mammal, notably a human, affected by a chronic myeloproliferative disorder, comprising administering to the affected individual a therapeutically effective amount of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof.
In a further aspect of the present invention, a medical kit for administering Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof is provided, comprising printed instructions for administering it according to the uses and methods of treatment set forth herein, and a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. In an additional aspect, the invention refers to a method for treating a human subject having a chronic myeloproliferative disorder, said method comprising: a) obtaining a nucleic acid sample from said human subject; b) determining the presence of the JAK2V617F mutation; and c) treating with Aplidine the JAK2V617F positive subjects.
On a further aspect the invention relates to a method for determining an increased likelihood of pharmacological effectiveness of treatment by Aplidine in a human subject diagnosed with a chronic myeloproliferative disorder comprising obtaining a nucleic acid sample from said human subject and determining the presence of the JAK2V617F mutation, wherein the identification of the JAK2V617F mutation indicates an increased likelihood of pharmacological effectiveness by Aplidine.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Platelet count in younger GATA- llow mice (9 month old) injected with one or two courses of Aplidine (60 μg/kg/day, i.p.) and/ or vehicle.
Figure 2. Platelet count in older GATA- llow mice (18 month old) injected with one course of Aplidine (60 mg/kg/day, ip) or vehicle.
Figure 3. Hematocrit levels in younger GATA- llow mice (9 month old) injected with one or two courses of Aplidine (60 μg/kg/day, ip) and/ or vehicle.
Figure 4. Hematocrit levels in older GATA- llow mice ( 18 month old) injected with one course of Aplidine (60 μg/kg/day, ip) or vehicle, k 10/24h= hematocrit level in mice group treated with vehicle one day after treatment cessation (day 10); ApIi 10/24h= hematocrit level in mice group treated with Aplidine one day after treatment cessation (day 10); k 16/7gg= hematocrit level in mice group treated with vehicle seven days after treatment cessation (day 16); ApIi 16/7gg= hematocrit level in mice group treated with Aplidine seven days after treatment cessation (day 16).
Figure 5. Bone marrow cellularity in younger GATA- llow mice (9 month old) injected with one or two courses of Aplidine (60 μg /kg/ day, ip) and/ or vehicle.
Figure 6. Bone marrow cellularity in older GATA- llow mice (18 month old) injected with one course of Aplidine (60 μg/kg/day, ip) or vehicle.
Figure 7. Bone marrow megakaryocyte number in younger GATA- llow mice (9 month old) injected with one or two courses of Aplidine (60 μg/kg/day, ip) and/ or vehicle.
Figure 8. Bone marrow megakaryocyte number (BMMN) in older GATA- llow mice (18 month old) injected with one course of Aplidine (60 μg/kg/day, ip) or vehicle, k 10/24h= BMMN in mice group treated with vehicle one day after treatment cessation (day 10); ApIi 10/24h= BMMN in mice group treated with Aplidine one day after treatment cessation (day 10); k 16/7gg= BMMN in mice group treated with vehicle seven days after treatment cessation (day 16); ApIi 16/7gg= BMMN in mice group treated with Aplidine seven days after treatment cessation (day 16).
Figure 9. Platelet count in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Platelet count was monitored at days 7 and 14 of each course of treatment. At the beginning of the assay the baseline levels of wild type and GATA- llow were 0.8 x 106 and 0.1 x 106 platelets/μL, respectively, and at the end of the assay these baseline levels were 0.75 x 106 and 0.2 x 106 platelets/μL.
Figure 10. Hematocrit level in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Hematocrit level was monitored at days 7 and 14 of each course of treatment. At the beginning of the assay the baseline levels of wild type and GATA- llow were 45% and 38%, respectively, and at the end of the assay these baseline levels were 48% and 43%.
Figure 11. Bone marrow cellularity in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively. At the beginning of the assay the baseline levels of wild type and GATA- llow 16 x 106 and 6 x 106 cells/ femur, respectively and at the end of the assay these baseline levels were 16 x 106 and 4 x 106 cells/ femur.
Figure 12. Bone marrow megakaryocyte number in 8- 10 month- old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
Figure 13. Bone marrow fiber deposition in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively. Figure 14. Bone marrow microvessel density (angiogenesis) in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively.
Figure 15. Bone marrow TGF-β mRNA levels in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively. 2- ΔΔCt is the amount of target mRNA normalized to the beta-2- microglobulin gene (housekeeping gene).
Figure 16. Bone marrow VEGF mRNA levels in 8- 10 month-old GATA- llow mice injected with two or four courses of Aplidine ( 100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle. Mice were sacrificed 14 days after the end of the second or fourth cycle, respectively. 2- ΔΔCt is the amount of target mRNA normalized to the beta-2- microglobulin gene (housekeeping gene).
Figure 17. GATAl expression in megakaryocytes and frequency of megakaryocytes in sections immunostained for GATAl and counterstained by hamatoxilyn-eosin, in bone marrow from 8- 10 month-old GATA- llow mice injected with four courses of Aplidine (100 μg/kg/day x 5d, q 2 Id, i.p.) or vehicle.
Figure 18. Effects of Aplidine on the clonogenic activity and the proliferation of cell lines; values shown are IC50 (mean+SD of at least 3 individual experiments). Figure 19. Effects of Aplidine on the clonogenic activity of progenitor cells from MPN patients; values shown are IC50 (mean+SD).
Figure 20. Effects of Aplidine on the proportion of BFU-E colonies harboring the JAK2V617F mutation obtained from JAK2V617F-mutant PMF patients. §: values represent the percentage (%) of colonies harboring the JAK2V617F mutation (V617F) or not (wt) of the total number of colonies genotyped (from a minimum of 20 to 50).
Figure 21. Effects of Aplidine on the differentiation of progenitor cells (CD34+) from PMF patients towards the megakaryocytic lineage (CD61+). Values are presented as the Mean+SD of five independent experiments.
Figure 22. Algorithm of treatment for primary myelofibrosis (PMF).
DETAILED DESCRIPTION OF THE INVENTION
In order to evaluate the efficacy of Aplidine and Aplidine analogues in the treatment of chronic myeloproliferative disorders (CMPDs), especially of those CMPDs wherein an increase in proliferation of abnormal megakaryocytes (MK) occurs, we initiated systematic studies wherein GATA- llow mice were treated with Aplidine. As general conclusion, we found that Aplidine effectively reverses or slows the progression of PMF. Thus, the present invention is directed to provide an efficacious treatment for chronic myeloproliferative disorders, especially for those wherein there is an accumulation of abnormal megakaryocytes. The term "treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above.
Aplidine is a cyclic depsipeptide with the following structure:
Figure imgf000020_0001
The term "Aplidine" is intended here to cover any pharmaceutically acceptable salt, solvate, hydrate, prodrug, or any other compound which upon administration to the patient is capable of providing (directly or indirectly) the compound as described herein. The preparation of salts, solvates, hydrates, and prodrugs can be carried out by methods known in the art.
Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N, N- dialkylenethanolamine, triethanolamine and basic aminoacids salts.
Any compound that is a prodrug of Aplidine is within the scope and spirit of the invention. The term "prodrug" is used in its broadest sense and encompasses those derivatives that are converted in vivo (ie. metabolized) to Aplidine. The prodrug can be hydro lyzed, oxidized, or otherwise react under biological conditions to provide Aplidine. Examples of prodrugs include, but are not limited to, derivatives and metabolites of Aplidine that include biohydrolyzable moeities such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Prodrugs can tipically be prepared using well-known methods, such as those described by Burger "Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and "Design and Applications of Prodrugs" (H. Bundgaard ed., 1985, Harwood Academic Publishers).
In addition, any drug referred to herein may be in crystalline form either as free compound or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art. Suitable Aplidine analogues include those compounds disclosed in WO 02/02596, WO 01 /76616, and WO 2004/84812. We incorporate by specific reference the content of these patent applications. The term "Aplidine analogue" is intended here also to cover any pharmaceutically acceptable salt, solvate, hydrate, prodrug, or any other compound which upon administration to the patient is capable of providing (directly or indirectly) an "Aplidine analogue" as described above.
Aplidine and Aplidine analogues for use in accordance with the present invention may be prepared following any of the synthetic processes disclosed in WO 02/02596, WO 01 /76616, and WO 2004/84812, which are incorporated herein by reference. In particular, we include by reference the compounds defined in the claims of those patent applications.
Pharmaceutical compositions comprising Aplidine, an Aplidine analogue, or a pharmaceutically acceptable salt thereof, may be formulated according to the chosen route of administration. The pharmaceutical composition of the invention can be administrated by any suitable route, including but not limited to oral, rectal, transdermal, ophthalmic, nasal, topical, vaginal or parenteral. Preferably, the pharmaceutical composition is formulated in order to be suitable for parenteral administration to a subject, e.g., a human being, preferably by intravenous, intramuscular, intraperitoneal or subcutaneous administration. Illustrative, non limiting examples of suitable formulations for parenteral administration are solutions, suspensions, emulsions, lyophilized compositions and the like. The administration of the pharmaceutical composition of the invention to a mammal in need thereof can be carried out by conventional means. Preferably, the administration of the pharmaceutical composition of the invention is by intravenous administration and includes an intravenous delivery through standard devices, e.g., a standard peripheral intravenous catheter, a central venous catheter, or a pulmonary artery catheter, etc. In any case, the pharmaceutical composition of the invention may be administrated using the appropriate equipments, apparatus, and devices which are known by the skilled person in art.
Preferably, Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof may be supplied and stored as a sterile lyophilized product, comprising the active ingredient and excipients in a formulation adequate for therapeutic use. In particular a formulation comprising the active ingredient and mannitol is preferred. Further guidance on pharmaceutical compositions of Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof is given in WO 99/42125, which is incorporated herein by reference in its entirety.
Administration of Aplidine, Aplidine analogues, a n d pharmaceutical compositions comprising the same is preferably by intravenous infusion. The infusing step is typically repeated on a cyclic basis, which may be repeated as appropriate over for instance 1 to 20 cycles. The cycle includes a phase of infusing the drug, and usually also a phase of not infusing the drug. Typically the cycle is worked out in weeks, and thus the cycle normally comprises one or more weeks of a drug infusion phase, and one or more weeks of non-infusion to complete the cycle. A cycle of 3 or 4 weeks is preferred, but alternatively it can be from 2 to 6 weeks. The infusion phase can itself be a single administration in each cycle of say 1 to 72 hours, more usually of about 1 , 3 or 24 hours; or an infusion on a daily basis in the infusion phase of the cycle for preferably 1 to 5 hours, especially about 1 or 3 hours; or an infusion every week or every two weeks in the infusion phase of the cycle for preferably 1 to 24 hours, especially about 1 , 3 or 24 hours. In one embodiment, we prefer a 1-hour weekly intravenous infusion for three consecutive weeks in a four-week cycle treatment schedule.
In another embodiment, we prefer a 3-hour intravenous infusion every two weeks in a four- week cycle treatment schedule.
The dose will be selected according to the dosing schedule, having regard to the existing data on Dose Limiting Toxicity, on which see for example the above mentioned WO 01 /35974 patent specification and the Phase I studies cited in the background of the invention. These documents are also incorporated herein in full by specific reference.
Representative schedules and dosages are for example:
a) about 3.75 mg/m2 body surface area, administered as 24-hour weekly intravenous infusion for three consecutive weeks in a four-week cycle treatment schedule; b) about 5 mg/m2 body surface area, administered as a 3-hour intravenous infusion every two weeks in a four-week cycle treatment schedule; c) about 3.2 mg/m2 body surface area, administered as a 1-hour weekly intravenous infusion for three consecutive weeks in a four-week cycle treatment schedule; d) about 5 mg/m2 body surface area, administered as a 24-hour intravenous infusion every two weeks in a four-week cycle treatment schedule; and e) about 1.2 mg/m2 body surface area, administered as a 1-hour infusion daily for 5 consecutive days in a three-week cycle treatment schedule. Schedule and dosages b, c) and d) are the most preferred to be used in the present invention. It is particularly preferred the dosing and administration regime defined in b), i.e. the administration of aplidine at about 5 mg/m2 body surface area, as a 3-hour intravenous infusion every two weeks in a four- week cycle treatment schedule.
Although guidance for the dosage is given above, the correct dosage of the compound may change according to the particular formulation, the mode of application, and the particular situs, patient and disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Dose delays and/ or dose reductions and schedule adjustments are performed as needed depending on individual patient condition and tolerance of treatments.
Aplidine, Aplidine analogues and pharmaceutically acceptable salts thereof may be used with other drugs to provide a combination therapy in the treatment of chronic myeloproliferative disorders. The other drug may form part of the same composition, or be provided as a separate composition for administration at the same time or at different time.
In an aspect, the present invention provides a method for treating any mammal, notably a human, affected by a chronic myeloproliferative disorders, comprising administering to the affected individual a therapeutically effective amount of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof.
In a preferred embodiment of the invention, said chronic myeloproliferative disorder is a BCR-ABL negative myeloproliferative disorder. The BCR-ABL fusion gene is a specific chromosomal abnormality that is due to a reciprocal translocation between chromosomes 9 and 22. The ABL gene from chromosome 9 joins to the BCR gene on chromosome 22, to form the BCR-ABL fusion gene. The changed chromosome 22 with the fusion gene on it is called the Philadelphia chromosome. Polycythemia vera (PV) , essential thrombocythemia (ET) and primary myelofibrosis (PMF) constitute the classic BCR-ABL negative myeloproliferative disorders.
In a further preferred embodiment, said chronic myeloproliferative disorder (CMPD) is characterised by an increase in the proliferation of abnormal megakaryocytes. The three classic BCR-ABL negative myeloproliferative disorders (PV, ET and PM) are characterized by an increase in the proliferation and some degree of clustering of abnormal megakaryocytes. Thus, more preferably the methods of the invention are used to treat PV, ET and PMF.
Accordingly, in one preferred embodiment, the chronic myeloproliferative disorder is primary myelofibrosis (PMF). PMF can show a variable degree of bone marrow fibrosis "myelofibrosis". According to the bone marrow features the disease can be classified in pre-fibrotic, early fibrotic or fibrotic PMF. Assessment of bone marrow fibrosis includes basic parameters such as cellularity and fiber content (Thiele et al., 2005, Haematologica, 90(8), 1128-32). In a further preferred embodiment, the methods of the invention are used for treating early stage primary myelofibrosis (pre-fibrotic or early fibrotic PMF) wherein fibrosis is not yet fully established.
In another preferred embodiment, the chronic myeloproliferative disorder is polycythemia vera (PV). In a further preferred embodiment, the chronic myeloproliferative disorder is essential thrombocythemia (ET).
It has been described that patients with either PV or ET can experience fibrotic disease transformation in which myelofibrosis is developed as the evolution of these CMPDs. The International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) agreed that the myelofibrosis that develops in the setting of either PV or ET will be referred to as post-polycythemia vera myelofibrosis (post-PV myelofibrosis) and post-essential thrombocythemia myelofibrosis (post- ET myelofibrosis), respectively (Mesa RA et al., 2007, Leukemia research, 31 , 737-740). These clinical conditions share many characteristics with PMF. Thus, in a further preferred embodiment, the methods of the invention are used for treating polycythemia vera (PV) having progressed to post-PV myelofibrosis and essential thrombocythemia (ET) which has progressed to post-ET myelofibrosis.
In a related aspect, the invention further provides the use of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a chronic myeloproliferative disorder.
In another related aspect, the invention provides Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, for use in the treatment of a chronic myeloproliferative disorder.
In another aspect, the present invention is also directed to a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, to be used in the treatment of a chronic myeloproliferative disorder. In a further aspect of the present invention, a medical kit for administering Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof is provided, comprising printed instructions for administering it according to the uses and methods of treatment set forth herein, and a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
In another preferred embodiment, the Aplidine, Aplidine analogue or pharmaceutically acceptable salt thereof for the methods, uses, pharmaceutical compositions and kits of the present invention, is Aplidine.
From the data in Example 3, it seems the JAK2V617F mutant cells are more sensitive to Aplidine than wild-type cells, for example in case of UKE- I and SET cells compared to bcr/abl positive K562 cell line. Furthermore, in the proliferation assay in liquid cultures, the murine cells Ba/ F3 cells transduced with the V617F allele were found more sensitive to Aplidine than their wild-type counterpart. In Example 4 are shown the effects of Aplidine on progenitor cells (CD34+) obtained from patients suffering of a chronic myeloproliferative disorder, particularly PV or PMF, wherein said patients were JAK2V617F mutated (5 PV patients and 4 or the 5 PMF). Cells obtained from PMF patients were the most sensitive to Aplidine. Furthermore, when single colony genotyping was performed to quantify proportion of hematopoietic colonies (BFU-E) harbouring the JAK2V617F mutation which grew in the presence of Aplidine, the available data suggest that, at least in some patients, Aplidine may preferentially inhibit the growth of V617F mutant clonogenic progenitors.
Accordingly, in a preferred embodiment of the invention, the mammal, preferably a human, suffering from a chronic myeloproliferative disorder is harbouring the JAK2V617F mutation. Preferably, the chronic myeloproliferative disorder is primary myelofibrosis (PMF). The gain of function V617F mutation of the Janus kinase (JAK) 2 gene (JAK2V617F mutation), has been associated with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PM) (A.Tefferi, 2008, American Journal of Hematology, 83(6), 491-7; A.Tefferi and JW Vardiman, 2008, Leukemia, 22, 14-22; and Baxter EJ et al., 2005, Lancet., 365 (9464): 1054- 1061). This mutation has provided a molecular explanation for the disregulated hematopoiesis typical of these disorders, a diagnostic test that distinguishes them from other types of myeloproliferative disorders, and an opportunity to develop targeted therapy that could potentially avoid the toxicities associated with the conventional chemotherapeutic agents currently employed in their treatment.
On another aspect the invention refers to a method for treating a human subject having a chronic myeloproliferative disorder, said method comprising: a) obtaining a nucleic acid sample from said human subject; b) determining the presence of the JAK2V617F mutation; and c) treating with Aplidine the JAK2V617F positive subjects.
On a further aspect the invention relates to a method for determining an increased likelihood of pharmacological effectiveness of treatment by Aplidine in a human subject diagnosed with a chronic myeloproliferative disorder comprising obtaining a nucleic acid sample from said human subject and determining the presence of the JAK2V617F mutation, wherein the identification of the JAK2V617F mutation indicates an increased likelihood of pharmacological effectiveness by Aplidine. The JAK2V617F mutation is present in the Janus kinase 2 (JAK2), a cytoplasmic protein-tyrosine kinase that catalyzes the transfer of the gamma-phosphate group of adenosine triphosphate to the hydroxyl groups of specific tyrosine residues in signal transduction molecules. It can be an homozygous or heterozygous mutation. A detailed description of this mutation, literature references for it and how to determine its presence is given in Tefferi et al, Mayo Clinic Proceedings 2005, July 205 80(7) 947-958.
The methods of the invention can be carried out in any type of sample from the patient, such as a biopsy sample, tissue, cell or fluid
(serum, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like). Preferably, peripheral blood cells or bone marrow samples will be examined.
In order to carry out the JAK2V617F mutation detection, several approaches are available such as PCR detection (Baxter EJ et al., 2005, Lancet., 365 (9464): 1054-1061), RNAase cleavage (Kambas et al., 2009, Eur. J. Haematol., 83(3), 215-219) or single-nucleotide polymorphism array (Knoops L. et al., 2009, Cancer Genet Cytogenet, 192(2), 102-4). In a preferred embodiment detection of the JAK2V617F mutation is performed by PCR, as illustrated in Example 4.
The following Examples further illustrate the invention. They should not be interpreted as a limitation of the scope of the invention.
To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about". It is understood that, whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/ or measurement conditions for such given value. In addition, unless otherwise stated, where values are given as ranges, the upper and lower values are specifically contemplated as preferred values.
EXAMPLES
In order to evaluate the efficacy of Aplidine and Aplidine analogues in the treatment of CMPDs, the activity of Aplidine has been assayed in a murine model of myelofibrosis: the GATA- llow mice. Mice carrying the hypomorphic Gatallow mutation express MK abnormalities similar to those observed in PMF patients and develop myelofibrosis with age, a syndrome that includes an increased angiogenesis process with striking similarities with that observed in human PMF.
Animal Care
Male wild-type and GATA llow mice were generated in the animal facility of Istituto Superiore di Sanita as described (Vannucchi et al., 2000, Blood, 95, 2559-2568). Two GATAllow (neodHS) mice (one female and one male of mixed C57 B1/6-SV 129 background) were crossed with CD l mice (Charles River, Calco, Italy) to generate a GATAllow colony, according to standard genetic protocols (Martelli F et al., 2005, Blood, 106, 4102- 13), that was kept at the animal facilities of the Istituto Superiore di Sanita. The mice were all genotyped by polymerase chain reaction (PCR) at birth for the absence of the deleted GATAl sequences (negative genotyping) and for the presence of the neo cassette (positive genotyping). Littermates whose genotype contained the GATAl region and did not contain the neo cassette were considered to be wild-type and used as negative controls. All the experiments were performed with sex- and age-matched mice under protocols approved by the institutional animal care committee. The mutants are also available from Jackson Laboratories (Bar Harbor, ME; JAX@Mice DATAbase- STOCK Gatal <tm2Sho>).
Because the GATA llow mutant mice develop myelofibrosis while aging, they were divided into 3 age classes: disease-free (1-6 months old), early myelofibrotic (8- 12 months old, when presence of the disease is detectable only in bone marrow), and myelofibrotic ( 15 months old to natural death, when the complete clinical picture of the human disease is manifested).
Control experiments were also performed on early and myelofibrotic mice, and age-matched littermates, as indicated. All the experiments were performed according to protocols approved by the institutional animal care committee.
Drug formulation Aplidine vials containing 0.5 mg of Aplidine and 25 mg of mannitol were reconstituted with 1 mL of a reconstitution solution. The composition of the reconstitution solution was Cremophor EL/ Ethanol/ Water for injection, 15%/ 15% /70% (v/v/v). The reconstitution solution was further diluted with 9 mg/mL (0.9%) sodium chloride solution up to a final concentration that contains in 100-200 μL the required amount of Aplidine to be injected to the animals. Control animals were injected i.p. with a vehicle solution consisting of a sodium chloride solution (0.9% w/v).
Example 1: Activity of Aplidine (60 ug/kg/d x 9d, ip) in the GATA-I low mice myelofibrosis model
Experimental design
Two different set of experiments were performed. A first set of experiments used younger GATAllow mice (9-month old) , when myelofibrosis is in its very early phases of development, corresponding to the cellular phase of the human disorder; these younger mice received either one or two cycles of Aplidine. In a second set of experiments, aged myelofibrotic GATA llow mice were employed (18- month old); these older mice received only one cycle of Aplidine. In both experiments age-matched sham injected GATAllow and wild-type controls were used.
Drug treatment GATAllow mice were individually weighted. Mice were then injected intraperitoneally (i.p.) with an Aplidine solution using a syringe with a needle of 26GA (Becton Dickinson S. A., Madrid, Spain) for 9 consecutive days. A vehicle or control group was treated in parallel with vehicle alone. The volume of the Aplidine solution injected in each animal was adjusted on the basis of the weight so that each animal received precisely 60 μg of Aplidine /kg/ day. After every Aplidine injection, mice were also injected, under the skin with 200 μL of 0.9% sodium chloride solution using again a syringe with a needle 26GA. This allowed for better recovery and prevented dehydration from lack of movement during the first few hours after Aplidine administration. Mice were sacrificed at days 10 and 16 for parameters evaluation (platelet count, hematocrit, bone marrow cellularity, etc). At least three mice were treated for each time point. Several animals were left recover for 4 weeks and then treated with a second cycle of Aplidine (60 μg/kg/day) or vehicle for 9 consecutive days (days 38-46); they were sacrificed 7 days after the last injection (day 53) and then parameters were analyzed. Body weight was monitored at sequential time points after treatment.
Materials and methods
- Hematologic values: platelet count and hematocrit. At appropriate time points, as described above, blood was collected from the retro-orbital plexus for evaluation of hematological parameters (hematocrit and platelet counts). Blood was collected with heparin- coated pipettes. The volume of blood collected from each animal was 800-1000 μL. 400-500 μL of blood were centrifuged at 3000 rpm for 15 minutes to separate plasma from blood cells. Plasma was recovered and kept frozen till its further analysis. The rest of whole blood (400-500 μL) was lysed with a cold lysis buffer (ammonium chloride at 0.83% w/v, potassium bicarbonate 0.1% w/v, Ethylenediaminetetraacetic acid (EDTA) 3.7% w/v pH 7.2-7.4) for 10 minutes at 4 0C and then centrifuged at 3000 rpm for 10 minutes and the pellet dissolved in TRIzol (Gibco).
1. Platelet count
The number of platelets was evaluated diluting 10 μL of whole blood in 90 μL of Stromatolytic agent for blood platelet counts (Stromatol®,
Mascia Brunelli Milano, Italy). After 20 minutes of incubation, few microliters of this solution were put in a Bύrker camera that was left for
10 minutes in a humidified environment. Platelets were recognized on the basis of the color and morphologic/ size characteristics and counted with an inverted microscope (Axiostar plus, ZEISS spa, Arese, MI, Italy).
2. Hematocrit
Hematocrit levels were evaluated by transferring 20-40 μL of whole blood into microhematocrit tubes that were centrifuged with a hematocrit-specific microcentrifugette 4203 (ALC International srl, Cologno Monzese MI, Italy), at 12.000 rpm for 10 minutes. After centrifugation, each micro hematocrit tube was measured with a decimeter to calculate the ratio between the volume of the tube occupied by red cells and that occupied by serum. The ratio between the two volumes was then expressed in percent. - Bone marrow cellularity
The number of cells in the marrow was calculated by diluting the marrow cells obtained by flushing the whole femur cavity with 10 mL of Iscove's Modified Dulbecco's Medium (IMDM, Gibco-BRL, Gaithsburg, MD, USA). The femur was held with sterile tweezers inside a Petri plate (60x15 mm) filled with 5 mL of medium to cut the extremities of the bone with a sterile surgical scissor. To flush the marrow cells, a needle (21GxI 1/2 0.45 x 13 mm) of a 5 mL syringe was inserted in one extreme of the bone cavity and the medium (IMDM) flushed into the femur cavity 5-6 times until all the marrow cells were cleaned from the cavity. The resulting cell suspension was filtered with a 100 μm Nylon Cell Strainer (BD Falcon, Erenbodegen, Belgium) and transferred in a 50 mL Falcon tube. 50 μL of cell suspension were diluted (dilution factor 1 :2) with Tripan Blue 0.4% (Sigma) to exclude dead cells and 10 μL of this solution were transferred, by capillarity, in a counter camera model Bύrker with a Pasteur pipette. The cells present in the 4 big quadrants of the camera were counted and the resulting number used to calculate the number of total cells/femur, according to the formula: Total cells/femur = number of cells/ quadrant x dilution factor x 104 x mL of cell solution.
A 50 mL Falcon tube with the cells solution were put in a centrifuge at 1 100 g for 5 minutes. The supernatant was eliminated and the pellet was washed with a Hank's balanced salt solution (HBSS) (Sigma- Aldrich, St Louis, MO, USA) and centrifuged at 1 100 g for 5 minutes. Cells were diluted to 106 of cells/ mL with TRIzol (Gibco) and carefully homogenized, left for 5 minutes and kept in a -80 0C refrigerator.
- Bone marrow megakaryocyte number
Femurs were carefully cleaned of adherent tissues, fixed with formalin (Sigma), decalcified and paraffin embedded according to standard procedures (Formigli L. et άL, J Oral Pathol Med. 1995 May; 24(5) :216- 20; F Martelli et άL, Blood, 15 December 2005, Vol. 106, No. 13, pp. 4102-4 1 13) ; and 2.5-3 μm sections were prepared. Bone marrow sections were routinely stained with hematoxylin-eosin. The number of megakaryocytes per section area was calculated in at least 5 High- Power Fields (HPF) (at 40Ox) on images that had been acquired from a photocamera connected to the microscope and transferred to computer, using Adobe Photoshop 7.0. Section area was calculated by visually tracing perimeter of the area. We used the image analysis system Leica Q5501W with color video camera DM RXA for light microscopy, using Axioskop Zeiss microscopy, equipped with a Acdhroplan 10x/ 0.25 or Achroplan 4Ox/ 0.65 objective
Results
1. Platelet count
An increase in platelet count was observed in all mice receiving Aplidine. In the group of younger mice the effect was evident 1 day after treatment cessation (day 10) and was more pronounced 7 days later (day 16) . Platelet increase was maintained seven days after the last injection in both, mice receiving a second course of treatment and mice receiving vehicle in the second course (day 53) (Figure 1) . An increase in platelet count was also observed in the group of older mice, where fibrosis is extensive. Platelet count increase was significant as compared to control mice injected with vehicle (Figure 2).
These data indicate that there is some degree of correction of one of the key features of myelofibrosis pathogenesis in these mice after treatment with Aplidine, i.e. dysmegakaryocytopoiesis.
2. Hematocrit levels In the group of younger mice a first course of Aplidine reduced hematocrit levels when the animals were sacrificed one day after treatment cessation (day 10); this may be explained by Aplidine toxicity. This effect was also observed in a multiple dose toxicity study in mice, showing that Aplidine (5 d, iv, 0.1 1-0.22 mg/kg/day) lowers hematocrit levels. However, after a 7 day recovery (day 16) an increase of hematocrit, although not statistically significant, was observed (from 37% to 44%) reaching almost normal levels (levels in control wild type mice). This increase in hematocrit levels was maintained, being still apparent at day 53 in the animals that received vehicle in the second course. A second course of Aplidine reduced hematocrit levels, again probably due to multiple dose toxicity (Figure 3).
In the group of older mice (Figure 4), hematocrit level was not altered significantly after the treatment with Aplidine. There was a slight reduction of the hematocrit after the treatment (day 10), probably due to haematological toxicity. Hematocrit reached almost the same level as in mice treated with vehicle after 7 days of recovery (day 16) .
3. Bone marrow cellularity
Bone marrow cellularity is typically reduced in GATA- llow mice as the consequence of fibrosis. In the group of younger mice, a first course of Aplidine induced a moderate increase in bone marrow cellularity which was more evident 7 days after treatment cessation (day 16) and even more at day 53 in the animals receiving vehicle in the second course. A second Aplidine course increased bone marrow cellularity to almost normal levels (levels in control wild type mice). The re was a normalization of total femur cellularity at the end of Aplidine treatment. Total cells/ femur increased from a median of 6x106 in untreated GATA- llow mice to 15x106 at 53 days after Aplidine treatment, as compared to a median value of 16xlO6 (p<0.01) in wild-type mice (Figure 5). In older mice a moderate increase in bone marrow cellularity was also observed on days 10 and 16, 1 and 7 days after Aplidine treatment, respectively (Figure 6).
The increase in bone marrow cellularity which is maintained and even increased with time is considered to be a positive effect of Aplidine treatment on the bone marrow deranged stroma.
4. Bone marrow megakaryocyte number There was an increase in bone marrow megakaryocyte number 7 days after treatment cessation (day 16) in the group of younger mice (Figure 7).
In the group of older mice (Figure 8) a significant change on the bone marrow megakaryocyte number after the treatment with Aplidine (day
10) was not observed. After the recovery period (day 16) there was a reduction in the megakaryocyte number. However, this decrease was less severe in the animals treated with Aplidine than in the animals treated with vehicle.
Example 2: Activity of Aplidine ( 100 μg/kg/d x 5d, q 21 d, ip) in the GATA-I low mice myelofibrosis model
Experimental design 8- 10 month old GATA- llow mice were used in this experiment. These young mice received either two or four cycles of treatment. Age-matched sham injected GATA- llow and wild-type controls were used.
Drug treatment GATAllow mice were individually weighted. Mice (10 animals) were then injected intraperitoneally (i.p.) with an Aplidine solution using a syringe with a needle of 26GA (Becton Dickinson S. A., Madrid, Spain) for 5 consecutive days, every 3 weeks. A vehicle or control group composed of 6 mice was treated in parallel with vehicle alone. The volume of the Aplidine solution injected in each animal was adjusted on the basis of the weight so that each animal received precisely 100 μg of Aplidine/kg/day. After every Aplidine injection, mice were also injected, under the skin with 200 μL of 0.9% sodium chloride solution using again a syringe with a needle 26GA. This allowed for better recovery and prevented dehydration from lack of movement during the first few hours after Aplidine administration. Body weight and blood parameters were monitored at days 7 and 14 of each cycle without sacrifice. At day 22, the animal underwent a second treatment cycle for further 5 days. At day 28 and 35, body weight and blood parameters were monitored again. At day 36 (14 days after finishing the second cycle), mice were sacrificed (5 animals in the Aplidine group and 3 animals in the vehicle group) and liver, spleen, tibias and femurs were surgically removed. The liver and spleen were weighted. Half of the liver and spleen was used for histological analysis (formalin 10% v/v). The other half of both organs was suspended in TRIzol (Gibco) for expression analyses with Real Time-PCR. One tibia was used for histologic analysis and the other tibia and the femurs was flushed with Ca++Mg++-free phosphate-buffered saline (HBSS) containing 1% v/v bovine serum albumin (BSA, Sigma). Marrow cells were counted and suspended in TRIzol for the Real Time- PCR analysis. The remaining animals were treated with two additional cycles of Aplidine (100 μg/kg/day) for 5 consecutive days; and at days 49, 56, 70 and 78 body weigh and blood parameters were monitorized and they were sacrificed 14 days after the end of the fourth cycle (day 79) and then parameters were analyzed.
Materials and methods The determination of platelet count, hematocrit levels, bone marrow cellularity and bone marrow megakaryocyte number was performed as described in Example 1.
- Bone marrow fibrosis
Bones were decalcified with acidified EDTA, pH 7.2 for 3-5 days depending on bone thickness and consistency, according to standard procedures (Formigli L. et at, J Oral Pathol Med. 1995 May; 24(5) :216- 20; F Martelli et at, Blood, 15 December 2005, Vol. 106, No. 13, pp. 4102-41 13). Consecutive 2.5- to 3-μM sections were stained with Gomori-silver (MicroStain MicroKit; Diopath, Bologna, Italy). Microscopic evaluations were performed with a DM RB (Das Microscope Research Biology) microscope (Leica, Heidelberg, Germany) set in a transillumination mode and images were acquired with the Image Manager (IM) 50 system (Leica) . For quantitative measurements of fibrosis, images of Gomori-stained sections were acquired at x 400 and processed with Adobe Photoshop (Adobe Systems, San Jose, CA) to calculate th e numb e r o f fib e rs-fiber bands intersecting 10 noncontiguous square lines of a 540-μm-length spaced grid in each microscopic section.
- Bone marrow microvessel density
For studies of microvessel density (MVD), a rabbit anti-mouse CD34 antibody from Cederlane was used (Anti-Mouse CD34, Purified (Clone MEC14.7) (rat IgG2a), Cedarlane Laboratories, dilution 1 :200). Antibody dilution and blocking of unspecific binding sites was performed with Ultra V- Block, code TA-060-UB, Thermo Scientific. Secondary labelled antibody incubation and stain development was performed using SuperPicTure Kit HRP Broad Spectrum, Histo-Line Laboratories, dilution 1 : 1 (Petrosyan K. et al., 2002, J Histotechnology, 25(4), 247- 250). MVD was calculated as the mean number of stained vessels per 40Ox high power field, calculating the mean of five random chosen areas. Counting was performed by two separate investigators in a blinded fashion.
- Cytokine mRNA levels For expression studies, RNA was purified from frozen bone marrow cell samples in Trizol (TriPure Isolation Reagent, Roche Applied Science) according to standard procedures (Chomczynski, P. and Sacchi, N. (1987) Anal Biochem 162: 156- 159). RNA was evaluated and quantified by the Nanodrop technology. Reverse transcription was performed using the TaqMan® Reverse Transcription Reagents (Applied Biosystems). A quantitative Real-Time RT-PCR procedure using the Assay-on-Demand from Applied Biosystems was employed for the quantitation of mRNAs for VEGFa (Applied Biosystems) and TGF-beta (Applied Biosystems), and control beta-2 microglobulin (Applied Biosystems).
- Determination of GATA- 1 expression by immuno-histochemistry Mice were analyzed at 79 days of Aplidine treatment.
Section of 4-6 μm were cut from paraffin embedded bone marrow samples and, after deparaffination, incubated with the anti-GATA- 1 monoclonal antibody (from Santa Cruz Biotechnology, Santa Cruz, CA) and the immunostaining developed with the Ultraystain polyvalent HRP Immunostaining kit (Ylem, Rome, Italy), as described by the manufacturer. Th e c h r o m o g e n w a s r e p r e s e n t e d by 3 . 3 '- diaminobenzedine (Sigma) and the development reaction occurred in the presence of H2O2 (0.03%) . Samples were counterstained with Hematoxylin and observed with light microscope (ZEISS AXIOSKOPE) equipped with a Coolsnap Videocamera to acquire images to analyze with the MetaMorph 6.1 Software (Universal Imaging Corp, Downingtown, PA).
The images were also used to calculate the frequency of morphologically recognizable megakaryocytes per mm2, the percentage of megakaryocytes that reacted with the Gatal antibody and the average intensity of the Gatal staining in the GatalP°s megakaryocytes. In each case, it was analyzed at least three field per mouse per a total of three untreated and three Aplidine treated Gatal low mice. The intensity of the Gatal immunostaining was determined with the MethaMorph program and expressed in pixels/cell. Values are expressed as mean (±SD) of these independent measurements and data obtained in untreated and Aplidine-treated mice compared by analysis of variance Anova with the Origin 6.1 program for Windows XP.
Results
1. Platelet count
An increase in platelet count was observed in all mice receiving Aplidine. Platelet increase was observed after the first cycle of treatment at day 15 and the increase was maintained throughout the subsequent cycles (Figure 9).
2. Hematocrit levels Changes in hematocrit level after the different cycles of treatment were not statistically considerable (Figure 10).
3. Bone marrow cellularity
Bone marrow cellularity was analyzed in mice that were sacrificed after 2 or 4 courses of Aplidine treatment. Two courses of Aplidine were not able to induce an increase in bone marrow cellularity; however, 4 courses of Aplidine increased bone marrow cellularity to almost normal levels (Figure 1 1).
4. Megakaryocyte number
Although not statistically significant, there was a trend towards less megakaryocyte number in bone marrow (Figure 12), liver and spleen. 5. Bone marrow fibrosis
Although not statistically significant, there was a trend towards a reduction in reticulin fibers /mm2. A reduction in bone marrow fiber deposition was observed after 4 courses of Aplidine treatment, as compared to vehicle treated animals (Figure 13).
6. Bone marrow microvessel density
A reduction in bone marrow microvessel density was observed after 2 courses of Aplidine treatment, as compared to vehicle treated animals. The effect in angiogenesis was more noticeable in animals sacrificed after 4 courses of Aplidine treatment, being the MVD reduced from 8+1.5 to 2.6+1.6 pixel arbitrary units (p<0.01) (Figure 14).
7. Cytokine mRNA levels mRNA levels for both TGF-beta and VEGF, measured by quantitative PCR, were significantly reduced in the bone marrow of Aplidine-treated mice (p <0.01 for both). A reduction in TGF-β mRNA levels was observed after two courses of Aplidine treatment, as compared to vehicle treated animals and the effect was maintained after 4 cycles of treatment (Figure 15). VEGF mRNA levels were also reduced after 2 courses of Aplidine treatment, as compared to vehicle treated animals; the effect was less noticeable after 4 courses of Aplidine treatment (Figure 16).
The observed inhibition of TGF-beta and VEGF expression, associated with reduced microvessel density, would suggest a possible activity of the drug in human PMF where levels of these two cytokines are abnormally increased.
8. Determination of GATA- I expression and bone marrow megakaryocyte frequency by immuno-histochemistry In Figure 17, it is shown that after the 4rth Aplidine treatment, there is an increase in Gatal expression in bone marrow megakaryocytes (34.2+2.6 vs 16.6+3.3 pixel unit/MK, p<.01). Moreover, the increment in blood platelet counts (Figure 9) is associated with a reduction in the frequency of megakaryocytes in the marrow (440+47 vs 636+37 MK/mm2 in treated and untreated mice, p<.05).
Finally, the frequency of megakaryocytes that do not react with the Gatal antibody (Gatal ne§ MKs) is slightly increased by Aplidine- treatment. However, the intensity of the immunostaining is greater in those megakaryocytes expressing Gatal in Aplidine-treated mice than in the vehicle control group. It is hypothetised that Gatal P°S megakaryocytes from Aplidine-treated mice progress in maturation more efficiently than those from control mice, which is associated with the increase in platelets counts, and are therefore selectively depleted from the marrow.
Discussion
Although the molecular lesion that reduces the GATA- I content in the megakaryocytes (MK) of the PMF patients is not known, the similarity between the maturation defects of the MK from the PMF patients and from GATA- llow mice suggests that agents that would restore MK maturation in mice would also restore MK maturation in PMF patients (Vannucchi AM et al., 2005, Am J Pathol, 167, 849-858). As such, these agents may cure those aspects of the human disease that are a direct consequence of the MK abnormalities. Overall, the obtained results lend support to the statement that Aplidine proved to have efficacy on some of the most characteristic pathologic abnormalities of the myelofibrotic trait that is displayed by GATA- llow mice. The improvement observed in several parameters after Aplidine treatment in the GATA- llow mice model, such as increase in platelet counts and related increase in megakaryocyte maturation and improvement of bone marrow cellularity, the reduction of bone fiber deposition and microvessel density in the bone marrow, and also the reduction of TGF-β and VEGFa mRNA levels (cytokines involved in the bone marrow stromal proliferation and neovascularisation, respectively); which possibly occurs as a result of improved megakaryocyte maturation, all speak in favour of the effectiveness of the treatment with Aplidine.
Example 3: Effects of Aplidine on the clonogenic activity and proliferation of cell lines
We evaluated the effects of Aplidine on the proliferation of human cell lines harboring homozygous (HEL and UKE- I) or heterozygous (SET2) JAK2V617F mutation. As a control, we used the BCR/ ABL mutant K562 cell line. The human erythroleukemia HEL, SET2 and K562 cell lines were purchased from DSMZ (German Resource Centre for Biological Material), and. UKE- I cells were a kind gift of Dr. W. Fiedler (Hamburg, Germany), (Fiedler W. et al., 2000, Cancer, 88 (2):344-351). UKEl cells were cultured in Iscove modified Dulbecco medium (IMDM) (Lonza, Ltd, Basel Switzerland) and HEL, SET2 and K562 cells were cultured in RPMI 1640 medium (Lonza, Ltd, Basel Switzerland). The antiproliferative activity of Aplidine was also studied in murine cells overexpressing the wild-type (Ba/F3-wt) or V617F mutant allele (Ba/ F3 V617F). Ba/ F3 cells were donated by Dr. R. Skoda (Basel, Switzerland), (Kralovics R. et al., 2005, N Engl J Med., 352 (17): 1779- 1790). These cells were cultured in RPMI 1 640 medium (Lonza, Ltd, Basel Switzerland). All medium were supplemented with 10% fetal bovine serum (Lonza, Belgium) (with the exception of SET2 cells which were supplemented with 20%), antibiotics and L-glutamine (200 mM in 0.85% NaCL solution). Medium for UKE- I also contained 10% horse serum (StemCell Techn.Inc, Canada) and 50 μM hydrocortisone (Sigma- Aldrich, Germany). Two different experimental approaches were used.
Agar clonogenic assay The agar colony assay explored the capacity of Aplidine to prevent clonogenic proliferation of the cell. Clonogenic assay for continuous cell lines was performed in 0.3% soft agar in Dulbecco's medium (Lonza, Ltd, Basel Switzerland) in the absence of growth factor, except in case of wild-type Ba/ F3 cells that were incubated in the presence of murine IL- 3 (50 pg/mL) (Miltenyi Biotech; Gladbach, Germany). Cells were plated at 103/mL in 35-mm dishes containing different final concentrations of Aplidine (from 0.01 to 10 nM), or no addition (control dishes) and allowed to incubated at 37°C in 5% CO2 atmosphere. Colonies were enumerated after 14 days using inverted microscopy (Nikon Eclipse TSlOO, Germany). The IC50 value, i.e., the concentration of Aplidine at which the number of colonies was reduced of 50% compared to control dishes, was calculated using plotted data.
Cell proliferation assay
In the second approach, a short-term (48-hr) proliferation assay in liquid cultures was performed. IxIO3 cells were plated into the wells of micro titer plates in 100 μL volume of RPMI 1640 (Lonza, Ltd, Basel Switzerland) with different concentrations of the drug. After 48 hr incubation, the tetrazolium salt WST- I (Roche Diagn . GmbH , Mannheim, Germany) was added to each well and incubated for 4 hr at 37°C in 5% CO2 atmosphere. The colored product was measured by spectrophotometry at 450 nm with reference wavelength at 650 nm. Experiments were performed in triplicate in three to seven independent experiments. The IC50, i.e., the drug concentration at which 50% inhibition of cell proliferation was observed, was determined using the Origin v7.0 software.
As shown in Figure 18, Aplidine was effective at very low nanomolar concentration in the prevention of the clonogenic potential of the different cell lines without overt differences among different cell lines.
Similar results were obtained in the proliferation assay. We found that Aplidine prevented cell growth with IC50 values of 1.0±0.3 nM for HEL, 0.5+0.03 nM for UKE- I , and 0.8+0.02 nM for SET2, that were all lower than 1 .5+0. 1 nM for the BCR/ABL mutated K562 cell line (P<.001 in case of UKE- I cells). Furthermore, in the proliferation assay in liquid cultures Ba/ F3 cells transduced with the V617F allele were found more sensitive to Aplidine (ICso= 0.03 + 0.0 1 nM) than their wild-type counterpart (IC5O= 0.4+0.03 nM; P<0.02). Overall, these data indicate that cells harboring JAK2V617F mutation are sensitive to Aplidine.
Example 4: Effects of Aplidine on the clonogenic activity and differentiation of progenitor cells (CD34+) from MPN patients
Blood samples from control subjects and from patients with Polycythemia Vera (PV) or Primary Myelofibrosis (PMF) , who were diagnosed according to the 2008 WHO criteria, were collected under an Institutional Review Board approved protocol and after obtaining an informed consent. Research was carried out according to the principles of the Declaration of Helsinki. Mononuclear cells were obtained from bone marrow aspirates or peripheral blood (PB) collected in preservative-free heparin after centrifugation over a Ficoll-Paque density gradient (Lympholyte, Cederlane, Canada). CD34+ cells were obtained by immunomagnetic selection (Miltenyi Biotech; Gladbach, Germany) according to manufacturer's instructions modified as described previously (Vannucchi AM et al., 2005, Am J Pathol., 167 (3):849-858). Purity of the isolated CD34+ cell population was evaluated by flow cytometry (FACScan, Becton Dickinson) after labeling with PE-HPCA2 anti-CD34 monoclonal antibody (Becton Dickinson).
Patient genotvping for the JAK2V617F mutation
Routine patient genotyping for the JAK2V617F mutation was performed on peripheral blood (PB) granulocytes separated by differential centrifugation over a Ficoll-Paque gradient (Lympholyte, Cederlane, Canada); contaminating red cells were removed by hypotonic lysis, and the cell pellet was processed for DNA purification using the QIAmp DNA blood Kit (Qiagen, GmbH, Germany). DNA was quantified with the NanoDrop technology (Wilmington, DE, USA) . Genomic analysis for JAK2V617F mutation was performed by an allele-specific (ASO) PCR using 75 ng granulocyte DNA, exactly as described by Baxter et al. (Baxter EJ et al., 2005, Lancet., 365 (9464): 1054- 1061). To evaluate whether the mutation was carried in the homozygous or heterozygous state, digestion of PCR products with BsaXl restriction enzyme (New England Biolabs, Hitchin, UK) was performed as described by Baxter et al.
Agar clonogenic assay The effects of Aplidine on the growth of BFU-E, CFU-GM and CFU-Mk from MPN patients were evaluated. All 5 PV patients and 4 of the 5 PMF patients analyzed were JAK2V617F mutated. Mononuclear cells from MPN patients or control subjects were plated at 105/mL for the growth of BFU-E and CFU-GM in 35-mm-diameter dishes in Methylcellulose (MethoCult, StemCell Technologies, Vancouver, Canada), without Aplidine or in the presence of different concentrations of Aplidine (0 to 10 nM), and in the presence of the following cytokines (all from Miltenyi Biotech; Gladbach, Germany): SCF 50ng/mL, IL-3 IOng/mL, IL-6 IOng/mL, GM-CSF IOng/mL, G-CSF IOng/mL and EPO 3U/mL. For the growth of CFU-Mk, an enriched fraction of CD34+ progenitor cells was used. CD34+ cells were plated at 5xlO4/mL in a 24-well plate in MegaCult-c Collagen and Medium with lipids (StemCell Technologies, Vancouver, Canada), in presence of the following cytokines (all from Miltenyi Biotech; Gladbach, Germany): Thrombopoietin 50ng/mL, IL-3 10ng/mL, IL-6 10ng/mL. Cultures were incubated at 37°C in a humidified 5% CO2 atmosphere, and colonies were enumerated at day 14 according to standard criteria. As shown in Figure 19, cells obtained from PMF patients presented significantly lower IC50 values than controls (Ctrl; P<.002) for all type of clonogenic progenitors; cells from PMF pts resulted also significantly more sensitive to Aplidine than those from PV patients (P<.02), while the difference between PV cells and controls (Ctrl) did not reach the significance level.
Single colony genotvping of BFU-E progenitors from PMF patients harboring the JAK2V617F mutation
Single colony genotyping of BFU-E progenitors was performed to quantify the proportion of hematopoietic colonies harboring the JAK2V617F mutation which grew in the presence of 1 nM Aplidine. For single colony genotyping, well delimited colonies of BFU-E progenitors were individually plucked off the methylcellulose, placed into each well of a 96-well plate, denatured at 99°C for 5 minutes, and directly processed for JAK2 genotyping by PCR, as described Baxter et al.. Initial data in 3 patients is available. As shown in Figure 20, in one patient the proportion of JAK2 -mutated BFU-E colonies decreased from 51% to 27% while no changes were observed in the other two patients. These data suggest that, at least in some patients, Aplidine may preferentially inhibit the growth of V6 1 7F mutant clonogenic progenitors.
Effect of Aplidine in the latest stages of differentiation of CD34+ cells from PMF patients To evaluate whether Aplidine also affected the latest stages of differentiation and maturation of megakaryocytes (MKs), the generation of CD61+ Mks was evaluated by a two-stage liquid culture system initiated with CD34+ cells purified from the peripheral blood (PB) of PMF patients. CD34+ cells purified from PMF patients and control subjects were plated at 105/mL in 500 μL volume in a 48-well plate in serum-free medium (SYN-H medium; AbCys Synergie, Paris, France) supplemented with the following recombinant cytokines (all from Miltenyi Biotech; Gladbach, Germany): SCF 5 (ng/mL), IL-3 (2 ng/mL), 11-6 (1 ng/mL), IL- I l (40 ng/mL) and TPO (50 ng/mL) for the first seven days of culture. At that time, cells were transferred to a 24-well plate and the culture volume was adjusted to 1 mL with Stem Alpha A serum free medium (STEM ALPHA, St Genis l'Argentiere, France) supplemented with the same cytokine as above. To enumerate CD61 + Mks, cells were recovered from the culture, labeled with CD41-PE and CD61-FITC (both from Becton Dickinson) and analyzed by flow cytometry using a FACSan (Becton Dickinson). As shown in Figure 21, it was found that the number of CD61 + cells was no different between cultures containing or not Aplidine, overall suggesting that the drug mainly affected early proliferation of Mk progenitors rather than influencing their differentiation.

Claims

1. A method for treating any mammal, notably a human, affected by a chronic myeloproliferative disorder, comprising administering to the affected individual a therapeutically effective amount of Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof.
2. The method according to claim 1 , wherein said chronic myeloproliferative disorder is a BCR-ABL negative myeloproliferative disorder.
3. The method according to claims 1 or 2, wherein said chronic myeloproliferative disorder is characterised by an increase in the proliferation of abnormal megakaryocytes.
4. The method according to claim 1 , wherein said chronic myeloproliferative disorder is primary myelofibrosis.
5. The method according to claim 4, wherein the primary myelofibrosis is an early stage primary myelofibrosis.
6. The method according to claim 1 , wherein said chronic myeloproliferative disorder is polycythemia vera.
7. The method according to claim 1 , wherein said chronic myeloproliferative disorder is post-poly cythemia vera myelofibrosis.
8. The method according to claim 1 , wherein said chronic myeloproliferative disorder is essential thrombocythemia.
9. The method according to claim 1 , wherein said chronic myeloproliferative disorder is post-essential thrombocythemia myelofibrosis.
10. The method according to any of the preceding claims, wherein the Aplidine or Aplidine analogue is Aplidine.
1 1. The method according to any of the preceding claims, wherein the patient is harbouring the JAK2V617F mutation.
12. The method according to any of the preceding claims, wherein the affected individual is a human patient.
13. The use of Aplidine, an Aplidine analogue or a pharmaceutical acceptable salt thereof, in the manufacture of a medicament for a method according to any of the preceding claims.
14. A pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or vehicle, to be used in a method according to any of claims 1 to 12.
15. Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, for use in a method according to any of claims 1 to 12.
16. A medical kit for administering Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, comprising printed instructions for administering it according to the method of any of claims 1 to 12, and a pharmaceutical composition comprising Aplidine, an Aplidine analogue or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or vehicle.
17. A method for treating a human subject having a chronic myeloproliferative disorder, said method comprising: a) obtaining a nucleic acid sample from said human subject; b) determining the presence of the JAK2V617F mutation; and c) treating with Aplidine the JAK2V617F positive subjects.
18. A method for determining an increased likelihood of pharmacological effectiveness of treatment by Aplidine in a human subject diagnosed with a chronic myeloproliferative disorder comprising obtaining a nucleic acid sample from said human subject and determining the presence of the JAK2V617F mutation, wherein the identification of the JAK2V617F mutation indicates an increased likelihood of pharmacological effectiveness by Aplidine.
19. The method according to any of claims 17 or 18, wherein said chronic myeloproliferative disorder is primary myelofibrosis.
PCT/EP2009/061820 2008-09-12 2009-09-11 Aplidine in the treatment of chronic myeloproliferative disorders WO2010029158A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP08380268 2008-09-12
EP08380268.6 2008-09-12
EP09382073.6 2009-05-20
EP09382073 2009-05-20

Publications (1)

Publication Number Publication Date
WO2010029158A1 true WO2010029158A1 (en) 2010-03-18

Family

ID=41397529

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/061820 WO2010029158A1 (en) 2008-09-12 2009-09-11 Aplidine in the treatment of chronic myeloproliferative disorders

Country Status (1)

Country Link
WO (1) WO2010029158A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8258098B2 (en) 2006-02-28 2012-09-04 Pharma Mar, S.A. Antitumoral treatments

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060178298A1 (en) * 2003-03-12 2006-08-10 Bertino Joseph R Antitumoral treatments

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060178298A1 (en) * 2003-03-12 2006-08-10 Bertino Joseph R Antitumoral treatments

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MESA RUBEN A: "New insights into the pathogenesis and treatment of chronic myeloproliferative disorders.", CURRENT OPINION IN HEMATOLOGY MAR 2008, vol. 15, no. 2, March 2008 (2008-03-01), pages 121 - 126, XP009126845, ISSN: 1065-6251 *
TEFFERI A ET AL: "Classification and diagnosis of myeloproliferative neoplasms: The 2008 World Health Organization criteria and point-of-care diagnostic algorithms", LEUKEMIA (BASINGSTOKE), vol. 22, no. 1, January 2008 (2008-01-01), pages 14 - 22, XP002560015, ISSN: 0887-6924 *
VERRUCCI M ET AL: "APLIDIN IMPROVES MEGAKARYOCYTOPOIESIS AND HALTS NEO-ANGIOGENESIS IN THE GATA1(LOW) MURINE MODEL OF MYELOFIBROSIS", HAEMATOLOGICA-THE HEMATOLOGY JOURNAL, vol. 94, no. Suppl. 2, June 2009 (2009-06-01), & 14TH ANNUAL MEETING OF THE EUROPEAN-HEMATOLOGY-ASSOCIATION; BERLIN, GERMANY; JUNE 04 -07, 2009, pages 266 URL - http://ww, XP009126805, ISSN: 0390-6078(print) 1592-8721(ele *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8258098B2 (en) 2006-02-28 2012-09-04 Pharma Mar, S.A. Antitumoral treatments

Similar Documents

Publication Publication Date Title
Arcasoy et al. Functional significance of erythropoietin receptor expression in breast cancer
Kabiri et al. Wnts are dispensable for differentiation and self-renewal of adult murine hematopoietic stem cells
Juntilla et al. AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species
Vannucchi et al. Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1low mice)
Allegrezza et al. Trametinib drives T-cell–dependent control of KRAS-mutated tumors by inhibiting pathological myelopoiesis
Kota et al. Aberrant signal transduction pathways in myeloproliferative neoplasms
ES2418433T3 (en) Modulation of catecholamine receptors
US8741640B2 (en) Expanding hematopoietic stem cells
US20170252362A1 (en) Methods &amp; compounds useful in hematopoietic stem cell medicine
Müller-Newen et al. Development of platelets during steady state and inflammation
US20180344777A1 (en) Method of modulating müller glia cells
JP2015134769A (en) Methods of inhibiting tumor cell aggressiveness using microenvironment of human embryonic stem cells
WO2012159044A1 (en) Stem cell mobilization and tissue repair and regeneration
US9034820B2 (en) Method and composition for modulating erythropoiesis
WO2010029158A1 (en) Aplidine in the treatment of chronic myeloproliferative disorders
CN110551688B (en) Composition for inducing reprogramming of somatic cells into hematopoietic stem/progenitor cells and promoting in-vitro expansion of hematopoietic stem/progenitor cells and application thereof
EP3052119B1 (en) Methods of modulating erythropoiesis with arginine vasopressin receptor 1b molecules
US20130072844A1 (en) Use of entrained neutrophils to treat metastatic and micrometastatic disease in at risk patients
EP4410964A1 (en) Medium composition for culturing urine-derived stem cell, method for isolating and culturing urine-derived stem cell using same, urine-derived stem cell with improved therapeutic function for renal disease, and cell therapy product composition containing same
WO2015174443A1 (en) Megakaryocytic differentiation induction and platelet production promoter containing interleukin 1α (il-1α)
CN112472695A (en) Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals
Sugiyama et al. Definitive hematopoiesis from acetyl LDL incorporating endothelial cells in the mouse embryo
CN107243006B (en) Use of AMD3100 in the manufacture of a medicament for the treatment and/or prevention of cachexia
WO2014126175A1 (en) Composition for preventing or treating diabetes
Weig et al. Impaired platelet function reduces myocardial infarct size in Gαq knock-out mice in vivo

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09782927

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09782927

Country of ref document: EP

Kind code of ref document: A1